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D E Selley

Publications and source records attributed to D E Selley.

29 records · Page 2Linked to original sources
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↗
PubMed1992

Effects of beta-endorphin on mu and delta opioid receptor-coupled G-protein activity: low-Km GTPase studies.

Human beta-endorphin 1-31 (beta-END) stimulated low-Km GTPase activity in a concentration-dependent and saturable manner in membranes prepared from the delta opioid receptor-containing hybrid cell line NG108-15 and from the mu opioid receptor-enriched human neuroblastoma cell line SK-N-SH. Naloxone and the delta-selective antagonist, ICI 174,864, blocked the stimulation of the GTPase activity produced by beta-END in NG108-15 cell membranes, whereas only naloxone inhibited the beta-END-induced stimulation in SK-N-SH cell membranes, suggesting that beta-END was acting through both mu and delta opioid receptors. Treatment of the cells with Bordetella pertussis toxin before the preparation of membranes blocked the stimulation of low-Km GTPase by beta-END in both cell lines. Activation of NG108-15 and SK-N-SH low-Km GTPase by beta-END was sodium-dependent, and lithium and potassium were poor promoters of this activation. These results demonstrate that beta-END stimulates the interaction of both mu and delta opioid receptors with B. pertussis toxin-sensitive G-proteins in SK-N-SH and NG108-15 cell membranes, respectively.

Cell Membrane↗
PubMed1989

Guanine nucleotide regulation of [125I]beta-endorphin binding to NG108-15 and SK-N-SH cell membranes: specific cation requirements.

Regulation of [125I]beta h-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only delta opioid receptors, and SK-N-SH, which contains predominantly mu opioid receptors. In contrast to the binding of the delta-selective agonist [3H][D-penicillamine2,D-penicillamine5]enkephalin to NG108-15 cell membranes, and of the mu-selective agonist [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin to SK-N-SH cell membranes, [125I]beta h-endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5'-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCl, [125I]beta h-endorphin binding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]beta h-endorphin binding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or manganese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]beta h-endorphin binding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [125I]beta h-endorphin binding by guanine nucleotides, and that the specificity of this cation requirement differs between the mu and delta receptor-containing cell lines.

Cations↗
PubMed1988

Guanine nucleotide regulation of [125I]beta-endorphin binding to rat brain membranes: monovalent cation requirement.

The binding of [125I]beta h-endorphin to rat brain membranes was investigated in the presence of GTP and guanylyl-5'-imidodiphosphate. In contrast to the binding of the mu-selective opioid agonist, [3H][D-Ala2,MePhe4,Glyol5]enkephalin, and the delta-selective opioid agonist, [3H][D-penicillamine2, D-penicillamine5]enkephalin, [125I]beta h-endorphin binding was not affected by GTP or guanylyl-5'-imidodiphosphate in a concentration-dependent manner in the absence of cations. However, in the presence of NaCl, the inclusion of either GTP or guanylyl-5'-imidodiphosphate resulted in a concentration-dependent inhibition of [125I]beta h-endorphin binding. This inhibition was significantly greater than the decrease in [125I]beta h-endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited [125I]beta h-endorphin binding in the presence of sodium, inhibition of [125I]beta h-endorphin binding by GTP was also observed in the presence of the monovalent cations lithium and potassium, but not the divalent cations magnesium, calcium, or manganese. The effect produced by GTP in the presence of NaCl was mimicked by GDP, but not by GMP or other nucleotides. Unlike [125I]beta h-endorphin, the binding of the putative sigma receptor agonist, (+)-[3H]SKF 10,047, was not significantly altered by GTP or guanylyl-5'-imidodiphosphate in the absence or presence of sodium.

Animals↗