Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (K-ATP) conductance in rat pancreatic A-cells

1. Capacitance measurements were used to examine the effects of the sulphon ylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secret ing rat pancreatic A-cells. 2. When applied extracellularly, tolbutamide stimulated depolarization-evok ed exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The c oncentration dependence of the stimulatory action was determined by intrace llular application through the recording pipette. Tolbutamide produced a co ncentration-dependent increase in cell capacitance. Half-maximal stimulatio n was observed at 33 mu M and the maximum stimulation corresponded to a 3.4 -fold enhancement of exocytosis. 3. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mM) and quinine (10 mu M). A similar stimulation was elici ted by 5-hydroxydecanoate (5-HD; 10 mu M), an inhibitor of mitochondrial AT P-sensitive K+ (K-ATP) channels. 4. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamid e- and Ca2+-induced exocytosis mere abolished by the H+ ionophore FCCP or b y arresting the vacuolar (V-type) H+-ATPase with bafilomycin A(1) or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to tha t obtained with tolbutamide. 6. We propose that during granular maturation, a granular V-type H+-ATPase pumps K+ into the secretory granule leading to the generation of a pH gradi ent across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant ex it of K+ through granular K+ channels with pharmacological properties simil ar to those of mitochondrial K-ATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown me chanisms, possibly involving granular alkalinization.