Signaling pathway underlying stimulation of L-type Ca2+ channels in rabbit portal vein myocytes by recombinant G βγ subunits

In previous studies, we (Callaghan B, Koh SD, and Keef KD, Circ Res 94: 626-633, 2004) have shown that voltage-dependent L-type Ca2+ channels (Cav) in portal vein myocytes are enhanced when muscarinic M2 receptors are activated with ACh. Current stimulation was coupled to the G protein subunit G beta gamma along with the downstream mediators phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), and c-Src. The present study was designed to determine whether the same second messenger pathway could be identified when exogenous recombinant G beta gamma subunits are introduced into cells. Smooth muscle myocytes were freshly isolated from rabbit portal vein, and Cav currents were recorded by using the patch-clamp technique. Dialysis of cells with recombinant G beta gamma (50 nM) significantly increased Cav currents (141%). Nifedipine (1 mu M) reduced both control and stimulated currents by similar to 90%. The enhancement of current by G beta gamma was equivalent to that produced by ACh (142%), whereas the PKC activator phorbol 12,13-dibutyrate (PdBu) gave rise to greater current stimulation (192%). Current stimulation with G beta gamma, ACh, and PdBu were not associated with changes in the voltage dependence of activation or inactivation. The PI3K inhibitor LY-294002 (20 mu M) reduced peak currents by 32% in cells dialyzed with G beta gamma, whereas the inactive analog LY-303511 resulted in a small but significant reduction in current (12%). The c-Src inhibitor PP2 (1 mu M) also significantly reduced currents (34%), whereas the inactive analog PP3 was without effect. These data provide further evidence for the hypothesis that G beta gamma leads to stimulation of Cav currents in rabbit portal vein myocytes via a signaling pathway that includes PI3K, PKC, and c-Src.