mu opioid receptor coupling to gi/o proteins increases

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Mu Opioid Receptor Coupling to Gi/o Proteins Increases During Postnatal Development in Rat

Brain

Jeffery N. Talbot1,2, H. Kevin Happe3, and L. Charles Murrin,

Department of Pharmacology and Experimental Neuroscience (JNT, HKH,

LCM), University of Nebraska Medical Center, Omaha, NE 68198-5800

JPET Fast Forward. Published on April 28, 2005 as DOI:10.1124/jpet.104.082156

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 28, 2005 as DOI: 10.1124/jpet.104.082156

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Running Title: Development of Mu Opioid Receptor G Protein Coupling

Address correspondence to L. Charles Murrin, 985800 Nebraska Medical Center,

Omaha, NE 68198-5800. Phone: (402) 559-4552; fax: (402) 559-7495; E-mail:

[email protected]

Pages:

Text – 23

Figures – 5

Tables – 1

References – 46

Word Count:

Abstract – 234

Introduction – 710

Discussion – 1418

Abbreviations Used: MOR, mu opioid receptors; DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol]-

enkephalin; α2AR, α2 adrenergic receptors; GppNHp, 5΄-guanylyl-imidophosphate;

PND, postnatal day.

Recommended Section Assignment: Neuropharmacology


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Abstract:

Mu opioid receptors are densely expressed within rat striatum and are concentrated in

anatomically discrete patches called striosomes. The density of striosomal mu

receptors remains relatively constant during postnatal development, but little is known of

their functional maturation. We examined the extent of G protein coupling by mu opioid

receptors in rat brain during development, focusing on striosomes within the striatum

because of receptor density. Mu receptors were quantified using [3H]DAMGO

autoradiography. Adjacent sections were analyzed for DAMGO-stimulated [35S]GTPγS

binding to assess mu receptor activation of Gi/o proteins. Striosomal mu receptor

expression increased only slightly between postnatal day 5 and adult. In contrast, mu

receptor-stimulated [35S]GTPγS binding increased from 0.13 fmol/mg tissue to 2.6

fmol/mg tissue over the same period, a 20-fold difference. The ratio of specific

DAMGO-stimulated [35S]GTPγS binding to [3H]DAMGO binding, representing the

relative number of G proteins activated per receptor, increased 19-fold between

postnatal day 5 and adult. Similar patterns were observed throughout the striatum and

other brain regions such as the nucleus accumbens although the extent of change

varied from region to region. These data indicate that mu opioid receptors exhibit

enhanced function in the adult rat brain compared to the neonate. These data also

suggest that this increase in G protein coupling is developmentally regulated and that in

the developing rat brain the density of mu opioid receptor expression may not

necessarily correlate with receptor activation of G proteins.


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Introduction

Mu opioid receptors (MORs) are heptahelical receptors expressed by neurons of

the central, peripheral, and enteric nervous systems. Nearly all of the biochemical

responses to MOR activation discovered thus far involve signal transfer through G

proteins, heterotrimeric proteins whose activity is regulated by guanine nucleotide

binding. Agonist-occupied receptors activate G proteins by increasing the frequency of

GTP binding to Gα, whereas intrinsic GTPase activity deactivates G proteins through

GTP hydrolysis (Gilman, 1987). MOR-activated G proteins alter cell function by

regulating adenylyl cyclase, Ca2+ transport, neurotransmitter release, inwardly rectifying

potassium channels, and the mitogen activated protein kinase (MAPK) cascade (Law et

al., 2000). The cellular responses elicited through MOR signal transduction lead to

numerous physiological effects such as analgesia, euphoria, respiratory depression,

and constipation (Dhawan et al., 1996). MOR agonists and antagonists are drugs of

choice for treatment of moderate to severe pain, drug dependence, pulmonary edema,

cough suppression, and diarrhea.

In adult mammals, high levels of MOR expression are found in the striatum, a

major component of the basal ganglia, where MORs are densely expressed on neurons

organized into anatomically distinct areas called patches or striosomes (Pert et al.,

1976; Atweh and Kuhar, 1977). Striosomes are complementary within the striatum to

areas of low MOR expression termed the matrix (Graybiel and Ragsdale, 1978). MOR

binding sites are first detected in the developing rat striatum at embryonic day 14.

Striosomal patterns of MOR binding are first observed at embryonic day 19-20 and

increase to near adult levels by birth reaching maximum levels within two weeks of

postnatal development (Kent et al., 1981; Recht et al., 1985).


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The [35S]GTPγS assay has been used as a measure of Gi/o protein activation.

This assay utilizes a radiolabeled GTP analogue that is poorly hydrolyzed to quantify

levels of Gα protein activated by specific receptors (Traynor and Nahorski, 1995;

Harrison and Traynor, 2003). Combining the [35S]GTPγS assay with autoradiography

allows G protein activation by specific receptors to be anatomically defined and

quantified (Sim et al., 1995; Happe et al., 2000). These techniques have been used to

evaluate the coupling of MORs to G proteins in the CNS as well as the relationship

between receptor binding and receptor-stimulated G protein activation. In the rat brain,

MOR agonists stimulate [35S]GTPγS binding to varying degrees in different brain

regions (Sim et al., 1996a; Tsuji et al., 1999). Childers and colleagues further evaluated

MOR/G protein coupling by determining the efficiency of G protein activation by MORs.

MOR-stimulated [35S]GTPγS binding was directly compared to MOR density

([3H]naloxone binding), resulting in an “amplification factor” or the relative number of G

proteins activated per receptor (Sim et al., 1996b; Maher et al., 2000). The relative

MOR/G protein coupling efficiency varied significantly between brain regions (Maher et

al., 2000). These studies suggested that signal transfer from MORs to G proteins is

regulated in a tissue-specific manner in the CNS.

For some systems, receptor expression during development appears to parallel

G protein activation, as might be expected. For example, during postnatal

development, increased α2 adrenergic receptor (α2AR) expression in the rat forebrain

parallels increases in α2AR-stimulated [35S]GTPγS binding (Happe et al., 1999). Also in

the rat, cannabinoid receptor expression corresponds with agonist-stimulated

[35S]GTPγS binding during embryonic development (Berrendero et al., 1998). However,

a number of studies suggest disparate G protein coupling by MORs during


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development. In several embryonic mouse brain regions, including the striatum,

radioligand binding to MORs is observed before MOR agonist-stimulated [35S]GTPγS

binding can be detected (Nitsche and Pintar, 2003). Also, sensitivity of MORs to the

GTP analog 5΄-guanylyl-imidophosphate (GppNHp) in postnatal day 27 rats is twice that

of animals at day 10, suggesting weaker MOR/G protein coupling in neonatal animals

(Windh and Kuhn, 1995). These data suggest that during development MOR binding

may not necessarily correlate with MOR-activated G proteins. The objective of the

current study was to determine whether MOR/G protein coupling changes during

postnatal development. We used receptor autoradiography and the [35S]GTPγS

autoradiographic assay to show that Gi/o protein activation by MORs increased

dramatically during postnatal development despite very small changes in MOR density,

indicating MORs exhibit increasing function with advancing development. These data

suggest that MOR-stimulated G protein coupling is a developmentally regulated process

and that in the developing rat brain MOR expression may not necessarily correlate with

MOR function.


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Methods:

Materials. [35S]GTPγS (1250 Ci/mmol) was obtained from Perkin Elmer Life Sciences,

Inc (Boston, MA). [3H]DAMGO (68 Ci/mmol) was purchased from Amersham

Biosciences (Piscataway, NJ). DAMGO, naloxone hydrochloride, and GTPγS were

purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). GDP was purchased

from United States Biochemical Corp. (Cleveland, OH). All other chemicals were

research grade.

Animals and Tissue Preparation. Sprague-Dawley rats (Sasco, Kingston, NY) were bred

in our colony. Litters were culled to nine pups and monitored for normal growth by body

weight. Brains were collected at postnatal days (PND) 5, 10, 15, 21, 30, and from

adults (Ad – determined by weight, 250 ± 25 g), rapidly frozen on dry ice, and stored

wrapped in Parafilm and foil at -80°C. Tissue sections were cut from brains at 16 µm

using a cryostat, thaw-mounted onto subbed slides, and stored at -20°C until use.

Coronal sections centered on the rostral, medial, and caudal regions of the striatum

were used (Plates 12-31: (Paxinos and Watson, 1986)). Procedures were in strict

accordance with The National Institutes of Health Guide for the Care and Use of

Laboratory Animals and were approved by the local IACUC Animal Care Committee.

Agonist-Stimulated [35S]GTPγS Binding Assay. The [35S]GTPγS assay was carried out

as previously described (Sim et al., 1995; Happe et al., 2001). Briefly, tissue sections

were re-hydrated in assay buffer (50 mM glycylglycine, 3 mM MgCl2, 1 mM EGTA, 100

mM NaCl, pH 7.45) for 10 min. Sections were then immersed in assay buffer containing

2 mM GDP for 15 min, followed by incubation for 210 min in assay buffer containing


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2 mM GDP, 0.25 mM dithiothreitol, and 0.1 nM [35S]GTPγS. Agonist-stimulated and

basal [35S]GTPγS binding were measured in the presence (stimulated) and absence of

3 µM DAMGO, a MOR-specific agonist. DAMGO-stimulated [35S]GTPγS binding was

also measured in the presence 5 µM naloxone to determine background. Non-specific

[35S]GTPγS binding was determined in the absence of agonist and the presence of 1 µM

unlabeled GTPγS. All incubations were performed at room temperature (25°C).

Immediately following incubations, tissue sections were washed twice (5 min

each) in ice-cold 50 mM glycylglycine buffer (pH 7.45) containing 0.25 mM dithiothreitol

and dipped once in ice-cold water. Sections were dried under a stream of cool air for

30 min and left at room temperature overnight. Sections were apposed to HyperFilm-

βMAX (Amersham Life Science, Arlington Heights, IL) for 1 day to 1 week and were

quantified by densitometric analysis with commercial 14C standards (American

Radiolabeled Chemicals, Inc., St. Louis, MO) that had been individually calibrated to

[35S]tissue standards (Miller and Zahniser, 1987). Data are expressed as fmol/mg

tissue.

[3H]DAMGO Autoradiography. Mu opioid receptors were analyzed with [3H]DAMGO

quantitative autoradiography using tissue sections adjacent to those used in the

DAMGO-stimulated [35S]GTPγS binding assay. Tissue sections were incubated at room

temperature with 3 nM [3H]DAMGO in 50 mM Tris-HCl, pH 7.45 for 45 min. Non-

specific binding was determined by the addition of 5 µM naloxone. Following

incubations, sections were washed twice (5 min each) in ice-cold 50 mM Tris-HCl and

briefly dipped once in ice-cold water. Sections were then treated identically to those

used in the [35S]GTPγS binding assay except Hyperfilm-3H autoradiographic film


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(Amersham) was used with previously calibrated commercial tritium standards.

Data Analysis. Films were developed using standard methods and analyzed using the

MCID-M5 system (Imaging Research, St. Catherines, Ontario, Canada). Four sets of

serial sections from rostral, medial, and caudal striatal regions of three to four rat brains

were assayed at each developmental age. Striosomes were identified from surrounding

matrix by the much greater density of MOR (4- to 5-fold). For each section, [3H]DAMGO

binding levels were measured in the matrix and in four striosomes. Corresponding

areas in adjacent tissue sections were measured for [35S]GTPγS binding. Basal values

of [35S]GTPγS binding were subtracted from DAMGO-stimulated binding, as previously

described (Happe et al., 2000).


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Results:

In preliminary studies we noticed an apparent discrepancy between MOR

expression ([3H]DAMGO autoradiography) and activation of G proteins by MORs

(DAMGO-stimulated [35S]GTPγS binding) during postnatal development. In the current

studies we examined in detail the relationship between these two. To anatomically

compare MOR levels with MOR coupling to Gi/o proteins in the developing rat brain,

adjacent tissue sections from PND5, 10, 15, 21, 30, and adult were used for either

DAMGO-stimulated [35S]GTPγS binding or [3H]DAMGO binding followed by quantitative

autoradiographic analysis. High levels of [3H]DAMGO binding were found in the

cingulate cortex, amygdala, thalamic nuclei, core and shell regions of the nucleus

accumbens, CA1 region of the hippocampus, and in striosomes of the caudate-putamen

(Fig. 1), in agreement with previous studies (Atweh and Kuhar, 1977; Quirion et al.,

1983). DAMGO, a MOR-specific agonist, significantly stimulated [35S]GTPγS binding in

these same regions in adjacent brain sections. In adults, there was a high degree of

anatomical correlation between [3H]DAMGO binding and DAMGO-stimulated

[35S]GTPγS binding, with both being greatest in striosomes. On the other hand, the

correlation was less precise at the early developmental ages studied (PND5 – PND15).

At these earlier ages [3H]DAMGO binding was similar in density to that observed in the

adult, whereas DAMGO-stimulated [35S]GTPγS binding was much weaker at the early

ages compared to the adult. This was most apparent in the striosomal regions of the

striatum, anatomically distinct areas of particularly robust MOR expression (Pert et al.,

1976; Atweh and Kuhar, 1977; Herkenham and Pert, 1981) and so we focused our

detailed studies on this area.

We used densitometric analysis to quantify MOR levels in striosomes from rostral


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areas of the striatum during development. [3H]DAMGO binding increased from 83 ± 1

fmol/mg tissue at PND5 to 112 ± 6 fmol/mg tissue at PND10 but did not change

significantly from PND10 through adulthood (Fig. 2). The modest increase in

[3H]DAMGO binding observed between PND5 and PND10 is in agreement with

previous autoradiographic studies indicating maximum levels of MOR density in

striosomes are reached before the second postnatal week (Kent et al., 1981). However,

no significant changes in [3H]DAMGO binding were observed after PND10.

By contrast, specific DAMGO-stimulated [35S]GTPγS binding increased

dramatically over the same time period, from 0.25 ± 0.01 fmol/mg tissue at PND5 to

3.10 ± 0.28 fmol/mg tissue in the adult, while basal (unstimulated) [35S]GTPγS binding

increased from 0.12 ± 0.01 to 0.71 ± 0.20 fmol/mg tissue (Fig. 3A). Specific DAMGO-

stimulated [35S]GTPγS binding (basal subtracted; Fig. 3B) showed a 20-fold increase

(0.13 ± 0.01 to 2.63 ± 0.05 fmol/mg tissue). The period between PND10 and PND15

saw the largest increase in [35S]GTPγS binding with incremental increases occurring at

each age measured thereafter. These data were also analyzed as the ratio of specific

DAMGO-stimulated [35S]GTPγS binding to [3H]DAMGO binding. This ratio represents

the relative amount of Gi/o protein activated per receptor, a reflection of the G protein

coupling efficiency of MORs under our assay conditions. In PND5 rats, the striosomal

MOR coupling efficiency was 0.15 ± 0.01 compared to 2.95 ± 0.46 in the adult, a 19-fold

increase (Fig. 4).

This same pattern of increasing MOR coupling efficiency during development

was also found in striosomes in the medial and caudal regions of the striatum.

Striosomal [3H]DAMGO binding remained relatively constant from PND5 to adulthood in

both the medial and caudal striatum (Table I). Striosomal DAMGO-stimulated


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[35S]GTPγS binding levels were comparable in the rostral, medial, and caudal regions of

the striatum at PND5, (0.13 ± 0.01, 0.19 ± 0.03, 0.22 ± 0.05 fmol/mg tissue,

respectively). However, greater differences were manifest between these same regions

during the later stages of postnatal development. In adult animals, rostral striosomes

had 60% greater DAMGO-stimulated [35S]GTPγS binding than medial striosomes (2.63

± 0.46 to 1.61 ± 0.25 fmol/mg tissue, respectively) and 240% greater than caudal

striosomes (2.63 ± 0.66 to 0.77 ± 0.27 fmol/mg tissue, respectively). There were 7- and

5-fold increases in MOR coupling efficiencies from PND5 to adulthood in medial and

caudal striosomes compared to the 19-fold increase in rostral striosomes (Table I). In

addition, MOR coupling efficiency in the caudal region of the striatum peaked earlier in

development (PND21) when compared to striosomes of the rostral and medial regions.

MOR/G protein coupling was analyzed in other areas of the CNS, as well. The

striatal matrix also exhibited increasing MOR/G protein coupling throughout

development, similar to striosomes, despite having considerably lower [3H]DAMGO

binding (data not shown). In a like manner, DAMGO-stimulated [35S]GTPγS binding

increased in the nucleus accumbens and the lateral septal nuclei during postnatal

development while there was little or no change in MOR density and MOR expression in

these regions was considerably lower than that observed in the MOR-rich striosomes

(Fig. 5A,B). DAMGO-stimulated [35S]GTPγS binding also increased during postnatal

development in areas where MOR density increased rather than remaining unchanged,

such as cortical layers IV and VI and cingulate cortex. The ratio of [3H]DAMGO binding

to DAMGO-stimulated [35S]GTPγS binding or the relative G protein coupling efficiency

of MOR increased to a variable extent during postnatal development from region to

region and this was not related to MOR density (Fig. 5C). Increased coupling efficiency


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was most clearly evident in areas where [3H]DAMGO binding changed very little during

postnatal development but DAMGO-stimulated [35S]GTPγS binding increased

dramatically, such as in striosomes, nucleus accumbens, and the lateral septal nuclei.


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Discussion:

In several brain regions, including striosomes, MOR expression reached adult

levels early in postnatal development, yet MOR coupling to Gi/o proteins (and

presumably to their associated signal transduction pathways) did not reach maximum

levels until adulthood. These data suggest there is a disparity between MOR

expression and receptor activation of G proteins during development. Changes in

MOR/G protein coupling appear to be the result of the increased activation of G proteins

by MORs rather than changes in either receptor density of or agonist affinity for MORs.

During postnatal development of the rat brain, DAMGO has been shown to bind with the

same affinity to a single high affinity population of MORs (Spain et al., 1985). DAMGO

labels the agonist high affinity state of MORs, the state which activates G proteins upon

agonist stimulation. A previous study has shown a parallel pattern of opioid receptor

development in striosomes using the antagonist, [3H]naloxone, which would label the

total receptor population (Kent et al., 1981). We did not see the drop in MOR density

during the second postnatal week found by Kent and colleagues. This may reflect a

difference in the number of high affinity receptors compared to total receptor number or

it may be due to differences in the methods of quantifying receptors.

Although our studies do not examine possible stimulation of delta opioid

receptors by DAMGO, we think it is unlikely they play a role in the current studies.

DAMGO has an affinity for DOR that is approximately three orders of magnitude less

than its affinity for MOR (Toll et al., 1998; Zhao et al., 2003). In addition it has been

shown that DAMGO at concentrations up to 10 µM produces no measurable

[35S]GTPγS binding in cells expressing only DOR (Toll et al., 1998; Alt et al., 2002).

Moreover, in demonstrating the MOR selectivity of DAMGO in rat brain tissue sections,


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Childers and colleagues showed under conditions similar to ours that DAMGO-

stimulated [35S]GTPγS binding is unaltered by DOR-selective and KOR-selective

inhibitors (Sim et al., 1996a).

Our findings are in agreement with other reports suggesting disparate coupling of

MOR to G proteins during development. Sensitivity of MOR to the GTP analog

GppNHp in PND27 rats was twice that of PND10 animals, suggesting weaker MOR/G

protein coupling in neonatal animals (Windh and Kuhn, 1995). More recently, MOR

mRNA expression was found to precede MOR-stimulated [35S]GTPγS binding in several

regions of the embryonic mouse brain, including the striatum (Nitsche and Pintar, 2003).

Taken together with our findings, these data indicate that the coupling of MORs to Gi/o

proteins is a developmentally regulated process. Low levels of G protein activation

early in developmental may result from partial G protein coupling of the entire receptor

population or the complete uncoupling of a subset of receptors. Regardless, our data

suggest MOR coupling to their cognate Gi/o proteins increases throughout the postnatal

developmental period. These data also suggest that, in the developing rat brain, MOR

expression does not temporally correlate with MOR function.

Several factors could be regulating this maturation of MOR signaling through G

proteins. Our data indicate that changes in MOR levels are responsible for little, if any,

of the increase in G protein coupling. It is possible that G proteins themselves may

regulate MOR signaling during development and a thorough examination of G protein

levels, by immunohistochemistry for example, will be necessary to determine this. Our

data (Fig. 3A and Table1) indicate that basal [35S]GTPγS binding increased significantly

during development, suggesting increased G protein expression. This increase in G

protein levels could contribute, at least in part, to increased coupling between MORs


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and G proteins. MORs activate multiple members of the family of Gαi/o proteins,

including Gαi1, Gαi2, Gαi3, Gαo1, and Gαo2 (Chan et al., 1995), and it has been

suggested that MORs activate each with varied efficacy and potency. For example,

MORs exhibit preferential coupling to Gαi3 in SH-SY5Y cells as measured by α-

azidoanilido[32P]GTP affinity labeling (Laugwitz et al., 1993). Also, in CHO cells, MOR

agonists stimulated incorporation of α-azidoanilido[32P]GTP into Gαi3 and Gαo2 with

greater potency than Gαi2, while the rank order of maximal labeling (efficacy) was Gαi2

= Gαo2 > Gαi3 (Chakrabarti et al., 1995). In addition, MORs fused to Gαi1 stimulated

[35S]GTPγS binding more efficiently than MORs fused to Gαi2 (Massotte et al., 2002).

These data suggest that MORs exhibit preferential coupling to different Gα subunits.

Therefore, it is possible that MOR signal transduction may be altered by the

complement of Gα subunits available for coupling. In this sense the G proteins

themselves may regulate MOR signal transduction through the developmentally-timed

and subunit-specific expression of G proteins in specific regions of the CNS.

Surprisingly, little has been reported regarding the developmental expression of

specific Gi/o α-subunits in specific anatomical areas. One study measuring Gα-subunit

expression in several regions of the rat brain, including hippocampus, thalamus, and the

cortex, demonstrated that during postnatal development levels of Gαi1, Gαi2, and Gαo1

increase, Gαo2 remains unchanged, and Gαi3 decreases (Ihnatovych et al., 2002).

These data suggest that Gα-subunit expression changes during development in a

subunit-specific fashion. Because MORs activate individual Gi/o α-subunits with varying

efficiencies, it also will be necessary to determine not only the relative levels of

expression of G proteins during development but also the relative contribution of each


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Gi/o α-subunit to the signal generated by MORs.

The contribution of pertussis toxin-insensitive G proteins, such as Gz, to the

DAMGO-stimulated [35S]GTPγS binding is not known. The expression of Gz relative to

Gi/o in the brain suggests that its contribution may be less than that of other G proteins

(Friberg et al., 1998), especially in light of the observation that both basal and

stimulated [35S]GTPγS binding is unaltered in mice lacking Gz (Laitinen, 2004).

Additional factors besides G proteins could serve as regulators of MOR/G protein

coupling during development. It is well established that RGS proteins (Regulators of G

protein Signaling) negatively regulate GPCR signaling by accelerating the GTPase

activity of the Gα subunit (Hollinger and Hepler, 2002). Several members of the RGS

protein family have been shown to negatively regulate MOR signaling in vitro and in vivo

(Potenza et al., 1999; Zachariou et al., 2003). In particular, expression of RGS2 in the

rat striatum appears to be highest during the early postnatal period (PND2 and PND10),

with a dramatic decrease in expression between PND10 and PND18 (Ingi and Aoki,

2002), a period which roughly parallels the large increases in MOR-stimulated

[35S]GTPγS binding observed in the rat striatum. Although RGS proteins are obvious

and otherwise attractive candidate regulators of G protein action, they are unlikely to

explain our current findings due to the hydrolysis-resistant and therefore “RGS-

insensitive” nature of GTPγS.

The non-visual arrestins regulate GPCR signaling by promoting uncoupling of the

receptor from its cognate G proteins and subsequent desensitization (Claing et al.,

2002). In the striatum, arrestin 2 (β-arrestin 1) increases throughout postnatal

development while arrestin 3 (β-arrestin 2) decreases slightly (Gurevich et al., 2002).

These data suggest that the influence of arrestin 2 on MOR signaling, from a purely


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kinetic standpoint, should increase throughout development, resulting in less rather than

more G protein signaling by MORs. Therefore, arrestin 2 is an unlikely mediator of

developmental increases in MOR-stimulated G protein activation. Decreasing arrestin 3

expression could enhance G protein activation, although the contribution of the small

decrease in arrestin 3 reported by Gurevich and colleagues relative to the near 20-fold

increase in MOR/G protein coupling during development is unknown. Thus, the exact

identity of the regulator(s) of these phenomena remains unclear.

With the advent of proteomic technology, it is possible to identify protein binding

partners for receptors such as the MOR. In many instances, regulators of GPCRs have

been identified that alter receptor signaling and, in so doing, provide yet another level of

signal transduction regulation (Brady and Limbird, 2002). Several recent reports

utilizing yeast two-hybrid methodology describe proteins that functionally interact with

MOR, including phospholipase D2 (Koch et al., 2003), filamin A (Onoprishvili et al.,

2003), periplakin (Feng et al., 2003), and that modulate either G protein signaling or

receptor internalization.. Proteins such as these may be differentially expressed during

development in such a manner as to regulate MOR signal transduction. The developing

rat brain appears be an ideal tissue in which to identify such proteins, given the nearly

20-fold difference in activation of G proteins by MORs in neonate vs. adult animals.

Such studies could yield valuable information regarding the developmental differences

in MOR signaling as well as pointing to as yet unidentified regulators of MOR signal

transduction.


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Footnotes:

This research was supported by the National Institutes of Health DA016346 and the

University of Nebraska Medical Center.

1 The current address of Jeffery N. Talbot is Department of Pharmacology, University of

Michigan Medical School, 1301 MSRB III, Ann Arbor, MI 48109

2This work was submitted to the University of Nebraska as partial fulfillment of the

requirements for the degree of Doctor of Philosophy. Portions of this work were

presented previously in abstract form as: Talbot JN, Happe HK, and Murrin LC. (2002)

Mu opioid receptor/G protein coupling efficiency increases in developing rat striatum.

The Pharmacologist, 44:A134.

3The current address of H. Kevin Happe is Department of Psychiatry, Creighton

University School of Medicine, 3528 Dodge St., Omaha, NE 68131


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Legends for Figures:

Figure 1. Development of MOR binding and MOR-stimulated G protein activation.

Representative autoradiograms of MOR-activated G proteins (DAMGO-stimulated

[35S]GTPγS binding: left) and of MORs (3 nM [3H]DAMGO binding: right) from adjacent

rat brain sections at postnatal days 5, 10, 15, 21, 30 and adult (Ad). Pseudo-color

images represent total binding. Sections are from the rostral striatum (Plates 12-15).

Figure 2. Specific [3H]DAMGO binding in striosomes of rostral striatum in rat

brains during postnatal development. Tissue sections harvested at postnatal day 5,

10, 15, 21, 30, and adult (Ad) were incubated with 3 nM [3H]DAMGO in assay buffer (50

mM Tris-HCl, pH 7.45). Naloxone (5 µM) was used to determine non-specific binding.

Data are presented as fmol [3H]DAMGO bound per mg tissue and are the means ±

S.E.M. of four striosomes from each of four tissue sections from each of three to four rat

brains. There are no statistically significant differences between the values in this table

using ANOVA for analysis. Values from Day 5 and day 10 were significantly different

when comparing only these two values (p < 0.01; Students t test).

Figure 3. [35S]GTPγS binding in striosomes of rostral striatum in rat brains during

postnatal development. Tissue sections harvested at postnatal day 5, 10, 15, 21, 30,

and adult (Ad) were incubated with [35S]GTPγS in the presence (stimulated) and

absence (basal) of 3 µM DAMGO in assay buffer. GTPγS (1µM ) was added to

determine non-specific binding. Data are expressed as fmol [35S]GTPγS bound per mg

tissue: basal and DAMGO-stimulated (A) and specific (basal subtracted) DAMGO-


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stimulated (B). Data are the means ± S.E.M. of four striosomes from each of four tissue

sections from each of three to four rat brains. In Fig. 3B each data set is significantly

different from the rest (p < 0.05) using ANOVA followed by the Tukey-Kramer Multiple

Comparisons Test except d5 vs. d10, d15 vs. d21, d21 vs. d30 and d30 vs. Ad.

Figure 4. Fold increase in relative G protein coupling efficiency of striosomal

MORs. The relative G protein coupling efficiency of striosomal MOR was determined

in rat brain sections harvested at postnatal day 5, 10, 15, 21, 30, and adult (Ad). Data

are expressed as the ratio of the mean specific [35S]GTPγS binding (fmol/mg tissue) to

the mean [3H]DAMGO binding (fmol/mg tissue). Data are normalized with PND5

relative coupling efficiency set to 1.

Figure 5. MOR binding, MOR-stimulated [35S]GTPγS binding, and the relative G

protein-coupling efficiency in various brain regions during postnatal

development. Specific [3H]DAMGO binding (A), DAMGO-stimulated [35S]GTPγS

binding (B), and relative G protein coupling efficiency of MOR (C) were determined in

various anatomical areas in rat brain sections harvested at postnatal day 5, 10, 15, 21,

30, and adult (Ad). Abbreviations: Str – striosomes; Ctx4 – 4th layer of frontal cortex;

Ctx6 – 6th layer of frontal cortex; Cng – cingulate cortex; Acb – nucleus accumbens

(shell and core); LSN – lateral septal nuclei. Data are presented as the means ± S.E.M.

from four tissue sections from each area of three to four rat brains.


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Table 1. Developmental Changes in MOR Levels and Function in Striosomes of the Medial and Caudal Striatal Regions

Striatal Regiona

Postnatal Ageb

[3H]DAMGO bindingc (fmol/mg

tissue)

Basal [35S]GTPγS bindingc,d (fmol/mg

tissue)

Agonist-stimulated

[35S]GTPγS bindingc,d (fmol/mg

tissue)

Coupling Ratioe

Fold Increase in Coupling

Ratio (Compared

to d5)

Medial d5 61.6 ± 3.1 0.13 ± .01 0.19 ± 0.03 0.32 ± 0.07 1.0 d10 76.7 ± 4.4 0.24 ± .02 0.31 ± 0.12 0.39 ± 0.12 1.2 d15 74.4 ± 18 0.47 ± 0.1 0.70 ± 0.31 0.80 ± 0.13 2.5 d21 68.0 ± 2.7 0.59 ± .03 1.22 ± 0.27 1.81 ± 0.33 5.7 d30 77.0 ± 7.4 0.61± .02 1.47 ± 0.06 1.97 ± 0.12 6.2 Ad 59.1 ± 9.6 0.62 ± .04 1.61 ± 0.25 2.34 ± 0.28 7.4

Caudal d5 55.5 ± 4.4 0.16 ± .01 0.22 ± 0.05 0.38 ± 0.05 1.0 d10 60.5 ± 7.8 0.27 ± .02 0.31 ± 0.13 0.44 ± 0.15 1.1 d15 68.3 ± 12 0.48 ± .05 0.65 ± 0.08 1.01 ± 0.13 2.6 d21 41.3 ± 1.1 0.50 ± .01 0.89 ± 0.22 2.20 ± 0.46 5.7 d30 49.2 ± 1.3 0.66 ± .08 1.23 ± 0.19 2.59 ± 0.43 6.8 Ad 40.8 ± 6.1 0.74 ± 0.2 0.77 ± 0.25 2.01 ± 0.76 5.3

a) Striatal regions were determined according to Paxinos and Watson (1986):medial - plates 22-24; caudal - plates 29-31. b) Postnatal age was measured from time of birth (within 12 hours); adult animals were determined by weight (250 ± 25 grams). c) Data are presented as the means ± S.E.M. of four striosomes from each of four tissue sections averaged from each of three to four rat brains. d) Basal [35S]GTPγS binding and DAMGO-stimulated [35S]GTPγS binding (stimulated minus basal) were measured from the same striosomes using adjacent tissue sections. e) Coupling Ratio is the ratio of DAMGO-stimulated [35S]GTPγS binding to [3H]DAMGO binding of individual striosomes multiplied by 100.


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mu opioid receptor coupling to gi/o proteins increases

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