SODIUM-FLUORIDE STIMULATES EXOCYTOSIS AT A LATE SITE OF CALCIUM INTERACTION IN STIMULUS-SECRETION COUPLING - STUDIES WITH THE RINM5F BETA-CELL LINE

In the insulin-secreting beta cell line RINm5F, sodium fluoride stimul ated exocytosis in a concentration (5-15 mM)- and temperature-dependen t manner. Depletion of aluminum with the chelator deferoxamine or addi tion of aluminum to the buffer failed to affect the NaF-stimulated ins ulin release. This suggests that stimulation of heterotrimeric G prote ins or inhibition of phosphatases or other enzymes by fluoroaluminate, an analog of the phosphate moiety, is not involved in the insulinotro pic action of NaF. Removal of extracellular Ca2+ suppressed the NaF-st imulated insulin release. However, nitrendipine, a blocker of L-type v oltage-dependent Ca2+ channels, did not inhibit the NaF-stimulated ins ulin release and NaF did not cause any changes in the cytosolic free c alcium concentration ([Ca2+](i)). Decreasing [Ca2+](i) with thapsigarg in or increasing [Ca2+](i) with ionomycin or a depolarizing concentrat ion of KCI resulted in suppression or enhancement of NaF-stimulated in sulin release, respectively. Furthermore, NaF enhanced Ca2+-induced in sulin release in electrically permeabilized RINm5F cells. These findin gs indicate that the effect of NaF on exocytosis is dependent on [Ca2](i), although NaF itself does not change [Ca2+](i). Inhibitors of pro tein kinase C, such as staurosporine and bisindolylmaleimide, in conce ntrations sufficient to block the effects of phorbol esters, did not a ttenuate the NaF-stimulated insulin release. Neither cellular cAMP con tent nor [H-3]arachidonic acid release was increased by NaF. NaF-stimu lated insulin release was synergistically enhanced by the activation o f protein kinases A and C. Finally, trifluoperazine, an inhibitor of c almodulin and other Ca2+-binding proteins, inhibited the insulinotropi c action of NaF in a concentration-dependent manner. Trifluoperazine ( 50 mu M) and W-7 (100 mu M) nullified the 10 mM NaF-stimulated insulin release. It is concluded that NaF evokes exocytosis by a novel mechan ism of sensitization to Ca2+, possibly on a Ca2+-responsive protein th at is sensitive to trifluoperazine and W-7, leading to exocytosis. Pro tein kinases A and C also act at this site or at a more distal point.