Glucose-induced changes in cytoplasmic free Ca 2+ concentration and the significance for the regulation of insulin release : Measurements with fura-2 in suspensions and single aggregates of mouse pancreatic β-cells

Abstract

The effects of glucose on cytoplasmic free Ca 2+ concentration, [Ca 2+] i, and insulin release were investigated using pancreatic β-cells isolated from obese hyperglycemic mice. Measurements of [Ca 2+] i were performed in cell suspensions in a cuvette and in single cell-aggregates in a microscopic system, using fura 2 and quin 2. Insulin release was studied from indicator loaded cells in a column perifusion system. In the presence of 1.28 mM extracellular Ca 2+, an increase in the glucose concentration from 0 to 20 mM had two major effects on [Ca 2+] i. Initially there was a decrease, which was immediately followed by a pronounced increase. At reduced extracellular Ca 2+, or when Ca 2+ influx was blocked, glucose induced only a decrease in [Ca 2+] i. With increasing intracellular concentrations of indicator, the effects of glucose on [Ca 2+] i were markedly reduced. Changes in [Ca 2+] i, similar effects being obtained in the cuvette and microfluorometric measurements, were paralleled by changes in insulin release. Insulin release from indicator loaded cells did not markedly differ from that of non-loaded controls, either with respect to rapidity or size in the response to the sugar. The addition of 20 mM glucose increased the efflux of fura 2, an effect that was not related to insulin release. Permeabilization of indicator loaded cells demonstrated a substantial amount of fura 2 bound intracellularly. Although the effects of glucose on [Ca 2+] i seemed to be similar in fura 2 and quin 2 loaded cells, the demonstrated leakage and possible intracellular binding should be considered before using fura 2 for measurements in pancreatic β-cells.