NEUROPEPTIDE RECEPTORS AND SECOND MESSENGERRESPONSES IN ASTROCYTES FROM RAT CENTRAL NERVOUS SYSTEM.

BY

JACQUELINE C. REID

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph. D)

IMPERIAL COLLEGE OF SCIENCE. TECHNOLOGY AND MEDICINE.

UNIVERSITY OF LONDON.

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r

ABSTRACT.

In order to identify astrocytes in culture and in vivo, a polyclonal antibody to glial fibrillary acidic protein, a specific astrocyte marker, was prepared. The protein was purified from bovine brain using hydroxylapatite chromatography, polyacrylamide gel electrophoresis and Western blot analysis. The antibody was further purified using DEAE Affi-gel chromatography.

In previous studies43, activation of the lithium-inhibited phosphoinositide response had been found when stimulating astrocytes wih supramaximal concentrations of neuropeptides. In this study dose-response curves were used to determine the ED50 values for some of these peptides in cultures of astrocytes from different brain regions. This data indicated that the PI responses were probably receptor-mediated, since it was found that a range of peptides were active at physiological levels (10”8 - 10“10 M) in both cortex and spinal cord. Regional and developmental differences in the PI responses, particularly of the tachykinins, were found. The spinal cord apparently developed earlier than the cortex. The levels of response, to the same stimulation, were found to be much higher in the spinal cord than those in the cortex.

Kinetic characteristics of the tachykinin receptors on astrocytes from different brain regions were determined using [^25I]BHSP, prepared and purified using HPLC

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procedures. Hot saturation, cold saturation and displacement experiments were carried out, using substance P, neurokinin A and neurokinin B as displacers. This data indicated that two binding sites of high (10-"11) and lower (10-8) affinity were found on both spinal cord and cortex astrocytes. However, the binding characteristics showed a similar disparity, as in the PI responses, between the brain regions. The level of receptors in the cortex was much lower than in spinal cord. The developmental profile of the spinal cord matched the PI response, but the cortex binding differed. In vivo binding experiments on lesioned brain sections were inconclusive.

The results are discussed with reference to factors which may contribute to the regional and developmental variations found.

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DEDICATION.

This study is dedicated to my mother.

With love, always.

SectionCONTENTS.

Pace1. INTRODUCTION 92. GLIAL FIBRILLARY ACIDIC PROTEIN 212.1 INTRODUCTION 212.2 MATERIALS AND METHODS 222.2.1. GFAP Isolation 222.2.1.1 Materials 222.2.1.2 Axonal flotation 232.2.1.3 Hydroxylapatite chromatography 242.2.2. Polyacrylamide gel electrophoresis 252.2.2.1. Gel recipes 252.2.2.2 . Stock solutions 262.2.2.3 . Sample preparation 262.2.2.4. Method 262.2.3. Western blotting 282.2.3.1 Method 282.2.3.2 . Staining of Western blots 302.2.4. Polyclonal antibody production 312.2.4.1. Method - bleeding 312.2.4.2 . Method - injections 312.2.4.3. Antibody testing 322.2.4.4. Antibody purification 332.2.5. Protein estimation 342.2.5.1. Stock solutions 342.2.5.2 . Reaction solutions 342.2.5.3 . Sample preparation 352.2.5.4. Method 352.2.6. sImmunofluorecence studies 352.2.6.1. Method - Sections 352.2.6.2. Method - Cultures 362.3. RESULTS 372.4. DISCUSSION 423. THE PHOSPHOINOSITIDE RESPONSE 453.1. INTRODUCTION 453.1.1. History 453.1.2. Mechanism of reaction 503.1.3. PI response in the nervous system 543.1.4. Role of PI system 583.1.5. Measurement of PI system 623.1.6. PI response of astrocytes 65

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3.2. METHODS 663.2.1. Tissue culture 663.2.1.1. Astrocyte media 663.2.1.2. Serum dialysis 673.2.1.3. Astrocyte culture 673.2.1.3.1. Cortex 683.2.1.3.2 . Spinal cord 693.2.1.3.3. Cerebellum 693.2.2.1. PI assay 693.2.2.2. DIV assay 713.2.2.3 . Separation of inositol phosphates 713.3. RESULTS 723.4. DISCUSSION 994. RECEPTOR BINDING STUDIES 1104.1. INTRODUCTION 1104.1.1. Astrocyte receptors 1104.1.2. Theoretical background 1114.2. METHODS 1244.2.1. High performance liquid chromatography 1244.2.2. Iodination of substance P 1244.2.3. Cell culture 1254.2.4. Binding assay 1254.2.5. Lesions 1264.2.6. Autoradiography / Immunocytochemistry 1274.3. RESULTS 1294.4. DISCUSSION 1485. DISCUSSION 1556. APPENDICES 1636.1. Appendix 1 - Tissue culture solutions 1646.2. Appendix 2 - Receptor binding theory 1656.3. Appendix 3 - HPLC programmes 1786.4. Appendix 4 - Abbreviations 1807. ACKNOWLEDGEMENTS 1838. REFERENCES 184

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LIST OF FIGURES AND TABLES.Page

Figure 1. Amino acid sequences of neuropeptides. 14 Figure 2a. Cortical astrocyte culture. 15Figure 2b. Spinal cord astrocyte culture. 15Figure 3. Diagram of Transblot apparatus. 29Figure 4. GFAP elution from Hydroxylapatite column. 38 Figure 5. PAGE and Western blots of GFAP. 40Figure 6. Purification of GFAP antibody. 41Figure 7. Structure of myo-inositol. 46Figure 8. The phosphoinositide reaction cycle. 51Figure 9. Time course of cortical SP-stimulated PI

response. 73Figure 10. Time course of spinal cord SP-stimulated

PI response. 74Figure 11. PI dose-response of SP in cortex. 76Figure 12. PI dose-response of NKA in cortex. 77Figure 13. PI dose-response of NKB in cortex. 78Figure 14. PI dose-response of EL in cortex. 79Figure 15. PI dose-response of BK in cortex. 80Figure 16. PI dose-response of VP in cortex. 81Figure 17. PI dose-response of OT in cortex. 82Figure 18. PI dose-response of SP in spinal cord. 83 Figure 19. PI dose-response of NKA in spinal cord. 84Figure 20. PI dose-response of NKB in spinal cord. 85Figure 21. Overview of spinal cord PI responses. 88Figure 22. Overview of cerebellar PI responses. 89Figure 23. Developmental SP-stimulated PI response. 90 Figure 24. Developmental NKA-stimulated PI response. 91 Figure 25. Developmental NKB-stimulated PI response. 92 Figure 26. Developmental EL-stimulated PI response. 93 Figure 27. Developmental BK-stimulated PI response. 94 Figure 28. IP separation of SP-stimulated cortex. 97Figure 29. IP separation of BK-stimulated cortex. 98Figure 30. Structure of Bolton-Hunter reagent. 116Figure 31. HPLC profile of substance P. 130Figure 32. HPLC separation of iodinated [125I]BHSP. 131 Figure 33. Time course of [125I]BHSP binding. 134Figure 34. Scatchard analysis of saturation studies

in spinal cord. 135Figure 35. Scatchard analysis of saturation studies

in cortex. 136Figure 36. Saturation binding of [125I]BHSP to

cortex. 138Figure 37. Saturation binding of [125I]BHSP to

spinal cord. 139Figure 38. Scatchard analysis of displacement

studies in spinal cord. 140Figure 39. Scatchard analysis of displacement

studies in cortex. 141Figure 40. Displacement profile of [125I]BHSP in

spinal cord. 143Figure 41. Displacement profile of [125I]BHSP in

cortex. 144Figure 42. Developmental profile of [125I]BHSP

binding. 1457

Figurei 43. Autoradiogram of [125I]BHSP bindingto brain sections. 147

Table 1. Properties of astrocytes 10Table 2. Astrocyte receptors. 48Table 3 . Summary of ED50 values. 86Table 4. Separation times and dissociation

constants. 118Table 5. Protease inhibitors. 121Table 6. Summary of kinetic data. 136ATable 2A. Summary of results showing purity of both

spinal cord and cortical neonatal cultures.Table 2B. Summary of results showing the purity of

spinal cord cultures in different media.Table 3A. Table illustrating the range of lipid counts

gained from SP dose-response curves on cortex and spinal cord cultures.

66A66B99A

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1. INTRODUCTION.

The aim of this study was to confirm and extend studies on the existence, and functional responses to stimulation of tachykinin receptors on cultured astrocytes from neonatal rat brain. The morphological intimacy of neurons and astrocytes suggests that these cells interact biochemically, and therefore that they communicate in some way. Neurons release neurotransmitter(s) on activation, and the presence of neurotransmitter receptors on astrocytes indicates their sensitivity to these substances. This may comprise a neuronal-glial signalling system. The detection and characterization of the effects of neurotransmitters/neuromodulators on glial cells would be useful in providing more information concerning a possible neuronal-glial link. Glial cells have been shown to release substances and to modulate the neuronal ionic microenvironment. These effects may additionally form a neuronal-glial-neuronal link.

Although several properties of astrocytes have been suggested (see Table 1), with varying degrees of experimental support, their full functional profile is, as yet, unknown. Historically, they were assigned a structural role in the physical support of neurons, hence the term 'neuroglia = nerve glue'. It has also been suggested that they are involved, in the repair and regeneration of neurons, since they retain the

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Table 1 Recognised astrocyte properties.

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TABLE 1. PROPERTIES OF ASTROCYTES.

1. STRUCTURAL SUPPORT

2. REPAIR AND REGENERATION

3. DEVELOPMENTAL GUIDANCE

4. POTASSIUM FLUX

5. UPTAKE AND RELEASE OF NEUROTRANSMITTERS

6. BLOOD-BRAIN BARRIER

7 . CNS IMMUNE RESPONSE

ability to divide throughout their life, and proliferate in areas of neuronal damage237. They have been traditionally ascribed a hindering role, in the imposition of the glial scar as a physical barrier to regeneration. However, it has recently been suggested that they may have a promotional role in providing a substratum for neuronal regeneration237. Astrocytes, from species which are able to regenerate neurons, have demonstrated laminin-immunoreactive glia in regenerating areas163. It is suggested that astrocytic laminin indicates a regenerating potential. Also, it has been suggested that orthogonal arrays of particles (OAP's), found on glia by freeze-fracture techniques in the olfactory system, may be related to the ability of astrocytes to affect neuronal regeneration175. The localization of these particles on astrocytic membranes apposed to the endothelial cells of the blood-brain barrier has also implicated them in the transport of substances to and from neurons152. However, because the relatively high electrical resistance of the glial membrane discourages direct diffusion of materials, a directed release and uptake of substances is more likely149.

A role for astrocytes has been indicated in developmental guidance of neurons234'65 by the formation of a glial palisade for subsequent neuronal organization, although this may not apply to all populations. Another property, which has been

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suggested, is that glia act as a spatial buffering mechanism via their K+ channels, providing a current loop through the intracellular and extracellular space, where a K+ gradient exists. Glial cells also contain voltage dependent Na+ and Ca2+, and Cl” channels25'26, suggesting an involvement in the modulation of the ionic composition of the extracellular fluid. They may also exert a modulatory effect by means of their ability to take up neurotransmitters by specific mechanisms296'143, and to release factors into the microenvironment. The control of these systems is presently uncharacterized, but there are indications that some release mechanisms may bereceptor-mediated223. Astrocytes can also be induced to express la antigens in vitro. suggesting a possible involvement in the immune response79.

Initially, it was necessary to review the methodological outlines for this study. Therefore, the use of cultured cells, as opposed to an in vivo study was considered. The former would provide a simple, easily prepared system, with a relatively homogeneous population of cells. It would also have the disadvantage of not necessarily providing a true facsimile of the in vivo situation, because of the exclusion of other central nervous system cell types. The latter method would obviate this problem, but in its stead would produce new problems. The foremost of these involves the difficulty of examining glial cells

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exclusively. In order to examine the receptor population on astrocytes their responses to certain neuropeptides wene. examined. Previous studies had defined many of the receptor-mediated cAMP responses102'284, while the examination of the phosphoinositide (PI) response of astrocytes was in its infancy217'43. The latter study had indicated that a range of neuropeptides produced a PI response in astrocyte cultures at supramaximal concentrations.Using this data it was decided to explore the nature of these responses to a limited range of peptides; both to determine if they were receptor-mediated, and to understand their physiological relevance. The choice of peptides was determined by their effectiveness in eliciting a significant PI response in astrocytes derived from at least one brain region. Of particular interest were the tachykinins, specifically, substance P (SP), neurokinin A and B (NKA, NKB), and to a lesser degree, eledoisin (EL), since these, despite their structural similarities (see Figure 1), showed distinct regional differences in their PI responses. Bradykinin (BK) demonstrated its suitability as a positive control in the measurements of PI response, because of the uniformly high response with all brain regions.Emphasis was placed on the cortical and spinal cord regions since these cultures provided the larger responses43 and also have the advantage that they can be prepared from animals at the same time point. Cultures from both regions (see Figure 2) were taken

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Figure 1. Amino acid sequences of neuropeptides used in this study. The structural similarity in N-terminal sequence of the tachykinins is of particular note.

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AMIND ACID SEQUENCES

11 10 9 8 7 6 5 4* 3 2 1S ubstance P: A rg - P ro - Lys - P ro - Gin - Gin - Phe - Phe - Gly - Leu - Met NH^

Neurokinin A: His - Lys - T hr - Asp - Ser - Phe - Val - Gly - Leu - Met nh^

Neurokinin B: Asp - Met - His - Asp - Phe - Phe - Val - Gly - Leu - Met nh^

Eleoloisin: pGlu - P ro - S er - Lys - Asp - Ala - Phe - lie - Gly - Leu - Met nh2

Braolykinin: A rg - Phe - P ro - Ser - Phe - Gly - P ro - P ro i > ID nh2

V a so p re ss in : Cys - T y r - Phe - Glu - Asp - Cys - P ro - A rg - Gly nh2

s------------------------------------- s□ xy to c in : Cys - T y r - lie - Gin - Asn - Cys - P ro - Leu - Gly NH^

S----------------------------------------------------------S

pGlu - His - P ro NH

Figure 2a. This photograph shows a neonatal cortical astrocyte culture at 12 DIV, stained by indirect immunofluorescence, with anti-GFAP/TRITC. Magnification x 288; 10 mm = 35 /im.

Figure 2b. This photograph shows a neonatal spinal cord astrocyte culture at 12 DIV, stained as Fig. 2a. Magnification x 288; 10 mm = 35 fim.

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from rats at 2 dpn, in order to eliminate any artefactual variations which may derive from differential ontogenetic profiles produced when cells from different time points are cultured107. This also allows the comparison of developmental profiles of PI response and binding, which may compare to reports of other systems249'107,286,215,10^

It is not yet technically feasible to co-localise specific astrocyte immunocytochemical markers and either radioligand binding at high resolution, or physiological responses in a normal in vivo situation. However, both radioligand binding and immunocytochemical labelling have been carried out in vitro186. Also the presence of /3-receptors has been shown on astrocytes both in vivo and in vitro186'109. Electron microscopic studies have revealed the presence of /3-AR-LI in vivo on astrocytes, and localized it on membranes apposed to neuronal synapses, implying a neuronal-glial link via /3-AR5. This lends credence to the theory that characteristics of the cultured astrocytes could parallel those in vivo. It was intended that the presence of receptors on astrocytes in vivo may be demonstrated using a combined autoradiographic and immunocytochemical approach on gliosed brain tissue. The effects of lesions on tachykinins and their receptors has been examined l8!/7 '45'H ? SP-IR in the substantia nigra, normally the highest in brain, is reduced by ibotenate

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lesions, and kainate lesion of the corpus striatum results in a depletion of the SP receptors in the substantia nigra, but induces an increase in the cortex181. Lesion studies in the spinal cord revealed that some SP-IR fibres survived dorsal rhizotomy, and these were assumed to be local interneurons. An electron microscopic study, also showed that SP-IR axons were in close association, and made synaptoid contacts with astrocytic processes in laminae I - III, suggesting that SP has activity, not only at synaptic junctions, but also with glial cells12.

The role of the tachykinins in the central nervous system been investigated since the discovery of substance P by von Euler in 1931287, and the recent discovery of the other mammalian tachykinins, NKA and NKB145. All tachykinins (mammalian and non-mammalian) can be divided into two groups based on their structural activities, which are derived from the aliphatic (NKA, NKB, EL, KASS) or aromatic (SP, PHY) nature of the amino acid in position *(Fig. I)177. As yet no clear function for these peptides has been determined. They show a similar range of biological activities, usually differing only in magnitude. Peripherally, tachykinins have potent effects on blood pressure, vasodilation, salivation and smooth muscle contraction126'114. Centrally, they increase locomotion, wet-dog shakes and grooming behaviour, and SP and NKA inhibit water intake; some of these effects

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are selective for specifictachykinins189, 69,99,114,221 a The effects of the tachykinin peptides on a series of bioassay systems have been reviewed73. In spinal cord, substance P has been suggested as a endogenous pain neurotransmitter, mediating its effect via specific tachykinin subtypes225'212'153, although this is contradicted in a recent series of papers84'3^/85,29 ̂ This confusion may be a reflection of the mediation of varying effects by different tachykinins through selected receptor subtypes. Alternatively, the responses produced by tachykinins may be mediated via modulation of the effects of other transmitters, for example, opioids, bradykinin, dopamine 153,256,14# similarly, the effects of SP may be modulated by other transmitters such as GABA 248. The study of endogenously released SP effects is complicated somewhat by the findings that, in several areas, SP is co-localized with other neurotransmitters, such as 5-hydroxytryptamine (5-HT), and calcitonin gene-related peptide (CGRP)294'86'290. CGRP and SP also demonstrate the modulation of transmitter effects by the co-released transmitter, since CGRP prolongs the SP effect by inhibiting the activity of a peptidase294. Consequently it is not possible to differentiate the effects of stimulation of neurons co-localizing more than one peptide/transmitter.

The tachykinins are derived from two genes;18

preprotachykinin A (PPTA) and preprotachykinin B (PPTB). PPTA gives rise, via alternative splicing of the gene to three mRNA's a-, /3-, and r-preprotachykinins, of which the first encodes SP, while the latter two both encode SP and NKA148'177. Recently, N-terminally elongated forms of both SP and NKA have been described270'110. They have been found to be widely and differentially distributed in brain tissues using immunoreactivity and radioimmunoassay techniques6'179'35'34'166'267'159'268* However, earlystudies of substance P distribution may encompass all the tachykinins because of the non-selectivity of the antisera35. Most recently the use of molecular biological techniques has enabled the demonstration of the distribution of the mRNA's for the tachykinin precursors, which has supported the earlier findings of regional variations289.

It has been generally assumed that the SP-immunoreactivity (SP-IR) examined in these studies derives from neuronal sources. This assumption must now be qualified, since SP-immunoreactive astrocytes have been detected near to striatal blood vessels in neonatal human brain191. Other peptides (Angiotensin I, Angiotensin II) have also been detected in cultured astrocytes from rat brain at the same concentrations as they are found in neurons118. This implies that conclusions drawn from peptide distribution studies of brain should not ignore the possible contribution of

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glial cells.

The distribution of SP receptors in brain has also been widely reported227 > 41' 16 / 207,59,275,242,159,17 ̂These generally incorporate the binding to all tachykinin subtypes, although some reports now demonstrate the regional variations in binding of the three tachykinin subtypes NK^, NK2, and NK3246'58. However, as with many other neurotransmitter systems, there appears to be a degree of mismatch between the receptor and peptide localizations117. No direct comparative studies have been carried out, but information drawn from separate studies seems to indicate differential localization of the individual tachykinins and their respective receptor subtypes289^246. The system is necessarily complicated by the range of affinities of the various tachykinins for the subtypes, the presence of multiple receptors, possibly within each subtype, and the fact that measurement at any one time point may not be representative, if there is developmental regulation of either the peptides or the receptors. This study was performed to examine the possibility of developmental and regional variations in SP responses in, and binding to astrocytes.

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2 . GLIAL FIBRILLARY ACIDICPROTEIN.

2.1. INTRODUCTION.

Glial fibrillary acidic protein (GFAP) is the major component of the glial form of the group of intermediate filaments52'71. These are so called because, in size (10 nm), they fall between microtubules (24 nm) and microfilaments ( 6 - 7 nm). The function of intermediate filaments (IFs) is not known, but it is possible that they have a role in stabilising cell shape33'56. However they possess certain biochemical properties which are useful when examining some cell types. One such property is the cell specificity of the IFs as suggested originally by the histological stains of Weigert (glia), 1895 and Cajal (neurones and glia), and subsequently demonstrated extensively27.

GFAP, as one would expect is found predominantly in glia, specifically, in astrocytes. The initiating factors for GFAP synthesis are unkown, but their distribution in the cytoplasm is apparently dependent on microtubules91. GFAP is a protein of MW 49 - 54000 (depending on source and method of analysis), synthesized by free polysomes. Recently, human cDNA clones were identified236 which coded for a 49000 MW protein, as previously described93. It is rapidly degraded into smaller, more acidic fragments, most of which are immunoreactive. The immunoreactive portion of

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the protein cannot apparently be easily identified.Several studies with monoclonal antibodies have produced varying results61. The molecule is generally well-conserved through different vertebrate species, although there are differences between the peripheral and central nervous system forms51'55. Consequently, it is an ideal immunological marker for astrocytes. It is particularly useful for marking damage in the nervous system, where reactive astrocytes rapidly become intensely immunoreactive. In disease states GFAP is increased, e.g. in multiple sclerosis plaques, in Alzheimers disease, in neurofibrillary tangles, and in CSF in hydrocephalus4 /70,213,279 a

I have used these properties of the protein to produce antibodies which selectively mark astrocytes in cell cultures.

2.2. MATERIALS AND METHODS.

2.2.1. G.F.A.P. Isolation

This is based on a modification of the method published by Liem162.

2.2.1.1. Material

Bovine brains and spinal cord were obtained fresh from a slaughterhouse, and were transported frozen in dry ice.

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They were stored at -80°C until use.

In initial experiments white matter from brains was used, however spinal cord was later used more successfully. The final preparation is described.

2.2.1.2. Axonal flotation

The white matter was dissected out, removing the outer arachnoid membrane, weighed, and homogenized in solution A (10 mM phosphate buffer pH 6.8, 0.1 M NaCl, 1 mM E.D.T.A.,2 mM P.M.S.F.) + 0.85 M sucrose, in a 1:4 ratio of tissue to buffer. This facilitated the separation of the enriched fraction of myelinated axons from other tissue, by centrifugation at 14,000 g for 15 minutes at 4°C. The floating pads of myelinated axons containing the intermediate filaments were removed, and manually rehomogenised in solution A + sucrose. Centrifugation and homogenization were repeated twice more as washing steps, finally rehomogenising in solution B (solution A + 1% Triton X-100), which lyses the axons. After stirring for 48 hours at 4°C, to remove myelin, this solution was layered on top of solution A + 0.85 M sucrose and centrifuged at 250,000 g for 45 minutes at 4°C. The resulting protein pellet was rehomogenized in solution A and retained, frozen, at -20°C until column separation.

Samples were taken at stages, in order to determine protein content for gel electrophoresis, and Western

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blotting.

2.2.1.3. Hvdroxvlapatite chromatography

Hydroxylapatite (BIO-RAD, Biogel HTP) was suspended in 10 mM phosphate buffer pH 7.4 and washed thoroughly. The column (26 cm x 2.5 cm) was equilibrated with three column volumes of 10 mM phosphate buffer + 8 M urea. The pellet suspension was thawed and centrifuged at 30,000 g for 45 minutes at 4°C. The resulting pellet was rehomogenized manually in 10 mM phosphate buffer + 8 M urea + 1% mercaptoethanol in order to dissolve the proteins. This was respun at 100,000 g for 30 minutes at 4°C to remove undissolved material, and the supernatant was applied to the column. Intermediate filament proteins are known to adsorb to the column even in the denatured state162. They were then separated by absorption chromatography, eluting the different proteins with increasing phosphate concentrations.

Proteins were eluted sequentially with 10 mM phosphate buffer pH 7.4, followed by 0.15 M phosphate buffer pH 7.0, then 0.3 M phosphate buffer pH 7.0; all contained 8 M urea. Fractions of 5 mis were taken, the absorption at 280 nm measured spectrophotometrically, and a protein profile plotted.

Fractions containing the three protein peaks were individually pooled and dialysed at 4°C against the

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appropriate phosphate buffer omitting 8 M urea and including 1 mM E.D.T.A. and 0.1 M NaCl, with one change of buffer.

Each dialysate was concentrated by ultrafiltration to 15 mis using an Amicon YM10 membrane (molecular weight cutoff at 10,000).

SDS-Polyacrylamide gel electrophoresis and Western blotting was performed on the three fractions.Tris-glycine SDS-PAGE was used in preference to phosphate SDS-PAGE, since tubulin and GFAP have been found to co-migrate on the latter244.

2.2.2. Polyacrylamide gel electrophoresis-^-^.

2.2.2.1. Gel Recipes for SDS-PAGE-^^.

Stock Solution Stackincr ael Resolvina ael3.75% 10%

Acrylamide/bisacrylamide30%:0.8% 2.5 ml 13.3 ml

Stacking gel buffer 5.0 " -Resolving gel buffer - 5.0 "10% SDS 0.2 " 0.4 "1.5% ammonium persulphate 1.0 " 2.0 "Distilled water 11.3 " 19.3 "TEMED 15 /il i—1o<N

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2.2.2.2. Stock solutions

Stacking gel buffer Resolving gel buffer Reservoir buffer

0.5 M Tris-HCl pH 6.8 3.0 M " " pH 8.80.25 M Tris + 1.92 M glycine

+ 1% SDS, pH 8.3

2.2.2.3. Sample preparation

The protein sample for analysis was diluted to a suitable concentration (single protein = 1 0 - 2 0 f ig ,

protein mixture = 40 - 60 f ig ) adding the following:-

Solution 10% SDSStacking gel buffer GlycerolBromophenol blue Mercaptoethanol Distilled water

Final concentration2.5 %0.0625 M

10 % 0.0033 %5 %

to volume

The mixture was then boiled for ten minutes and centrifuged (bench centrifuge, maximum rpm) for ten minutes.

2.2.2.4. M e t h o d ^ l.

All equipment (BIO-RAD) was washed very thoroughly before use.

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The glass plates were placed together, separated only by plastic spacers (1.5 or 0.75 mm). After alignment, they were placed in the pouring stand and locked down onto the bottom silicon pad, which maintains an unbroken seal.

The resolving gel was mixed, omitting TEMED, and degassed for one minute. The TEMED was added, the mixture stirred with a glass rod, then poured immediately between the plates, avoiding formation of any bubbles, and checking the base for leaks. A 3 cm gap was left at the top of the gel for the stacking gel. The resolving gel was overlaid with 0.1% SDS solution to a depth of 2 - 3 mm. The gel was left to set, usually overnight, before preparing the stacking gel in the same way. The overlay was poured off and the stacking gel poured between the plates to the top. The comb was then inserted, initially at an angle, to avoid trapping air bubbles.

After leaving to set for 30 minutes, the comb was removed and the wells overlaid with reservoir buffer (diluted 1/10 from stock). The samples and standards were prepared and loaded into the wells. The gel was then placed in the electrophoresis tank and the electrodes connected to the tank and the power pack. Gels were run routinely at 120 V until the bands passed into the resolving gel, then increased to 200 - 250 V until a good resolution was achieved.

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The gel was removed from the apparatus, cut down to a manageable size, then either stained or used for Western blotting.

Staining was carried out by placing gel in staining solution (0.1% Coomassie blue in water:methanol:acetic acid in ratio of 5:5:2.) for 4 - 6 hours, or overnight. The gel was then destained in propan-2-ol (12.5%), acetic acid (10%) for 2 - 3 hours. Gels were retained until drying in a solution of 30% methanol, 3% glycerol.

The gels were then dried under vacuum, using a BIO - RAD gel slab dryer, for 1 - 2 hours.

Photographs of dried gels were taken routinely.

2.2.3. Western blotting^^-.

2.2.3.1. Method

The transfer buffer (25 mM Tris-HCl, 192 mM glycine,2 0 % methanol) was prepared during the gel electrophoresis, and cooled to 4°C. The gel was cut down and a piece of nitrocellulose cut to match. The nitrocellulose, Scotchbrite pads and 3 MM paper were all presoaked in transfer buffer.

The gel was rinsed in buffer and placed in the Transblot apparatus as shown in the diagram (Fig. 3),

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Figure 3. Diagrammatic representation of Transblot apparatus (Bio-Rad).

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TRANSFERBUFFER

NITROCELLULOSE

TRANSBLDTCELL

\\\

\\X\

\ .

\A .

\A .

\

CLAMP

SCOTCHBRITE

3MM PAPER

GEL

ensuring that no air bubbles were trapped.

The reservoir was filled with 2 - 2.5 1 of buffer, the lid attached and the power supply connected. The equipment was run at 20 V for 1 8 - 2 0 hours.

After blotting, the gel was removed and stained as previously described. The nitrocellulose paper was removed and placed in 10 mM Tris-saline pH 7.4 until required for staining.

2.2.3.2. Staining of Western blot.

The nitrocellulose paper strips were incubated in blocking buffer (10 mM Tris-saline pH 7.4) + 5 % BSA for 30 minutes shaking, and 30 minutes still.

They were labelled initially with rabbit polyclonal anti-GFAP (Courtesy of Dr. J. Newcombe, Institute of Neurology, London) diluted 1/200, or rabbit polyclonal anti-tubulin diluted 1/50 in buffer + BSA, or with no primary antibody at all, in the control, for 45 minutes shaking, 30 minutes still. The strips were then washed in buffer for 5 x 10 minutes, shaking.

They were then labelled with swine anti-rabbit peroxidase diluted 1/150 in buffer + BSA for 60 minutes. The strips were washed in buffer for 3 x 5 minutes, then PBS for 3 x 5 minutes.

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f

The strips were developed in DAB (25 mg/ 100 ml PBS) + 60 fJLl H 2 0 2 until bands were distinct. The developing solution was removed, and the strips washed with PBS for 3 x 5 minutes, then distilled water for 2 x 5 minutes, before drying between filter paper.

Stained blots were routinely photographed since the results were prone to fading.

2.2.4. Polyclonal antibody production.

2.2.4.1. Method - bleeding

The rabbit used for polyclonal production was bled (10 ml) prior to any treatment. This pre-immune bleed was clotted at 37°C for 1 hour then stored at 4°C overnight before centrifugation to separate serum (bench centrifuge, maximum rpm). This procedure was also carried out for all post-injection bleeds. Serum was frozen at -20°C initially, then stored at -80°C until apportioned for use.

2.2.4.2. Method - injections.

The GFAP isolate (1 mg in 500 f i l ) was mixed with 120 /il of 10% SDS (1% final concentration) and boiled for 5 minutes, according to the method of Dahl and Bignami52.This has been reported to increase the immunogenicity of the protein. 600 n l of complete Freunds adjuvant was then

31

added and mixed immediately prior to injection.

The rabbit was immunized with 3 weekly injections, each being split between the neck and both hind quarters.

2.2.4.3. Antibody testing.

The specificity of the antibody was tested by running a 10% SDS-PAG of:-Lane 1 - low molecular weight markers " 2 - rat cerebellar homogenate 150 mg/ml" 3 - " ” " 15 mg/ml" 4 - GFAP" 5 - rat cerebellar homogenate 1.5 mg/ml

Lanes 7 - 10 as for 2 - 5

Rat cerebella were manually homogenized in PBS and diluted to produce protein for this gel separation.

I

Lanes 1 5 were stained with Coomassie blue asdescribed, ft

Lanes 7 - 1 0 were blotted as described, for 18 hours at 20 V, and stained using the polyclonal, as produced above, as the primary antibody.

The titre of the antibody was checked by examining immunofluorescent staining of 1 0 fim rat cerebellar sections at dilutions of 1/100, 1/250, 1/500, 1/1000, of the GFAP

32

polyclonal antibody as primary antibody and goat anti-rabbit TRITC as secondary antibody, for fixed and unfixed sections.

2.2.4.4. Antibody purification.

The immunoglobulin fraction of the polyclonal antibody was purified from the crude rabbit serum in order to eliminate excessive background staining. This was effected using the DEAE Affi-gel Blue gel method as described by the manufacturers, BIO-RAD.

Initially, the crude rabbit serum was dialysed against column buffer (0.02 M Tris-HCl pH 8.0, 0.028 M sodium chloride, 0.02% sodium azide), for three days at 4°C with three changes of buffer. It was then centrifuged at 8,800 g for fifteen minutes at 4°C.

The column was prepared using 3.7 ml gel (fresh) per ml of serum. It was prewashed with five bed volumes of 0.1 M acetic acid pH 3.0, containing 1.4 M sodium chloride and 40% isopropanol (v/v), in order to elute residual dye. The column was equilibriated with more than twelve column volumes of column buffer. The serum was applied to the gel and eluted with column buffer. Fourteen fractions of initial sample volume were collected and assayed spectrophotometrically at 280 nm for the protein peak. The peak fractions were pooled and concentrated down to half the initial sample volume using a Minicon concentrator

33

(Amicon). The column was regenerated with three bed volumes of 2 M sodium chloride in column buffer.

The titre of the purified antibody was checked against the crude, dialysed antibody by incubation on fixed 1 0 /xm sections of rat brain at a range of concentrations from 1/50 - 1/5000 in P.B.S. + 0.3% B.S.A.. In order to compare crude and purified (concentrated) antibody, the latter was initially diluted 1/2. Staining was visualized by incubating all slides with goat anti-rabbit TRITC at 1/100 dilution.

Purified antibody stocks were stored at -80°C. When in use they were kept at 4°C.

2.2.5. Protein estimation-3^-.

2.2.5.1. Stock solutions.

1) 0.189 M Na2 C03 in 0.1 M NaOH.2) 1% Cu S04 .7H2 0.3) 2% Na+K+Tartrate.

2.2.5.2. Reaction solutions.

A) 1 ml of (2) + 1 ml of (3).B) 50 mis of (1) + 1ml (A).C) 2.5 mis Folins reagent + 3.5 mis distilled water.

34

2.2.5.3. Sample preparation.

The protein sample was diluted 2/5 in 0.25 M NaOH, and placed in water bath at 50°C for at least 45 minutes. It was then diluted with an equal volume of 0.1 M NaOH

2.2.5.4. Method.

2 0 0 n l of the sample solution was taken and 1 ml of solution B was added, mixed and left for 10 minutes. To this, 100 f i l of solution C was added, it was mixed immediately and left for a further 30 minutes. Standards ranging from 0 - 300 f iq /m l were made up from BSA in 0.1 M NaOH and treated in the same way.

The absorbance at 500 nm was measured spectrophotometrically. A standard curve was constructed and the resulting protein concentrations calculated using the LOWRY computer programme.

2.2.6. Immunofluorescence staining

2.2.6.1. Method; Sections.

The sections were fixed with 4% paraformaldehyde in PBS for 20 - 30 minutes, then rinsed in PBS for 3 x 5 minutes. Sodium borohydride (0.5 mg/ml) was applied to the sections for 3 x 2 minutes before rinsing again as stated. This has been found to decrease the natural background

35

*

Primaryimmunofluoresence of the aldehyde groups2 1 0

antibodies were applied, appropriately diluted in PBS +0.3% BSA, or omitted in the case of controls, for 45 - 60 minutes. The sections were rinsed again before applying the secondary antibody (swine anti-rabbit linked rhodamine or goat anti-mouse linked fluorescein), diluted 1/50 in PBS + BSA. They were again rinsed prior to mounting in Citifluor (City University), and sealing the coverslip with clear nail varnish.

2.2.6 .2. Method: Cultures.

The culture coverslips were carefully removed from dishes to humid chambers and rinsed immediately with Astrocyte medium (AM) at 37°C. The primary surface antibody was applied, diluted appropriately in AM or omitted, for controls, for 45 - 60 minutes. The coverslips were then rinsed with AM, 3 x 2 minutes, before applying the secondary antibody, diluted 1/50 in AM for 4 5 - 6 0 minutes. They were then rinsed in PBS 3 x 2 minutes, before fixing in 4% paraformaldehyde in PBS for 15 minutes.

They were rinsed in PBS, and sodium borohydride was applied as for the sections. After another rinse in PBS they were rinsed once and then incubated in acid alcohol (95% ethanol, 5% acetic acid) at -20°C, for 15 - 20 minutes. The coverslips were rinsed very thoroughly with PBS before application of anti-GFAP diluted 1/50 in PBS + 0.3% BSA, or buffer + BSA alone for controls, for 45 - 60

36

minutes. After rinsing in PBS as before, swine anti-rabbit linked rhodamine diluted 1/150 was applied for 45 - 60 minutes. Finally, the coverslips were rinsed in PBS, mounted on slides with Citifluor and sealed with nail varnish.

2.3. RESULTS.

Initially several mechanical problems were encountered with the procedure which caused the loss of a lot of tissue in the initial stages. However, these were overcome and, finally, the elution profile of the hydroxlapatite column was demonstrated (Fig. 4). This showed several peaks of varying heights. The first and strongest peak was between fractions 11 and 30, eluted with 10 mM buffer. This was considered to be the peak which contains the GFAP162. The second peak, between fractions 56 and 85, eluted by 0.15 M buffer was thought to contain tubulin, the most common contaminant of GFAP isolations. The third and final peak, was thought to contain the neurofilament proteins, and was eluted by 0.3 M buffer between fractions 95 and 131. The protein concentration of the various stages and fractions was assayed and is shown in the table below.

37

Figure 4. Elution profile of hydroxylapatite column, showing protein peaks corresponding to GFAP, Tubulin and NF. The latter peak is spread, probably because of the different NF subunits.

38

GFAP elution profileHydroxylapatite column

Protein concn.SamplePrecolumn supernatant 530 jig/mlPellet from 14,000 g run 6 . 6 mg/mlMyelin pad from 14,000 g run 16.8 "GFAP fraction 875 /ig/mlTubulin fraction 463 f ig /m l

Neurofilament fraction 190 n g /m l

The final volume of each of the fractions was about 15 mis, giving a final recovery of about 13 mg of purified GFAP. This was a purification factor of 4061 over the original tissue.

The electrophoretic pattern of the proteins is shown in Figure 5. This shows that the GFAP has a molecular weight of about 54,000 with the major breakdown product at about 40,500. When co-electrophoresed with pure tubulin (Courtesy of Roy Burns, Dept, of Pure and Applied Biology, Imperial College of Science, Technology and Medicine), the GFAP bands migrated differently. Also, when the tubulin was incubated with the rabbit anti-GFAP polyclonal, the Western blots showed no cross-reaction. In contrast the blots of GFAP incubated with rabbit anti-GFAP (Fig. 5) showed several positive bands, corresponding to the main protein and its degradation products50.

The original rabbit anti-GFAP produced was found to be specific for glial filaments when dual staining with RT97 (anti-neurofilament antibody) was carried out on sections.

39

Figure 5. Purification of GFAP demonstrated by PAGE and Western blotting. The two main bands on the Western blot correspond to GFAP and its main breakdown product, at 54 kd and 40.5 kd. The other blot shows the control reaction using BSA instead of anti-GFAP antibody.

40

G W LHW

i

Figure 6 . Elution profile of polyclonal anti-GFAP antibody from DEAE Affi-gel Blue gel (BIO-RAD). The single peak indicates the Ig fraction.

41

<D

Cfl

O

(A

C

O

<D CtJ

CM 0

0 O

C

E

GFAP antibody elution profile

However, the background staining with TRITC was found to be unacceptably high and further purification of the polyclonal antibody was performed. The protein profile of the polyclonal antibody as eluted from the column is shown in Figure 6 . Fractions 4 - 6 inclusive were pooled. The remaining fractions were discarded. The immunostaining showed that the titre of the antibody had increased and also that the background had been reduced to an extremely low level.

Antibody concn. Before After

1/50 +++ ++++1 / 1 0 0 ++ (+) ++++1/250 + +++1/500 - +++1 / 1 0 0 0 - ++1 / 2 0 0 0 - ( + )1/5000 - -

The purified antibody could consequently be used at a dilution of 1/500 and 1/200, for clear staining of sections and cultures, respectively.

2.4. DISCUSSION.

This preparation used a modification of the axonal flotation method described by Yen et al304. It is likely that this method relies on the high level of GFAP

42

expression in fibrous astrocytes, and similarity in properties of the intermediate filaments, NF and GFAP. The substituted use of bovine spinal cord instead of whole brain was found, by trial and error, to produce a cleaner preparation in the initial stages. The isolation of the GFAP as detailed by Liem1 6 2 was used. Some modifications have been instituted based on his results. These have concentrated on the recovery of GFAP rather than neurofilaments. The column chromatography was adapted by only eluting the fractions which were shown to produce peaks. An improvement as suggested by Liem, the inclusion of ATP in the reassembly stage, was not incorporated since the improvement in recovery for GFAP was small. Also the added expense could not be justified.

Alternative methods of isolation have been described, particularly those of Dahl and Bignami group. However, these methods have not produced a high-yield preparation, uncontaminated by tubulin, as would be required for the production of a specific polyclonal antibody 54'244.

The production of the polyclonal antiserum was also improved by using the modification in which 1 % sodium dodecyl sulphate increases the immunogenicity of the protein. Also, by purifying the polyclonal antibody, a much clearer visualization of glial filaments in the stained sections and cultures, was possible. In this study a much smaller amount of GFAP was found to be effective in producing the antibody than in other papers52. A

43

polyclonal antibody was produced rather than a monoclonal antibody since, as described in the introduction, some monoclonal antibodies show inefficient recognition of the molecule. Also, polyclonal antibodies usually produce a higher signal, related to their recognition of a larger number of epitopes. These problems may now be overcome by the introduction of molecular biology techniques, which have produced specific cDNA clones of the protein236. These will allow production of large amounts of pure protein and also use of in situ hybridization techniques for localization.

44

3. THE PHOSPHOINOSITIDE RESPONSE.

3.1. INTRODUCTION.3.1.1. History

Myo-inositol, seen in Figure 7, in the preferred 'chair' conformation, was first described in the 1850's.It was recognised as a membrane component of bovine brain in the 1940's in the form of the phospholipid phosphatidylinositol, and at the same time the presence of inositol phosphates was detected. The biosynthetic and degradative pathways of inositol were initially elucidated to show a cyclic pattern, in the 1950's, and this work continues. Although the major pathways are considered to be relatively well characterized, their components, and the minor pathways are not. For example, the identity and properties of various biosynthetic and degradative enzymes are largely unknown.

The paper by Hokin & Hokin, 1953, first suggested a role for inositol phospholipids12? In their experiments they showed that 32P uptake into phospholipids was selectively increased by acetylcholine stimulation in brain slices and of protein secretion from exocrine pancreas.The increase in incorporation was localized to phosphatidylinositol (PI). It was at this stage that the increases in the activity of phosphoinositidase C and diacylglycerol kinase were suggested, but these theories

45

Figure 7. Structure of myo-inositol in favoured chair conformation, from Fisher & Agranoff77. Phospholipids are formed by an phosphoesteric link through position 1 , to a stearoyl-enriched sn-1, arachidonyl-enriched sn-2 DAG.

46

(on

For almostwere not followed up until very recently124, ten years the role of the PI response was obsessively linked to the secretory process. This monopoly was broken after a direct correlation between rapid cell proliferation and rapid PI turnover was demonstrated82.

Another clear link was repeatedly shown between external stimuli and the PI response. This suggested a membrane linked receptor-stimulated PI hydrolysis66'154.The hydrolysis was found to be catalysed byphosphoinositidase C (PLC)125'138, but the substrate of the reaction was only later correctly recognised as PI(4,5)P2146'193• The products of the reaction were shown to be diacylglycerol and PI(1 ,4 ,5 )P3 . Both of these formed a critical part of the pathway, and more importantly, they were later seen to fulfil the role of second messengers. In this way, the PI pathway became a complementary second messenger system to the well-characterized cyclic AMP system. The complementarity was accentuated when it was found that the two systems operated almost entirely through different sets of receptors (see Table 2)2 2 • 2 5 7 . The receptor-linked second messenger systems also provided a means of classifying subtypes of receptors on the basis of their secondary activations193.

It was soon recognised that phospholipid labelling was secondary to the very rapid PI hydrolysis by PLC as the initial reaction after stimulus167. Consequently, the sequential dephosphorylation to IP2 and IP was easily

47

Table 2. Astrocyte receptors and second messenger functions.

48

ASTROCYTE RECEPTORSReceptors

Beta., -adrenoceptor Alpha., -adrenoceptor Alpha2 - adrenoceptor M -muscarinic cholinergic M2 - muscarinic cholinergic H ^ histam ine H 2 ‘ histamine D i-dopam ine D 2 -dopamine GABAa GABAb Quisqualate A ^ a d e n o s in e ?A 2 “adenosineBenzodiazepineAngiotensin IIVIPACTHPTHCalcitonin

S econ d m essenger References

CAMP (+) 186PI 216, 67CAMP (-) 75, 67PI 198cAMP (-) 198PI • 127, 60

127cAMP 129

Ca2+129

(+) 30Ca2* (-) 3PI 141, 88cAMP (- ) 130cAMP (+) 284, 13Ca2* 19PI 2 3 3cAMP (+) 42cAMP (+) 75CAMP '(+) 170CAMP (+) 170

ASTROCYTE RECEPTORS (cont.)Receptors S econ d m essenger References

Glucagon CAMP (+) 147Serotonin PI, Ca2* 128, 291Somatostatin cAMP (- )? 75, 2 4 3Atrial natriuretic peptide cGMP (+) 87Bradykinin PI 43Vasopressin PI 43Oxytocin PI 43Sub stan ce P PI 43Neurokinin A PI 43Neurokinin B PI 43Opioid ? 37, 176Prostaglandins cAMP (+) 75, 2 8 4Insulin ? 46Triiodothyronine ? 214, 2 2 6

demonstrated21. The speed of the response was demonstrated by the finding that the secondary labelling occurs within two seconds. An important practical advance occurred when it was discovered that this pathway could be inhibited by the inclusion of lithium in the incubation media20. The Li+ acts to inhibit IP phosphatase, thus blocking the cycle and causing a build-up of IP^. This finding was used practically in many studies to examine the effect of stimuli on various tissues, since the measurement of IP apparently showed a direct correlation to the stimulus. Recent work has complicated this effect, since it is now thought that an agonist-sensitive pool of PI exists, which is not affected by Li+ .

Within the last few years the field of PI has ramified considerably. It was only recently that the position of PI(1 ,4 ,5 )P3 had been challenged as the most prominent second messenger of the inositol phosphates. The existence of IP4 IP5 , IPg, cyclic inositol phosphates and a variety of isomeric forms have been demonstrated and their role and relevance as second messengers in their own right proposed. Also, the interfaces between the cyclic AMP and PI systems have been investigated. Calcium and guanine nucleotide binding proteins ("G" proteins) have roles in the signalling process which have yet to be fully explored or understood. It should be remembered that PI turnover occurs normally within cells, and that its activity is only increased by external stimuli. Finally, it is still not known how any of the second messenger systems are finally

49

linked to changes in gene expression which effect cellular responses.

3.1.2. Mechanism of reactions.

A suggested scheme of the reactions is shown in Figure 8 . The established reactions form a complex cycle which becomes further involved when the postulated, but as yet unproved reactions are included.

The main pathway is initiated when phosphatidic acid is converted to CDP-diacylglycerol by CTP-PAcytidyltransferase. This then combines with myo-inositol to form phosphatidylinositol using the enzyme PI synthetase. These reactions both occur in the endoplasmic reticulum, prior to the transfer of the PI to the plasma membrane using a PI specific transfer protein. It is then sequentially phosphorylated by specific kinases, i.e. PI kinase, PI(4)P kinase. The activity of these has been found to be increased by polyamines and product-inhibited by PIP2173. The product of this reaction, PI(4,5)P2 may possibly enter at least three different pathways. The main pathway involves the action of phospholipase C (polyphosphoinositide phosphodiesterase) producing I (1 ,4,5)P 3 and diacylglycerol, the critical step in the process. The latter may then stimulate the activity of protein kinase C, while the former has been proposed to mobilize Ca2+ from intracellular stores263. It is at this point that the effect of the external stimulus is

50

Figure 8. The current understanding of the phosphoinositide cycle1'11'201. PLC is shown to be activated here in only one reaction of the system, however the activation is universal, and can be recognised by the release of DG. Abbreviations given here are described in full in Appendix 4.

51

STIMULUS

Kcl:2,a4)P3I9 I I

I(ci:2,3,4,5)P4AY

ADP <K U p.ATP^l VI(cl:2,4,5)P3DG<̂

yPK4,5)P2

"M

RECEPTDR

G-PRDTEIN

DG<jy N < 7

<■ATP ADP

K14,5)P3

ATI

1(1,3,4)R

j O " I(1,3,4,5)P,4--------- PI(3,4,5)P■7/ AY' ATP

" < ^ adpJ v

IP ,A Y ' ™

P‘ ^ ADPAv IP.

DG

communicated to the pathway by means of the receptor and its associated G protein to the PLC. The receptors which are linked in this way are generically referred to as calcium-mobilising receptors, because of the later actions of the IPs.

At this point there are two possible pathways available to I(1 ,4 ,5 )P3 . The first of these involves sequential dephosphorylation to myo-inositol via I(1,4)P2 and I(4 )P3

catalysed by specific phosphatases. The first step is catalysed by I(1 ,4 ,5 )P3 5-phosphomonoesterase. This enzyme is also active in the cyclic pathway, but there the Km value is much higher48. The last two steps in the non-cyclic pathway are sensitive to inhibition by Li2+, enabling investigators to measure the receptor-activated PI response by measuring the build-up of I(4)P162. The alternative pathway involves the recently discovered soluble enzyme, I (1,4,5) trisphosphate 3-kinase in the production of I (1,3,4,5)P416'135. This reaction is Ca2+-dependent and the product is thought to act synergistically with I(1 ,4 ,5 )P3 to release intracellular pools of Ca2+133.

The next step in the pathway is the dephosphorylation of I(1,3,4,5)P4 to I(1,3,4)P3 catalysed by 5-phosphomonoesterase. I(1,3,4)P3 is metabolised by two different pathways. The first utilisespolyphosphoinositide phosphatase (inositol polyphosphate 1-phosphatase - a Li+ sensitive enzyme which accounts for

52

the reported build-up of I(1 ,3 ,4 )P3 and I(1,4)P2 in some stimulated systems) to produce I(3,4)P2 . This enzyme also acts on I(l,4)P2to produce I(4)P1132. The second pathway involves conversion to I(1,3)P2 by action of inositol polyphosphate 4-phosphatase11. As yet there is no evidence that 1(1,4,5)P3 could be derived from 1(1,3,4,5)P4 in the same manner. No significant role has yet been determined for 1(1,3,4)P3 . The remaining dephosphorylation steps in this pathway have yet to be defined absolutely but there is evidence that I(3)P;j_ is produced by a specific Mg2+-dependent 4-phosphatase from I(3,4,)P211. However, the same group have also shown that I(1,3)P2 and 1 (1 )?^ can also be formed. It is known that the major IP2 formed in stimulated cells is I(1,4)P2, but that the proportion of the other IP2s varies. This variety may well be caused by the differing Li+ sensitivities and Mg2+ requirements of the specific enzymes.

Peripheral to the major pathways are other minor metabolic routes. It has been suggested that I(1,3,4,5)P4

may be the precursor for more polar inositol phosphates such as IP5 and IP6 which have been demonstrated in GH4

cells 121. It has also been suggested that PI(3 ,4 ,5 )P3 may be derived from 1 (1 ,3,4,5)P4 and that its breakdown may be a source of DG201. However, the existence of this isomer has been disputed and should be accorded appropriate importance.

53

Another pathway is that of the cyclic inositol phosphates. These are known to derive from the direct action of PLC on PI, PI(4)P, and PI(4,5)P248. The cyclic forms are not apparently directly converted to the non-cyclic forms until the 5- and 4-phosphates are removed and the cyclic diester bond cleaved. Instead, they constitute a separate pathway which is initiated by the action of I(1,4,5)P3 5-phosphomonoesterase on I(cl:2,4,5)P3

to form the biologically inactive I(cl:2,4)P2 . This is broken down to I(cl:2 )P^,which is further degraded to I(1)P^ by Inos 1,2 cyclic hydrolase. It has been found that this enzyme cannot metabolize the other cyclic inositol phosphates. Consequently the cyclic pathway links to the main pathway only at the formation of I(l)P^. It would seem that some of the enzymes responsible for similar reactions in the non-cyclic IPs are not able to act upon the cyclic forms e.g.I(1,4,5)P3 3-kinase. It is thought that I(c(1 :2 ,,4,5)P3 may also have a role similar to that of I(1,4,5)P3 in the mobilisation of calcium, and may, in fact be the major IP3 formed in platelets stimulated by thrombin.

The final link of all pathways is the re-formation of myo-inositol from the IP^s. This is achieved by the action of inositol monophosphate phosphatase.

3.1.3. The PI response in the nervous system.

A major problem in investigating the PI response is the54

complexity of nervous tissue, and the difficulty in isolating the response to a particular cell type. Cells from the nervous system were some of the first used to examine the functioning of this response. Since then, the brain has been examined extensively, usually in the form of brain slices, synaptosome preparations or, more recently, cell cultures. It was initially thought that the PI response had some particular significance in the CNS because of the large amounts of myo-inositol and IPs found in nervous tissue. However, the response is no longer considered to be tissue-specific, since it has been shown in many different tissues e.g. platelets, parotid gland, liver, thyroid, smooth muscle etc.. Also, it was found that PI had a role in membrane protein anchorage for Thy-1 and myelin basic protein; the latter explains the high concentrations present in myelin fractions171'303. Also it is possible that a novel second messenger is released by the activation of PLC by insulin, which releases an inositol phosphate glycan that regulates cAMP-PDE247. It is possible that these form a pool of PI which is agonist-insensitive. The PI response may also have a role in neuronal plasticity, since it has been found to be modulated by a growth-associated, axonally transported protein, GAP-43, in its phosphorylated state285. This may form a link with the complex systems of growth and neuroplasticity.

One of the major characteristics of the CNS PI system is its sensitivity to Ca2+. Calcium is a regulator of many

55

cell functions particularly the activation/inactivation of key enzymes. It is vital to the cells survival that Ca2+ levels are controlled, since excess levels could lead to the activation of Ca2+-dependent proteases which could cause damage. Under normal conditions it is controlled by four main mechanisms: 1). the Ca2+-dependent plasma membrane ATPase pump; 2). the plasma membrane Na+/Ca2+ exchange mechanism; 3). the mitochondrial Ca2 +-pump; and4). the ATP-dependent Ca2+ pump in the ER1. The role of Ca2+ in neurons is particularly important, since extracellular Ca2+ influences the threshold and the current-voltage relations of the membrane potential while at normal levels it stabilizes the membrane. Also, Ca2+ enters the neuron during the impulse, and this is responsible for neurotransmitter secretion at terminals. Intracellular Ca2+ also affects the potassium conductance by regulating a potassium channel, and in some cells it may augment or replace the rising phase of the action potential149. The complementary DG/PKC system acts oppositely by inhibiting the hyperpolarization24'80. This system has also been implicated in memory processes in molluscs, and could, theoretically apply to mammalian nerve cells23.

It was long considered that an increase in the cytoplasmic Ca2+ level was the primary response after receptor activation, and that the PI response was a secondary response. This was supported by the knowledge that PLC was a Ca2+-dependent enzyme, and that the

56

cationophore A23187 alone could induce a small PI response. However, it was found that Ca2+ deprivation did not abolish the PI response. Also, the realization that PIP2

hydrolysis was the primary step after receptor activation supported the hypothesis that Ca2+ activation was, in fact, a secondary response. It has been discovered recently that Ca2-W kireleased from endoplasmic reticulum by I(1 ,4 ,5 )P3

and I(cl:2,4,5)P3 , the latter being most active298. This work was supported by the recent recognition and characterization of a specific IP3 receptor from rat cerebellum and cortex168'266'297. Also Irvine has suggested that 1 (1 ,3,4,5)P4 and I(1 ,4 ,5 )P3 may act synergistically in a Ca2+-dependent Ca2+-regulatory process. This involves the control by IP4 of the amount of Ca2+ release which can be stimulated by IP3 133. It has also been suggested that IP3 production may reflect a high-energy status in the cell, since the process is energy-expensive, and Ca2+ is released into an environment which has to be capable of removing excess Ca2+, a process which is also ATP-dependent. Under stimulating conditions, the increased rate of the PI cycle may contribute to the characteristically high metabolic rate of brain tissue81.

As was previously mentioned, the localization of the PI response has yet to be clearly shown. There have been conflicting reports concerning the relative importance of neuronal and glial contributions to experiments where both are present. Some studies addressed this issue by producing 'homogeneous' cell cultures, but it seemed that

57

the measurement of stimulation was idiosyncratic to different laboratories95'217. However, several groups have now independently identified clear PI responses in glial and neuronal cells, so it must be deduced that the response is present in both, but that their relative importances are still unknown217'218'43'8.

Contrary to earlier belief, it is now thought that the PI response in neurons is predominantly located postsynaptically. This is based on lesion experiments which have shown the abolition of the response after ibotenate or kainate application, which destroys the cell bodies and interneurons. This contrasts with findings that surgical and 6 -OHDA lesions do not affect the PI response80. These results are difficult to interpret without the ability to localize individual responses to a particular cell type.

Clinically, lithium has been used for many years as a treatment for manic-depression. The mechanism of action is still unclear, although obvious parallels have been drawn between its modulation of the PI system and its clinical effect. Other CNS drugs have been found to interact with PI system. Both tricyclic antidepressants and neuroleptics are competitive antagonists at Pi-linked sites140'239. Malfunctioning of the PI system in nerves has also been implicated in diabetic neuropathy292.

3.1.4. Role of PI system.

58

The functional role of the PI system must be defined mainly by our current understanding of it as a second messenger system. It has been shown that at least three and probably more secondary and tertiary messengers are produced by the activation of the system. The second messengers referred to are I(1,4,5)P3 and DG. The role of the former has been described earlier, along with that of the tertiary messenger (in this system), Ca2+, whose release it stimulates.

DG is an equally important second messenger since it connects the activation of lipid functions to the activation of proteins via the stimulation of PKC. The DG released from the hydrolysis of PI(4,5)P2, PI, and PI(4)P1, may activate PKC, lowering its affinity for Ca2+. In animals this DG is predominantly enriched in stearoyl residues at the sn- 1 position, and arachidonoyl residues at the sn-2 position. This unusual pattern was found in all phosphatidylinositols in brain115'274. It is interesting to note that PKC is not activated specifically by this configuration of fatty acids in DG, but that it may be activated by a variety of forms of 1,2-DG194.

It has been argued that DG is a less important second messenger than IP3 since phosphatidylserine and Ca2+ are also required for activation of PKC. In some senses it does only provide a feedback system for IP3 , since it modulates calcium signalling and other receptor mechanisms. It effects this modulation in two ways: 1) by means of

59

inhibition of PI(4/5)P2 hydrolysis, thus decreasing the release of I(1,4,5)P3 and consequently Ca2+; and 2) by increasing the activity of the calcium pumps. However, this role is essential and leading, as it does, to the phosphorylation of cell proteins, may provide a vital link from the activation of the pathway to modulation of the cell functions. At the moment the task of identifying these proteins and their functions is still to be accomplished. Until this is completed the true significance of this system remains hidden.

DG may also have importance as a source of arachidonate for prostanoid synthesis. The sequential action of PLC and DG lipase or the direct action of PLA2 on Pis would release the arachidonate for incorporation into prostanoid synthesis1. This may form an amplification system, since certain arachidonate metabolites stimulate the PI response. Alternatively, it may provide a communication system with nearby cells which contain appropriate receptors. In some cells the metabolites may participate in cGMP formation, which may lead to activation of the calcium pump77.However, a recent study has suggested that receptor agonists provide only a small, transient increase in the intracellular arachidonic acid levels, and that inositol phospholipids in astrocytes do not provide a source for eicosanoid synthesis219.

Clearly, the PI system does not work in isolation from other signalling systems in the cell. In fact cAMP and

60

Ca2+ have been found to modulate the alternate systems extensively. Calcium is not the only regulator of the cAMP response derived from PI. As described earlier, in liver a Pi-linked carbohydrate moiety forms an IP-glycan, after hydrolysis, which has been found to regulate the activity of cAMP-PDE. Another common link between the PI and cAMP systems is the mediation of the responses through G proteins. The identity of these G proteins differs between the two systems, and in fact, within eachsystem89'28'204^169'165. It has been found that activation of either system may modulate the activity of the other, often conversely208'72. As yet no direct inhibitory stimuli have been located for the PI system, so it may be that the inhibitory effects of the cAMP system at other levels provide a necessary feedback208'72. Cyclic AMP has a regulatory effect on several of the enzymes which participate in the PI system, such as the protein kinases, and phosphoprotein phosphatase.

The role of the PI system has been discussed quite extensively, but its effects on cellular function have not yet been mentioned. In fact it is difficult to summarize these since each cell has a specialized function and activation of the PI system leads to different effects in each cell. Indeed more problems arise in attempting to directly ascribe such effects to the PI system itself, since usually, activation of the PI pathway and evidence of a physiological response are examined in the light of receptor stimulation. In this way they could easily be

61

viewed as parallel rather than serial effects, especially when the known complexity of the system is considered. However, among the effects which are attributed to jthe PI system are: mitogenesis and oncogenesis293; fertilization; regulation of cytoskeleton-membrane interactions38;' cell motility155; phototransduction; glycogen breakdown; secretion of amylase, insulin, histamine and prolactin; smooth muscle contraction; platelet aggregation; and DNA synthesis22.

As with other systems, there is still much to discover and understand about this system, particularly concerning its regulation, but most importantly, how the activation of the system is linked to the final cellular effects.

3.1.5. Measurement of PI response.

The measurement of the PI response has been gradually improved since the importance of the PI system was realized. As would be expected, the earlier studies laid an equal emphasis on the measurement of the lipids and phosphates. Since the acceptance of the stimulation of the PI system as an index of receptor activation, most studies have concentrated on the measurement of the inositol phosphates. Also, the measurement of lipids frequently does not reflect the activation of the system, because PIP and PIP2 levels are maintained by inositide kinases. The innovation of a lithium-amplified method allowed investigators to examine the increased turnover of inositol

62

phosphates in a sensitive assay20.

Measurement of the response is dependent on the pre-labelling of the system with radio-label. In most common use is [3 H], but if conditions preclude its use, [3 2 P] can be used. The latter provides a measure of incorporation into PA so it is essential that the system is specific for inositide level alteration. IP2 , IP3 , and IP4

can be separated from other cell constituents most simply by ionophoresis on paper or cellulose acetate, using a Na+/oxalate system. Visualization of the results is effected by autoradiography. Standards are routinely included because of the variable nature of the separation251.

The most common method of measurement was first introduced by Berridge20. It is most frequently used with [3 H]myo-inositol because of the masking effects of ATP, other nucleotides and 3 2 [P04]. The method involves Dowex anion exchange chromatography; the column being in the formate form. The inositol phosphates are separated by sequential elution using; distilled water, 60 mM ammonium formate in 5 mM disodium tetraborate, then increasing concentrations of ammonium formate in 0.1 M formic acid.It is important to be aware of the identity of individual fractions before initiating studies. In most studies, it is sufficient to separate the total IP's from the other cell components. In this case, sequential elution is not required, and all the IP's can be 'batch' eluted with 1.0 M

63

Ammonium formate in 0.1 M formic acid. Although the technique is easily performed, there are certain pitfalls which should be avoided. The levels of inositol phosphates present in cells are small compared to the amount of inositol. Therefore, after applying the sample, it is important to wash the Dowex column with a large volume of water or unlabelled 5 mM myo-inositol to remove it. Unfortunately, this method does have a drawback, especially with respect to the most recent work carried on the PI cycle. The assay separates inositol,glycerophosphoinositol, IPlf IP2 , IP3 , and IP4. It does not distinguish between the various isomers within the groups e.g. I(1 ,4 ,5 )P3 and I(1,3,4)P3. In most assays, however, the precise isomeric composition of the response is not required.

In the initial isomeric studies9 paper chromatography provided a successful separation method. However, in recent years, the use of HPLC has increased dramatically as the method of choice. The use of HPLC is at the moment is limited in the range of inositol phosphates that may be separated in one experiment. The most promising method for adaptation is that of Irvine134, since it allows separation of cyclic IP's, IP3 's , IP5 , IPg and some IP2 's. For these experiments the inclusion of internal standards is essential134'48. The cyclic forms provide an added difficulty in that they are converted to the non-cyclic forms by acid extraction, so this must be avoided. When large numbers of samples are required to be assayed

64

relatively quickly, a new technique exists which separates individual IP's, and isomers of IP3 3 0 2 in 5 - 6 minutes per sample. It utilises the separating ability of ACCELL QMA anion-exchange SEP-PAKS. It does not provide the detailed separation of the HPLC systems, and some problems have been found with the reproducibility of manufacture of the SEP-PAKS.

The mass measurement of IP's would be a more accurate method of determining turnover, but this cannot be accomplished using standard radiolabels because of the problems of pool sizes and isotopic equilibrium. However, new methods involving dephosphorylation of IP's and consequent measurement of their inositol content by gas chromatography may provide a possible technique240. An alternative, and less time-consuming, method has been suggested, using GC/mass spectrometry followed by FAB255. Despite the requirement for complex equipment, the latter method promises the sensitivity and adaptability required to analyse all IP's.

3.1.6. PI response in astrocytes.

The existence of the PI response specifically in astrocytes is a recent discovery. It was probably not found earlier because astrocytes were not considered to have receptors for many years. As such, any role they may have had in the generation of the PI response from brain slices or mixed cultures was ignored. Also some

65

investigators reported that the response from glial cells was very small compared to that of neurons95. It is now known that astrocytes respond to several stimuli by effecting an increase in PI turnover216.

Earlier experiments carried out by Dr. Andrzej Cholewinski in this laboratory have shown a PI response to several peptides in different brain regions43. These responses were elicited by supramaximal concentrations of peptides. From this data it was not possible to determine if the response was specifically receptor-mediated, or if it was mediated by other indirect means. A range of peptides that had elicited responses in this initial study were targetted. This study aimed to examine the PI responses in more detail, especially with regard to their possible physiological significance and regional differences.

3.2. METHODS.

3.2.1. Tissue Culture.

3.2.1.1. Astrocyte Media.

Media were produced fresh about once every six weeks from DMEM powdered media (Imperial Labs.), containing 4.5g/L glucose and L-glutamine without sodium pyruvate and sodium bicarbonate. The media also contained no L-valine and twice the concentration of D-valine of standard DMEM.

66

Developmental profile of cortex.DIV Total no . of cells % GFAP +ve NO. Of fields3 40 ± 6 78 ± 4 97 191 ± 20 88 ± 4 910 322 ± 28 91 ± 4 912 346 ± 33 96 ± 2 3Summary of culture purity.Cortex.DIV Total no. of cells % GFAP +ve No. of12 144 ± 8 99 ± 0.5 412 96 ± 12 98 ± 1.2 411 85 ± 8 97 ± 2.5 412 178 + 24 99 ± 0.4 411 133 ± 18 96 ± 1.9 411 100 ± 10 89 ± 0.6 4Spinal cord.DIV Total no. of cells % GFAP +ve No. of12 102 + 17 98 ± 0.6 412 129 ± 14 98 + 0.9 312 98 ± 2 88 ± 7.4 312 49 ± 16 97 ± 2.0 412 137 ± 16 98 ± 0.7 5

f i e l d s

f ie lc £ s

Table 2A. This table shows the developmental purity profile of astrocytes in cortical cultures. The mean cell counts and percentage purity of several experiments for both cortex and spinal cord cultures are also shown. Cell counts are determined as mean ± SEM coverslips. Astrocytes areidentified as cells which stain positively with rabbit anti-GFAP/TRITC, as compared with controls.

Purification of spinal cord cultures.Days in D,L-valine DMEM D-valine DMEMculture

GFAP Thyl .1 GFAP Thyl .16 69.0 + 3.7 22.8 + 3.3 77.9 + 1.2 '14.1 + 1.98 71.0 + 2.7 22.8 + 3.4 86.7 + 0.9 5.7 + 1.610 71.3 + 3.1 25.3 + 2.6 91.2 + 0.6 3.4 + 0.312 73.4 + 3.6 20.7 + 2.2 92.1 + 1.0 2.7 + 0.6Table 2B. This indicates the improvement in purification of spinal cord cultures which is effected by changing the culture medium to D-valine at 5 DIV, 8 DIV and 10 DIV. Results are expressed as mean + SEM of four experiments for 6, 8, and 10 DIV and three experiments for 12 DIV. In each experiment ten random fields were counted. This table is taken from Cholewinski et al, 198944.

This latter modification was introduced as a method of decreasing the number of fibroblasts present in the spinal cord cultures74/!̂ In the case of cultures for use in PI assays, further modifications of the medium were incorporated. Initially, Hams F-10 was substituted for DMEM during the labelling period to increase the specific activity of the label. Later a further modification improved this by using a custom-made inositol-free variation of the D-valine medium. Sodium bicarbonate (3.7 g/L) was added fresh to the media when they were prepared.

Astrocyte medium (AM) was prepared by adding, when required, L-valine 95 mg/L (Sigma), 1% gentamicin (as gentamicin sulphate, Flow Labs.), and 10% foetal calf serum (Imperial Labs.). When D-valine DMEM was required, dialysed serum was used.

3.2.1.2. Serum dialysis.

Dialysis tubing was prepared by boiling in four changes of 0.19 M sodium carbonate, 13.4 mM EDTA . The serum was dialysed for three days against normal saline (1:50, serum:saline), one change per day. After this it was filter-sterilized and heat-inactivated at 56°C for one hour.

3.2.1.3. Astrocyte culture.

These methods were standard for all experiments using67

astrocyte cultures except that for PI assays the cells were plated onto round 13 mm No. 2 glass coverslips coated with poly-L-lysine.

Usually cultures were grown up for twelve days in D/L-valine media with media changes at five and ten days in vitro (DIV). Spinal cord cultures were changed to D-valine at 5 DIV and 10 DIV. If cultures were to be used for PI assays they were changed to Hams F-10 inositol-free D- or D/L-valine media as appropriate at 10 DIV.

3.2.1.3.1. Cortex.

Newborn rat pups ( 1 - 3 days postnatal) were decapitated, the brain exposed and the cortices lifted out onto a small chopping board. They were chopped in two directions at right angles to each other. The tissue was suspended in EBS + trypsin + BSA + DNAase (see Appendix 1 for Tissue culture solutions), and incubated at 37°C for fifteen minutes. An equal volume of AM was added and the tissue centrifuged at 650 g for five minutes. The supernatant was aspirated, the pellet resuspended in EBS + BSA + DNAase and triturated with a Pasteur pipette twenty-five times to produce a single cell suspension. After settling for five minutes, the supernatant was removed, fresh EBS + BSA + DNAase was added and the trituration process was repeated for ten passes on the settled tissue. The supernatants were pooled and centrifuged as before. After aspiration of the supernatant

68

the cells were resuspended in AM to a concentration of 4 x 1 0 5 cells per ml and plated out (0.5 ml/well) in 24-well plates (Flow Labs.), previously coated with poly-L-lysine (5/ig/ml) containing 0.5 ml of AM. When larger plates were used a proportionally larger number of cells was plated out.

3.2.1.3.2. Spinal cord.

The animals were decapitated as before, and the skin was dissected away along the spinal column. An incision was made at the base of the tail and the spinal cord exposed by cutting along the spinal column fom the tail up to the pad of adipose tissue below the neck. The spinal cord was removed by running a fine pair of forceps up the column. The meninges were stripped off and the tissue treated as described as above.

3.2.1.3.3. Cerebellum.

The animals were decapitated and the brains exposed.The cerebellum was removed by cutting it away in front and behind, then lifted out with scissors. It was then treated as the cortex.

3.2.2.1. Phosphoinositide assay.

The cells were labelled with 0.5/1.0 /xCi/ml of [3 H] myo-inositol in Hams F-10 or inositol-free DMEM medium 48 -

69

72 hours prior to the assay43.

Medium was aspirated and cells washed twice with incubation medium (Hams F-10, 25 mM HEPES, 10 mM Lithium chloride, 28.6 mM sodium hydrogen carbonate, 5 mM magnesium sulphate, and fresh; 0.4 mg/ml bovine serum albumin Type V, 40 /zg/ml bacitracin, 1.6 /Ltg/ml leupeptin, 0.86 /ig/ml chymostatin) at 37°C, before pre-incubating for 30 minutes. In single point assays, peptides were used at a final concentration of 1 juM; in other experiments, to determine the ED5 0 , peptides were used at a range of concentrations between 1 pM and 100 /xM. In all experiments, bradykinin was included, as a separate point, at a concentration of 10” 5 M, as a positive control, since it shows a good response in all regions to be examined43.

After incubating with peptide(s) for 45 minutes ( 5 - 90 minutes for time-course experiments), the reaction was stopped by removing the coverslips to an ice-cold chloroform/methanol mix (2:1 ratio). The coverslips were then broken, 0.5 ml of distilled water added, the contents mixed and then the two phases allowed to separate. 2 0 0 fi 1 samples of the upper (lipid) phase were removed and allowed to evaporate before addition of scintillant (Aquasol-2,NEN) and counting. 0.5 ml samples of the aqueous phase were taken, then 3 mis of distilled water and 0.5 mis of 50% Dowex anion-exchange resin (AG 1-X8, 100 - 200 mesh, formate form, BIO-RAD) were added. The resin was then washed with 3 mis of 5 mM myo-inositol four times, before

70

1

the inositol phosphates were batch eluted with 1 M ammonium formate in 0.1 M formic acid. Scintillant is added to the eluant and counted. In all cases controls were included. Each point is the mean of quadruplicate samples.

3.2.2.2. DIV assay.

Cells were usually used at about 12 DIV, when approaching confluency, except in this assay when the development of response over days in vitro was examined.In this case cells were assayed from 3 DIV to 28 DIV.

3.2.2.3. Separation of inositol phosphates.

Cells were plated directly onto 60 mm plates for this experiment, in order to increase the yield of IP's. The protocol was as described as above except for the following alterations. The incubation medium was identical except that LiCl was omitted, since it has been found that it's inclusion tends to selectively increase IP^ with smaller increases in IP2 and IP3 up to 30 seconds incubation1.Also a time- and concentration-dependent inhibition of IP3

accumulation was found specifcally in rat cerebral cortex15. The cells were incubated with peptide for only 15 seconds, and ice-cold methanol was added to the plates to stop the reaction. This was then added to the chloroform. After dilution with an equal volume of distilled water the mixture was then applied to a pre-washed 1 ml column of the Dowex resin and the inositol

71

phosphates eluted by the following solutions:-

1 .

2.3.4.5.6 .

Distilled water60 mM Ammonium formate,150 mM 0.4 M1.0 M2.0 M

5 mM disodium tetraborate 0.1 M formic acid

ii ii it

it it ti

i i i i i i

1 ml fractions were collected, scintillant added and then counted.

3.3. RESULTS.

Time-course experiments (Figs. 9, 10) indicated a rapid increase in the rate of turnover in the first fifteen minutes of the assay, but that by 30 - 45 minutes the rate of turnover was constant. This occurred in both the spinal cord and cortex cultures. Routinely all experiments were carried out at 45 minutes to produce a significant response. The control and stimulated levels were compared in order to check that desensitization of the response, as would be evidenced by a decrease in the rate of increase of

level compared to control, had not occurred. There was also no apparent decrease in control levels which might suggest a depletion of the inositol pools. This was confirmed by measurements of the decrease in lipid counts in the developmental studies, which showed average decreases of 2.5% and 4.6% in cortex and spinal cord, respectively. An interesting finding is the differences in both control and

72

Figure 9. Time course of SP-stimulated PI response in cortex cultures (12 DIV) over 90 minutes. Substance P was used at 10" 6 M.

Representative of two experiments, each point in quadruplicate.

73

PI stimulation time courseSubstance P on Cortex

DPM (Thousands)

D Control * SP

was used at 10_g m . [^H]Inositol phosphates released were separated on a Dowex column, and counted.

Representative of two experiments, each point in quadruplicate.

Figure 10. Time course of SP-stimulated PI response inspinal cord cultures (12 DIV) over 90 minutes. Substance P

74

PI stimulation time courseSubstance P on Spinal Cord

DPM (Thousands)

D Control * SP

stimulated dpm levels in the spinal cord and cortex. The spinal cord control level is approximately three times that of the cortex at t = 5 minutes, and maintains this difference up to 90 minutes. The stimulated levels follow a similar pattern.

The dose response curves produced are shown in Figures 1 1 - 2 0 inclusive. Each of these is representative of a number of experiments as shown in Table 3. With the exception of NKA in spinal cord, all the peptides show an ED5 0 between 1 0 - -1-0 and 1 0 “8 . The standard errors are quite large in some cases, reflecting the innate variability of this system of measurement and the subjective nature of the production of the ED5 0 curves. (^5c£

The dose-response curves are measured as percentages of the control value since the variation in cpm/dpm values between experiments was too great to allow direct comparison. In comparing percentages the small errors contributed by differences between cultures in terms of protein concentration and amount of label added were eliminated. Also, the procedure was modified at certain stages but the comparisons remained true even when the cpm/dpm values were dramatically different. There was no difference in the percentage stimulation when cpm or dpm was measured. Increases in the dpm levels were introduced at an early stage by using two coverslips per point. This was abandoned on the introduction of inositol-free DMEM since the dpm levels were easily measurable with only one

75

EDgo's were calculated as the 'half-maximal' response i.e. (maximum stimulation - minimum stimulation) / 2 (ED5 Q) only provides an estimate of the KD since the PI response in intact cells is used as an index. If 'spare receptors' are present, or a threshold concentration must be reached before activation of the system, the ED5 Q will deviate from the KD .

If a maximal response has not been reached, as it may appear, from some of the representative graphs, the ED5 0 which is determined by this method, will be artificially decreased. The experiments which may indicate this phenomenon involve NKA in spinal cord and cortex and oxytocin in cortex. As can be seen from Table 3, these demonstrate a lower affinity than the other peptides and this may indicate that the peptides are acting as partial agonists on receptors specific for other peptides of similar structure e.g. oxytocin on vasopressin receptors, and NKA on NK^ or NK2 receptors, be of equivalent importance.

A major disadvantage of this method of analysis is that the curves are fitted manually, and therefore prone to subjective variations. When a graph was fitted manually to a selection of curves, the range of ED5 Q values calculated showed very little variation.

A large number of the experiments carried here use the batch elution method, to extract IP's from other substances. It is understood that this method is effective only in looking at I P 1 's, since recovery of the inositol polyphosphates by this method is poor. However, in this study we are only concerned with a maximal indication of response of the whole system, not with the constituents of that response, and this method is sufficient for these requirements.

Representative of two experiments, each point in quadruplicate.

Figure 11. Dose-response of SP-stimulated PI response incortex astrocytes (12 DIV). ED50 = 1.9 nM.

76

Dose-response of SP stimulationCortex

220% - r

200%

Per 180%centa 160%9eofcontr01

140% - -

120% - -

100% - -

80%

0 -11 -10 -9 -8 -7 -6

Log {Concentration (M)

□ Mean ■ SEM

Figure 12. Dose-response of NKA-stimulated PI response incortex astrocytes (12 DIV). ED5 0 = 56.1 nM.

Representative of two experiments, each point in quadruplicate.

77

Dose-response of NKA stimulationCortex

300%

50%

0 -11 -10 -9 -8 -7 -6 -5

Log {Concentration (M)

■ SEM □ Mean

Representative of two experiments, each point in quadruplicate.

Figure 13. Dose-response of NKB-stimulated PI response incortex astrocytes (12 DIV). ED5 0 = 57.1 nM.

78

Dose-response of NKB stimulationCortex

195%

75%

0 -10 -9 -8 -7 -6

Log {Concentration (M)

□ Mean ■ SEM

Representative of two experiments, each point in quadruplicate.

Figure 14. Dose-response of EL-stimulated PI response incortex astrocytes (12 DIV). ED5 0 = 4.2 nM.

79

Dose-response of EL stimulationCortex

Log {Concentration (M); □ Mean ■ SEM

Representative of two experiments, each point in quadruplicate.

Figure 15. Dose-response of BK-stimulated PI response incortex astrocytes (12 DIV). ED5 0 = 0.61 nM.

80

Dose-response of BK stimulationCortex

percentageofcontr01

0 -11 -10 -9 -8 -7 -6 -5 -4Log {Concentration (M)

□ Mean ■ SEM

Representative of three experiments, each point in quadruplicate.

Figure 16. Dose-response of VP-stimulated PI response incortex astrocytes (12 DIV). ED5 0 = 0.54 nM.

81

Dose-response of VP stimulationCortex

450% - r

400% - -

350%

300%

250% - -

200% - -

150% —

100% —

50%

0 -12 -11 -10 -9 -8 -7 -6Log {Concentration (M)

□ Mean ■ SEM

Figure 17. Dose-response of OT-stimulated PI response incortex astrocytes ( 1 2 DIV). ED5 0 = 5 . 6 nM.

Representative of two experiments, each point in quadruplicate.

82

r

Dose-response of OT stimulationCortex

300%

250%Percent 200% ageofc 150%ontro

100%

50%

0 -11 -10 -9 -8 -7 -6Log {Concentration (M)}

□ Mean ■ SEM

Representative of-three experiments, each point in quadruplicate.

Figure 18. Dose-response of SP-stimulated PI response inspinal cord astrocytes (12 DIV). ED5 0 = 0.05 nM.

83

Dose-response of SP stimulation Spinal cord

380%

330%

280%

230%

180%

130%

80%

0 -11 -10 -9 -8 -7 -6 -5Log {Concentration (M)

□ Mean ■ SEM

Representative of two experiments, each point in quadruplicate.

Figure 19. Dose-response of NKA-stimulated PI response inspinal cord astrocytes (12 DIV). ED50 = 291 nM.

84

Dose-response of NKA stimulationSpinal Cord

0 -10 -9 -8 -7 -6 -5 -4Log {Concentration (M)}

Figure 20. Dose-response of NKB-stimulated PI response inspinal cord astrocytes (12 DIV). ED5 0 = 0.32 nM.

Representative of four experiments, each point in quadruplicate.

85

Dose-response of NKB stimulationSpinal Cord

Percentageofcontr01

170%

160%

150%

140%

130%

120%

110%

100%

90%

80%

□ B

0 -12 -11 -10 -9 -8 -7 -6 -5Log {Concentration (M)

□ Mean ■ SEM

Table 3. Summary of ED5 0 values determined from PI dose-response curves.

8 6

TABLE 3 ASTROCYTE RECEPTOR SUMMARY

REGION PEPTIDE £^50 MEAN e d 50+ s *E.CORTEX SUBSTANCE P 1.9 nM 1 . 1 + 0.85 nM

0.27 nMNEUROKININ A 56.2 nM 56 + 0.07 nM

56.1 nMNEUROKININ B 57.1 nM 36 + 2 1 nM

14.3 nMELEDOISIN 2.05 nM 3.1 + 1 . 1 nM

4.2 nMBRADYKININ 0.35 nM 0.48 + 0.13 nM

0.61 nMVASOPRESSIN 0.61 nM 0.51 + 0.06 nM

0.39 nM0.54 nM

OXYTOCIN 14.8 nM 1 0 . 2 + 4.6 nM5.6 nM

SPINAL CORD SUBSTANCE P 1 . 8 nM 1 . 1 + 0.54 nM1.5 nM0.05 nM

NEUROKININ A 291 nM 332 + 41 nM373 nM

NEUROKININ B 0.14 nM 1.35 + 1.15 nM0 . 1 2 nM0.32 nM4.8 nM

coverslip. When the cells were incubated with either inositol-free DMEM or low-inositol Hams F-10, no significant difference was found between the control or stimulated levels in either cortex or spinal cord, in either medium. The only problem in presenting the data in this form is that it masks the differences in dpm levels between the two areas. This can be illustrated by the fact that in the time course experiment (Figs. 9, 10) the control dpm value at 45 minutes is 519 ± 158 dpm for cortex and 12366 ± 1026 dpm for spinal cord.

In order to gain an overview of the PI response, a few experiments were carried out which examined the response in spinal cord and later, in the cerebellum, to a range of peptides. Representative examples are shown in Figures 21, 22. This demonstrates further the differences in response to peptides based on regional localization.

In view of the difference in the levels of response between the cortex and the spinal cord, a developmental study was carried out. This involved the examination, at a series of time points, of the response in sister cultures of the two regions to selected peptides. The results of this study are shown in Figures 23 - 27 inclusive. The pattern varies between peptides but there are some outstanding consistencies. In all the experiments, as in the time course experiments, there is clear evidence of a higher response in spinal cord at all time points. The only exception to this is at 15 DIV for neurokinin B

87

Figure 21. Overview of peptide-stimulated PI responses in spinal cord astrocytes at 12 DIV. All peptides are used at 10" 6 M. S.E.M. are designated by

Representative of two experiments, each point in quadruplicate.

8 8

PI stimulation of spinal cord

0 50 100 150 200 250 300 350 400Percentage of control

Figure 22. Overview of peptide-stimulated PI responses in cerebellar astrocytes at 12 DIV. All peptides are used at 10-*6 M. S.E.M. are designated by ' +

Representative of two experiments, each point in quadruplicate.

89

PI stimulation of cerebellum

0 200 400 600 800 1000 1200 1400 1600Percentage of control

Figure 23. Developmental SP-stimulated PI response in cortex (left) and spinal cord (right) up to 28 DIV. Substance P was used at 10~ 6 M. Control turnover = 100%^ each point in quadruplicate.

90

Substance P PI stimulationP

DIVs ^ l Cortex \//A Spinal cord

Figure 24. Developmental NKA-stimulated PI response in cortex (left) and spinal cord (right) up to 28 DIV. Neurokinin A was used at 10- 6 M. Control turnover = 100%. each point in quadruplicate.

91

i

•-►

3 0

0

-*

»0

C

DC

QJ

D—

-Z

SC

DO

-'i

CD

T)

Neurokinin A PI stimulation

DIVCortex V/A Spinal cord

Figure 25. Developmental NKB-stimulated PI response in cortex (left) and spinal cord (right) up to 28 DIV. Neurokinin B was used at 10“ 6 M. Control turnover = 100%.

each point in quadruplicate.

92

30

0

-**

O

<D

(QD

)*-*

,3®

0“

iCD

"D

Neurokinin B PI stimulation

DIVCortex \//A Spinal cord

Figure 26. Developmental EL-stimulated PI response in cortex (left) and spinal cord (right) up to 28 DIV. Eledoisin was used at 10“6 M. Control turnover = 100%.

each point in quadruplicate.

93

*-*■ 3

O O

O

©

<Q

ft)»

^3®

O-«

©“0

Eledoisin PI stimulation

DIVCortex Y//A Spinal cord

Figure 27. Developmental BK-stimulated PI response in cortex (left) and spinal cord (right) up to 28 DIV. Bradykinin was used at 10” 6 M. Control turnover = 100%.

each point in quadruplicate.

94

•-►3

0 0

—•

* O

CD(Q

£13—

►3C

DO

~«C

D“D

Bradykinin PI stimulation

DIVCortex V/A Spinal cord

stimulation. As described earlier, this does not indicateSthat the total reponse is higher in cortex, but that atA

this point, the percentage increase over control is higher.

Bradykinin is singular in the way in which the alterations in level of response is mirrored in the two regions, although the spinal cord remains higher. It also seems to reach peak levels at a later time point (12 DIV) than the other peptides examined.

In the spinal cord SP, NKA, NKB and eledoisin all show a similar pattern of rapid increase to maximum levels by about 8 DIV. This level of stimulation is maintained at least until 21 DIV. There may then be a decrease, but the trend is not clearly established because of lack of data beyond 28 DIV. There is no collective trend for the cortex stimulation by these peptides. SP stimulation shows the same trend as does BK in the cortex, except that the peak activity occurs at 15 DIV as opposed to 12 DIV. NKB, NKA and eledoisin show a more complex pattern. The response increases up to 8 DIV, then shows a small decrease at 12 DIV. There is a further increase to maximum at 15 DIV, followed by a rapid decrease to 28 DIV.

An interesting feature of the response in cortex is that the response is apparently depressed below control levels at 3 DIV and 28 DIV for all these peptides. This clearly contrasts with the spinal cord response, where the smallest response, at 3 or 28 DIV, is still 24% above the

95

control level.

The analysis of the batch response was carried out by looking at the fast PI response at fifteen seconds. This was examined using both bradykinin, as positive control, and substance P (Figs. 28, 29). In both cases it is possible to see that the level of all inositol phospates is elevated above control levels (no stimulation), although to different degrees. The level of IP^ in the stimulated cells indicates that the breakdown of IP3 to IP2 and IP! has already begun. The individual peaks are quite broad for IP2 and IP3 , which indicates the probable presence of more than one isomer. A continuous gradient would allow separation of the isomers which elute close together, since they have similar properties in this system. The bradykinin profile indicates that the levels of inositol phosphates eluting after IP3 are also elevated. This may suggest the presence of IP4 , IP5 , or IP6. The position of the IP3 peak was identified using [3 H]IP3 , kindly donated by Dr. M.J.O. Wakelam, University of Glasgow. Using the same system, the peak eluted between fractions 47 and 52.

96

Figure 28. Separation of [3 H]inositol phosphates produced by SP-stimulation of cortical astrocytes. [3 H]Inositol phosphates released were separated on a Dowex column, and counted. RePresentative of two experiments

*

97

Substance P PI stimulationSeparation of inositol phosphates

DPM (Thousands)

■e— Control — Substance P

Figure 29. Separation of [3 H]inositol phosphates produced by BK-stimulation of cortical astrocytes. [3 H]Inositol phosphates released were separated on a Dowex column, and counted. Representative of two experiments.

98

3.4. DISCUSSION.

The existence of the PI response in astrocytes has previously been described in detail, using adrenergic and muscarinic agonists216. It has been reported using peptide agonists, and the relevance of these responses is demonstrated here by their activity at physiologically significant concentrations of agonists. The separation of the inositol phosphates indicates that the IP degradation follows the main pathway as suggested earlier (Figs. 28,29) .

The measurement of the PI response used the method as described by Cholewinski43. Certain modifications were made to the method to improve the sensitivity of the assay. Initial experiments produced statistically viable results, but it was considered that the low cpm levels throughout could mask variations, because of the increased weighting on such small values. Therefore, the levels were increased by using two coverslips per point. Also dpm measurements were substituted for cpm measurements, since these eliminated the possible small sample-to-sample variations in quenching. In order to decrease the diluting effect of the myo-inositol in the DMEM ( 7 mg/1), Hams F-10 medium (0.54 mg/1) was used during incorporation. This was later improved further by using inositol-free DMEM. The degree of the responses was not affected by these changes. This suggests that decreasing the available inositol at this stage does not have a limiting effect on the response.

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%-inositol labelled lipids (PI) in SP-stimulated cultures-,Cortex cultures.SP (M) DPM ;± SEM n DPM ± SEM n0 4649 + 234 4 1679 ± 212 410"11 4234 ± 67 4 1088 + 62 410"10 4191 ± 307 4 1807 ± 160 410" 9 2795 ± 380 4 1493 ± 125 410“ 8 2757 ± 363 4 1714 ± 194 410" 7 3241 ± 272 4 1711 ± 235 410“ 6 4619 ± 283 4 1776 ± 210 310" 5 4659 ± 245 4 2025 ± 34 3Spinal cord cultures.,.SP (M) DPM :± SEM n DPM ± SEM n DPM ± SEM n0 2996 ± 187 4 4891 ± 850 4 69682 ± 2118 410"11 3121 ± 357 4 4502 ± 536 4 49385 ± 6030 410"10 3279 + 455 4 4728 ± 628 4 53513 ± 3433 410" 9 3918 ± 177 4 4430 ± 499 4 42740 + 1986 410" 8 2488 + 191 4 5176 ± 517 4 53312 + 2003 410" 7 2638 + 196 4 5252 ± 424 4 56284 + 3452 410- 6 2419 ± 343 4 5211 ± 137 4 60070 ± 2154 410" 5 3132 + 305 4 5477 ± 623 4 48213 ± 3213 4

Table 3A. 'This table shows the variation in lipid :recoverybetween experiments, which prevents pooling of the raw data. Within each experiment, the decrease in lipid pool content is not remarkable, indicating that this is not a limiting factor for activation of the PI cycle. As shown, each experiment is carried out in quadruplicate where possible. The counts are derived from 200 ul samples of the upper phase of the extraction mixture.

It seems therefore, that the time-courses indicate that with SP, desensitization of the system occurs after 15 minutes in cortical cultures. While in spinal cord cultures, desensitization may occur before five minutes, or the turnover rate may be much slower than in cortex. In contrast to this finding, is that of Torrens et al, 1989^5, wh0 find no desensitization up to 60 minutes incubation, using SP at 10“^M. This suggests that there is either a concentration threshold at which desensitization occurs, or that the rate of desensitization is concentration-dependent. It would be interesting to test this phenomenon at a range of concentrations to determine either the threshold concentration or the rate.

The rate of IP production was biphasic in form but it continued to increase, up to 90 minutes. This compares well with the time-courses of PI response gained using NaF, calcium and GTPrS in brain membranes94. The change in the rate of increase may be thought to indicate partial desensitization of a population of receptors. (.5^^ Desensitization of PI response has been reported^ substance P in parotid acinar cells 188,271^ This was attributed to either desensitization, peptide breakdown or IP degradation. The addition of fresh peptide at a higher concentration had no effect, implying that desensitization had occurred. The affinity of the receptors involved was found to be unchanged, while the number decreased. No cross-desensitization was detected with the muscarinic cholinergic agonist, methacholine. Also the effect was not dependent on calcium mobilization, or affected by PKC activation265. However, IP3 turnover induced by substance P was found to be reduced by PKC activation, suggesting a possible role in negative feedback of the stimulation. In fact, phorbol esters have recently been shown to mediate down-regulation of astrocyte receptors coupled to PI metabolism by the same mechanism as agonists220. This information shows that the desensitization effect is not mediated via the common components of the system such as PLC, G-proteins or calcium effects. This suggests that the effect is a direct result of receptor sequestration or removal, as evidenced by the decreases in available receptor number. As will be seen in the next chapter,

100

there was no evidence of desensitization by receptorremoval over this time interval, in the time course of[125i ]b r s p binding which implies that desensitization occurred by

another method. This suggests that this system differs from the parotid acinar cells in the method of desensitization.

Alternatively, the the second phase may indicate a limiting effect of the phospholipid pools. However, examination of the labelled lipid pools indicated that at least 75 % of the pool in the activated system, compared to control levels, was unaffected.

If densensitization of the mechanism had occurred, by a mechanism other than receptor removal, the errors in the ED50s would indicate an artificially lowered value for the apparent Kp, i.e. higher affinity. This was considered to be unlikely since the binding data shows that the ED50s were, in fact, generally higher, i.e. lower affinity, than the K^'s found.

Bradykinin has previously shown similar responses to those described here, in cerebral microvascular endothelial cells (EC5 0 = 0.6 - 1.0 nM)64. Also it has demonstrated a pertussis toxin-insensitive PI stimulation in NG108-15 and NG115-401L cell lines211'97'136. In fact bradykinin also shows a pertussis toxin-sensitive inhibition of adenylate cyclase in NG108-15 cells1 2 2 and pertussis toxin-sensitive PI stimulation in a neuroblastoma x dorsal root ganglion

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sensory neuron cell line (F-ll)83. This suggests that the response is not mediated via G-̂ , the G-protein associated with inhibitory stimulation of the adenylate cyclase system. This supports the theory that different G-proteins mediate the effects of the adenylate cyclase and PI systems.

Vasopressin stimulation of the PI response has been reported in hippocampal slices of rat brain, giving an ED5 0

= 7.1 nM259. This response was decreased by V^ receptor antagonists, implying a subtype-specific response.Oxytocin had no effect in this tissue, as opposed to the response found in the cultures of cortex. This indicates that the PI response may not be linked to all subtypes of a specific receptor. In fact this property may be used to categorize receptors and their subtypes.

In considering the possibility of the presence of different subtypes of receptors, the identity of the subtypes may be elucidated by studying the potencies of a range of agonists. In these experiments, a range of tachykinins have been examined and their order of potency may indicate the presence of a specific subtype.Tachykinin receptors are classified according to the following order of potency230•116:-

NK^ SP > NKA > EL > NKBNK2 NKA > NKB > EL > SPNK3 NKB > EL >NKA > SP

102

In these studies the following orders of potencies are shown:-

Cortex SP > EL > NKB > NKASpinal cord SP > NKB » NKA

The differences between the peptides are quite small in some cases, but it would seem that the evidence points to NK^ receptors being present in both tissues. However, this does not encompass the possibility that more than one receptor subtype may be present and contributing to the response or binding the ligands without responding, which would affect the calculated ED5 0 values.

Until recently, most measurements of PI hydrolysis were carried out on brain slices or mixed cell preparations.The relative importance of the glial contribution was reported to be small, when using muscarinic or adrenergic stimuli96. This finding has been extended to apply to all stimuli on glial cells by some authors. However, the importance of the response recorded in glial cells is demonstrated both here and elsewhere216'217'43. Histamine-mediated increase in PI turnover provides a good example. Histamine was found to stimulate PI turnover in developing rat brain via H^-receptors4 7 . It was of particular interest because, in the early stages of development, 25% of the response was found not to be mediated via the receptor. Later experiments by the same

103

group showed a localization of the PI response in glial cells, but without the uncoupled response8 . Another interesting feature of these studies was the developmental progression of the response47. The pattern of the histamine stimulated PI hydrolysis in cortical slices was similar to that of the tachykinin stimulation of spinal cord in its initial increase, followed by a plateau and a slight decrease at about 20 dpn. No other parallels can be drawn between the two situations because of our ignorance of the source of the response in the slice preparation. However, this study did draw attention to the variability of the EC5 0 values throughout development, which was a facet not included in my experiments.

The level of response gained in these experiments is thought to depend on a number of features. The primary factors being the amount of receptors present and the availability of the pools of PI intermediates.Consequently variations in the level of response between regions and time points may indicate variations in these elements.

In order to explore this situation in more detail, it becomes necessary to determine the source of variation in response between peptides and regions. It has been suggested that there is conservation of the mechanism of peptide action187. This does not preclude variations in the individual components of such a system. It would therefore seem sensible to examine these components to

104

determine if the regional and/or developmental variations can be localized to one part of the mechanism, or if they exist throughout the system. Initially the receptors could be examined; a study of the tachykinin receptors in these cultures is contained in the next chapter.

In the next instance, the variations in the G-protein linkage should be considered. The G-proteins are made up of three subunits; alpha, beta and gamma. The alpha subunits embody the specific activity of the molecules, forming the pertussis toxin substrates, while the beta-gamma subunits are generally interchangeable. The role of G-proteins has been extensivelydescribed89'205'204'28. There are seven known G-proteins, of which Gs is positively linked to adenylyl cyclase, while two forms of G^ are negatively linked. There are also two isoforms of G-j- which are positively linked to cGMP-PDE, with a major role in phototransduction. As yet no functional link for G0 or Gp have been described but it is possible that one or both of these may be linked to the PI system. A recent review by Lo & Hughes, suggests the possibility of multiple G-proteins mediating the PI response169. They also include a novel G-protein, Gc , sensitive to cholera toxin, which may mediate bradykinin and vasopressin receptors. Gp has been isolated from placenta, platelets and possibly in bovine brain288, G0 has been found to be plentiful in brain261. Also it has recently been localized specifically in neurons and glia32. It shows regional differences in glia since there are high

105

levels in cortex and colliculi, while levels in cerebellar astrocytes are very low. A 40 kDA protein constituted the major substrate for pertussis toxin in glial cells. This has been found in several other tissues but a common identity has yet to be established. The differential regional localization of the G-proteins may explain the differences in PI responses.

These mediators may also explain the developmental differences elucidated, since they also show marked differences during development. In chick atria there are two pertussis toxin substrates, < 2 3 9 and a42. The amount of the former shows a rapid increase at 4 days in oyp, compared to the amount of the latter100. In 3T3 cells, 0 : 3 9

initially decreases then increases at the onset of differentiation. The other substrate, a 4 1 showed no effect. It seems likely that some variations during development occur in the G-proteins mediating the PI response in astrocytes. Coupled to the fact that different alpha-subunits have different biochemical properties, this may provide a basis to explain the differences in responsiveness.

Following the line of investigation suggested, the next component to be examined is the enzyme PLC. PLC has been grouped with adenylate cyclase as the central components of their respective second messenger systems98. PLC is coupled to the G-proteins described above, as has been described for SP in the rat parotid gland271, and for VP in the WRK-1

106

cell line9 8 . The action of PLC has been described earlier. The enzyme cannot be activated directly by agonists, but requires the presence of guanyl nucleotides. When PLC is activated by agonists it becomes sensitive to intracellular [Ca2+]. PLC activity is also under tonic inhibitory control of the common beta-gamma subunits of G-proteins, and by an unidentified pertussis toxin-sensitive G-protein in bovine retina137. In this tissue, variations in sensitivities of PLC substrates to the light stimulus, toxins and GTP<fs, were found. This suggested the possibility of multiple forms of PLC, with variable G-protein regulation137. This theory was supported by a study which isolated three different isozymes of PLC in bovine brain245. They exhibit variations in molecular weight, specific activity dependent on pH, and inhibition by BSA. Substance P has also exhibited a dual mechanism of PI hydrolysis which may be accounted for by these variations40. In cerebral cortex slices, low concentrations (65 pM) of SP have shown preferential hydrolysis of 1 -acyl(predominantly stearoyl)-2 -arachidonylglycerophospoinositol, while higher concentrations (0.65 uM) induce a non-specific hydrolysis. The mechanism of this reaction is not clearly understood, but could be related to differential PLC activation or G-protein linkage to the PLA2 system. These factors suggest a possible basis for variation on the PI response. It would be interesting to determine if there are regional or cell-specific localizations of the specific isozymes, and if any developmental correlates could be found.

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The possibility of agonist receptor variation was mentioned earlier. However, these are not the only species of receptor involved in the response. The recent characterization of both IP3 and IP4 receptors from brain membranes opens a new area for the investigation of the mechanism and control of the PI response. The IP3 receptor has now been characterized from cerebellar membranes, and autoradiographically identified in brain sections300. It apparently shows a high degree of regional variation in receptor number; the highest amount being found in the cerebellum, with lower levels in the cortex. [3H]IP4 binding to rat cerebellar membranes has elucidated IP4 sites distinct from IP3 sites as designated by affinity for substrates, sensitivity to calcium and, most interestingly, regional variations in brain and other tissues273. The highest binding of IP4|vas found in hippocampus and cerebellum. As yet no developmental studies have been carried out on these binding sites, but their differential distribution and their co-operative roles in the PI response provides yet another variable factor for consideration.

In conclusion, although this data clearly demonstrates the ability of astrocytes to respond to physiological concentrations of peptides by increasing the turnover of IPs, it is by no means clear how this response is functionally related. The ability of astrocytes in vivo specifically to demonstrate a similar response has yet to

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be shown. The difficulties inherent in such a study are considerable, since current techniques do not permit the co-localization of the biochemical response and specific identification of cell type in the ;Ln vivo situation. It is more likely that the detection of the presence of receptors, which are indicated by the responses found here, and could be co-localized with identifying immunochemical markers, will provide a more viable alternative starting point.

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4. RECEPTOR BINDING STUDIES

4.1. INTRODUCTION

4.1.1. Astrocyte receptors.

Despite the early discovery of astrocytes and the more recent recognition of their importance in the nervous system, it was only in the 1970's that the possibility of their possessing receptors was examined. Early studies merely demonstrated the effect of 'neurohormones' on glial cells in culture284. The demonstration of specific receptors for these substances has been reported only in the last few years. However, this field has now expanded considerably, and has been recently reviewed144'197. A summary of the current list of glial receptors is in Table 2.

Cell culture of glial cells has enabled investigators to examine these cells specifically, separated from the other cell types of the nervous system, and constitute the method of choice. Cell lines derived from glia are particularly useful, since they require far less maintenance than primary cultures 223. However, these preparations do not necessarily accurately reflect the in vivo situation, especially in case of cell lines, which, by their nature, have incorporated abnormal properties. In order to examine astrocytes selectively, it is necessary to label them with specific antibodies, a technique which has

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only been successfully performed in vitro186, or to destroy all other cell types, selectively, with neurotoxins, and then examine binding. The latter, however, does not allow for binding of the radioligands to the membranes of the dead cells. It is known that, in areas of neuronal damage, glial cells will proliferate. This property was exploited, in order to examine the binding of the radioligand in vivo. The glial scar may be induced by several means; such as stab wounds, neurotoxic injury, electrolysis, laser damage. It was considered that lesions caused by injection of the neurotoxin kainic acid would be most suitable for this purpose, because of previous experience with this method. The stab method is the simplest, but it suffers from the disadvantage that fibroblasts are frequently introduced to the lesion and these also proliferate. The laser and electrolytic methods require sophisticated and expensive equipment which limit their availability.

4.1.2. Theoretical background to receptor binding.

The aim of this study was to examine and characterize the receptors for substance P on astrocytes. The concept of 'receptors' was first introduced by P. Erhlich, although the term itself was initially coined by J.N. Langley. The most important principle of this concept was that of selectivity, from which came the quotation: "Corpora non agunt nisi fixata" (Agents cannot act unless bound). The earliest work was carried out by examining the

1 1 1

drug-response link, and consequently, the initial description of the agonist-receptor interactions relied heavily on investigations of drug action. Langley made intuitive assumptions concerning the formation of complexes between drugs and receptive substances, the dependence on the concentrations of the drugs and their affinities for the receptive substances. He also suggested that the system was saturable. These were pre-emptive of any algebraic characterization of the interactions.

The quantitation of these descriptions was carried outby A.J. Clark. He postulated that the rate of formation ofthe complexes was dependent on the concentrations of thedrug and the receptor, and that the rate of breakdown of

*

the complex formed was proportional to the number of complexes present. He then suggested that the number of receptors occupied was proportional to the response. This implied that the maximum response would only be produced if all the receptors were occupied, a situation which is seldom achieved experimentally, but that the occupation of all the receptors would not necessarily produce a maximum response. It was at this point that Stephenson suggested the concept of 'efficacy' as defined by these points: 1 ). The maximum effect may be produced by less than the maximum number of receptors; 2). The response is not linearly proportional to the number of receptors occupied; 3). Different drugs have different capacities to initiate a response, and thus occupy different numbers of receptors to produce an equal effect. The explanation of antagonism was

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defined as the occupation of the receptor with no effect. Experimental data also elucidated the phenomenon of receptor reserve or 'spare' receptors. This describes the situation where a response is produced without full receptor occupation. This situation is of particular use in neurotransmission since a rapid response can be gained from a very low agonist concentration.

The allostery theory was postulated to account for certain deviations from the simple discrete receptor interactions, such as non-linear coupling between receptor occupation and response. It suggested that two interconvertible states of the receptor existed, active and inactive, and that the ligand had differing affinities for the two states. The ligand would bind to the active, higher affinity state thus displacing the equilibrium towards that state. This would be seen as positive co-operativity. An antagonist would bind to the inactive state, causing a similar displacement.

As described earlier, the most important property of receptors is their specificity. For physiological effects, this is determined using dose-response curves to examine the order of potency. From this data, the ED5 0 /EC5 0 can be determined, and used as an index of potency. Specificity can also be demonstrated by using selective blockade by antagonists. However, problems may arise if the antagonists are not simply competitive, but also elicit a functional response. Unfortunately, it is not possible to

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determine the receptor density from this type of study. In order to ascertain this information, the easiest methodology is that of radioligand binding.

In the simplest form, the situation is assumed to be a single ligand exhibiting fully reversible binding to a homogeneous population of sites. This would behave according to simple mass action law, as follows;

klD + R v==- DR.................................1

k2

D = drug/hormone/agonist ( D* = labelled drug)R = receptor DR = drug-receptor complex k! = association rate constant k2 = dissociation rate constant

The derivation of various methods of measurement of the kinetic constants and their merits is contained in Appendix 2 .

Primarily, it is necessary to choose an appropriate radio-isotope for the study. There are four commonly available isotopes; 1 4 C, 3 H, 1 2 5 I, and 3 2 P. Tritium is very useful in many cases because of its long half-life (ti/ 2 = 12.3 years), and availability. Also biological activity is frequently unchanged by tritiation. However,

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its major disadvantage, which it shares with 14C, is its low specific activity. Even at maximum incorporation and receptor occupancy, this could only provide cpm/mg protein in the 103 range. The large amount of tissue required for a measurable level disqualifies these isotopes for studies with low receptor density or small amounts of tissue. 32P is also useful in certain applications, but, despite its high specific activity, it has a relatively short half-life (ti/ 2 = 14.3 days) which entails frequent replacement and consequent technical complications. From this brief summary, it can be seen that 125I is the isotope of choice. It has a manageable half-life of 60.2 days and a relatively high specific activity, which enables accurate measurement at low receptor density. It has certain disadvantages in its requirement of a tyrosine residue or an unsaturated cyclic system for incorporation. If these structures are not present in the native compound, or the protein is particularly sensitive, it is possible to incorporate the radiolabel by conjugation, via an active ester link, with iodinated Bolton-Hunter reagent (Fig. 30). In some cases 125I has also been shown to abate or destroy the native activity of the agonist. Therefore it is essential to determine that the synthesized radioligand is binding at the same specific sites as the unlabelled agonist. One of the advantages of using 125I ligands is that the samples require no pre-treatment e.g. scintillants before counting.

The most frequently determined parameters of binding are the affinity of the receptor and the receptor density.

115

Figure 30. Structure of Bolton-Hunter reagent, showing position of iodine on the benzyl ring. The peptides form an amide link to the reagent.

116

CJO

J

OJ

The former is usually described by the equilibriumdissociation constant or KD/ since its derivation assumes the system to be in equilibrium. The latter is termed bMAX* According to the above equation, we can determine this constant by measurement of the D*R complex.Therefore, this must be resolved experimentally from D*, the free ligand. Several methods of separation are available, each having individual merits and demerits.They can be categorised as applying to particulate (centrifugation, filtration) or soluble (equilibrium dialysis, gel filtration chromatography, precipitation, adsorption) systems. The separation of D* and D*R must be carried out according to the restrictions of the kinetic parameters. Low-affinity receptor/agonist complexes dissociate more quickly than high-affinity receptors, and consequently require very fast separation times. This is illustrated in Table 4.

Equilibrium dialysis is a powerful separation technique for soluble preparations, which uses two chambers of equal volume separated by a porous membrane. This allows the passage of D* but not R. At equilibrium or steady-state, equal volumes removed from both sides do not affect the equilibrium. This method is useful for low-affinity receptors, since D*R does not dissociate before measurement, thus avoiding non-equilibrium artefacts. Gel filtration is useful for sensitive singular experiments but problematical for multiple samples. Technically, both methods are tedious, the former because of cumbersome

117

Table 4. Separation times of ligand from receptor, related to Kp values.

118

TABLE 4. SEPARATION TIMES.

K d _ £ M 1 Seoaration time f0.15t1/2JL—

1 0 “ 1 2 1 . 2 days1 0 - 1 1 2 .9 hoursio“10 17 min.1 0 “ 9 1.7 min.

H O1 00 1 0 sec.

H O1

0 . 1 sec.

VO1OH

0 . 0 1 sec.

(* Assumes Association rate constant 106 M 1 sec 1 )sec 1 )

equipment, and the latter because o^its requirement for constant low temperatures .̂ Cn both cases there is a high level of non-specific binding. The technique of precipitation bases its success on the rapidity and completeness of precipitation, a problem with some small molecules since these do not precipitate effectively. Adsorption is carried out using inert supports such as talc and charcoal. The nature of this method excludes its use with large D*R complexes. Centrifugation is frequently used to separate the unadsorbed and adsorbed material.Care must be taken to ensure that the dissociation time is considered, and that no more than 10% of the D*R complex is lost.

The alternative method of separation is that for particulate preparations. Centrifugation is fast and effective, membranes are pelleted within seconds. Discontinuous or continuous systems may be used; the former increases the probability of D*R dissociation while the latter risks trapping of ligand in the pellet. This may be removed by washing, but the risk of D*R dissociation is increased. Filtration is a commonly used method, which is particularly useful when there are large numbers of samples. It has a minimum time limitation of 15 seconds, thus restricting it to receptors with KD < 10” 8 M. The other disadvantage is the frequent occurrence of non-specific binding to filters. This can be largely eliminated by pre-incubation of filters with anti-adsorbents e.g 0 .1 % polyethyleneimine or displacers

119

e.g. 2 0 0 fiK isoprenaline (p -receptors) . Experimentally, the time limitations are slightly more flexible since, it is known that reduction of temperature to 4°C immediately prior to separation decreases the dissociation rate of the complex. Filtration incorporates this decrease since the filter is under vacuum, below ambient temperature. Care should be taken to ensure that the affinity of the receptor does not change at lower temperatures.

Another alternative to these methods is binding to whole cell preparations. This has the advantage of most closely resembling the natural environment of the receptor, and is thus comparable with studies of receptor function.It is important, in this method, that the cells are washed thoroughly, so that the free ligand is removed. It is usual to solubilize the cells using 1% Triton X-100 or 2M sodium hydroxide, in order to measure the bound ligand. Also, these assays are usually carried out at lower temperatures than the in vivo situation, to avoid any possibility of active transport of the ligand.

The experimental protocol has to be adapted to fit each situation. This is particularly important for neuropeptide receptors, since the ligands are frequently susceptible to degradation by proteases in homogenate and whole-cell preparations104'295^260/174'280. It is important to avoid this because of the effects on the kinetic measurement and the possible interference of breakdown products. Peptide ligands are usually protected by adding protease

120

Table 5. Protease inhibitors and their specificities 104

121

TABLE 5. PROTEASE INHIBITORS.

INHIBITOR ENZYME SPECIFICITY

Antipain PapainAprotinin Serine proteases (e.g kallikrein)Bacitracin Broad spectrum (undefined)Benzamidine Serine proteasesBestatin AminopeptidaseCaptopril Angiotensin-converting enzymeChymostatin Chymotrypsin-likeEDTA Metalloendopeptidases,

carboxypeptidasesLeupeptin Trypsin, papain, cathepsin BPepstatin Pepsin, renin, cathepsin DPMSF Serine proteasesPhosphoramidon MetalloendopeptidasesPuromycin AminopeptidaseSulfhydryl reagents Thiol endopeptidasesThiorphan Enkephalinase

inhibitors281* A range of these are available, applying to

specific enzymes in some cases or acting against a broad spectrum (Table 5). It is usually found that a 'cocktail' of inhibitors is most effective. In fact, chymostatin (50 /xg/ml) has been reported to specifically inhibit proteolysis of substance P39. Alternatively, a protease-resistant analog of the compound can be used, providing it shows the same kinetics and selectivity as the unprotected compound. It is also important to maintain a constant optimum temperature. Peptide assays show relatively rapid kinetics, so assays are often carried out at room temperature, or 4°C when stability is poor or the kinetics are too fast for precise measurement at higher temperatures. In order to determine non-specific binding it is most effective to use a native agonist/antagonist at 1 0 0

- 1000 times expected KD. Ideally, an antagonist should be used, since this demonstrates binding without effect, and should not activate any cellular processes, which may affect binding parameters. This poses particular problems in this study since no specific, selective antagonists for the tachykinin receptor subtypes are yet available.

The three major problems with characterizing peptide receptors are:-

1) . No specific binding is observed because of ligand degradation or interference with the site from buffer, anti-peptidases, enzymatic instability, or conversion to low affinity site.

2) . High non-specific binding is found. This can be122

corrected by decreasing the incubation temperature or adding an anti-adsorptive treatment. Ligand may bind to intracellular membranes. This can be circumvented by use of intact cells.

3). There may be a mismatch between the pharmacological characterization of the biological response and that of the binding site.

The method of measurement of radioligand binding has not yet been mentioned. Indeed, it would seem to be a relatively simple matter to count the radioactivity in cpm/dpm in an appropriate counter. However, this does not take into account the situations where relative binding is the required information. This occurs when experiments are carried out on tissue slices or sections, with the intention of determining tissue, regional, cellular or sub-cellular location. In these cases, the integrity of the tissue must be maintained. The ability of the high-energy emissions to react with photographic film or emulsions is exploited here. After binding, the sections are apposed to specifically formulated photographic film (Ultrofilm, LKB; 3 H-film, Amersham) or coverslips dipped in photographic emulsion. Silver grains appear over the areas of bound D*, indicating, after development, the areas of high and low density of binding sites directly. This method can be quantified, using radioactive standards. Alternatively, sections can be 'dipped' in photographic emulsion and examined microscopically. The latter method is particularly useful if the sections have been stained or

123

immunochemically marked prior to binding, since direct correlation of cellular structures and binding sites is possible.

4.2. METHODS.

4.2.1. High Performance Liquid Chromatography (HPLC).

This was carried out in order to check the purity of commercially supplied substance P. The HPLC apparatus was set up using an ultraviolet spectrophotometer as a detector, set at 230 nm. Programme 1 (see App. 3) was run at 2mls/minute, and then substance P was injected onto the C 1 8 /^Bondapak (Waters) column. Programme 2 was run and the elution profile recorded. Programme 3 was then run to return the system to standby.

4.2.2. Iodination of Substance P.

All solvents were filtered and degassed prior to use with the HPLC apparatus. [1 2 5 I]Bolton-Hunter reagent wasobtained from Amersham International. The benzene was removed by evaporation by dry nitrogen in the sealed vial, leaving the reagent in dimethylformamide. The H.P.L.C. apparatus was set up, the flow rate set to 2 mls/minutey and programme 1 was carried out (see App 3) . 2 0 /xl substance P(0.2 mg/ml in HPLC grade water) was added to the reagent, mixed carefully and left for 60 minutes on ice. After this time 10 (jlI of freshly made and filtered 0.1 M borate buffer

124

pH 8.5 was added to the mixture and all of the radioiodinated material was taken up in a Hamilton syringe and injected onto the C1 8 /zBondapak column. Programme 2 was started and samples were collected every 30 seconds. After the peptide had eluted, 2 f i l samples were taken, counted and the peak fractions pooled. Mercaptoethanol was added to a final concentration of 5 mM, then 2/zl samples were counted to determine the concentration and recovery of iodinated substance P. Programme 3 was run on the HPLC apparatus. The [1 2 5 I]Bolton-Hunter conjugated substance P was stored at -80°C until use.

4.2.3. Cell cultures.

These were prepared as described in section 2.2.6 .2.

4.2.4. Binding assay.

Medium was aspirated from cultures and replaced by incubation medium (Hams F-10, pH 7.4, 25 mM HEPES, 28.6 mM sodium hydrogen carbonate, 5 mM magnesium sulphate, and fresh; 0.4 mg/ml bovine serum albumin Type V, 40 /xg/ml bacitracin, 1 . 6 f ig/ml leupeptin, 0 . 8 6 /zg/ml chymostatin) at 25°C. In saturation assays cold substance P was added to half of the wells to a concentration of 1 /zM. In displacement assays, the displacer was added at a range of concentrations. The cells were pre-incubated for 10 minutes before addition of [1 2 5 I]BHSP, either at a range of concentrations, in saturation assays, or at 0.1 nM

125

otherwise. The incubation was carried out at ambient temperature (22°C - 27°C) for 55 minutes. It was stopped by aspirating the medium and washing the cells twice with three volumes of ice-cold incubation medium. The cells were detached with 2 M sodium hydroxide, and transferred to tubes for counting. Data was resolved using the EBDA and LIGAND non-linear regression analysis curve-fitting analysis programs of Munson and Rodbard196.

Cell cultures were commonly used at 12 - 15 DIV except for examination of the development of binding with time in culture, when they were used from 3 DIV up to 28 DIV.

4.2.5. Lesions.

These were carried out by the Mr. David Green, the curator of the Imperial College Biochemistry Dept. Animal House. The animals were anaesthetised with 11 oxygen (02), 500cm3 nitrous oxide (NO), 3% halothane, all per minute.The anaesthesia was maintained during the operation by 500cm3 0 2 250 cm3 NO, 1 - 2 % halothane, per minute. The animals were restrained in a stereotactic apparatus and the respiration monitored. The head was shaved, swabbed with alcohol and an incision made along the sagittal axis. The tissue covering the bone is gently removed using a periosteal elevator. The dura was exposed by drilling through the skull at a position -4 sagittal, 4 lateral,from the bregma. The cortex was then injected with 2 /xl of kainic acid (2 mg/ml) at a depth of 1.5 mm below dura. The

126

hole was sealed with Ethicon bone wax, and the skin closed with sutures. Post-operatively, the animals were injected with 0.5 ml/ kg body weight of Temgesic (0.3 mg/ml), an analgesic, and monitored continually for several hours.

4.2.6. Autoradiography / Immunocvtochemistrv.

Adult rats were stunned, transcardially perfused then decapitated. The brains were removed, cut down, mounted on cork blocks with Tissue-Tek, and then frozen in isopentane at -15°C. The size of the blocks was decreased as much as possible, without omitting the lesioned site, since there is then much less chance of the block cracking on freezing. 1 0 /m sections of rat cortex and striatum were cut and thaw-mounted on slides 'subbed' in a 0.5% gelatine, 0.05% potassium chrome alum solution. The sections were stored, desiccated at -20°C until use.

An initial study, based on the methods of McCarthy1 8 1

was carried out combining binding with the /3-receptor ligand, [1 2 5 I]ICYP (30 pM), or [1 2 5 I]BHSP (0.1 nM) , and staining with GFAP antibody and TRITC. In this study, sections were initially fixed with 4% paraformaldehyde in 50 mM tris-buffered saline (TBS), pH 7.4. After rinsing in TBS, incubating in sodium borohydride to eliminate excess flourescence from remaining aldehyde groups, and rinsing again, primary antibodies ( aGFAP, aNF) were bound to the sections (TBS + 0.3% BSA). After a further rinse, binding of radioligand and secondary antibodies ( TRITC/FITC) was

127

carried out for 90 minutes at room temperature. To determine non-specific binding for /3-receptors and substance P receptors, 500 fXM. D/L isopropanol and 1/xM substance P were included. The peptide binding incubation medium also contained 5 mM MgCl2, 0.4 mg/ml BSA, 0 . 8 6 /ig/ml chymostatin, 40 /ig/ml bacitracin, 1.6 /ig/ml leupeptin. All slides were rapidly rinsed three times in 10 mM TBS at 4°C, before drying and dipping in K5 photographic emulsion.

In order to dip the slides, the emulsion was melted and diluted 50% with distilled water at 43°C. Slides were then dipped, allowed to dry, and exposed for 7 days at 4°C. Development was effected using D19 developer for 3.5 minutes, stopping the reaction in 1 % acetic acid, hardening, then fixing with 25% sodium thiosulphate. After a rapid wash, the slides were dried in a cool air stream, mounted in PBS/glycerol + DABCO, and the coverslips sealed with clear nail varnish for later microscopic examination.

In later experiments the following protocol was used. Alternate sections in a consecutive series were pre-incubated in 50 mM Tris-HCl pH 7.4 + 0.02% bovine serum albumin for 15 minutes. Incubation was carried out in buffer as above with, additionally, 5 mM magnesium sulphate + 0.86/xg/ml chymostatin + 1.6 /ig/ml leupeptin + 40 /xg/ml bacitracin, at room temperature for 45 minutes. All incubations contained 1 nM [1 2 5 I]BHSP; non-specific incubations also included 1 /iM ’cold' substance P. Incubations were stopped by draining off the incubation

128

medium and rinsing the slides five times for 30 seconds in a large volume of ice-cold pre-incubation buffer. The sections were rapidly dried in a cold air-stream and stored, desiccated, overnight at 4°C.

Slides were apposed to Hyperfilm 3H (Amersham) in the dark, and allowed to develop at 4°C for 14 days, also in the dark. The film was developed using the X-Omat system.

Sections were immunofluorescently stained with aGFAP + aNF (RT97)/ TRITC + FITC as described in section 2.2.6.The slides selected were the alternates in a consecutive series with the autoradiographs.

4.3. RESULTS.

High performance liquid chromatography carried out on commercial substance P showed a single peak eluted at around 26 % Acetonitrile (Fig. 31). This shows that the peptide used for the iodination had not broken down or oxidised at this point.

The profile of the fractions from the iodination of SP is shown in Figure 32. This shows that elution of [1 2 5 I]BHSP occurs at about 33% Acetonitrile. This shift is caused by the addition of the Bolton-Hunter reagent (Dr H.P. Too, previously at MRC Molecular Neurobiology Unit, Cambridge; personal communication). A small peak is seen before the major SP peak, which is believed to consist of

129

Figure 31. Profile of HPLC separation of commercially acquired substance P; the single peak showing the purity of the preparation.

Flow rate = 2 ml/min.; U.V. Measurement = 230 nm; A = HPLC-grade water + TFA (0.1%); B = Acetonitrile + TFA (0.1 %); Chart rate = 30 cm/hour; Sample = Substance P, 20 ul (1 mg/ml) in HPLC-grade water.

130

Figure 32. Profile of HPLC separation of iodinated [125j j b h s p . The fractions forming the highest peak were pooled for use in binding studies. The small peak preceding the main peak is believed to consist of [125j ]b h s p oxidised during the iodination.

131

Elution profile of iodinated BHSP

21 26 31 36 41 46 51Fractions

oxidised SP. It is essential that no free N groups are permitted to come into contact with the BH reagent since it would then react. The pooling of the single peak ensures that no impurities are present to interfere with binding. Since the reagents act, under these conditions, to produce the mono-iodinated form, the specific activity is equivalent to that of the original iodinated reagent.Using this information it is simple to calculate the concentration of the ligand present in the final solution:-

Recovery of f-l^ n BHSP.Mean CPM + s.e.m. = 1210859 + 156705 (n=4)Counter efficiency = 60.08%DPM = 2015411 (1 DPM = 4.5 X 10“ 7 jLtCi)=> 0.907 fJLCi/ 2

=> 0.453 JLlCi/ /xl = 453 \iC i/ml

Recovered amount = 1.75 mis.=> 792.75 Mci recovered

% Recovery = 79.3 %

132

Concentration.Specific activity @ day 0 = 2050 Ci/mmol.Specific activity @ day -11 = 1.119 x 2050

= 2294 Ci/mmol.ljitCi = 1/2294 nmol. = 1/453 ml.Cone. = 453/2294 nmol/ml

= .197 nmol/ml = 197.5 nM

in 1.75 mis.

The time course of binding is shown in Figure 33. This shows that binding reaches a peak at around 55 - 60 minutes incubation. After this point it decreases, probably because of breakdown of the ligand, despite the inclusion of peptidase inhibitors. However the rate of decrease is quite slow, so no large variations in binding would be expected by a small change in binding time. Preliminary binding experiments had indicated a KD = 7 /iM in assays where no peptidase inhibitors were included. The non-specific binding remains constant, after the initial deviations, as would be expected with a constant concentration of ligand.

The saturation data is expressed in Figures 34, 35 and Table 6 . From these we can see that, in the spinal cord, the computer best fit suggests the presence of a single binding site? KD = 1.7 x 10“ 1 0 ± 5.9 x 10“ 1 0 M, = 214± 23.3 fmoles/ mg protein. While in the cortex, a two-site fit is suggested; K^'s of 2.74 x 10- 1 1 ± 2.58 x 10”11, and

133

Figure 33. Time course of [1 2 5 I]BHSP (0.1 nM) binding tocortex cultures ( . 12 DIV) over 90

minutes.

Representative of two experiments, each point in quadruplicate.

134

Substance P bindingTime course

[125I]BHSP bound (Dpm)

■ *— Total binding — Non-specific bindingSpecific binding + S.E.M.

Figure 34. Scatchard analysis of saturation study of [125i ]b h s p binding to spinal cord astrocytes (12 DIV) showing a single site. NSB was determined by addition of 1 /xM SP. This representative graph was produced using the LIGAND programme from three co-analysed experiments^ each point in quadruplicate.

135

BO

UN

D/F

RE

E

0.273

0.227

0.182

0.136

0.099

0.045

BOUND (fmoles/mg protein)

Figure 35. Scatchard analysis of saturation study of [125I]BHSP binding to cortex astrocytes (12 DIV) showing a two-site fit. NSB was determined by addition of 1 /LtM SP. This representative graph was produced using the LIGAND programme from three co-analysed experiments^ each point in quadruplicate.

136

BOUND/FRF.E

0.128 KD1 = 2.74 x 10"11 ± 2.58 x 10-11 M

KD2 = 1.65 x 10"9 ± 1.28 x 10"9 M

0.106 - 1 BMAX1 = 16.8 ± 9.6 fmoles/mg protein

1 BMAX2 = 59.7 ± 31.4 fmoles/mg protein

0.085

\ •

0.064 _

0.043 _

0.021 _

r v•

• •--- -~~i----------1----------1--------- 1 ~~ ' .10.1 20.1 30.2 40.3 50.4

BOUND (fmoles/mg protein)

Table 6. Summary of data determined from saturation and displacement studies.

136A

TABLE 6 SUMMARY OF KINETIC DATASPINAL CORDFrom saturation studies.Kd 1.7 x 1CT10 ± 5.9 x lO-11 M

bMAX 214 ± 23.3 fmoles/mg

From displacement studies.Kd HA 1.38 x 10“10 ± 4.51 x 10“10

LA 7.45 X 10“8 ± 1.28 X 10“7 M Bm a x 114 ± 22 fmoles/mg

LA 3864 + 1698 fmoles/mg

1^50SP 4.16 X 10"9 ± 1.54 X 10“9NKB 1.43 X 10“8 ± 1.10 X 10“8NKA 1.53 X 10"8 + 4.95 X 10"9Spinal cord SP > NKA, NKB Cortex SP > NKA, NKB

CORTEX

HA 2.74 X 1CT11 i 2.58 X 10-11 LA 1.65 X 10“9 ± 1.28 X 10“9 MHA 16.8 + 9.6 fmoles/mg LA 59.7 + 31.4 fmoles/mg

M HA 1.70 X 10-11 ± 1.55 X 10-11 MLA 8.24 x 10 "8 i 1.27 x 10“7 MHA 5.15 + 1.14 fmoles/mgLA 4840 + 2280 fmoles/mg

3.54 X 10"10 ± 1.02 x 10“10

5.13 X 10“8 ± 4 .48 x 10“8 M5.12 X 10“8 i 1.98 X 10“9

4.16 nM > 15. 3 nM, 14.3 nM0.35 nM > 51. 2 nM, 51.3 nM

1.65 x 10~9 ± 1.28 X 10“9 M, Bj^x's of 16.8 ± 9.6, and 59.7 ± 31.4 fmoles/mg protein. Saturation curves of the two areas showed that binding was beginning to plateau in most experiments, although complete saturation was not seen at the highest concentrations (Figs. 36, 37). The levels of binding in the two regions showed a considerable difference. Also the percentage of non-specific binding in the spinal cord was much lower than in the cortex.

In order to conserve ligand, further experiments were carried out using cold displacement of 0.1 nM [125I]BHSPwith 1/liM substance P. These experiments provided more data

<*

for Scatchard plots. However, the data gave a different fit for spinal cord (see Figure 38). The computer best fit in this case suggested a two-site fit; with KD 's of 1.38 x 10“10 ± 4.51 X 10-10, and 7.45 X 10"8 ± 1.28 x 10-7 M, bMAx 's 114 - 23/ and 3864 ± 1698 fmoles/mg protein.Since the range of concentrations in these experiments was much larger than in the saturation experiments, it encompassed the second site. The displacement data from the cortex also gave a two-site best fit (Fig. 39), as before; with Kd 's of 1.7 x 10"11 ± 1.55 xlO"11, and 8.24 x 10"8 ± 1.27 x 10“7 M, and Bj^x's of 5.15 ± 1.14, and 4840 ± 2280 fmoles/mg protein. The second site here shows a considerable difference from that of the saturation data. The increased range probably also accounts for this variation. The standard errors are very large, in some cases. This is caused by the nature of the computer program. Each calculation involves only two fixed values

137

Figure 36. Saturation of [125I]BHSP binding sites in cortical astrocytes (12 DIV). NSB was determined by addition of 1 /xM SP.

Representative of two experiments, each point in quadruplicate.

138

Substance P BindingCortex

[125IJBHSP Bound (pM)

■S” Specific binding Non-specific binding ■*“ Total binding

Figure 37. Saturation of [125I]BHSP binding sites in spinal cord astrocytes (12 DIV). NSB was determined by addition of 1 /xM SP.

Representative of two experiments, each point in quadruplicate.

139

Substance P BindingSpinal cord

[125IJBHSP Bound (pM)

Figure 38. Scatchard analysis of displacement study of [1 2 5 I]BHSP binding to spinal cord astrocytes (12 DIV) showing a two-site fit. 0.1 nM [1 2 5 I]BHSP was displaced with increasing concentrations of SP. This representative graph was produced using the LIGAND programme from six co-analysed experiments j eocV' 'p©\rvt in

140

BOUN

D/FR

EE

BOUND (fmoles/mg protein)

Figure 39. Scatchard analysis of displacement study of [125 i]BHSP binding to cortex astrocytes (12 DIV) showing a two-site fit. 0.1 nM [1 2 5 I]BHSP was displaced with increasing concentrations of SP. This representative graph was produced using the LIGAND programme from six co-analysed experiments j each po\nT io c|vAcxcV̂ upUca[i:-e .

141

BOUN

D/FR

EE

BOUND (fmoles/mg protein)

and at least two variables, so when several experiments are co-analysed, the variability is necessarily very high.

Displacement experiments were also carried out using NKA and NKB as displacers (Figs. 40, 41). These gave IC50 values for SP, NKA and NKB in both regions. The order of potency was the same in both regions; SP > NKA > NKB.However, the absolute values of the 1 0 5 0 's differed]

1slightly:-

Spinal cord SP 4.16 nM > NKB 14.3 nM > NKA 15.3 nM Cortex SP 0.35 nM > NKA 51.2 nM > NKB 51.3 nM

The developmental study (Figure 42) showed a rapid increase in binding in spinal cord up to a peak at 12 - 15 DIV, after which it declined slowly. By contrast, binding in the cortex showed very low initial binding, which increased very slowly to reach a plateau level at 15 -21 DIV. The levels of binding in the two regions also differed considerably, as seen in the saturation curves; the range in spinal cord being between 16 and 99 fmoles/mg protein, and 1 - 2 2 fmoles/mg protein in cortex. This indicates at least a five-fold difference in levels at 1 2

DIV where saturation and displacement studies were carried out. This almost certainly applies to the high affinity sites in each case, since the developmental study was carried out using 0.1 nM [1 2 5 I]BHSP. At this concentration little binding to the lower affinity sites would be expected. The values in this study are corrected for ligand

142

Figure 40. Displacement of 0.1 nM [1 2 5 I]BHSP byNKB, from spinal cord astrocytes (12 DIV). IC5 0

were SP = 4.16 nM, NKA = 15.3 nM, NKB =14.3 nM.C ^ (_ O = (rp =■ )

SP, NKA, values

143

Log [Displacer]

PERC

ENTA

GE B

OUND

NKB

Figure 41. Displacement of 0.1 nM [1 2 5 I]BHSP by SP, NKA,NKB, from cortex astrocytes (12 DIV). IC5q values were SP= 0.35 nM, NKA = 51.2 nM, NKB = 51.3 nM.

(n - 6 ) ('ri - 3 ) (/o = 3 )

144

I Displacer I

PERC

ENTA

GE B

OUND

NJ

O'

00

O

(NJ

c c

c o

c o

NKA

Figure 42. Developmental profile of binding [1 2 5 I]BHSP (0.1 nM) to spinal cord and cortex astrocytes up to 28 DIV. NSB was determined by addition of 1 fiM SP.

Representative of two experiments, each point in quadruplicate.

145

Substance P bindingDevelopmental study

SPECIFIC BINDING (fmoles/mg protein)

D Spinal cord Cortex

decay and protein concentration, in order to directly compare time points.

Initial autoradiographic studies were unsuccessful in showing binding of [1 2 5 I]BHSP to the lesioned sections of rat brain. However, (3-receptor binding could be seen over the expected area of the lesion. Unfortunately, the morphology of the sections was badly affected by the necessary treatments, so no clear binding of the GFAP antibody could be seen. An area of high TRITC density was present over the expected area of the lesion, but there was no way to demonstrate a clear link between the glial cells and the /3-receptor binding. It was found that the damage was caused during the final rinse of the developing stage. This implied that the binding information was valid, but that the immunofluorescent staining was void. At this point this method was abandoned for routine autoradiography. The latter method used alternate slides for autoradiography and immunofluorescence. Unfortunately, only a limited number of experiments were performed and no clear glial scar was identified, either by eye or by GFAP immunofluorescence. However, [1 2 5 I]BHSP binding to the sections did show the characteristic pattern of binding to the various brain regions as reported (Fig. 43). There was no increased levels of binding in the cortical areas where the lesion was expected. It is possible that, either the glial scar failed to develop, or that it was missed in sectioning of the brain.

146

Figure 43. Autoradiogram showing characteristic binding of [1 2 5 I]BHSP to rat brain sections. NSB was determined by inclusion of 1 /xM SP in the incubation medium.

147

4.4. DISCUSSION.

These experiments have shown that by using a pure ligand prepared by HPLC, it has been possible to characterise the binding sites present on astrocytes in culture. In culture, two binding sites for substance P have been demonstrated in both spinal cord and cortex. The data from saturation studies and displacement studies confirm the presence of the high-affinity site in both cases. The differences between the two methods of measurement demonstrate the limitations inherent in the assumptions made in the data analysis. The saturation study only demonstrated a single site in spinal cord and underestimated the number of low-affinity sites in the cortex. This was caused because the second sites in both regions had not reached saturation at the maximum ligand concentration. The consequent displacement studies, with the larger concentration range, elucidated the low-affinity sites. It is clear, by comparing the kinetic data, that the high-affinity sites differ considerably, both in affinity and in receptor density. The high-affinity site in the cortex has a higher KD and lower Bj^x than that of the spinal cord. The lower affinity sites both have very similar affinities and receptor densities. It could be suggested that the second site described in both these regions, is, in fact, the same site.

The data from displacement studies of SP, NKA and NKB also suggests a regional variation. The ten-fold

148

As can be seen from figures 34 and 35, the Scatchard plots are curvilinear, suggesting multiple binding sites or negative co-operativity. The computer analysis suggested a single binding site for the spinal cord data, but this was probably caused by the fact that the binding sites were not fully saturated ( see fig. 37). Later experiments using higher concentrations of SP successfully demonstrated the presence of two sites. In the cortex, Hill analysis of the graphs ( n = 4), indicated that, if resolved into two lines ( nHj^ = 0.93 + 0.08, nHj^ = 0.91 + 0.06), each may represent a single population of binding sites.

A major problem of the computer analysis is that it produced results which had large SEM values, thus suggesting a possible large range for the mean values. This is probably caused by the inherent limitations of the program itself, and the system which we attempted to characterise. It has been reported^0® that when multiple sites are present, a large error factor is incorporated when the affinities of the two sites are separated by a factor of less than 1000, and equal proportions of the sites are present.In this case, in both regions the affinities of the two sites are separated by a factor of 100 or less. Also the lower affinity site, in both cases, has a much higher density of binding sites which may cause further complication. The errors would certainly be decreased by the performance of a larger number of experiments e.g. 10 - 20 per region (although the cost of this would be prohibitive).

A further limitation of this program is that it requires estimates of the and KD, and then uses these to determinethe best fit. In this case a problem arises, since it has recently been determined that the SP binding sites in the cortex are inducible, by a factor present in serum. This then suggests that there will be variations between experiments carried out on different cultures. Since the computer analysis involves all the experiments carried out, it is not possible to give a precise estimate of the as required, and this incorporates an errorinto the system in the initial stages of analysis of both kinetic parameters.

difference between the IC5 0 's of SP compares to the kinetic values found in saturation and displacement Scatchard analyses. The other tachykinins demonstrate the same order of potency but there are differences in the IC5 0 values for the two regions. The order of potency for the different tachykinin subtypes was stated in Chapter Two. The order of potency shown by the IC5 0 values is:-

Cortex SP > NKA > NKBSpinal cord SP > NKB > NKA

This shows that the high-affinity sites are both of the NK^ subtype.

Considering this data, the time-course and developmental studies must be presumed to apply predominantly to the high-affinity sites, since the concentration of ligand was at least 1 0 0 times lower than the Kj) of the low-affinity sites. This would also account for the differences in the levels of binding seen in the developmental study. However, it does not account for the differences in the developmental profile. The spinal cord apparently shows a rapid increase in specific binding while development in the cortex is comparatively slow. If we postulate that in vitro development parallels _in vivo development, this suggests that the spinal cord astrocytes develop earlier than those in the cortex. Previous in vivo studies in whole brain have demonstrated high binding in the late embryonic stages228. This then decreases

149

gradually to adult values by 14 days post-natal (dpn). The overall receptor density decreases with age but, there is considerable redistribution of binding sites. In the cortex, initially low to moderate binding seems to become more defined in the cortical laminae by 21 dpn228. In the spinal cord, ontogenetic studies have shown a similar gradual decrease in levels of substance P receptors , and the redistribution of the sites. However, specific loci have been shown not to be fully developed by 8 dpn4 1 .These studies would suggest that there are regional differences in binding, not only between the two regions but also, within them.

Many studies have been carried out to determine the distribution of tachykinin binding sites in brain and other regions2 2 7 *41,229,57,36,207,59,275,242,17,159 ̂ However,several of these have relied on the theory that the ligand is binding to only one site or to all sites with equal affinity. Since the elucidation of different subtypes of tachykinin receptors, it is now understood that the tachykinin ligands act preferentially but not exclusively on the binding sites. The molecular mechanisms of action of the specific subtypes have recently been described250. It is therefore necessary to take this into account when examining tachykinin receptor binding. Indeed, a recent study has reported on the differential localization of NK^, NK2 and NK3 receptors in the cerebral cortex58. This also demonstrated that the development of the subtypes varies. The NK^ subtype is present mainly in the superficial

150

cortical layers, increasing in density between 6 dpn and 14 dpn, but apparently decreased in the adult. The variation in receptor density between the spinal cord and cortex is also supported by the finding that NK^ receptors are the predominant subtype in the spinal cord while NK2 and NK 3

receptors are predominant in the cortex230. The presence of 'Nl^-like' receptors, has also been reported in spinal cord and cortex membranes when using [1 2 5 I]BHEL as the ligand131. It is possible that the low-affinity sites found in both regions are of this subtype. A very recent study using several radiolabelled tachykinins has attempted to localise the different receptor subtypes in rat brain, and concluded that there was no evidence for the presence of NK 2 subtype246.

Substance P receptors have previously been reported in neonatal cultures of mouse astrocytes, where the kinetics showed a single site with a (derived from kinetic studies) of 0.034 nM and (from dissociation studies) of 0.33 nM and Bj^x 1 7 0 fmoles/mg protein277. This equates with the ten-fold difference between the two methods of analysis found in rat cortex. It also suggests that there may be some species differences in the substance P binding, since only a high-affinity site was found in the cortex.It is also possible that differences between studies are more dependent on the incubation conditions during binding, or in this case between the material under examination.

Despite the lack of success in demonstrating binding to151

A comparison of the results from this study and the published paper indicates a high affinity site, of similar magnitude, in both the rat and mouse cortex. However, they apparently found no evidence of a second, lower affinity site. This may derive from interspecies differences or that in mouse the second site is of much lower affinity and was therefore not detected even at maximum saturation of the high affinity site. If the latter was true, it is likely that the site is too low affinity to be physiologically relevant to the levels of peptide naturally present. It is not possible to fully compare the results of the two studies since no SEM's were given for the iterative non-linear regression analysis. However, the results suggest an affinity of 0.27 - 0.38 nM ( excepting the kinetic analysis, 0.034 nM). This is about ten-fold lower than the estimate of 0.017 nM found in this study. The conclusion of Torrens et al, 1986, was that although they could not give a definitive value for the affinity of the site, a high affinity site, specific for substance P, was present. In the same way, I can conclude that high affinity receptors of the NK^ subtype are present in both spinal cord and cortex, in differing densities, and also there is evidence for the existence of lower affinity binding sites for SP, but that the charcteristics of this site have not yet been elucidated.

In a very recent paper by the same group^5, they have also demonstrated that the receptors are of the NK^ subtype and are linked to PLC. They have also found considerable inter-experimental variations in the cortical responses. They have attempted to standardise these results variously as % of control, % of SP response at 10“^M, and ratio of stimulated IP's to free [̂ H]Inositol. Using Student's t-test it was found that EC5 q 's of NKA and NKB were significantly different (0.01>p>0.001) from the results found here in rat cortex. This could be caused by differences in species or system. There was no significant difference (SP,NKA, p>0.5; NKB, 0.5>p>>0.1) found between IC5 0 values of the two studies.

1S\ ^

astrocytes in vivo. this has shown that the biological properties of the ligand were not affected by iodination. Also, it is probable that this study could be successful when certain technical difficulties are overcome. It is also hoped that this technique could be extended to examine binding to lesions in spinal cord. From the initial experiments, where limited success was gained with /3-receptor binding but not with substance P receptor binding, it could be concluded that the (3-receptors are present on reactive astrocytes, while substance P receptors are not. Studies on spinal cord substance P receptors have shown that electrolytic lesions had no effect on receptor density113. This was also the case with electrolytic and 6 -OHDA lesions of the afferent input to the interpeduncular nucleus, an area which receives dense SP and cholinergic projections. In this study both neonatal and adult lesions had no effect on binding of [1 2 5 I]BHSP or [1 2 5 I]BHEL199. This data implies that removal of substance P from a region does not affect the receptor density. It is therefore interesting that increased neonatal exposure to substance P has a positive effect on the receptor numbers in some brain regions103.

It is known that the tachykinin receptors in brain are linked to the PI response via a G-protein linkage182'43'184'269. The activity of these receptors, like other peptide receptors209'241, is also modulated by the presence of divalent cations (Mn2+, Mg2+) and the concentrations of these ions, separately, or in concert,

152

may cause significant alterations in the kinetics of the sites254'158. It is also important, when using astrocyte cultures, that the heterogeneity of astrocytes present be considered. It has been demonstrated that there are at least two types of astrocyte, based on their antigenic development profile231'232. Although the cultures used in these experiments have been shown to be purified for astrocytes, they have not been characterised as to the relative proportions of Type I or Type II cells present. Morphologically, the cells predominantly appear to show the epithelioid shape associated with Type I astrocytes at confluency, in cortex cultures. In spinal cord cultures, there is a much higher degree of heterogeneity of cell phenotype44. However, the proportion of these cells does seem to vary through development, so it could be argued that the tachykinin receptors are only present on one type of astrocyte, or that the tachykinin receptor subtypes are specific for the different types of astrocytes. Since the regional enrichment of the different types of astrocytes has not been examined, it is possible that certain types are concentrated in specific CNS regions.

This study has characterised the high-affinity sites identified, but although the low-affinity sites have been characterised kinetically, no other information has been shown. The problem with the low-affinity sites is that the experiments carried out on the high-affinity sites cannot be applied to them in this system because of the interference of the high-affinity sites. Ideally, the

153

activity of the high-affinity sites could be negated or abolished. Similar experiments as described could be carried out using radioiodinated ligands selective for the other subtypes e.g. [1 2 5 I]BHEL or [1 2 5 I]BHNKB, [1 2 5 I]BHNKA. If specific antagonists were available, these could be used to eliminate the high-affinity sites. Selective agonists are a relatively recent innovation to this field and if made available, these could be used301'157'156. Otherwise, anti-idiotypic antibodies specific for the selected subtypes could distinguish them49. These theories assume that the low-affinity sites constitute a different subtype, whereas it may be that the low-affinity site is also of the same subtype, but in a different conformation.Low-affinity substance P receptors have been described in neuronal cell lines, but have not yet been fully characterized238. However, it may be that the tachykinin subtypes may soon be directly characterised. Recent studies have isolated the substance P binding protein from rat brain1 8 3 and bovine brainstem202. Also, innovations in molecular biology have enabled the expression of functional receptors in frog oocytes by injection of mRNA161'108.This has provided the investigators with sufficient material to establish the structure and sequence of the NK2

receptor184. This method seems to provide a useful alternative to the direct isolation of receptors in their native environment. It also facilitates the examination of inaccessible or low density populations of receptors.

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5. DISCUSSION.

In this study, the presence of high-affinity tachykinin receptors on neonatal cultures of rat astrocytes from both cortex and spinal cord, has been demonstrated. These receptors, of the NK^ subtype, are apparently linked to the PI system, possibly via a G-protein, although no evidence for the latter mechanism is presented here. It is also probable that cortical astrocytes possess similarly-linked receptors for other peptides, i.e. vasopressin, oxytocin, bradykinin, and that these are active at physiologically relevant concentrations. The cerebellum and spinal cord have also demonstrated PI responses to a range of peptides at supramaximal concentrations, which may indicate the presence of appropriate receptors in these areas. It has not been possible to demonstrate the specific localization of the tachykinin receptors to astrocytes in vivo. in lesioned tissue. It seems that /3-adrenergic receptors appear to be present on reactive astrocytes, but no substance P receptor binding could be detected. This implies that either no receptors are present, or that incubation conditions were inappropriate for ligand-receptor binding. If, in fact, no receptors were present, this may indicate that substance P receptor binding does not occur on astrocytes in vivo, or that it does occur, but not on reactive astrocytes. Another interesting finding is that the levels of certain peptidases found in astrocytes are increased by kainate262. This may correlate with the situation in damaged CNS,

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implying that since peptides are then broken down more quickly, they are less important in, reactive, as opposed to normal astrocyte function. Correspondingly, the peptide receptors would not then be induced on reactive astrocytes.

It has also been demonstrated that there are significant regional variations in both receptor binding and activation, in terms of kinetics and development. In considering the PI response, a fundamental difference between the two regions was that of reproducibility. While the results from spinal cord were consistent, those from the cortex, particularly in relation to substance P, have shown considerable variation. This is also supported by results from this laboratory, reported by Dr.Cholewinski and D. Marriott, where SP elicited a very small or statistically insignificant PI response in cortex43. A possible cause for this variation has been suggested by recent findings that, in serum-free cultures of cortical and spinal cord astrocytes, there is a marked increase in the SP-induced PI response (Dr. Cholewinski, personal communication). This would suggest that factors in serum may suppress the expression of the receptors or that constituents of the serum-free medium induce the expression of receptors. The binding experiments revealed that the receptor density of the high-affinity SP receptors was very low compared to that of the spinal cord, which accounted for the lower level of PI response.

When the developmental profiles of the PI response and156

the SP receptor binding were compared, it was expected that they would show a similar pattern of fluctuation. This was found not to be the case in cortical cultures. In spinal cord, the pattern was of binding was comparable to the PI response, indicating a direct correlation between rec eptor occupation and response. In cortex, binding and response were both low, at the earliest time points, but while the PI response describes a 'normal distribution'/around a peak at 15 DIV, SP binding increases slowly to a plateau at about the same time point. The cause of the decrease in the PI response is unknown, but it indicates an indirect occupancy-response relationship, which suggests an uncoupling of the receptor-mediated PI response. As described earlier, the differences in the PI responses may not devolve completely on the receptor variations, but may also be affected by alterations of the coupling to the PI system, or the factors within the system.

An interesting finding in these experiments was the statistically significant decreases, with respect to the control, in the PI response at 3 DIV (0.5 >> P > 0.1) and 28 DIV (0.05 > P > 0.01), as determined by Students' t-test. This suggests some form of inhibitory control of the PI system, by an unknown mechanism. The developmental pattern of the spinal cord response suggests that these receptors are more important in the first 3 - 4 weeks of development, before the number of receptors decreases. In the cortex, the situation, as described above, is more complex. These receptors indicate a delayed development of

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this region, suggesting that their role continues beyond 28 DIV. This parallels the earlier development of the neuronal systems of the spinal cord compared to the cortex. These receptors may then support the theory that astrocytes act in the development and guidance of neurons, and that the tachykinin receptors may be involved in the communication of this effect.

The plasticity of astrocytes in vitro and in vivo has been demonstrated in severalreports195'253,139,37,112,203,164, ^nd it may be that the regional differences found relate to this property of astrocytes. It has been suggested that the methods of isolation and separation, substratum, growth medium, region of derivation, and developmental time point are all factors which affect glial plasticity107. An examination of the literature reveals a mass of reports concerning morphological changes induced by various means, but few, if any, of these have specifically characterized the cells undergoing these changes. Many of these studies discuss the morphological changes in terms of the 'state of differentiation' of astrocytes. Some reports have suggested that specific properties are linked to astrocyte maturation215; this reasoning could also be applied to receptor functions. As yet, no definitive outline of the properties of astrocytes throughout development has been drawn up, because of the complications of interspecies, and interregional differences, with particular regard to apparently specialised astrocytes. A review, however, has

158

summarised the current understanding264.

Alternatively, the receptor variations may appertain to the cellular heterogeneity of the cultures. It has been suggested that different populations of astrocytes exist232'206'68, and that they may be differentiated by the expression of cell-surface antigens. If this is accepted, it is surely a small step to hypothesise that distinct populations may express receptor diversity. In these studies, although the cultures were determined to be enriched in GFAP-positive cells, indicating relatively pure astrocyte preparations, no attempt was made to determine the proportion of type I and type II cells present.However, it was clear that the morphology of the cells in spinal cord was quite different to that of the cortical astrocytes. Therefore, the regional and developmental differences may be linked to the presence of specific receptors on subtypes of astrocytes which are differentially induced in the two areas.

Having established the presence of tachykinin receptors on astrocytes it is necessary to consider the possible functional effects of activation of these receptors. Of the receptors which have currently been identified on astrocytes, most of these have been associated with second messenger functions (see Table 1). A wide selection of agents have also been linked to physiological, although not necessarily receptor-mediated, responses, such as:- DNA synthesis92'258'299'252'90'190; glycogenolysis243; release

159

of factors272'283'9 '185'200; morphological changes203'258'92'76'2 '106; protein production2 8 3 and phosphorylation203; ion transport120'142; and enzyme activities203'150'2 . Manthorpe et al, have produced a comprehensive summary of agents and their effects in a recent review180. The actions of certain peptides e.g. VIP, have been found to be multiple in effect, although these may be related more to the effects of its second messenger, cAMP243'178. Astrocytes also have other effects in modulation of the extracellular environment224'235, and the activities of enzymes in neighbouring cells, particularly at the blood-brain barrier18, in neuronal-glial interactions6 3 ' 6 5 especially in areas of regional specialization78'282'222'111. It would be interesting to determine which of the effects are modulated by the activation of the tachykinin receptors.

The regional and functional specialization of astrocytes may also be one of the factors which account for the regional differences in receptor parameters and response. The cells obtained from the cortex are derived from a region of neuronal circuitry responsible for the complex higher mental faculties, and the function of specific neurons and astrocytes from this area is largely uncharacterized. The role of the PI system, as activated by the tachykinin receptors, may be less important here than in the spinal cord, where the astrocytes are surrounded by neurons directly responsible for motor and sensory afferents and efferents. It would therefore be

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expected that astrocytes from different regions may be required to respond preferentially to specific stimuli, and would have a corresponding receptor population. It should also be remembered that the astrocytes used in these studies are derived from the complete spinal cord and cerebral cortex, and that regional variations in binding of tachykinins within these structures have been reported2 2 9 ,4 1 .

In conclusion, this study has demonstrated the presence, kinetics, second messenger association, regional and developmental diversity of substance P (NKq̂) receptors on astrocytes from cerebral cortex and spinal cord. It has also indicated that receptor diversity may also exist in other brain regions for other neuropeptides. As would be expected, this study has proposed several new lines of investigation which would provide evidence for some of the theories advanced here. The most important of these, is the determination of the presence of these receptors in vivo, in normal or gliosed tissue. It would then be of interest to determine the in vivo developmental profile, for comparison with the in vitro study. The possible function of these receptors may then be indicated more clearly. This could also be determined by examining the gross effects of the neuropeptide(s) on selected parameters, such as growth, enzyme activities, ionic flux, morphology, etc., in cultured astrocytes. It would also be important to characterise these cultures in terms of specific astrocyte types, and to determine the cellular

161

localization the receptors using combined autoradiography and immunocytochemistry. Generally, knowledge of the mechanism of the PI response with respect to G-proteins, PLC, IP3 and IP4 receptors, enzyme activities, pooling of IP's , and, most importantly, its link to cellular effects, would provide vital information concerning its control and effects in all cell types. It is to be hoped that the collection of this information will eventually elucidate the function of receptors on astrocytes, and possibly the function of astrocytes themselves.

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APPENDICES

163

6 . 1 . Appendix 1. - Tissue Culture solutions.

Solution 1.

Earles balanced salt solution:- 116 mM sodium chloride5.4 mM potassium chloride0.8 mM magnesium sulphate1.0 mM sodium dihydrogen phosphate1.8 mM calcium chloride2 . 6 mM sodium hydrogen carbonate5.6 mM glucose 0 .0 0 1 % phenol red

+ 0.3% bovine serum albumin + 0 .0 0 2 % deoxyribonuclease

Solution 2.

Solution 1 + 0.025% trypsin+ 0.04% deoxyribonuclease

6 . 2 . Appendix 2.- Receptor binding theory.

RECEPTOR MODELS.

Occupancy.

Assumptions:- i) simple bimolecular reactionii) no interaction with other binding sites

iii) doesn't change binding characteristics upon binding of the ligand

Two-state model (Hill plot).

This is adapted for allosteric activation. It suggests different affinities for the receptor depending on binding when the ion channel is open/closed

Mobile/Floating receptor model

The receptor may interact with a number of 'effector' substituents in the plane of the membrane, e.g. catecholamines, prostaglandins, glucagon, ACTH all independently stimulate adenylate cyclase. This implies that receptors are freely diffusible in membrane.

This theory accounts for non-linear Scatchard plots,Hill plots with -ve co-operativity, and spare but equivalent receptors, i.e. those having two different affinities, only one of which coincides with ED5 0 .

165

DERIVATION.

[H] + [R] == [HR] — — response

k_i = KD = [H] [R] ............... .................... 1

ki [HR]

H = free hormone: R = free receptor : HR = hormone-receptor complex

Assumes [RH] a response.

Q = a [HR] .............................................2

Q = response/pharmacological effect.Also assumes that unoccupied receptors have no

response, and that a maximal response is achieved when all receptors are occupied.

Qm a x = a Cr t ] .......................................... 3

Rt = Total receptor number.

RT = [R] + [HR] ....................................... 4

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From these equations we derive:-

[RH] = [H] 5

Rt Kd + [H]

Dividing 2 by 3:-

Q = [HR] ........................................... 6

Qm a x rT

Substituting:-

Q = Qm a x [ h 1 7

Kd + [H]

c.f. Michaelis - Menten eqn.

From (5), [RH] = B; [H] =F; RT = B^x-

B = bMAX f ............................................. 8

F + Kd

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Rearranging:-

B = bMAX ” B ........................................... 9

F KD

This is 'known' as the Scatchard equation, although it is actually derived from Rosenthal, since Scatchard assumed that R was known.

Plot B vs. B to give a slope of and an abscissa intercept

F

of BMAX*

Hill plot.

This quantitates deviations from classic mass action, rectangular hyperbola behaviour. It is the graphical application of the sequential ligand binding model.

It is important to monitor the ratio of RT :KD because when Rip » Kd this manifests falsely as apparent positive co-operativity. It is best if RT < 0.1KD

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Data should be between 30% - 70% of total binding.< 3 0 % : possibly co-operativity is not symmetrical with

occupancy e.g. if threshold value.> 7 0 % : high receptor occupancy exaggerates the experimental

error in:- B

bMAX “ B

Derivation.

KD1L + R = = ( L - R ) x 10

KD2( L - R ) x + L = = ( L - R ) 2 11

*Dn( L - R )n-i + L == ( L - R )n 12

B = bMAX [L]n 13

k 'D + [L]n

n = theoretical no. of ligand binding sites per receptor molecule; k 'd = composite of KD and interaction factors which determine alteration of KD at each discrete binding step.

169

BKd + B[L]n = Bm a x [L]n 14

KD = [ L ] n ( B j ^ x - B ) 15

B

log KD = n log [L] + log ( B ^ x - B ) 16

B

log B = n log [L] - log KD 17

( bMAX “ B )

Plot log B vs. log F

( BMAX “ B )

slope = nH ( Hill coefficient)abscissa intercept, log B = 0 = k 'd (apparent KD)

( bMAX " B )

When nH = 1, k 'd = KD .

njj = 1 ; single species of receptor exhibiting simple reversible bimolecular binding which obeys mass action laws. If multiple species are present, they have identical

r

170

*»

affinity for the ligand and are non-interacting.

njj > 1 ; positive co-operativity.

nH < 1 ; negative co-operativity, or multiple non-interacting binding sites for one ligand, or multiple interconvertible affinity states.

I C 50

This enables the calculation of KD of competititors from competition binding profiles. It assumes that Scatchard or Rosenthal plots used to derive KD of receptor are linear, and values are constant for changing RT and [L*].

= Kd for competitor

Ki = IC5 0 ............................ 18

1 + [L*] / Kd

IC5 0 = n ( KD + [L*t] + Rt - 3/2 [LR] )

= Ki + n( [L*t ] + Rt - 3/2 [LR] ) ....................19

When ( L ip + Rip ) < Kq , IC^q ~ ^i*

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These equations rely on the accuracy of the measurements of Rrp and [L*<p] .

Rs = co-operativity index, indicates deviations from 'normal steepness'.

s0.9 = 0.9/0.1 = 81 ........................ 20

S0 . i 0.1KD / 0 .9Kd

RS < 81 positive co-operativity

Rs > 81 negative co-operativity, or multiple binding sites.

E C 5 0

This is used for quantitation of potency of competitive agonists. It is defined as the concentration of a competitor which effectively competes for 50% of radioligand binding.

Plot measurement of specific binding e.g. % control of SB vs. log1 0 [competitor]

log [LR]^ = n log [I] + n log EC5 0 .........2 1

[LR] - [LR]i

172

[LR] = binding in absence of competitor [LR]^ = binding in presence of competitor

KD apparent = EC5 on

E C 5 0 = K i ( 1 + [ L * ] ) ..............................................................................................2 2

k d *

Kd* = Kd for radioligand

Assumptions.

1) L* must associate with a single population of receptors, obey mass action law, as must competitors.2 ) [L*] = CL*]free# This makes the EC5o very difficult to quantify, so usually IC5 0 estimates are made.3) [R] « KD* / Ki4) The measured specific binding is an accurate reflection of [L*R]5) Steady-state binding is attained by L* and I at all concn

E D 5 0

Dose-response curves are otherwise known as ED5 0

curves. If a threshold value needs to be reached the ED5 0 > Kd . If spare receptors are present i.e. max response at less than RT , KD > ED5 0 .

173

ed50 ~ CH 3(0.5)

[Ht ] = [H] + [RH]

Ch t 3o .5 = k d + ° * 5 [RT ]

This assumes that the concentration of bound drug is negligible, if RT » k d

BASIS FOR RECEPTOR IDENTIFICATION.

Kinetics.

a) Reversible binding.b) Saturable binding - NSB determined by 100 x KD to determine concentration of displacer. Any materials used should be checked for excessive binding of ligand e.g. 125I to glass.

- no of sites (spare receptors?) .c) Competitive bindingd) Association - time course for biological effect (this may be more complex than simple bimolecular reaction.) Is it physiologically relevant?e) Dissociation. - according to occupancy theory, this should correspond to washout biological effect.f) Artefacts may appear from hydrolysis of peptide ligand, pre-binding, active uptake etc. These should be eliminated by manipulation of the incubation conditions.

174

Distribution

a) Tissueb) Brain regional location.c) Cellular location - neurons/astrocytes/oligodendrocytes.d) Species differencese) Subcellular localizationf) Corresponds to biological function - PI, cAMP response.g) Binding without function - uncoupled receptors.

Pharmacology.

1) Quantitative correlation - dose-response.2) Agonists/antagonists change binding affinity - spare receptors may give agonists greater potency e.g. in the case of the muscarinic system, receptors have similar potencies but show a 1 0 0 -fold difference in absolute potencies depending on spare receptors.3) Stereospecificity - also artefacts sometimes appear from the binding of ligands to inert materials, occasionally exhibiting pharmacological specificity. Dopamine receptors distinguish between (+) and (-) isomers of LSD.4) Multiple sites - ligand binds to multiple classes of sites but only one is receptor of interest e.g LSD binds to multiple receptors,non-receptor sites and also opioid receptors.5) Blank - should be 1 0 0 X KD / IC5 0

- it is best not to use cold ligand but natural ligand or175

different specific agonist/antagonist.- if an excessively high concentration of displacer is used, there may be erroneous results (lower KD)6 ) Antibodies to the receptor may be used if the receptor molecule has been purified. Otherwise, anti-idiotypic antibodies may be prepared from the natural ligands.

Tissue linearity.

This demonstrates the absence of artefacts e.g. receptor/ligand degradation. Downward curvature doesn't necessarily identify the wrong receptor but may give a false value for Upward curvature is less common,caused by inappropriate choice of blank or loss of binding sites during separation.

Temperature dependence.

If binding is strongly temperature dependent it implies that active transport is involved. If the temperature of the incubation is greater than 40°C, breakdown of the ligand-receptor complex ensues. If binding is increased at higher temperatures, this implies that a covalent chemical reaction of the ligand, or its breakdown products, has occurred with the receptor.

176

pH and ion effects.

Many neurotransmitters have Na+-*dependent binding, except GABA which has Na+-dependent uptake. The effects are individual for each receptor, but generally correspond to physiological concentrations. Certain ions must be removed before binding can be measured e.g. Cl” inhibits strychnine binding to glycine receptors. Some receptors have a mixed response to different ions e.g. SP binding is increased by Mn2+, Mg2+, and decreased by Zn2+, Co2+.

The pH value is usually held at physiological levels although maximum binding may require slight variations to encompass the stability/ solubility of the ligand.Extremes of pH should eliminate binding.

177

6.3. Appendix 3. - High Performance Liquid Chromatography.

Programme 1.

Solvents. A: HPLC-grade water + 0.01 % Trifluoric Acid B: Methanol

Time______ %B________ Duration

0 1 0 0 55 0 3035 0 1 0

45 END

Programme 2.

Solvents. A: HPLC-grade water + 0.01% TFA B: Acetonitrile + 0.01 % TFA

Time______ %B________ Duration

0 0 55 50 3035 50 1 0

45 0 3075 0 580 END

178

Programme 3.

Solvents. A: HPLC-grade water + 0.01% TFA B: Methanol

Time______ %B________ Duration

0 0 55 1 0 0 3035 1 0 0 1 0

45 END

179

6.4. Appendix 4.- Abbreviations

6 -OHDA 6 -hydroxydopamineAM Astrocyte mediumATP Adenosine trisphosphateBH Bolton-Hunter reagentBK BradykininBSA Bovine serum albuminCNS Central nervous systemCPM Counts per minuteCSF Cerebrospinal fluidCTP Cytidine trisphosphateDAB DiaminobenzidineDG DiacylglycerolDIV Davs in vitroDMEM Dulbecco's modified Eagles mediumDNA Deoxyribonucleic acidDPM Decays per minuteDPN Days post-natalEBS Earles balanced salt solutionEDTA Ethylene diamine tetra-acetic acidEL EledoisinER Endoplasmic reticulumFITC Fluorescein isothiocyanateGAP Growth-associated proteinGC Gas chromatography

180

GFAP Glial fibrillary acidic proteinGTP Guanine trisphosphateHPLC High performance liquid chromatographyI(cl:2 )P Cyclic inositol phosphateIF Intermediate filamentsIP Inositol phosphateKASS KassininMW Molecular weightNKA Neurokinin ANKB Neurokinin BNSB Non-specific bindingOAP Orthogonal arrays/assemblies of particlesOT OxytocinPAGE Polyacrylamide gel electrophoresisPA Phosphatidic acidPBS Phosphate buffered salinePDE PhosphodiesterasePG ProstaglandinPHY PhysalaeminPi PhosphatePI PhosphatidylinositolPIP Phosphatidylinositol phosphatePKC Protein kinase C

p l a 2 Phospholipase A 2

PLC Phospholipase CPMSF Phenylmethylsulphonyl fluoride

181

PPTA Preprotachykinin APPTB Preprotachykinin BRNA Ribonucleic acidSB Specific bindingSDS Sodium dodecyl sulphateSP-IR Substance P-immunoreactivitySP Substance PTEMED N ,N ,N ' ,N '-tetramethylethylenediamineTFA Trifluoroacetic acidTRH Thyrotropin-releasing hormoneTRITC Tetramethylrhodamine isothiocyanateTX ThromboxaneVP Vasopressin

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7 ACKNOWLEDGEMENTS.

I should like to thank all the members of Dr. G.P. Wilkin's research group at Imperial College for their discussions. I am also extremely grateful to Mr. P. Packer, and Midnight Design Ltd. for the use of their facilities and their expertise. Finally, my thanks to Dr. Wilkin for his guidance and patience.

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