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NEUROPHARMACOLOGY
Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska
Received for publication
December 13, 2004
Accepted
April 27, 2005.
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Abstract |
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S) binding to assess µ receptor activation of Gi/o proteins. Striosomal µ receptor expression increased only slightly between postnatal day 5 and adult. In contrast, µ receptor-stimulated [35S]GTPS binding increased from 0.13 to 2.6 fmol/mg tissue over the same period, a 20-fold difference. The ratio of specific DAMGO-stimulated [35S]GTPS 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 µ opioid receptors exhibit enhanced function in the adult rat brain compared with 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 µ opioid receptor expression may not necessarily correlate with receptor activation of G proteins.
, 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 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 to 20 and increase to near adult levels by birth, reaching maximum levels within 2 weeks of postnatal development (Kent et al., 1981; Recht et al., 1985).
The [35S]GTPS assay has been used as a measure of Gi/o protein activation. This assay uses a radiolabeled GTP analog that is poorly hydrolyzed to quantify levels of G protein activated by specific receptors (Traynor and Nahorski, 1995; Harrison and Traynor, 2003). Combining the [35S]GTPS 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]GTPS 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]GTPS 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 seems to parallel G protein activation, as might be expected. For example, during postnatal development, increased 2 adrenergic receptor expression in the rat forebrain parallels increases in 2 adrenergic receptor-stimulated [35S]GTPS binding (Happe et al., 1999). Also in the rat, cannabinoid receptor expression corresponds with agonist-stimulated [35S]GTPS binding during embryonic development (Berrendero et al., 1998). However, a number of studies suggest disparate G protein coupling by MORs during development. In several embryonic mouse brain regions, including the striatum, radioligand binding to MORs is observed before MOR agonist-stimulated [35S]GTPS binding can be detected (Nitsche and Pintar, 2003). In addition, sensitivity of MORs to the GTP analog 5'-guanylyl-imidophosphate 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]GTPS 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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Materials and Methods |
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S (1250 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). [3H]DAMGO (68 Ci/mmol) was purchased from Amersham Biosciences, Inc. (Piscataway, NJ). DAMGO, naloxone hydrochloride, and GTPS were purchased from Sigma-Aldrich (St. Louis, MO). GDP was purchased from U.S. 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 day (PND) 5, 10, 15, 21, and 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 Institutional Animal Care and Use Committee.
Agonist-Stimulated [35S]GTPS Binding Assay. The [35S]GTPS assay was carried out as described previously (Sim et al., 1995; Happe et al., 2001). Briefly, tissue sections were rehydrated in assay buffer (50 mM glycylglycine, 3 mM MgCl2, 1 mM EGTA, and 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 2 mM GDP, 0.25 mM dithiothreitol, and 0.1 nM [35S]GTPS. Agonist-stimulated and basal [35S]GTPS binding were measured in the presence (stimulated) and absence of 3 µM DAMGO, a MOR-specific agonist. DAMGO-stimulated [35S]GTPS binding was also measured in the presence 5 µM naloxone to determine background. Nonspecific [35S]GTPS binding was determined in the absence of agonist and the presence of 1 µM unlabeled GTPS. All incubations were performed at room temperature (25°C).
Immediately after 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-
MAX (Amersham Biosciences, Inc.) for 1 day to 1 week and were quantified by densitometric analysis with commercial 14C standards (American Radiolabeled Chemicals, St. Louis, MO) that had been individually calibrated to 35S tissue standards (Miller and Zahniser, 1987). Data are expressed as femtomoles per milligram of tissue.
[3H]DAMGO Autoradiography. The µ opioid receptors were analyzed with [3H]DAMGO quantitative autoradiography using tissue sections adjacent to those used in the DAMGO-stimulated [35S]GTPS 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. Nonspecific binding was determined by the addition of 5 µM naloxone. After 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]GTPS binding assay except Hyperfilm-3H autoradiographic film (Amersham Biosciences, Inc.) 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, ON, 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]GTPS binding. Basal values of [35S]GTPS binding were subtracted from DAMGO-stimulated binding, as described previously (Happe et al., 2000).
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Results |
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S binding) during postnatal development. In the current study, 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, and 30, and adult were used for either DAMGO-stimulated [35S]GTPS 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]GTPS 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]GTPS 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]GTPS binding was much weaker at the early ages compared with 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.
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). However, no significant changes in [3H]DAMGO binding were observed after PND10.
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S 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, whereas basal (unstimulated) [35S]GTPS binding increased from 0.12 ± 0.01 to 0.71 ± 0.20 fmol/mg tissue (Fig. 3A). Specific DAMGO-stimulated [35S]GTPS 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]GTPS binding with incremental increases occurring at each age measured thereafter. These data were also analyzed as the ratio of specific DAMGO-stimulated [35S]GTPS 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 with 2.95 ± 0.46 in the adult, a 19-fold increase (Fig. 4).
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S binding levels were comparable in the rostral, medial, and caudal regions of the striatum at PND5 (0.13 ± 0.01, 0.19 ± 0.03, and 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]GTPS 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 with the 19-fold increase in rostral striosomes (Table 1). In addition, MOR coupling efficiency in the caudal region of the striatum peaked earlier in development (PND21) compared with striosomes of the rostral and medial regions.
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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]GTPS binding increased in the nucleus accumbens and the lateral septal nuclei during postnatal development, whereas 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. 5, A and B). DAMGO-stimulated [35S]GTPS 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]GTPS 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 was most clearly evident in areas where [3H]DAMGO binding changed very little during postnatal development but DAMGO-stimulated [35S]GTPS binding increased dramatically, such as in striosomes, nucleus accumbens, and the lateral septal nuclei.
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Discussion |
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). DAMGO labels the agonist high-affinity state of MORs, the state that 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 with 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 
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 3 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]GTPS 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, Childers and colleagues showed under conditions similar to ours that DAMGO-stimulated [35S]GTPS 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 5'-guanylylimidophosphate 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]GTPS binding in several regions of the embryonic mouse brain, including the striatum (Nitsche and Pintar, 2003). 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 development 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; Table 1) indicate that basal [35S]GTPS 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 and G proteins. MORs activate multiple members of the family of Gi/o proteins, including Gi1, Gi2, Gi3, Go1, and Go2 (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 Gi3 in SH-SY5Y cells as measured by -azidoanilido[32P]GTP affinity labeling (Laugwitz et al., 1993). In addition, in Chinese hamster ovary cells, MOR agonists stimulated incorporation of -azidoanilido[32P]GTP into Gi3 and Go2 with greater potency than Gi2, whereas the rank order of maximal labeling (efficacy) was Gi2 = Go2 > Gi3 (Chakrabarti et al., 1995). In addition, MORs fused to Gi1 stimulated [35S]GTPS binding more efficiently than MORs fused to Gi2 (Massotte et al., 2002). These data suggest that MORs exhibit preferential coupling to different G subunits. Therefore, it is possible that MOR signal transduction may be altered by the complement of G 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 subunits in specific anatomical areas. One study measuring G subunit expression in several regions of the rat brain, including hippocampus, thalamus, and the cortex, demonstrated that during postnatal development levels of Gi1, Gi2, and Go1 increase; Go2 remains unchanged; and Gi3 decreases (Ihnatovych et al., 2002). These data suggest that G subunit expression changes during development in a subunit-specific manner. Because MORs activate individual Gi/o 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 Gi/o subunit to the signal generated by MORs.
The contribution of pertussis toxin-insensitive G proteins, such as Gz, to the DAMGO-stimulated [35S]GTPS 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]GTPS 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 regulators of G protein signaling (RGS) proteins negatively regulate GPCR signaling by accelerating the GTPase activity of the G 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 seems 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 that roughly parallels the large increases in MOR-stimulated [35S]GTPS 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 GTPS.
The nonvisual 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 (-arrestin 1) increases throughout post-natal development, whereas arrestin 3 (-arrestin 2) decreases slightly (Gurevich et al., 2002). These data suggest that the influence of arrestin 2 on MOR signaling, from a purely 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 using yeast two-hybrid methodology describe proteins that functionally interact with MOR, including phospholipase D2 (Koch et al., 2003), filamin A (Onoprishvili et al., 2003), and 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 seems 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 versus 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 |
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ABBREVIATIONS: MOR, µ opioid receptor; [35S]GTPS, 5'-O-(3-[35S]thio)triphosphate; CNS, central nervous system; DAMGO, D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; PND, postnatal day; Ad, adult; RGS, regulators of G protein signaling; GPCR, G protein-coupled receptor.
1 Current address: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI. 
2 Current address: Department of Psychiatry, Creighton University School of Medicine, Omaha, NE.
Address correspondence to: Dr. L. Charles Murrin, 985800 Nebraska Medical Center, Omaha, NE 68198-5800. E-mail: cmurrin{at}unmc.edu
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