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 cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated 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 uptake and an increased intragranular hydrostatic pressure.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000276:immunology / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005089:Exocytosis / Q000187:drug effects*; D005905:Glyburide / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects*; D051379:Mice; D008815:Mice, Inbred Strains; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug / Q000378:metabolism; D064233:Sulfonylurea Receptors; D014044:Tolbutamide / Q000494:pharmacology*