title: Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells.
abstract: 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 microM 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 microM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 microM), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) 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 KATP 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.
authors.affiliation: Laboratory of Islet Cell Physiology, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.
authors.forename: M
authors.initials: M
authors.lastname: Høy
authors.forename: H L
authors.initials: HL
authors.lastname: Olsen
authors.forename: K
authors.initials: K
authors.lastname: Bokvist
authors.forename: K
authors.initials: K
authors.lastname: Buschard
authors.forename: S
authors.initials: S
authors.lastname: Barg
authors.forename: P
authors.initials: P
authors.lastname: Rorsman
authors.forename: J
authors.initials: J
authors.lastname: Gromada
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D004553:Electric Conductivity; D005089:Exocytosis / Q000187:drug effects*; D005934:Glucagon / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007476:Ionophores / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008954:Models, Biological; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D013453:Sulfonylurea Compounds / Q000494:pharmacology; D014044:Tolbutamide / Q000494:pharmacology*; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D007476:Ionophores; D008565:Membrane Proteins; D015221:Potassium Channels; D013453:Sulfonylurea Compounds; C117780:mitochondrial K(ATP) channel; D005934:Glucagon; D014044:Tolbutamide; D011493:Protein Kinase C; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D011188:Potassium; D002118:Calcium