GTP-DEPENDENT INHIBITION OF INSULIN-SECRETION BY EPINEPHRINE IN PERMEABILIZED RINM5F CELLS - LACK OF CORRELATION BETWEEN INSULIN-SECRETION AND CYCLIC-AMP LEVELS

The mechanism by which .alpha.2-adrenergic agonists inhibit exocytosis was investigated in electrically permeabilized insulin secreting RINm5F cells. In this preparation .alpha.2-adrenoceptors remain coupled to adenylate cyclase, since basal- and forskolin-stimulated cyclic AMP production was lowered by epinephrine and clonidine by 30-50%. Cyclic AMP levels did not correlate with the rate of insulin secretion. Thus, at low Ca2+, forskolin enhanced cyclic AMP levels 5-fold without eliciting secretion, and Ca2+-stimulated secretion was associated with decreased cyclic AMP accumulation. Epinephrine (plus propranolol) inhibited Ca2+-induced insulin secretion in a GTP-dependent manner. The maximal inhibition (43%) occurred at 500 .mu.M GTP. Clonidine also inhibited Ca2+-stimulated secretion. Replacement of GTP by GDP or by the nonhydrolyzable GTP analog guanosine 5''-(3-O-thio)triphosphate as well as treatment of the cells with pertussis toxin prior to permeabilization abolished epinephrine inhibition of insulin secretion. Pertussis toxin did not affect Cs2+-stimulated secretion. Insulin release stimulated by 1,2-didecanoly glycerol was also lowered by epinephrine suggesting an effect distal to the activation of protein kinase C (Ca2+/phospholipid-dependent enzyme). These results taken together with the ability of epinephrine to inhibit ionomycin-induced insulin secretion in intact cells suggest that .alpha.2-adrenergic inhibition is distal to the generation of second messengers. A model is proposed for .alpha.2-adrenoceptor coupling to two effector systems, namely the adenylate cyclase and the exocytotic sites in insulin-secreting cells.