Effects of inhibitors of guanine nucleotide synthesis on membrane potential and cytosolic free Ca2+ levels in insulin-secreting cells.
Adenine nucleotides play an important role in the control of membrane potential by acting on ATP-sensitive K+ (K(ATP)) channels and, in turn, modulating the open probability of voltage-gated Ca2+ channels in pancreatic islet beta-cells. Here, we provide evidence that guanine nucleotides (GNs) also may be involved in the modulation of these events in vivo. GNs were depleted by treatment of HIT-T15 cells with mycophenolic acid (MPA). Resting membrane potential was more depolarized in cells treated for 3 and 6 hr with MPA than in control cells, and this effect was inhibited by diazoxide. After 6 hr of exposure to MPA, basal cytosolic free Ca2+ concentrations ([Ca2+]i) were elevated by 20%. Increments in [Ca2+]i induced by submaximal concentrations of K+ (10-15 mM) or bombesin were enhanced by > 50%. Opening K(ATP) channels with diazoxide lowered basal [Ca2+]i in MPA-treated cells to normal and abrogated the enhanced [Ca2+]i responses. However, an L-type Ca2+ channel blocker only abolished the enhanced [Ca2+]i response to stimuli and had no effect on the elevated basal [Ca2+]i, in contrast to EGTA, which obliterated both, implying that the latter was due to Ca2+ influx via non-L-type Ca2+ channels. These effects on ion fluxes were attributable specifically to GN depletion, since guanosine, which restores GTP content and the GTP/GDP ratio, but not adenosine, prevented all MPA-induced ion changes; furthermore, the latter were mimicked by mizoribine (a structurally dissimilar GTP synthesis inhibitor). It is concluded that, in addition to adenine nucleotides, GNs might contribute to the modulation of K(ATP) channels in intact beta-cells. In addition, GN depletion appeared to be able to reduce stimulated insulin secretion by a mechanism largely independent of the changes of ion fluxes observed above.
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Cardiovascular Research Institute, National University Medical Institutes, National University of Singapore, Singapore. nmiligd@nus.edu.sg
Luo
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Metz
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003600:Cytosol / Q000187:drug effects / Q000502:physiology; D003981:Diazoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000096:biosynthesis / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009173:Mycophenolic Acid / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000502:physiology; D011685:Purine Nucleotides / Q000378:metabolism; D012263:Ribonucleosides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D006150:Guanine Nucleotides; D007328:Insulin; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011685:Purine Nucleotides; D012263:Ribonucleosides; C010052:mizoribine; D009173:Mycophenolic Acid; D003981:Diazoxide; D002118:Calcium