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 stud ied in RINm5F cells. Stimulation was associated with mobilization of C a2+ from thapsigargin-sensitive intracellular stores and elevation of the cytosolic Ca2+ concentration ([Ca2+](i)). However, when the elevat ion of [Ca2+](i) was blocked by prior treatment of the cells with thap sigargin 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 stimulati on of release. The role of protein kinase C (PKC) was next investigate d. Carbachol-stimulated insulin release was unchanged by phorbol ester -induced down-regulation of PKC, at a time when the stimulation of rel ease by 12-O-tetradecanoylphorbol-1 3-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 (stauros porine, bisindolylmaleimide, or 1-O-hexadecyl-2-O-methylglycerol), car bachol stimulated insulin release normally. Thus, the carbachol activa tion of PKC was also dissociated from the stimulation of insulin relea se. Finally, the effect of carbachol was examined in PKC-down-regulate d cells in the simultaneous presence of thapsigargin. Carbachol still stimulated insulin release normally. It is concluded that carbachol st imulates insulin release in RINm5F cells by a novel mechanism that doe s not involve the elevation of [Ca2+](i) or the activation of PKC. The action of carbachol appears to be exerted at a ''distal site,'' beyon d the point of increased [Ca2+](i), in stimulus-secretion coupling.