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Mechanism of activation of nonselective cation channels by putative M 4 muscarinic receptor in guinea‐pig chromaffin cells
Author(s) -
Inoue Masumi,
Imanaga Itsusei
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb13243.x
Subject(s) - muscarine , oxotremorine , muscarinic acetylcholine receptor , chemistry , protein kinase c , endocrinology , biophysics , medicine , receptor , biochemistry , kinase , biology
1 Mechanisms involved in the generation of nonselective cation currents ( I NS ) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2 Bath application of muscarine (0.1–30 μ m ) produced an inward I NS with or without a transient outward current at −40 mV, whereas oxotremorine (0.06–60 μ m ) induced I NS alone. Rectangular hyperbolas with EC 50 S of 2.01 and 0.21 μ m were fitted to muscarine‐ and oxotremorine‐induced I NS s, 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 I NS 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 I NS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine I NS 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 I NS , whereas compound 5, a potent inhibitor of calmodulin‐dependent kinase II (CaM kinase II), produced no evident inhibition. 5 HA 1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine I NS with an IC 50 of 163 μ m , 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 I NS 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 I NS to the right in a parallel manner. The K D S for pirenzepine and AF‐DX 116 were estimated to be 13 n m (95% confidence interval, 11–16 n m ) and 365 n m (283–470 n m ), respectively. 7 These results suggest that muscarine efficiently produces I NS , probably through binding to the M 4 subtype, that intracellular Ca 2+ has a facilitating, but not an essential role in the generation of I NS , and that neither CaM kinase II nor protein kinase C is involved.