The involvement of a conserved serine (Ser196 at the ,I-, Ser177 at the -, and Ser187 at the K-opioid To study G protein activation, adenylyl cyclase assays on CHO cells stably expressing the xtb2 chimeric receptors or the analogously mutated MOR-1 and DOR-1 (MORS196L and DORS177L, respectively) and electrophysiological studies of GIRK1 channel activation using Xenopus laevis oocytes coexpressing the mutant opioid receptors and the GIRK1 channel were performed. Additionally, back mutation of the iu82 TM4 leucine to the wild-type serine abolishes the antagonist's agonistic activity, demonstrating the importance of this serine residue over a unique tertiary structure adopted by the chimeric receptor. MePhe4,enkephalin; TIPPi, H-Tyr-Tic[q,CH2NH]Phe-Phe-OH (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid).
We report here that the M1, M3 and M5 muscarinic acetylcholine receptor subtypes that have been shown to couple to phosphoinositide hydrolysis also activate the mitogen-activated protein kinase (MAPK). Pharmacological characterization as well as mechanistic details of the activation pathway are presented. Carbachol-induced MAPK activation was time- and concentration-dependent at all subtypes. Pharmacological characterization of the MAPK response revealed that McN-A-343 was a partial agonist at the M1 and M3 subtypes, and that pilocarpine was a partial agonist at the M3 and M5 receptors. Carbachol-mediated MAPK activation at these receptor subtypes was pertussis toxin and wortmannin insensitive. By contrast, both agents significantly inhibited carbachol-induced MAPK activation by the M2 muscarinic receptor subtype. Furthermore, two independent single point mutations in the M1 receptor attenuated carbachol-induced activation of MAPK. Activation of MAPK at the M1, M3 and M5 muscarinic receptor subtypes was not dependent on intracellular or extracellular Ca2+, but was partially dependent upon protein kinase C. These data suggest that activation of MAPK by M1, M3 and M5 muscarinic receptors involves protein kinase C-dependent and independent pathways.
This study shows that activation of M1 muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)‐K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca2+ concentration and nNOS activity. We examined the relationship between receptor‐mediated increases in intracellular Ca2+ concentration and activation of nNOS over both short and long intervals using guanosine 3′,5′‐cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca2+ from intracellular stores in both the CHO M1/nNOS transfected cells and in neuroblastoma (N1E‐115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M1 muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l‐[3H]arginine to l‐[3H]citrulline as well as cGMP formation following stimulation of M1 muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca2+ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca2+.
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