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 poten tial by acting on ATP-sensitive K+ (K-ATP) channels and, in turn, modulatin g the open probability of voltage-gated Ca2+ channels in pancreatic islet p -cells. Here, we provide evidence that guanine nucleotides (GNs) also may b e involved in the modulation of these events in vivo. GNs were depleted by treatment of HIT-T15 cells with mycophenolic acid (MPA). Resting membrane p otential 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 o f exposure to MPA, basal cytosolic free Ca2+ concentrations ([Ca2+](i)) wer e elevated by 20%, Increments in [Ca2+](i) induced by submaximal concentrat ions of K+ (10-15 mM) or bombesin were enhanced by > 50%. Opening K-ATP cha nnels with diazoxide lowered basal [Ca2+], in MPA-treated cells to normal a nd abrogated the enhanced [Ca2+](i) responses. However, an L-type Ca2+ chan nel blocker only abolished the enhanced [Ca2+], response to stimuli and had no effect on the elevated basal [Ca2+](i), in contrast to EGTA, which obli terated both, implying that the latter was due to Ca2+ influx via non-L-typ e Ca2+ channels. These effects an ion fluxes were attributable specifically to GN depletion, since guanosine, which restores GTP content and the GTP/G DP ratio, but nut adenosine, prevented all MPA-induced ion changes; further more, the latter were mimicked by mizoribine (a structurally dissimilar GTP synthesis inhibitor). It is concluded that, in addition to adenine nucleot ides, GNs might contribute to the modulation of K-ATP channels in intact be ta-cells. In addition, GN depletion appeared to be able re, reduce stimulat ed insulin secretion by a mechanism largely independent of the changes of i on fluxes observed above. (C) 2000 Elsevier Science Inc.