Role of Ca2+ in apoptosis evoked by human amylin in pancreatic islet β-cells

Abstract

The objective of these studies was to clarify the role of Ca2+ in the mechanism of death evoked by human amylin (hA) in islet β-cells. hA forms fibrils in vitroand islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet β-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 μM) 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 Ca2+-dependent and Ca2+-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 μM. Direct measurements of the cytoplasmic free Ca2+ concentration ([Ca2+]i) in fura-2 acetoxymethyl ester (AM)-loaded β-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca2+]i. Modulators of Ca2+ regulation were tested for their effects on hA-induced β-cell apoptosis. Ca2+ ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca2+-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca2+]i. Neither the Ca2+ channel blocker verapamil, the extracellular Ca2+ chelator EGTA nor the cytosolic Ca2+ buffer bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (‘BAPTA’)/AM protected β-cells from hA-evoked apoptosis. Prolonged incubation of β-cells with a lethal dose of hA altered neither the basal [Ca2+]i nor the thapsigargin-induced release of Ca2+ from intracellular stores. Furthermore, 45CaCl2 uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca2+ homoeostasis do have a significant role in certain forms of β-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet β-cells.