Unusual carbachol responses in RINm5F cells: evidence for a "distal" site of action in stimulus-secretion coupling

SH Tang, GC Yaney and GW Sharp

Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.

The mechanisms by which carbachol stimulates insulin release were studied in RINm5F cells. Stimulation was associated with mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and elevation of the cytosolic Ca2+ concentration ([Ca2+]i). However, when the elevation of [Ca2+]i was blocked by prior treatment of the cells with thapsigargin or with the anticalmodulin agents W-7 or W-13, the effect of carbachol to stimulate insulin release was unchanged. Thus, the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. The role of protein kinase C (PKC) was next investigated. Carbachol-stimulated insulin release was unchanged by phorbol ester-induced down-regulation of PKC, at a time when the stimulation of release by 12-O-tetradecanoylphorbol-13-acetate was abolished. Similarly, when the effect of 12-O-tetradecanoylphorbol-13- acetate to stimulate release was blocked by each of three separate PKC inhibitors (staurosporine, bisindolylmaleimide, or 1-O-hexadecyl-2-O- methylglycerol), carbachol stimulated insulin release normally. Thus, the carbachol activation of PKC was also dissociated from the stimulation of insulin release. Finally, the effect of carbachol was examined in PKC-down-regulated cells in the simultaneous presence of thapsigargin. Carbachol still stimulated insulin release normally. It is concluded that carbachol stimulates insulin release in RINm5F cells by a novel mechanism that does not involve the elevation of [Ca2+]i or the activation of PKC. The action of carbachol appears to be exerted at a "distal site," beyond the point of increased [Ca2+]i, in stimulus- secretion coupling.

Volume 47, Issue 4, pp. 863-870, 04/01/1995
Copyright © 1995 by American Society for Pharmacology and Experimental Therapeutics

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