G protein activation by different μ-selective opioid agonists was examined in rat thalamus, SK-N-SH cells, and μ-opioid receptor-transfected
mMOR-CHO cells using agonist-stimulated guanosine-5â²- ...O -(γ-thio)-triphosphate ( 35 SGTPγS) binding to membranes in the presence of excess GDP. d -Ala 2 , N -MePhe 4 ,Gly 5 -olEnkephalin (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 μ 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 agonist-stimulated 35 SGTPγS binding revealed two major components responsible for agonist efficacy differences. First, differences in the K D values of agonist-stimulated 35 SGTPγS 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 B max value of agonist-stimulated 35 SGTPγS 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 μ-opioid agonist efficacy
is determined by the magnitude of the receptor-mediated affinity shift in the binding of GTP (or 35 SGTPγS) versus GDP to the G protein and by the number of G proteins activated per occupied receptor.
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.
Abstract
Gi/Go proteins are uncoupled from receptors by ADP-ribosylation with pertussis toxin (PTX). However, PTX treatment of δ 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-32PNAD+-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 35SGTPγS 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.
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.
The binding of 125Ibeta 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, ...3HD-Ala2,MePhe4,Glyol5enkephalin, and the delta-selective opioid agonist, 3HD-penicillamine2, D-penicillamine5enkephalin, 125Ibeta 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 125Ibeta h-endorphin binding. This inhibition was significantly greater than the decrease in 125Ibeta h-endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited 125Ibeta h-endorphin binding in the presence of sodium, inhibition of 125Ibeta 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 125Ibeta h-endorphin, the binding of the putative sigma receptor agonist, (+)-3HSKF 10,047, was not significantly altered by GTP or guanylyl-5'-imidodiphosphate in the absence or presence of sodium.
Neurotransmitter receptors contain two main functional components: a ligand binding domain, which specifically recognizes the neurotransmitter, and a signaling component, which translates the binding ...of the neurotransmitter (or its agonists) into a physiological response. Radioligand binding assays represented a breakthrough in understanding the properties of these receptors in brain at the level of the ligand binding site, and the development of techniques like in vitro autoradiography of receptor binding allowed high-resolution analysis of the neuroanatomical localization of these receptors. However, these methodologies provide no information regarding the signal transduction component of these receptors, and in particular, cannot provide a true picture of the biological activity of receptors and of the efficacy of neurotransmitters and their agonists to produce a biological response. Even more recent methodological developments, like in situ hybridization of receptor mRNA, provide excellent localization of receptor gene expression, but also give little information about receptor coupling to intracellular signaling mechanisms. In order to address this question, techniques must be developed to allow for in vitro localization of receptor-mediated signal transduction.
