Effect of secretagogues on cytosolic free Ca 2+ and insulin release at different extracellular Ca 2+ concentrations in the hamster clonal β-cell line HIT-T15

Abstract

We have examined the relationship between extracellular Ca 2+, cytosolic free Ca 2+ and insulin release in the clonal β-cell line HIT-T15. Glucose-stimulated insulin release was dependent on the extracellular Ca 2+ concentration in a dose-related manner; the threshold medium Ca 2+ concentration for glucose-stimulated insulin release was 0.5 mM. Both forskolin and 12- O-tetradecanoylphorbol 13-acetate (TPA) increased insulin release in the presence of glucose at all extracellular Ca 2+ concentrations tested (0.1–2.5 mM) but not in the absence of Ca 2+. Thus, the threshold medium Ca2+ concentration for glucose-stimulated insulin release was reduced to 0.1 mM by forskolin or TPA. Step-wise increases in the medium Ca 2+ concentration in the presence of an initiator of insulin release resulted in a dose-related increase in cytosolic free Ca 2+. In the presence of 10 mM glucose, cytosolic free Ca 2+ in HIT cells was increased from 60 ± 5 nM in Ca 2+-free medium to 290 ± 46 nM in medium containing 2.5 mM Ca 2+. The effects of increasing extracellular Ca 2+ in the presence of 40 mM K + were similar but considerably more pronounced. Inclusion of either TPA or forskolin in the incubation medium had no significant effect on the steady-state cytosolic free Ca 2+ levels in the absence of glucose but in the presence of 10 mM glucose forskolin caused modest (11–18%) increases in steady-state cytosolic free Ca 2+ levels at extracellular Ca 2+ concentrations of 0.25 mM or above. In contrast, in the presence of glucose TPA significantly reduced the steady-state levels of cytosolic free Ca 2+ by 17–21% at extracellular Ca 2+ concentrations of 0.25 mM or above. These data provide further evidence that insulin release mediated by activation of β-cell protein kinases involves primarily an increase in sensitivity of the secretory system to intracellular Ca 2+.