Unusual carbachol responses in RINm5F cells: evidence for a "distal" site of action in stimulus-secretion coupling.
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.
Tang
S H
SH
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
Yaney
G C
GC
Sharp
G W
GW
D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D006160:Guanosine Triphosphate / Q000502:physiology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D008564:Membrane Potentials / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin
D007328:Insulin; D013449:Sulfonamides; D013729:Terpenes; D016718:Arachidonic Acid; C017967:W 7; D019284:Thapsigargin; D006160:Guanosine Triphosphate; D002217:Carbachol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium