title: Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells.
abstract: 1. Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2. Bath application of muscarine (0.1-30 microM) produced an inward INS with or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 microM) induced INS alone. Rectangular hyperbolas with EC50s of 2.01 and 0.21 microM were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3. In 36% of the cells exposed to Ca(2+)-free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca(2+)-free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A-23187 was added to normal solution. 4. W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5. HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 microM, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13-dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6. The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine INs to the right in a parallel manner. The KDS for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively.7. These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INs, and that neither CaM kinase II nor protein kinase C is involved.
authors.affiliation: Department of Physiology, School of Medicine, Fukuoka University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Inoue
authors.forename: I
authors.initials: I
authors.lastname: Imanaga
mesh_terms: D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000494:pharmacology; D002412:Cations / Q000378:metabolism*; D002838:Chromaffin System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D009116:Muscarine / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D002412:Cations; D007473:Ion Channels; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D000001:Calcimycin; D010095:Oxotremorine; D009116:Muscarine; D011493:Protein Kinase C; D002118:Calcium