Signaling pathway underlying stimulation of L-type Ca2+ channels in rabbit portal vein myocytes by recombinant Gbetagamma 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 Ca(2+) 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 Gbetagamma 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 Gbetagamma 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 Gbetagamma (50 nM) significantly increased Cav currents (141%). Nifedipine (1 microM) reduced both control and stimulated currents by approximately 90%. The enhancement of current by Gbetagamma 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 Gbetagamma, ACh, and PdBu were not associated with changes in the voltage dependence of activation or inactivation. The PI3K inhibitor LY-294002 (20 microM) reduced peak currents by 32% in cells dialyzed with Gbetagamma, whereas the inactive analog LY-303511 resulted in a small but significant reduction in current (12%). The c-Src inhibitor PP2 (1 microM) also significantly reduced currents (34%), whereas the inactive analog PP3 was without effect. These data provide further evidence for the hypothesis that Gbetagamma leads to stimulation of Cav currents in rabbit portal vein myocytes via a signaling pathway that includes PI3K, PKC, and c-Src.
Callaghan
Brid
B
Dept. of Physiology and Cell Biology, Univ. of Nevada, School of Medicine, Reno, NV 89573, USA.
Zhong
Juming
J
Keef
Kathleen D
KD
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011169:Portal Vein / Q000166:cytology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014665:Vasodilator Agents / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D044388:GTP-Binding Protein gamma Subunits; D011994:Recombinant Proteins; D014665:Vasodilator Agents; D015240:Phorbol 12,13-Dibutyrate; D009543:Nifedipine; D000109:Acetylcholine