Sensitivity to dihydropyridines, ω-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca²⁺ channels in rat insulinoma and human pancreatic β-cells

Abstract

High-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma (RINm5F) and human pancreatic β-cells were tested for their sensitivity to dihydropyridines (DHPs), ω-conotoxin (ω-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent K _D<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 μM and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca²⁺ agonists and antagonists in human β-cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 μM and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca²⁺ channels in these cells. In RINm5F cells, but not in human β-cells, DHP-resistant currents were sensitive to ω-CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a “residual” current (25–35%) with similar voltage-dependent characteristics and Ca²⁺/Ba²⁺ permeability. Noradrenaline (10 μM) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of ω-CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and ω-CgTx-resistant current although intracellularly applied guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 μM). Both actions were prevented by intracellular perfusion with guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic β-cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during β-cell activity.