Regulation of insulin release, membrane potential, transmembrane 45Ca fluxes and cytoplasmic free Ca2+ concentration, [Ca2+](i) was examined using suspension of transplantable NEDH rat insulinoma cells previously cultured for 2-3 days to eliminate necrotic tumour cells and counter prior hypoglycaemia. Insulinoma cells displayed a resting [Ca2+](i) of 94 ± 8 nM (n = 17) and released 104 ± 15 ng insulin 10-6 cells (n = 7) during 60 min incubation with uptake of 2.7 ± 0.2 nmol 45Ca 10-6 cells (n = 7). High concentrations of glucose did not affect membrane potential, transmembrane 45Ca fluxes [Ca2+](i) or insulin release by insulinoma cells. K+ at 25 mM depolarised the plasma membrane, induced a small increase in 45Ca efflux and increased [Ca2+](i) by 65%. This model action was not associated with demonstrable effects on 45Ca uptake and insulin release. The effect of 25 mM K+ on [Ca2+](i) was counteracted by D-600, but this blocker of voltage-activated Ca2+ channels and verapamil lacked effects on transmembrane 45Ca fluxes and insulin release. The Ca2+-calmodulin antagonist, trifluoroperazine, was also without effect on 45Ca fluxes and insulin release. Ca2+ ionophore ionomycin increased [Ca2+](i), whereas A23187 and X537A did not affect transmembrane 45Ca fluxes. Moreover, insulin release was independent of extracellular Ca2+ over the range 0-20.4 mM despite marked affects on transmembrane 45Ca fluxes and a greater than 4-fold change of [Ca2+](i). Dibutyryl cyclic AMP increased insulin release by 55% without affecting transmembrane 45Ca fluxes or [Ca2+](i). The phosphodiesterase inhibitor, theophylline, also enhanced insulin release by 10-36% with no change of 45Ca uptake. The effectiveness of theophylline was independent of extracellular Ca2+ over the range 0-10.2 mM. These results indicate that inappropriate Ca2+ regulation is a key pathogenic feature underlying the inappropriate insulin secretion of rat insulinoma cells.
ASJC Scopus subject areas
- Cancer Research