identification and characterization of pipecolic acid binding sites in mouse brain

Neurochemical Research, Vol. 10, No. 5, 1985, pp. 691-702

IDENTIFICATION AND CHARACTERIZATION OF PIPECOLIC ACID

BINDING SITES IN MOUSE BRAIN

MARIA D.C. GUTIERREZ* AND EzIo GIACOBINI Department of Pharmacology

Southern Illinois University School of Medicine

Springfield, Illinois 62708

Accepted January 2, 1985

Pipecolic acid (PA, piperidine-2-carboxylic acid) is the major product of lysine metabolism in the mammalian brain (Giacobini et al., 1980). In this study we have characterized the binding of [3H]PA to P2 fraction membranes and its distribution in the mouse brain. The binding was found to be saturable (70 nM), temperature and Na + and C1 dependent. A high affinity binding site with an apparent KD of 33.2 nM and a Bma• of 0.2 pmol/mg protein was demonstrated. The regional distribution of [3H]PA specific binding in mouse brain showed the highest concentration in cerebral cortex, thalamus and olfactory bulb. Unlabeled PA (10-3-10-11 M) displaced specific binding of [3H]PA in a concentration dependent manner. Out of several substances tested, only proline showed a similar pattern of displacement. Pre-incubation of the membrane preparation with GABA (10 3_ 10-11 M) resulted in either an increase or decrease of [3H]PA binding depending on the concentrations of GABA and PA. These results suggest a modulatory action of GABA on PA binding sites. The postnatal development of [3H]PA specific binding was studied in the whole brain of the mouse. [3H]Pipecolic acid binding increased progressively (8-fold) from one day after birth to 16 days. Following this developmental peak, the binding decreased gradually to 30 days at which age, adult values were attained.

INTRODUCTION

Earlier research has pointed out the possible role of several amino acids as synaptic transmitters or modulators (1). More recently, major lysine

* Present address: Maria D.C. Gutierrez, Instituto Nacional de Neurologia y Neurocirujia, Laboratorio de Neuroquimica, Insurgentes sur No. 3877, Mexico 2, D.F., Mexico.

Send correspondence to: Ezio Giacobini, Department of Pharmacology, S.I.U. School of Medicine, P.O. Box 3926, Springfield, IL 62708.

691 0364-3190/85/0500-0691504.50/0 �9 1985 Plenum Publishing Corporation

692 GUTIERREZ AND GIACOBIN1

metabolites, such as pipecolic acid (PA) (2,3), L-a-aminoadipic acid (L- a-Aaa) (4) and dicarboxylic acids such as quinolinic acid (5) have been shown to possess neurotransmitter-like characteristics. In the case of PA, the evidence is mainly at the presnaptic level: its regional distribution and its synaptosomal localization (6); its high affinity uptake in synaptosomes (7) and its K+-induced, C a 2+ dependent release (8). So far, neither characterization of postsynaptic binding sites of PA nor interaction of PA with the membrane receptors of putative transmitters have been reported. This imino acid has been shown to depress the firing of cortical and hippocampal neurons when applied iontophoretically (9-10). Intracerebral administration of PA in rat produced sedation, sleeping wave pattern in the electroencephalogram, and other behavioral changes (11). These results suggest the presence of receptor sites for PA in the CNS.

However, the pharmacological effect produced by PA may not nec- essarily be a result of direct transmitter action, but it may be produced by its interaction with an established neurotransmitter systems. Recent reports of cyclic GABA analogs, especially the six-member ring piperidine-4-sulfonic acid and isonipecotic acid with potent and specific GABA agonist activity (12), suggest the possibility that an alicyclic compound such as PA may act as an endogenous ligand for GABA receptor.

This hypothesis is supported by recent studies in rats, showing that PA o r GABA electrophoretically-applied inhibit unit activity of cortical and hippocampal pyramidal neurons (I0). Takahama et al. (13) have reported that: (1) PA inhibitory effect was blocked by electrophoretic application of bicuculline but not by strychnine; (2) the inhibitory action of proline and nipecotic acid was little affected by bicucuUine; (3) simultaneous injection Of PA with a low current facilitated the GABA response, whereas the glycine response was only slightly facilitated by PA; and, (4) the~de- pressive actions of PA, and GABA as well, were not affected in Ca ~+ free, high Mg 2+ medium. These results suggest a relationship between the postsynaptic action of PA and GABA receptors.

In this study a high affinity binding site for PA has been characterized in mouse brain and its possible relationship with the GABA system has been investigated. A preliminary report of this study was published by Giacobini and Gutierrez (14).

EXPERIMENTAL PROCEDURE

Reagents. [3H]-Pipecolic acid hydrochloride (3H]PA (2.98 Ci/mmol) obtained by custom tritium labeling (New England Nuclear Corp., Boston, MA) was routinely monitored and purified if necessary as described below. D,L-PipecoIic acid, L-proline, a-Aaa, glutamate, aspartate, glycine and piperidine were from Sigma Chemicals (St. Louis, MO). The GABA

PIPECOLIC ACID BINDING 693

analogues (RS)-nipecotic acid, isonipecotic acid, 4,5,6,7-tetrahydroisoxyzolo-[5,4C]-pyri- din-3-ol (THIP), guvacine and isoguvacine were graciously donated by Dr. A. Schousboe (Panum Institute, University of Copenhagen, Denmark).

Purification of[3H]Pipecolic Acid. [3H]-Pipecolic acid ([3H]-PA) was purified by ion exchange column chromatography according to Piez et al. (15). An AG50W ion exchange column (22 • 1 cm) was used. The column was loaded with 5 ml of [3H]PA (50 mCi) in 0.1N HCI to be eluted with 10 ml 0.1 N HC1, 20 ml 0.1 N HC1 pH 3.42 and finally with 0.5 N HCt pH 3.88. Aliquots of I ml were collected after 20 ml with the last eluent. Pipecolic acid was identified by determining radioactivity in each aliquot.

The specific activity of [3H]PA was determined using dinitrophenol-PA as a standard. Binding Experiments. Preparation of Crude P2 Fraction Membrane. C57/BL10 adult male mice (20-30 g) were

decapitated, the brain was removed, weighed and homogenized in 10 vol. ice-cold 0.32 M sucrose in 50 mM Tris-HC1 buffer, pH 7.6. A crude P2 fraction was prepared according to Whittaker (16). The homogenate was centrifuged at 1,000 g for 10 rain, the pellet discarded and the supernatant centrifuged at 20,000 g for 20 mira The resultant crude mitochondrial pellet (P2) was resuspended in Tris-HCl buffer, pH 7.6 and dispersed with a Polytron for 15 sec. The homogenate was centrifuged at 40,000 g for 10 rain at 4~ The supernatant was discarded and the pellet resuspended and centrifuged under the same conditions. The pellet was resuspended in six volumes of 50 mM l'ris-HC1 buffer containing 100 mM NaCI and 1 mM MgCI~, pH 7.6. For regional distribution, the brain was dissected into nine regions.

[3H]Pipecotic Acid Binding Assay. Fifty/xl of [3H]PA were added to disposable test tubes and the assay was initiated by addition of homogenate containing 1-2 mg protein in 0.44 ml. The tubes were incubated in triplicate for 2 hr at 4~ Nonspecific binding was determined by addition of unlabeled PA at a final concentration of 1 mM prior to addition of the homogenate. Incubation was stopped by addition of 4 ml ice-cold Tris-HC1 buffer, pH 7.6, followed by rapid filtration through Whatman GF/B filters. The filters were rinsed twiCe with 4 ml ice-cold Tris-HCl, buffer pH 7.6. Filtration and rinsing were completed within 20 sec. The radioactivity remaining on filters was counted in a Beckman liquid scintillation counter (Model LS 5800) after addition of 10 mt Ready-Solv EP (Beckman) at 45% efficiency. The difference between the amount of radioactivity bound in the presence of unlabeled PA (nonspecific binding) and in its absence (total binding) represented the specific binding. This specific binding was abolished if the membranes were preincubated at 80~ for 10 rain. The radioactivity present on filters without tissue was less than 2%. A centrifugation procedure was compared with filtration. Although specific binding was similar in both cases, with centrifugation, nonspecific binding was significantly increased. Ouabain (10 -4 M) did not affect this binding.

Protein concentration of the resuspended membrane preparation was determined by the method of Lowry et al. (17).

In preliminary experiments, the conditions of the assay, tissue concentration, time, temperature and buffer Were established. The Na +-dependent specific binding of [3H]PA was linear up to 2.5 mg of membrane proteir~/ml. Therefore, all experiments were performed within this linear range.

The membrane-bound radioactivity following 2 hrs of incubation at 4~ was analyzed by thin layer chromatography (TLC) in an isopropanol-ammonia system (70:22). No metabolism of [3H]-PA was detected under these conditions.

Saturation curves for [3H]PA binding were estimated using [3HJPA concentrations from 20-110 riM. Kinetic constants and Eadie-Hofstee analysis of the [3H]PA binding were determined as described by Zivin and Waud (18). Ion-dependency, brain regional distribution and displacement of [3H]PA specific binding by structurally related comounds were deter-

694 GUTIERREZ AND GIACOBINI

4000.

Total 3000-

Nonspecific E o .

"o 2000-

1000 Specific

l b 2o 30 40 s'o 6'0 70 80 90 1 6 o l i o

[3H-PAl nM

Fro. 1. Saturation curve of [3H]PA specific binding to crude P2 fraction membranes. Crude P2 fraction membranes were prepared as described in Experimental Procedure. Homogenate was incubated with different concentrations of [3H]PA in the nM range, Nonspecific binding was determined in the presence of unlabeled PA (10 -3 M). Specific binding is the difference between total and nonspecific binding.

mined under similar conditions. Postnatal development (1, 3, 7, 10, 16, 22, 25, 30, 60 day old mice) of [3H]PA binding was determined using crude Pz fraction membranes as described above.

Uptake Studies. Uptake of radioactive [3H]PA to synaptosomal and crude P2 fraction membrane preparations of brain tissue were assayed as described by Nomura et al. (7). Unlike in the synaptosomal fraction, no measurable uptake was detected in the crude Pz fraction under the same experimental conditions.

RESULTS

Na§ [3H]PA specific binding was saturable at 70 nM (Fig- ure 1). Nonspecific binding was found to be unsaturable.

Eadie-Hofstee analysis of these data indicated a high affinity binding with a single population of Na---dependent binding sites (Figure 2). An apparent KD value of 33.2 nM and a Bmax value of 0.213 pmol/mg protein were determined. Hill plot analysis of these data was linear (r = 0.97) with a slope of 1.08, indicating the absence of cooperative interactions.

Various cations were tested for their ability to modify the [3H]PA specific binding (Figure 4). NaC1 increased specific binding approximately 2.5-fold in a concentration-dependent manner (Figure 3). N a 2 S O 4 did not modify PA binding. Specific binding was increased with increasing Na §


.3 �84 ~o

<[ .EE .2,

K D = 33 .2nM Bmax = .213 p m o l / m g p r o t S D I=rad = 0.095

1 2 3 4 5 6

Bound/Free x 103

Fjr3. 2. Eadie-Hofstee Plot of [3H]pA specific binding to crude P2 fraction membranes. Analysis of the saturation curve of [3H]PA specific binding. SD Erad = Coefficient of good- ness of fit (18).

concentration. In the presence of Li + and Cu 2 +, specific binding was decreased (Figure 4). On the other hand, MgCI2 and CaCI2 (1 raM) did not change PA binding (Figure 4).

Regional distribution of [3H]PA specific binding showed the highest concentration in cerebral cortex followed by thalamus, olfactory bulb,

25

A

,,,, +, < + I s

++1o

TRIS-HCI N a c l KCl LiCI MgCl 2 CaC I 2 C u C I 2

[100 raM] '. ~ [ l m M ] ,

F]G. 3. Effect of Na + on [3H]PA specific binding. Binding assay was conducted at 40 nM of [3H]PA in the presence of several NaCI concentrations. Nonspecific binding was determined by adding 1 mM of unlabeled PA to the assay. Values represent the means --2- SD of at least three separate experiments.


3 0 .

om~ 2 0 -

t

"-" 1 0

0 25 50 7'5 1 O0

[Na+] (raM) F[o. 4. Effect of monovalent and divalent cations on [3H]PA specific binding. The binding assay was conducted at 40 nM [3H]PA without cations added (Tris-HC1) and in the presence of 100 mM or 1 mM individual salts. Nonspeci f ic binding was determined in the presence of 1 mM unlabeled PA. The results are means _+ SD of four separate experiments. * P < 0.001, * P < 0.01.

hippocampus, and cerebellum. No binding was detected in corpus stria- turn, midbrain and pons medulla (Table I).

The effect of a series of GABA analogues and structurally related compounds to PA on [3H]PA (37 nM) binding is shown in Table II. D,L-PA (10 5 M) and L-proline (10 -5 M) equally inhibited high affinity [3H]PA specific binding. On the other hand~ glutamate (10 5 and 10 3 M) showed only a low potency in displacing [3H]PA specific binding. No significant effect could be detected with other compounds. The pattern of inhibition of D,L-PA and L-proline was similar (Table III).

When the membrane preparation was incubated 10 min prior to the binding assay in the presence of GABA (10 ~-10 -3 M) the effect of GABA on [3H]PA binding varied depending on both PA and GABA concentrations (Table IV). At higher concentration of unlabeled PA (10 -3 M), no effect of GABA was detectable at any concentration, however, at lower concentrations of PA (10 -5 and 10-6), [3H]PA binding either increased or decreased depending on GABA concentration. A higher concentration of GABA (10-3-10 5 M) decreased specific binding, but more interesting, its lowest concentration (10- ~ M) increased specific binding.


T A B L E I

REGIONAL DISTRIBUTION OF [3H]PA BINDING TO CRUDE P2 FRACTION IN MOUSE

BRAIN

Specific binding Region (fmol/mg protein)

Olfactory bulb 3.73 _+ .07 Cerebral cortex 10.78 _+ 2.42 Corpus striatum not detectable Thalamus 4.12 -+ .68 Midbrain not detectable Hippocampus 1.68 -+ .35 Cerebellum .67 _+ .46 Ports and medulla not detectable

The results which represent the specific binding at 40 nM of [3H]PA are means _+ SD of four separate experiments. The conditions of the assay were the same as for the saturation study in the presence of 100 mM NaCI.

T A B L E II DISPLACEMENT OF [3H]PIPECOLIC ACID BINDING BY STRUCrUaALLu RELAYED

COMPOUNDS

Percent of displacement of [3H]pipecolic acid specific binding

Compound 10 -5 M 10 3 M

D,L-Pipecolic acid 40.2 100 L-Proline 39.6 102 c~-Aminoadipic acid 0 9.1 Glutamate 6.4 73.5 Aspartate 3.7 16.3 Glycine 14.4 57.1 Piperidine 7.0 17.1 Nipecotic Acid 13.2 39.3 3,-Aminobutyric acid 0 0 Isonipecotic acid 0 1.6 TH1P 0 2.1 Guvacine 0.8 20.1 Isoguvacine 5.4 7.0

Total binding was determined in the presence of 37 nM of [3H]PA. One hundred percent displacement was determined in the presence of 10 3 M unlabled PA. All other values were determined as percentages of this displacement.

698 G U T I E R R E Z A N D GIAC OBINI

T A B L E I l l

PERCENT OF DISPLACEMENT OF [ 3 H ] P A SPECIFIC BINDING IN THE PRESENCE OF

VARIOUS CONCENTRATIONS OF UNLABELED LIGANDS*

[M] Pipecolic acid Proline

l0 3 100 99.2 10 -5 40.2 39.6 10 -7 15.7 6.9 l0 -9 8.8 0 10 ii 5.6 1.8

* Total binding was determined in the presence of 37 nM of [3H]PA. One hundred percent d isplacement was determined in the presence of l0 -3 M unlabeled PA. All other values were determined as percentages of this displacement. Crude P2 fraction membrane preparations were prepared as described in Exper imental Procedures .

During postnatal development, [3H]PA specific binding increased 8-fold from day 1 after birth to a peak at day 16. Then, specific binding decreased gradually to adult levels at day 30 and remained constant (Figure 5).

DISCUSSION

The existence of a high affinity, Na +-dependent PA binding site has been demonstrated in this study. Its KD (33.2 nM) is in the range seen for putative amino acid neurotransmitters such as glutamate, aspartate and GABA (14). A significant increase in PA binding was observed in the

T A B L E IV

PERCENT DISPLACEMENT OF SPECIFIC BINDING OF [ 3 H ] P A AFTER PREINCUBATION

WITH G A B A *

Molar Concentra t ion of GABA

[PAl (M) 0 10 3 10-5 10 7 10-9 10-11

10 -3 100 97.1 • 8.7 93.3 • 7.6 120.3 +_ 10.2 104.2 • 8.3 101.9 -- 5.2 10 -5 41.0 • 3.1 25.0 • 4.3 16.9 • 4.9 36.7 • 5.3 34.8 • 3.1 64.0 _+ 7.1 10 6 10.6 • 2.2 0 0 0 11.0 • 4.0 37.5 • 3.2

* Crude Pz fraction membranes (2.5 mg/ml) were preincubated 10 minutes with different concentra t ions of GABA. [3H]PA (37 nM) was then added and binding was determined in the presence of three concentrat ions of unlabeled PA. 10 -3 M PA without G A B A is a s sumed to displace all specific binding; other values are calculated relatively to this figure. Resul ts are means + SE of three separate exper iments in triplicates.

PIPECOLIC ACID B I N D I N G 699

70

o / 1 10 e-_ 0 -, O 50 O~.

O. '" 40 = O EE

30 " - - - -~

20

10

// ; -~ 1'o 1'6 ~;2 2s a'o go

DAYS

FIG 5. [3H]PA specific binding during postnatal development . Crude P2 fraction membranes of m o u s e brain at different ages were prepared as described in Exper imental Procedure. The binding assay was conducted at 40 nM [3H]PA and nonspecific binding was determined in the presence of 1 mM unlabeled PA. Values represent the means _+ SD of at least three separate exper iments .

presence of NaC1, but not with Na2SO4, This result indicates that both Na § and C1 ions must be present in order to obtain maximal binding. PA is taken up into mouse brain synaptosomes by both a low and a high affinity transport system (7). The high affinity sites have a strict require- ment for Na § and are ouabain-sensitive. Thus, the Na +-dependent binding site for PA reported in this study could represent binding to a re- uptake site. However, we excluded this possibility for the following rea- sons. First we were unable to demonstrate PA uptake under the same conditions of PA binding assay. Secondly, binding was ouabain- insen- sitive indicating no energy-dependence. Finally, Nomura et al. (7) reported a KM value of 3.9 ixM for the high affinity transport of PA; a value 100 times higher than the KD obtained in this study.

Displacement of PA binding by proline was similar to that by PA (Table III). The close structural relationship between PA and proline suggests that these two imino acids might share the same binding site. The mutual inhibition of PA and proline binding (10 .3 M, data not shown) favors such interpretation. However, the lack of specific inhibitors of PA binding prevented us to further examine such a possibility. It is interesting to note


that proline and PA have been postulated to share the same transport system (7, 19).

Neurophysiological studies in rat (10, 13) have suggested a possible relationship between PA and GABA. Although PA exerts an inhibition of neuronal firing in cerebral cortex and hippocampus, the present results suggest that GABA and PA do not share the same binding sites. In fact, displacement of [3H]PA binding with either GABA or some of its analogues was not observed and PA did not displace [3H]GABA Na+-in - dependent binding. In addition, nipecotic acid, a specific inhibitor of GABA uptake did not influence either PA uptake in synaptosomal preparations (7) or PA binding in the crude P2 fraction. Nevertheless, it seems that GABA might exert a modulatory effect on [3H]PA binding (Table IV). Although GABA (10 -3 M) shows only a weak displacement of PA binding, preincubation of neuronal membranes with GABA (10 -1~ M) increases PA binding (14).

Our data on the regional distribution of [3H]PA binding sites suggests that PA receptors exist which are localized in specific regions of the mouse brain and may have particular kinetic constants. Given the composition of the membrane fraction we used we could not localize more specifically PA binding sites to nerve cells soma, nerve endings or to glial cells. On the other hand, the binding distribution found by us correlates with the distribution of A l-pyrroline-2-carboxylate reductase in mouse brain (20), the enzyme which catalyzes the reduction of A ~-piperidine-2-carboxylate, resulting in the formation of PA. This pattern of distribution contrasts with the regional distribution of PA in rat and mouse brains (6) and with the "in vivo" accumulation of [3H]-PA following i.p. injection (unpub- lished). This is not surprising since these parameters may express distinct properties of PA-containing neurons such as accumulation of the product in the cell body and terminals of homologous cells, versus presence of binding sites on heterologous neurons. In line with this notion is the ob- servation that the concentration of muscarinic cholinergic receptor sites in various brain regions correlates only to a limited extent with endogenous acetylcholine levels (21), and norepinephrine (NE) sensitive cyclic AMP accumulation associated with postsynaptic NE receptors does not parallel levels of endogenous NE in various brain regions (22). There are no available data regarding regional variations in the uptake of PA in adult mouse brain, therefore, it is not possible to judge if PA binding correlates with uptake distribution or not.

In conclusion, our study has for the first time identified a high affinity Na+-dependent binding site for PA which was saturable, unevenly dis- tributed in the brain, displaceable by specific compounds and analogues.


This binding shows a characteristic developmental pattern. These data support the role of PA as a brain-intrinsic neuromodulator (3).

ACKNOWLEDGMENTS

The skillful technical assistance of Mrs. E. Woodruff is acknowledged. The authors wish to thank Mrs. D. Smith for the careful typing and editing of the manuscript. Supported in part by a Central Research Committee grant No. 3-84 from S.I.U. School of Medicine to E.G.M.d.C.G. was supported by a fellowship from the Academic Formation Program from the National Autonomous University of Mexico.

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identification and characterization of pipecolic acid binding sites in mouse brain

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