Subcellular B cell calcium and insulin secretion in vitro. Comparative ultracytochemical studies after glucose stimulation and cyproheptadine inhibition

Abstract

Using the ultracytochemical pyroantimonate technique different patterns of calcium containing precipitates were found in the B cells of the isolated perfused rat pancreas under conditions of stimulated and inhibited insulin secretion. The calcium specificity of the ultracytochemical method was assessed by perfusion with a EGTA containing calcium-free medium, which markedly reduced the extent of precipitation. Perfusion with 20 mM D-glucose over a period of 30 min resulted in calcium distribution patterns which could be related to the biphasic insulin release. The calcium patterns differed significantly in their quality and quantitative morphometry from those after 5 mM D-glucose or cyproheptadine (CPH) perfusion (20 mM D-glucose plus 0.1 mM CPH). After 3--5 min of 20 mM glucose perfusion there was an increased calcium precipitation along the inner side of the B cell membranes. After 20--30 min an additional increase in precipitation was found in the cytoplasmic matrix and in the secretory granules. B cells in a CPH-inhibited state of secretion and also after perfusion with 5 mM glucose lacked these findings. The data suggest that an increase in the membrane associated calcium may induce the first phase of insulin secretion by triggering the exocytosis of peripheral granules, while the cytoplasmic calcium may be involved in long term regulation of insulin release.