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Biomedical subjects

D E Selley

Publications and source records attributed to D E Selley.

At least 19 recordsLinked to original sources
PubMed2001

Inhibitory effects of SR141716A on G-protein activation in rat brain.

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), a cannabinoid CB(1) receptor antagonist, has inverse agonist effects in cannabinoid CB(1) receptor-expressing cell lines, brain and peripheral organs. These studies characterized SR141716A-inhibited G-protein activity by measuring [35S]GTPgammaS binding. Maximal inhibition of basal [35S]GTPgammaS binding in cerebellar membranes was 50%. The EC(50) value for inhibition of [35S]GTPgammaS binding was 4.4 microM, whereas the K(e) for inhibition of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2)-stimulated [35S]GTPgammaS binding was 0.6 nM. [35S]GTPgammaS autoradiography was used to examine the regional specificity of SR141716A inhibition. SR141716A inhibited basal [35S]GTPgammaS binding in all regions examined, with inhibition ranging from approximately 20% in caudate-putamen to 40% in hippocampus. These studies demonstrate that SR141716A is a competitive antagonist at nanomolar concentrations, whereas it inhibits basal receptor-mediated G-protein activity at micromolar concentrations. These data suggest that the apparent inverse agonist effect is either not cannabinoid CB(1) receptor-specific or that SR141716A is binding to different sites on the cannabinoid CB(1) receptor to produce inverse agonist versus competitive antagonist effects.

Animals↗
PubMed2000

Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain.

In previous studies from our laboratory, chronic noncontingent morphine administration decreased mu opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, mu opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg. kg(-1). d(-1). mu opioid-stimulated [(35)S]GTPgammaS and [(3)H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [(35)S]GTPgammaS autoradiography also examined other G-protein-coupled receptors, including delta opioid, ORL-1, GABA(B), adenosine A(1), cannabinoid, and 5-HT(1A). In brains from heroin self-administering rats, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in mu-activated G-proteins, [(3)H]naloxone binding demonstrated increased mu opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in mu receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and mu receptor binding. No effects on agonist-stimulated [(35)S]GTPgammaS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease mu-stimulated [(35)S]GTPgammaS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.

Animals↗
PubMed2000

Relationship of mu opioid receptor binding to activation of G-proteins in specific rat brain regions.

This study investigated the relationship between mu receptor binding and mu agonist activation of G-proteins in the rat brain. To directly compare agonist potencies in receptor binding (K(i) values) and G-protein activation (K(s) values), both agonist-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) and [(3)H]naloxone binding assays were conducted under identical conditions, using the full mu agonist [d-Ala(2), N-Me(4), Gly(5)-ol]-enkephalin (DAMGO). DAMGO exhibited biphasic competition of [(3)H]naloxone binding and stimulation of [(35)S]GTPgammaS binding in most regions. Whereas the high-affinity component represented a large percentage (50-80%) of total receptor sites, the high-affinity component of DAMGO-stimulated [(35)S]GTPgammaS binding was much lower, <30% of the total, and in most regions significant stimulation of [(35)S]GTPgammaS binding did not occur until the high-affinity binding sites were completely occupied. Moreover, the low-affinity potencies for DAMGO in receptor binding and G-protein activation were the same across different regions. Receptor-transducer amplification factors were calculated by the ratio of the apparent B(max) of net agonist-stimulated [(35)S]GTPgammaS binding to the B(max) of receptor binding. Amplification factors for the nine regions examined were relatively high and varied significantly across regions, from a ratio of 8 in the thalamus to 38 in the cortex, suggesting that the efficiency of mu opioid receptor coupling to G-proteins varies across brain regions.

Analgesics, Opioid↗
PubMed2000

Region-specific changes in 5-HT(1A) receptor-activated G-proteins in rat brain following chronic buspirone.

5-Hydroxytryptamine(1A) (5-HT(1A)) receptors, which activate inhibitory G-proteins, are implicated in psychiatric disorders including anxiety and depression. Studies suggest that chronic 5-HT(1A) receptor agonist administration alters 5-HT(1A) receptor function, but the effect of chronic treatment on 5-HT(1A) receptor-activated G-proteins is unclear. In this study, agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding was examined following chronic administration of buspirone. Brains were processed for [35S]GTPgammaS autoradiography using R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) for 5-HT(1A) receptors or baclofen for GABA(B) receptors. Net 8-OH-DPAT-stimulated [35S]GTPgammaS binding was decreased by 25-30% in the septum and dorsal raphe nucleus of buspirone-treated animals. No significant changes in 8-OH-DPAT-stimulated [35S]GTPgammaS binding were found in the prefrontal, entorhinal or cingulate cortices or hippocampus in buspirone-treated rats. GABA(B) receptor-stimulated [35S]GTPgammaS binding was increased by 25% in the hippocampus, with no significant changes in any other region examined. These results demonstrate region-specific alterations in 5-HT(1A) and GABA(B) receptor-activated G-proteins following chronic buspirone treatment, which may contribute to the clinical effects of this drug.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2000

Effects of sodium on agonist efficacy for G-protein activation in mu-opioid receptor-transfected CHO cells and rat thalamus.

1. Sodium ions inhibit spontaneous G(i)/G(o)-coupled receptor activity and promote agonist-induced responses in vitro. The effects of sodium on the relative efficacy of opioid agonists for G-protein activation was measured by guanosine-5'-O-(gamma-(35)S)-triphosphate ([(35)S]-GTPgammaS) binding in membranes from two mu-opioid receptor-containing systems: CHO cells stably transfected with mouse mureceptors (mMOR-CHO cells) and rat thalamus. 2. NaCl inhibited basal [(35)S]-GTPgammaS binding in both systems, and this effect was partially mimicked by KCl. In mMOR-CHO membranes, net [(35)S]-GTPgammaS binding stimulated by partial but not full agonists was inhibited by NaCl with a potency that was inversely proportional to agonist efficacy. Monovalent cations were required for agonist-stimulated [(35)S]-GTPgammaS binding in this system, and increasing NaCl concentrations magnified relative efficacy differences among agonists. 3. In thalamic membranes, which contain a lower receptor:G-protein ratio than mMOR-CHO cells, similar monovalent cation effects were observed, with two exceptions: (1) [(35)S]-GTPgammaS binding stimulated by both full and partial agonists was inhibited by NaCl; and (2) monovalent cations were not required to observe agonist-stimulated [(35)S]-GTPgammaS binding. 4. Basal [(35)S]-GTPgammaS binding stimulated by the absence of monovalent cations resembled that of agonist-stimulated binding and was blocked by pretreatment of mMOR-CHO cells with pertussis toxin. 5. These results indicate that sodium inhibits spontaneous and agonist-occupied mu receptor-mediated G-protein activation in a manner inversely proportional to the efficacy of the agonist, and that spontaneous mu receptor activity and the relative efficacy of partial agonists acting at these receptors are both increased by increases in the stoichiometric ratio of receptors:G-proteins.

Animals↗
PubMed1998

Cannabinoid receptor agonist efficacy for stimulating [35S]GTPgammaS binding to rat cerebellar membranes correlates with agonist-induced decreases in GDP affinity.

The relationship between GDP and cannabinoid-stimulated [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding was investigated in rat cerebellar membranes. Kinetic analyses showed that [35S]GTPgammaS binding reached steady-state levels and that the association rate was increased by the agonist WIN 55212-2 proportional to the concentration of GDP. Dissociation of [35S]GTPgammaS occurred with two rates (t1/2 = 7 and 170 min), and WIN 55212-2 increased the proportion of sites exhibiting the faster rate. Without GDP, [35S]GTPgammaS bound to membranes with high and low affinity, and WIN 55212-2 had no effect. With 30 microM GDP, [35S]GTPgammaS bound to low and intermediate affinity sites, and WIN 55212-2 induced high affinity [35S]GTPgammaS binding without affecting low affinity sites. GDP competed for high affinity [35S]GTPgammaS binding with high and intermediate affinity in the absence of WIN 55212-2 and with high and low affinity in the presence of WIN 55212-2. Cannabinoid ligands displayed differential abilities to maximally stimulate [35S]GTPgammaS binding in the presence of GDP. Efficacy differences among ligands increased with increasing GDP concentrations. GDP competition curves revealed that agonists induced low affinity GDP Ki values that were proportional to agonist Emax values, indicating that agonist efficacy is determined by displacement of GDP from G-proteins.

Animals↗
PubMed1998

mu-Opioid agonist-stimulated [35S]GTPgammaS binding in guinea pig hypothalamus: effects of estrogen.

mu-Opioid receptors play a critical role in the regulation of the female reproductive cycle, and estrogen modulates the coupling of mu-opioid receptors to a potassium channel in the basal hypothalamus (BH) of the female guinea pig. Therefore, we ascertained the distribution of mu-opioid receptors in the BH with autoradiography using the mu-opioid selective agonist [3H]DAMGO. In addition, we investigated the effects of estrogen on DAMGO- or the GABAB receptor agonist baclofen-stimulated [35S]GTPgammaS binding in the BH. Based on the high density of mu-opioid receptors, but the lack of effects of estrogen on [35S]GTPgammaS binding, we conclude that mu-opioid receptor interaction with its G-protein is not the target of estrogen's actions.

Animals↗
PubMed1998

Endomorphin-stimulated [35S]GTPgammaS binding in rat brain: evidence for partial agonist activity at mu-opioid receptors.

Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at mu-opioid receptors. In the present study, the effect of endomorphin-1 on mu receptor-coupled G proteins was compared with that of the mu agonist DAMGO by using agonist-stimulated [35S]GTPgammaS binding in rat brain. [35S]GTPgammaS autoradiography revealed a similar localization of endomorphin-1- and DAMGO-stimulated [35S]GTPgammaS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [35S]GTPgammaS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [35S]GTPgammaS binding. Differences in maximal stimulation of [35S]GTPgammaS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [35S]GTPgammaS binding revealed a lower apparent Bmax value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [35S]GTPgammaS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the mu-opioid receptor in brain.

Animals↗
PubMed1998

Opioid inhibition of adenylyl cyclase in membranes from pertussis toxin-treated NG108-15 cells.

Gi/Go proteins are uncoupled from receptors by ADP-ribosylation with pertussis toxin (PTX). However, PTX treatment of delta opioid receptor-containing NG108-15 cells reduces, but does not eliminate, opioid inhibition of adenylyl cyclase. The present study explored potential mechanisms of this residual inhibition. Overnight treatment of NG108-15 cells with 100 ng/ml PTX eliminated both PTX-catalyzed [adenylyl-32P]NAD+-labeling of G proteins and agonist stimulation of low Km GTPase in membranes. Although PTX-treatment decreased the maximal opioid inhibition of adenylyl cyclase by 50-65%, the inhibition that remained was concentration-dependent and antagonist-reversible. This inhibition persisted in the absence of GTP (even though opioid inhibition of adenylyl cyclase in untreated membranes was GTP-dependent), but was eliminated by hydrolysis-resistant guanine nucleotide analogs, indicating that G-proteins were still involved in the coupling mechanism. However, assays of agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP indicated that PTX pretreatment eliminated stimulation of guanine nucleotide exchange by opioid agonists. These results suggest that in membranes from PTX-treated NG108-15 cells, a subpopulation of G proteins may transduce an inhibitory signal from agonist-bound opioid receptors without involvement of guanine nucleotide exchange.

Adenosine Diphosphate Ribose↗
PubMed1998

Signal transduction correlates of mu opioid agonist intrinsic efficacy: receptor-stimulated [35S]GTP gamma S binding in mMOR-CHO cells and rat thalamus.

This study examined the signal transduction correlates of mu opioid agonist efficacy in two systems: mu receptor-transfected mMOR-CHO cell and rat thalamic membranes. The potency and maximal stimulation of [35S]GTP gamma S binding by various agonists was measured in the presence of excess GDP and compared with receptor binding affinity under identical assay conditions. Results showed that the relative maximal stimulation produced by these agonists was greater in mMOR-CHO cell than in rat thalamic membranes; some drugs that were full agonists in mMOR-CHO cells were partial agonists in the thalamus, and some partial agonists in the transfected cells were full antagonists in the thalamus. Furthermore, there was receptor reserve for G-protein activation by some agonists in mMOR-CHO cell membranes, but no receptor reserve was detected in rat thalamic membranes. Saturation analysis of agonist-stimulated [35S]GTP gamma S binding revealed that full agonists produced both a higher Bmax and apparent affinity of [35S]GTP gamma S binding than partial agonists. Correlation of the Bmax and KD of agonist-stimulated [35S]GTP gamma S binding with agonist intrinsic efficacy revealed only a moderate correlation with either parameter alone, but a highly significant correlation (r > 0.9) with a combination of the two parameters (Bmax/KD). These results suggest that the intrinsic efficacy of agonists at G-protein-coupled receptors is determined primarily by the ability of the agonist-occupied receptor to promote high-affinity GTP binding to the G-protein and to catalytically activate a maximal number G-proteins.

Animals↗
PubMed1997

Opioid receptor-coupled G-proteins in rat locus coeruleus membranes: decrease in activity after chronic morphine treatment.

The nucleus locus coeruleus is involved in the expression of opiate physical dependence and withdrawal, and has been characterized extensively with regard to chronic morphine-induced alterations in biochemical and electrophysiological responses. In the present study the effects of chronic morphine treatment on opioid receptor-coupled G-protein activity was investigated in membranes from rat locus coeruleus. Opioid agonists stimulated low Km GTPase activity with pharmacology consistent with mu receptors. Chronic morphine treatment resulted in decreases in both basal and opioid-stimulated low Km GTPase activity, with no change in the percent stimulation by agonist. The decrease in low Km GTPase activity appeared to be due to a decrease in the Vmax of the enzyme, with no change in the Km for GTP hydrolysis. These results were confirmed by assays of basal and opioid receptor-stimulated [35S]GTP gamma S binding in the presence of excess GDP. Thus, chronic morphine treatment apparently decreased inhibitory G-protein activity in the locus coeruleus without producing any detectable desensitization. These results suggest a potential adaptation at the receptor/transducer level which may contribute to other biochemical changes produced in the locus coeruleus by chronic morphine treatment.

Analgesics↗
PubMed1997

Effects of intracerebroventricular administration of beta-funaltrexamine on DAMGO-stimulated [35S]GTP-gamma-S binding in rat brain sections.

Intracerebroventricular administration of beta-funaltrexamine (beta-FNA) reduces the density of mu opioid receptors as measured by in situ autoradiography by 40-50% throughout the brain, with little regional variation [Martin et al. (1993) J. Pharmacol. Exp. Ther. 267:506-514] Recently an assay has been developed to study opioid stimulation of [35S]GTP-gamma-S binding autoradiographically in situ using slide-mounted brain sections [Sim et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:7242-7246]. The present study was undertaken to determine the effect of mu opioid receptor alkylation on G protein activation by the mu opioid agonist DAMGO. Animals were injected intracerebroventricularly with 40 nmol of beta-FNA or saline and sacrificed 24 hours later. DAMGO stimulated [35S]GTP-gamma-S binding with an anatomical specificity consistent with the localization of mu opioid receptors. The percent stimulation by DAMGO ranged from approximately 50 to 100% in the regions studied. beta-FNA significantly decreased G protein activation by DAMGO in regions that are consistent with its reported long-lasting and insurmountable antagonism of the antinociceptive (medial thalamus, central gray) and reinforcing (nucleus accumbens) effects of mu opioid agonists [Adams et al. (1990) J. Pharmacol. Exp. Ther. 255:1027-1032; Martin et al. (1995) J. Pharmacol. Exp. Ther. 272:1135-1140]. However, the effects of beta-FNA were not equal in all brain regions. This may indicate regional differences in the coupling efficiency of mu opioid receptors with G proteins, or in the effects of beta-FNA on mu opioid receptor binding or on mu opioid receptor-stimulated G protein activity.

Animals↗
PubMed1997

Acute and chronic effects of opioids on delta and mu receptor activation of G proteins in NG108-15 and SK-N-SH cell membranes.

To compare activation of G proteins by opioid receptors, opioid agonist-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding in the presence of excess GDP was assayed in membranes from NG108-15 (delta) and SK-N-SH (primarily mu) cells. Basal [35S]GTP gamma S binding consisted of a single class of low-affinity sites (KD 400-500 nM). Addition of agonists produced a high-affinity site 100-300-fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10-fold higher for SK-N-SH mu receptors than for NG108-15 delta receptors. Chronic delta agonist ([D-Ser2]-Leu-enkephalin-Thr; DSLET) treatment of NG108-15 cells resulted in an 80% loss of DSLET-stimulated [35S]-GTP gamma S binding within 1 h. Morphine treatment of SK-N-SH cells decreased mu agonist ([D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; DAMGO)-stimulated [35S]GTP gamma S binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.

Analgesics↗
PubMed1997

mu-Opioid receptor-stimulated guanosine-5'-O-(gamma-thio)-triphosphate binding in rat thalamus and cultured cell lines: signal transduction mechanisms underlying agonist efficacy.

G protein activation by different mu-selective opioid agonists was examined in rat thalamus, SK-N-SH cells, and mu-opioid receptor-transfected mMOR-CHO cells using agonist-stimulated guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTP gamma S) binding to membranes in the presence of excess GDP. [D-Ala2, N-MePhe4, Gly5-ol]Enkephalin (DAMGO) was the most efficacious agonist in rat thalamus and SK-N-SH cells, followed by (in rank order) fentanyl = morphine > > buprenorphine. In mMOR-CHO cells expressing a high density of mu receptors, no differences were observed among DAMGO, morphine or fentanyl, but these agonists were more efficacious than buprenorphine, which was more efficacious than levallorphan. In all three systems, efficacy differences were magnified by increasing GDP concentrations, indicating that the activity state of G proteins can affect agonist efficacy. Scatchard analysis of net agon stimulated [35S]GTP gamma S binding revealed two major components responsible for agonist efficacy differences. First, differences in the KD values of agonist-stimulated [35S]GTP gamma S binding between high efficacy agonists (DAMGO, fentanyl, and morphine) and classic partial agonists (buprenorphine and levallorphan) were observed in all three systems. Second, differences in the Bmax value of agonist-stimulated [35S]GTP gamma S binding were observed between DAMGO and morphine or fentanyl in rat thalamus and SK-N-SH cells and between the high efficacy agonists and buprenorphine or levallorphan in all three systems. These results suggest that mu-opioid agonist efficacy is determined by the magnitude of the receptor-mediated affinity shift in the binding of GTP (or[35S]GTP gamma S) versus GDP to the G protein and by the number of G proteins activated per occupied receptor.

Animals↗
PubMed1996

Differences in G-protein activation by mu- and delta-opioid, and cannabinoid, receptors in rat striatum.

Receptor activation of G-proteins can be measured by agonist-stimulated [35S]GTP gamma S binding in the presence of excess guanosine diphosphate (GDP). To determine whether opioid and cannabinoid receptor-mediated G-protein activation correlate with their receptor densities, this study compared opioid- and cannabinoid-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTP gamma S) binding with the corresponding Bmax values of receptor binding in rat striatum. Scatchard analysis revealed that the Bmax of cannabinoid receptor binding was approximately ten times higher than that of mu- or delta-opioid receptor binding. However, comparable levels of cannabinoid- and mu- and delta-opioid-stimulated [35S]GTP gamma S binding were observed in the caudate-putamen by [35S]GTP gamma S autoradiography in brain sections. Scatchard analysis of net agonist-stimulated [35S]GTP gamma S binding in membranes showed that the Bmax of cannabinoid-stimulated binding was only twice that of mu- or delta-opioid-stimulated binding. Thus, the calculated amplification factors for mu- and delta-opioid receptors are seven times that of cannabinoid receptors.

Animals↗
PubMed1996

Effects of chronic morphine administration on mu opioid receptor-stimulated [35S]GTPgammaS autoradiography in rat brain.

Chronic opiate administration results in the development of tolerance and dependence, but the regulation of mu opioid receptor function during this process is not clearly understood. To localize changes in mu opioid receptor-coupled G-protein activity in various brain regions after chronic morphine treatment, the present study examined mu opioid agonist-stimulated [35S]GTPgammaS binding to brain sections by in vitro autoradiography. Rats were treated for 12 d with increasing doses (10-320mg . kg-1 . d-1) of morphine. Control rats were injected with either saline or a single acute injection of morphine (20 mg/kg). mu opioid-stimulated [35S]GTPgammaS binding was measured by autoradiography of brain sections in the presence and absence of the mu opioid-selective agonist DAMGO. In rats injected with a single acute dose of morphine, no significant changes were detected in basal or agonist-stimulated [35S]GTPgammaS binding in any region. In sections from chronic morphine-treated rats, however, DAMGO-stimulated [35S]GTPgammaS binding was reduced significantly compared with control rats in the following brain-stem nuclei: dorsal raphe nucleus, locus coeruleus, lateral and medial parabrachial nuclei, and commissural nucleus tractus solitarius. No significant changes were observed in several other brain regions, including the nucleus accumbens, amygdala, thalamus, and substantia nigra. These data indicate that chronic morphine administration results in reductions in mu opioid activation of G-proteins in specific brainstem nuclei involved in physiological homeostasis and autonomic function, which may have implications in the development of opiate tolerance and physical dependence.

Animals↗
PubMed1996

Cannabinoid receptor stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding in rat brain membranes.

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [35S]GTP gamma S binding in rat brain membranes. Assays were conducted with 0.05 nM [35S]GTP gamma S, incubated with rat cerebellar membranes, 1-30 microM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [35S]GTP gamma S binding increased with increasing concentrations of GDP, with 10-30 microM GDP providing approximately 150-200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [35S]GTP gamma S binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [35S]GTP gamma S binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [35S]GTP gamma S binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [35S]GTP gamma S autoradiography. These results demonstrate that [35S]GTP gamma S binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.

Animals↗