PubMed · 6328917
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M Rodbell. 1984. Structure-function problems with the adenylate cyclase system.. https://pubmed.ncbi.nlm.nih.gov/6328917/
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Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the kappa-opioid receptor, displays very low potency in competing with binding of [3H]diprenorphine to or inhibiting adenylate cyclase via mu-, delta- and kappa-opioid receptors. Tritium-labeled nociceptin ([3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL 1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL 1 receptor, equilibrium binding of [3H]nociceptin is highly specific, saturable (Bmax in the range 1.3-1.8 pmol/mg protein) and of high affinity (Kd approximately equal to 0.1 nM). It is selectively decreased in the presence of Na+ ions and/or of the GTP analog 5'-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14-endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [3H]nociceptin at the ORL 1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL 1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.
5-HT activates the peristaltic reflex and is the neurotransmitter of a subset of myenteric interneurons. Hyperpolarizing afterpotential (AH)/type 2 neurons respond to 5-HT with a long-lived depolarization that is caused by the inhibition of a Ca(2+)-activated K+ conductance (gKCa). This effect is mediated by a G-protein-coupled receptor, 5-HT1P. 5-HT1P agonists specifically activate G alpha o, the immunoreactivity of which was found to be highly abundant and membrane-associated in almost all enteric neurons. Responses of hyperpolarizing AH/type 2 neurons to 5-HT were inhibited by intracellular injection of GDP beta S or anti-G alpha o Fab fragments but were potentiated and prolonged by intracellular GTP gamma S. Responses to 5-HT were antagonized by pertussis toxin, downregulation of protein kinase C (PKC) and inhibitors of phosphatidylcholine phospholipase C (PC-PLC), PKC (including pseudosubstrate peptides, chelerythrine, and the alpha/beta isoform-specific inhibitor Go 6976), protein kinase A (PKA), and adenylate cyclase. Responses to 5-HT were mimicked by activators of PKC, and 5-HT induced a concentration-dependent increase in the membrane-associated PKC activity in isolated myenteric ganglia. Immunocytochemical studies suggested that the most abundant isoforms of PKC in enteric neurons are alpha and delta. These data suggest that signal transduction of the 5-HT1P-mediated slow response to 5-HT involves activation of PC-PLC by G alpha o to liberate diacylglycerol, which stimulates PKC (most likely alpha). PKC probably activates adenylate cyclase, which through cAMP, activates PKA. Activation of both PKA and PKC lead to closure of gKCa.
We studied the activities of the monocyte chemoattractant proteins MCP-1, MCP-2 and MCP-3 on human embryonic kidney 293-EBNA cells transfected with the MCP-1 receptor (CC CKR2B). At 4 nM, MCP-2 induced a Ca2+ influx which was as potent as that with MCP-1 at 4 nM, although the increase by MCP-2 became saturated at higher concentrations. In addition, all three MCPs showed dose-dependent inhibition of adenylyl cyclase activity stimulated by forskolin (IC50 values: 0.3 nM for MCP-1, 7 nM for MCP-2, and 1.5 nM for MCP-3). In conclusion, our data indicate that MCP-2 can exert its effects through the MCP-1 receptor, CC CKR2B.