Inhibition of calcium-induced insulin secretion from intact HIT-T15 or INS-1 beta cells by GTP depletion.

Using intact rat islets, we previously observed that GTP depletion (achieved through the use of mycophenolic acid or other synthesis inhibitors) impedes nutrient- but not K+-induced insulin secretion. It was concluded that a proximal nutrient-dependent step in stimulus-secretion coupling (but not the process of Ca2+-induced exocytosis itself) is modulated by ambient GTP levels. To examine Ca2+-dependent steps further in intact beta cells, INS-1 cells (which synthesize GTP and ATP similarly to rat islets) and HIT-T15 cells (whose synthesis of purine nucleotides is different) were studied following cell culture for 1-18 hr in various concentrations of mycophenolic acid (MPA) or mizoribine (MZ). Both agents profoundly reduced GTP content (mean: -78%) and lowered the GTP/GDP ratio by an average of -73%; concomitantly, MPA or MZ reduced insulin secretion induced by 10 mM glucose, 30 or 40 mM KCl, or 100 microM tolbutamide, independent of any changes in cell viability, insulin content, ATP content, the ATP/ADP ratio, or cytosolic free Ca2+ concentrations. In INS-1 cells (which appear to have normal nucleobase transport and "salvage" pathway activities), guanine (but not adenine) restored GTP content, the GTP/GDP ratio, and Ca2+-induced secretion. In HIT cells, the phosphoribosylation of exogenous guanine or hypoxanthine is defective; however, provision of 500 microM guanosine (but not adenosine) reversed the effects of MPA. We conclude that, at least in certain situations, a requisite role for GTP in the distal step(s) of exocytosis can be demonstrated.