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

Marianne Høy, Hervør L. Olsen, Krister Bokvist, Karsten Buschard *, Sebastian Barg †, Patrik Rorsman † and Jesper Gromada

1. Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca²⁺-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells.

2. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4·2-fold without affecting the whole-cell Ca²⁺ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 µM 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 µM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 µM), an inhibitor of mitochondrial ATP-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, tolbutamide- and Ca²⁺-induced exocytosis were abolished by the H⁺ ionophore FCCP or by arresting the vacuolar (V-type) H⁺-ATPase with bafilomycin A₁ or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide.

6. We propose that during granular maturation, a granular V-type H⁺-ATPase pumps H⁺ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H⁺ is facilitated by the concomitant exit of K⁺ through granular K⁺ channels with pharmacological properties similar 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 mechanisms, possibly involving granular alkalinization.

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