Relative Importance of Extracellular and Intracellular Calcium for the Two Phases of Glucose-Stimulated Insulin Release: Studies with Theophylline*

Abstract

Whether theophylline (2 mM) is able to restore a biphasic release of immunoreactive insulin (IRI) on glucose stimulation was investigated with perfused rat islets, when Ca entry into .beta. cells in prevented by omission of extracellular Ca (> 20 .mu.M) or addition of an antagonist of Ca influx (D 600 [.alpha.-isopropyl-.alpha.-(N-methyl-N-homoveratryl-.gamma.-aminopropyl)-3,4,5-trimethoxyphenylacetonitrile]). Ca omission at the time of glucose stimulation (300 mg/100 ml) reduced IRI release to a small 1st phase, further diminished by ethyleneglycol-bis(.beta.-aminoethyl ether) N,N''-tetraacetic acid (EGTA). Ca withdrawal 5 min before glucose increase completely prevented secretion. Theophylline addition augmented the reduced rapid phase of release, but failed to restore it when it was completely suppressed. The methylxanthine permitted reappearance of a late phase of IRI release except when EGTA was present. Theophylline introduction during glucose stimulation in the absence of Ca was followed by a rapid, although partial, restoration of IRI release. This effect was inhibited by EGTA and reduced after prolonged washing of the islets in the absence of Ca. When glucose-stimulated IRI release was completely suppressed by D 600 (50 .mu.M), theophylline did not restore a rapid phase but permitted the appearance of a late secretory phase. Glucose metabolism by islet cells was slightly reduced only after perifusion and incubation in the absence of Ca and presence of EGTA. These results suggest that a Ca influx into .beta. cells in required for glucose to trigger a rapid phase of IRI release, whereas mobilization of intracellular Ca by theophylline, from a pool of limited size, can partially compensate for the absence of extracellular Ca in the late phase of insulin release.