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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
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↗
PubMed1995

In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding.

Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors.

Animals↗
PubMed1994

Calcium and cAMP mediated stimulation of Fos in cultured hypothalamic tyrosine hydroxylase-immunoreactive neurons.

Immediate-early genes, such as c-fos, couple extracellular signals to genetic changes in the cell. We have previously demonstrated that depolarization with 50 mM KCl increases Fos immunoreactivity in hypothalamic tyrosine hydroxylase (TH) and oxytocin immunoreactive (-ir) neurons in primary culture. This Fos activation occurs within 1.5-2 h in TH-ir cells. In the present study, we examined the effects of depolarization, glutamate receptor activation and adenylyl cyclase stimulation on Fos-ir to determine the possible mechanism(s) of Fos activation in TH-ir neurons. Hypothalamic cultures were treated with KCl, glutamate or forskolin, and Fos and TH were visualized immunocytochemically. Forskolin increased the percentage of Fos/TH-ir neurons in a dose-dependent manner, with a maximal stimulation of 53.4 +/- 4.5% Fos/TH-ir neurons at 30 microM forskolin. The dose-response curve for glutamate was steep, with a maximal stimulation of 24.8 +/- 2.1% Fos-ir neurons at 100 microM. 50 mM KCl resulted in 50.0 +/- 0.8% Fos/TH-ir neurons. Pretreatment with verapamil decreased KCl induced Fos-ir by 57%, glutamate by 65% and forskolin by 39%. Combined drug administration demonstrated significant additivity between forskolin and glutamate, and forskolin and KCl, however, no significant additivity was found with KCl and glutamate. The results are discussed in terms of cAMP and calcium mediation of the Fos response to these stimuli.

Animals↗
PubMed1994

D2 inhibition of stimulated Fos immunoreactivity in cultured tyrosine hydroxylase-ir hypothalamic neurons.

We have previously demonstrated that Fos immunoreactivity can be stimulated by KCl, forskolin or glutamate in cultured tyrosine hydroxylase-immunoreactive (TH-ir) hypothalamic neurons. The present study was performed to determine whether agents that regulate dopaminergic activity, particularly D1 and D2 receptor agonists, modulate the intracellular cascade leading to Fos expression. Dissociated hypothalamic cultures were prepared from neonatal rats. The cultures were treated with D1- or D2-specific agonists, followed by KCl, forskolin or glutamate. Cultures were fixed after 2 h and immunocytochemically stained for tyrosine hydroxylase and Fos. Pretreatment of the cultures with the D2 agonist LY163502 inhibited KCl- and forskolin-stimulated Fos-ir in TH-ir neurons in a saturable dose-dependent manner. The maximal effective dose was 30 microM LY163502, which decreased Fos-ir by 23% in cultures treated with 50 mM KCl and by 33% in those treated with 30 microM forskolin. The D2 agonist had no effect on glutamate-stimulated Fos-ir. LY163502 inhibition of Fos-ir was blocked by D2 antagonist or Bordetella pertussis toxin pretreatment which demonstrates that the effect is mediated by D2 receptor activation of an inhibitory G protein. Treatment of the cultures with the D1 agonist SKF82526 had no effect on basal or stimulated levels of Fos-ir. These results demonstrate that in neonatal TH-ir hypothalamic neurons the D2 receptor system may regulate levels of the immediate-early gene product Fos and, therefore, subsequent genetic expression in these neurons.

Animals↗
PubMed1993

Modification of G protein-coupled functions by low-pH pretreatment of membranes from NG108-15 cells: increase in opioid agonist efficacy by decreased inactivation of G proteins.

Low-pH pretreatment increases opioid agonist efficacy in inhibiting adenylyl cyclase in brain membranes. The mechanism of this effect was examined in membranes from cultured NG108-15 cells. Pretreatment of NG108-15 membranes at pH 4.5 before assay at pH 7.4 produced the following modifications in G protein-mediated signal transduction: 1) decreased activation of adenylyl cyclase by Gs, 2) increased maximal inhibition of opioid agonist binding by sodium and by guanine nucleotides in the presence of sodium, and 3) increased maximal inhibition of adenylyl cyclase by agonists for G(i)-coupled receptors. These results are similar to those previously observed in rat brain membranes. The mechanism by which low-pH pretreatment increased receptor-mediated inhibition of adenylyl cyclase was investigated further by examining low-Km GTPase activity in low-pH-pretreated NG108-15 cell membranes. Low-pH pretreatment decreased basal and agonist-stimulated low-Km GTPase activity maximally in the absence of sodium and minimally in the presence of 120 mM NaCl. This change was due to a decrease in the Vmax of the enzyme, with no change in the Km for GTP, indicating that GTP hydrolysis was decreased without any decrease in the affinity of the G protein for GTP. Scatchard analysis revealed no decrease in the Bmax for high affinity opioid agonist binding, and Western blot analysis with a G(i)-specific antibody revealed no loss of G(i) protein, in low-pH-pretreated membranes. Moreover, concentration-effect curves for GTP in supporting opioid inhibition of adenylyl cyclase showed that low-pH pretreatment increased inhibition by the agonist only at GTP concentrations equal to or greater than the Km for GTP hydrolysis by the low-Km GTPase. Taken together, these results indicate that the efficacy of receptor-mediated inhibition of adenylyl cyclase can be increased by decreasing the maximal inactivation rate of G(i) subsequent to its activation by the receptor.

Adenylyl Cyclase Inhibitors↗
PubMed1993

BW373U86: a nonpeptidic delta-opioid agonist with novel receptor-G protein-mediated actions in rat brain membranes and neuroblastoma cells.

BW373U86 is a potent and highly selective nonpeptidic agonist for delta-opioid receptors. To determine its ability to couple with G protein-linked second messenger systems, this study examined the effects of BW373U86 on the inhibition of adenylyl cyclase and the stimulation of low-Km GTPase activity. In rat striatal membranes, BW373U86 inhibited basal adenylyl cyclase activity in a GTP-dependent manner, with maximal inhibition levels similar to those of the prototypic delta agonist [D-Ser2,Thr6]Leu-enkephalin (DSLET). However, BW373U86 was approximately 100 times more potent than DSLET in inhibiting adenylyl cyclase. Analysis of the inhibitory activity across 10 brain regions revealed that both low and high concentrations of BW373U86 inhibited adenylyl cyclase activity in a manner similar to that of DSLET. Inhibition of adenylyl cyclase by BW373U86 was delta receptor selective, because the delta receptor-selective antagonist naltrindole was significantly more potent than naloxone and the mu receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 was ineffective in blocking BW373U86 inhibition. BW373U86 also inhibited adenylyl cyclase activity in membranes prepared from NG108-15 cells, with an IC50 value 5 times lower than that of DSLET. This increased potency was not observed in concentration-effect curves for agonist-stimulated low-Km GTPase in NG108-15 membranes. BW373U86 is a competitive inhibitor of [3H]diprenorphine at delta receptors of NG108-15 cell membranes. However, unlike DSLET, BW373U86 displacement of [3H]diprenorphine binding to NG108-15 cell membranes was not affected by sodium and guanine nucleotides. This lack of GTP effect on binding apparently produced slow dissociation rates for this agonist, because naltrindole was less potent in blocking BW373U86 inhibition of adenylyl cyclase when membranes were preincubated with this agonist. These results demonstrate the novel finding that the binding of a full agonist to a G protein-coupled receptor is not regulated by GTP, and they also show how the lack of regulation in receptor binding affects agonist potency.

Adenylyl Cyclase Inhibitors↗