Effect of secretagogues on cytosolic free Ca2+ and insulin release in the hamster clonal β-cell line HIT-T15

Abstract

We have examined the effect of secretagogues on cytosolic free Ca²⁺ (Ca_i) in the hamster clonal β-cell line HIT-T15 using the Ca²⁺-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Ca_i in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mm increased Ca_i 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 Ca_i to 139 ± 6 nm. Ca_i 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 Ca_i to 407 ± 48 nm. The increases in Ca_i 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 Ca_i 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 Ca_i but sensitize the secretory mechanism to Ca²⁺.