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 (ac hieved through the use of mycophenolic acid or other synthesis inhibit ors) impedes nutrient- but not K+-induced insulin secretion. It was co ncluded that a proximal nutrient-dependent step in stimulus-secretion coupling (but not the process of Ca2+-induced exocytosis itself) is mo dulated by ambient GTP levels. To examine Ca2+-dependent steps further in intact beta cells, INS-1 cells (which synthesize GTP and ATP simil arly to rat islets) and HIT-T15 cells (whose synthesis of purine nucle otides is different) were studied following cell culture for 1-18 hr i n 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 redu ced insulin secretion induced by 10 mM glucose, 30 or 40 mM KCl, or 10 0 mu M tolbutamide, independent of any changes in cell viability, insu lin content, ATP content, the ATP/ADP ratio, or cytosolic free Ca2+ co ncentrations. In INS-1 cells (which appear to have normal nucleobase t ransport 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 hypoxan thine is defective; however, provision of 500 mu M 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. (C) 1997 Elsevier Science Inc.