M1 muscarinic receptors stimulate rapid and prolonged phases of neuronal nitric oxide synthase activity: involvement of different calcium pools.

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.

M1, M3 and M5 muscarinic receptors stimulate mitogen-activated protein kinase.

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.

Activation of neuronal nitric oxide synthase by M2 muscarinic receptors associated with a small increase in intracellular calcium.

We investigated the coupling of the M2 muscarinic acetylcholine receptors expressed in Chinese hamster ovary cells to activation of neuronal nitric oxide (NO) synthase. Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an indirect measure of the generation of NO in Chinese hamster ovary cells. The muscarinic agonist carbachol induced marked time- and concentration-dependent enhancement of the activity of NO synthase.

mu-opioid receptor regulates CFTR coexpressed in Xenopus oocytes in a cAMP independent manner.

The objective of this study was to characterize the signaling mechanisms of the mu-opioid receptor in its coupling to the cystic fibrosis transmembrane conductance regulator (CFTR) when coexpressed in Xenopus oocytes. Because oocytes do not contain endogenous cAMP-regulated ion channels, the cAMP-modulated CFTR was coexpressed with receptors as a 'reporter' channel. Agonist treatment of oocytes coexpressing mu-opioid receptors, beta2-adrenergic receptors and CFTR produced Cl- currents in a dose-related manner and immunocytochemical analysis confirmed receptor expression.

Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists.

The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel.