ABSTRACT
We have examined the effect of secretagogues on cytosolic free Ca2+ (Cai) in the hamster clonal β-cell line HIT-T15 using the Ca2+-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Cai in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mm increased Cai by 36%, from 89 ± 4 to 121 ± 6 nm (mean ± s.e.m., n = 23). Further raising the medium glucose concentration to 10 mm increased Cai to 139 ± 6 nm. Cai was maximum and plateaued at 4 min after each addition of glucose. Addition of 40 mm K+ to the medium rapidly depolarized the HIT cells and increased Cai to 407 ± 48 nm. The increases in Cai in response to glucose or K+ were blocked by the simultaneous presence of verapamil (50μm). Stimulation by glucose or K+ also increased insulin release in parallel incubations of Quin 2-loaded HIT cells. Carbamylcholine chloride, forskolin or the phorbol ester 12-O-tetradecanoylphorbol acetate had no significant effect on Cai in glucose-stimulated HIT cells monitored 5 min after the addition of each test agent, despite increasing insulin release by 241, 239 and 216% respectively. These data support the hypothesis that potentiators of insulin release which activate cAMP-dependent protein kinase or protein kinase C do not increase Cai but sensitize the secretory mechanism to Ca2+.
