Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

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.

Original publication

DOI

10.1111/j.1469-7793.2000.00109.x

Type

Journal article

Journal

J Physiol

Publication Date

15/08/2000

Volume

527 Pt 1

Pages

109 - 120

Keywords

Animals, Calcium, Cell Culture Techniques, Electric Conductivity, Exocytosis, Glucagon, Hydrogen-Ion Concentration, Ionophores, Islets of Langerhans, Male, Membrane Potentials, Membrane Proteins, Models, Biological, Pituitary Gland, Potassium, Potassium Channels, Protein Kinase C, Proton-Translocating ATPases, Rats, Rats, Inbred Lew, Rats, Sprague-Dawley, Sulfonylurea Compounds, Tolbutamide, Vacuolar Proton-Translocating ATPases