The objective of these studies was to clarify the role of Ca(2+) in the mechanism of death evoked by human amylin (hA) in islet beta-cells. hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet beta-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 microM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca(2+)-dependent and Ca(2+)-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 microM. Direct measurements of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca(2+)](i). Modulators of Ca(2+) regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca(2+) ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca(2+)-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca(2+)](i). Neither the Ca(2+) channel blocker verapamil, the extracellular Ca(2+) chelator EGTA nor the cytosolic Ca(2+) buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal [Ca(2+)](i) nor the thapsigargin-induced release of Ca(2+) from intracellular stores. Furthermore, (45)CaCl(2) uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca(2+) homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.