ENHANCED STIMULUS-SECRETION COUPLING IN POLYAMINE-DEPLETED RAT INSULINOMA CELLS - AN EFFECT INVOLVING INCREASED CYTOPLASMIC CA(2+), INOSITOL PHOSPHATE GENERATION, AND PHOROBOL ESTER SENSITIVITY

To extend previous observations on the role of polyamines in insulin p roduction, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RIN m5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhi bitor difluoromethylornithine (DFMO), which led to an increase in cell ular insulin and ATP contents. Analysis of different parts of the sign al transduction pathway revealed that insulin secretion and the increa se in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K+-induced d epolarization were markedly enhanced in DFMO-treated cells. These effe cts were paralleled by increased voltage-activated Ca2+ currents, as j udged by whole-cell patch-clamp analysis, probably reflecting increase d channel activity rather than elevated number of channels per cell. D FMO treatment also rendered phospholipase C in these cells more sensit ive to the muscarinic receptor agonist carbamylcholine, as evidenced b y enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic r eceptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentra tions suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to c ontrol cells. The stimulatory effects of glucose or the cyclic AMP rai sing agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treat ed cells, there was no secretory response or altered increase in [Ca2]i in response to the drug in these cells. It is concluded that partia l polyamine depletion sensitizes the stimulus-secretion coupling at mu ltiple levels in the insulinoma cells, including increased voltage-dep endent Ca2+ influx and enhanced responsiveness to activators of phosph olipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of poly amine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studie s of beta cell insulin secretion.