Measurements of membrane potential, transmembrane ⁴⁵Ca fluxes, cytoplasmic free Ca²⁺ concentration and insulin release by transplantable rat insulinoma cells maintained in tissue culture

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.