CHRONIC EXPOSURE TO TPA DEPLETES PKC-ALPHA AND AUGMENTS CA-DEPENDENT INSULIN-SECRETION FROM CULTURED RAT ISLETS

The insulin secretory responses of rat islets to glucose (15 mM), 12-O -tetradecanoylphorbol 13-acetate (TPA; 500 nM), and potassium (30 mM) were determined from perifused islets cultured for 22-24 h in CMRL-106 6 medium (control cultured) or islets cultured in the additional prese nce of 500 nM TPA. Islet content of protein kinase C alpha (PKC alpha) and serine and threonine phosphoprotein patterns were also monitored after the culture period. Compared with freshly isolated islets, cultu ring alone had no adverse effect on the capacity of TPA or 30 mM potas sium to stimulate secretion or on the islet content of PKC alpha. In a greement with previous studies, culturing in TPA reduced the islet con tent of immunoreactive PKC alpha by >95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPA slightly improved the insulin secretory r esponse to 15 mM glucose compared with control-cultured islets; howeve r, sustained rates of 15 mM glucose-induced secretion from these islet s were significantly less than the responses of freshly isolated islet s. Islets cultured in TPA responded to 30 mM potassium with a markedly amplified insulin secretory response that was abolished by nitrendipi ne. Enhanced phosphorylation of several islet proteins was also observ ed in TPA-cultured islets compared with control-cultured islets. These findings demonstrate that culturing alone impairs glucose-induced sec retion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture me dium. Sustained phosphorylation of several islet proteins in TPA-cultu red islets may account, at least in part, for augmented calcium-depend ent secretion.