The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis > 5-fold when applied at a cy toplasmic Ca2+ concentration of 0.17 mu M. This effect was not detectable i n the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), The stimulatory acti on could be antagonized by the sulfonamide diazoxide, by the Cl--channel bl ocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intrace llular application of the antibody JSB1 [originally raised against a 170-kD a multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of th e mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secreto ry granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea re ceptors also present in the granules. We conclude that (i) tolbutamide stim ulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high -affinity sulfonylurea receptor in the secretory granules; and (ii) a granu lar 65-kDa mdr-like protein mediates the action. The processes thus initiat ed culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptak e and an increased intragranular hydrostatic pressure.