Effect of intracellular alien chelators in secretagogue-induced release

Abstract

The only sensor that selectively detects [Ca2+]j at the release sites is the Ca2+ receptor that triggers secretion. In order to study the temporal aspects and Ca2+ dependence of insulin secretion we have used the cell-permeant Ca2+ chelators EGTA-AM and BAPTA-AM. These chelators have a similar equilibrium affinity for calcium signal at pH 7-2 and allow the use of a band pass filter for [Ca2+]i signals. Mouse pancreatic islets obtained by collagenase digestion were loaded with 100 uM EGTA-AM or BAPTA-AM during 60 min. Insulin release was assayed using a multi-islet perifusion chamber (volume, 75 #1; flow, 1 ml min-'). Experiments were done in triplicate. Pre-incubation with 100 #M EGTA-AM and BAPTA-AM completely inhibited 100 /SM carbachol-induced release. On the other hand, while BAPTA was a good blocker of 22 mM glucose-induced insulin release, EGTA caused a negligible effect. Furthermore, tolbutamide (100 #M)- and K+ (15 mM)- induced insulin release were unaffected by pre-incubation with EGTA, but blocked by BAPTA. The better efficiency of BAPTA compared with EGTA apparently results from the faster calcium-binding kinetics of BAPTA and suggests that the calcium-binding molecule that triggers release binds calcium in considerably less than 200-400 ,us and should be located very close to calcium channels. On the other hand, both EGTA and BAPTA were efficient inhibitors of carbachol-induced potentiation of insulin release, suggesting that Ca2P mobilized from intracellular stores has to diffuse for a period greater than 200-400 ,us before reaching the target.