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

¹Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada; and ²Department of Anatomy, Physiology, and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, Alabama

Submitted 26 April 2006 ; accepted in final form 20 July 2006

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 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 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 (50 nM) significantly increased Cav currents (141%). Nifedipine (1 µM) reduced both control and stimulated currents by 90%. The enhancement of current by G 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, ACh, and PdBu were not associated with changes in the voltage dependence of activation or inactivation. The PI3K inhibitor LY-294002 (20 µM) reduced peak currents by 32% in cells dialyzed with G, whereas the inactive analog LY-303511 resulted in a small but significant reduction in current (12%). The c-Src inhibitor PP2 (1 µM) also significantly reduced currents (34%), whereas the inactive analog PP3 was without effect. These data provide further evidence for the hypothesis that G leads to stimulation of Cav currents in rabbit portal vein myocytes via a signaling pathway that includes PI3K, PKC, and c-Src.

vascular calcium channel; G protein subunits; tyrosine kinase