Involvement of Pertussis Toxin - sensitive G - proteins in the Hormonal Inhibition of Dihydropyridine - sensitive Ca 2 + Currents in an Insulin

Adrenaline inhibits insulin secretion via pertussis toxin-sensitive mechanisms. Since voltage-dependent Ca”+ currents play a key role in insulin secretion, we examined whether adrenaline modulates voltage-dependent Ca2+ currents of the rat insulinoma cell line, RINm5F. In the whole-cell configuration of the patchclamp technique, dihydropyridinebut not o-conotoxin-sensitive Ca2+ currents were identified. Adrenaline via a2-adrenoceptors inhibited the Ca2+ currents by about 50%. Somatostatin which also inhibits insulin secretion was less efficient (inhibition by 20%). The hormonal inhibition of Ca2+ currents was not affected by intracellularly applied CAMP but blocked by the intracellularly applied GDP analog guanosine 5’-0-(2thiodiphosphate) and by pretreatment of cells with pertussis toxin. In contrast to adrenaline and somatostatin, galanin, another inhibitor f insulin secretion, reduced Ca2+ currents by about 40% in a pertussis toxin-insensitive manner. Immunoblot experiments performed with antibodies generated against synthetic peptides revealed that membranes of RINm5F cells possess four pertussis toxin-sensitive G-proteins including Gilt Gi2, Go2, and another Go subtype, most likely representing Gol. In membranes of control but not of pertussis toxin-treated cells, adrenaline via a2adrenoceptors stimulated incorporation of the photoreactive GTP analog [cx-~~PIGTP azidoanilide into pertussis toxin substrates comigrating with the a-subunits of Gi2, Go2, and the not further identified Go subtype. The present findings indicate that activated a2-adrenoceptors of RINm5F cells interact with multiple Gproteins, i.e. two forms of Go and with Gi2. These Gproteins are likely to be involved in the adrenalineinduced inhibition of dihydropyridine-sensitive Ca2+ currents and in other signal transduction pathways contributing to the adrenaline-induced inhibition of insulin secretion.