Regulation of L-type Ca2+ channels in rabbit portal vein by G protein αs and βγ subunits

1. The effect of purified G protein subunits alpha(s) and beta gamma on L-type Ca2+ channels in vascular smooth muscle and the possible pathways involved were investigated using freshly isolated smooth muscle cells from rabbit portal vein and the whole-cell patch clamp technique. 2. Cells dialysed with either G alpha(s) or G beta gamma exhibited significant increases in peak Ba2+ current (I-Ba) density (148% and 131%, respectively) compared with control cells. the combination of G alpha(s) and G beta gamma further increased peak I-Ba density (181%). Inactive G alpha(s) and G beta gamma did not have any effect on Ca2+ channels. 3. The stimulatory effect of G alpha(s) on peak I-Ba was entirely abolished by the protein kinase A inhibitor Rp-8-Br-cAMPS, or the adenylyl cyclase inhibitor SQ 22536. On the other hand, the stimulatory response of Ca2+ channels to G beta gamma was not affected by the protein kinase A inhibitors Rp-8-Br-cAMPS and KT 5720, or by the Ca2+-dependent protein kinase C inhibitor bisindolylmaleimide 1, but was completely blocked by the protein kinase C inhibitor calphostin C. Pretreatment of cells with phorbol 12-myristate 13-acetate for over 18 h prevented the stimulatory effect of G beta gamma on peak I-Ba. In addition, acute application of phorbol 12,13-dibutyrate enhanced peak I-Ba density in control cells, which could be entirely blocked by calphostin C. 4. These data indicate that enhancement of Ba2+ currents by G alpha(s) and G beta gamma can be attributed to increased activity of protein kinase A and protein kinase C, respectively. No direct membrane-delimited pathway for Ca2+ channel regulation by activated G(s) proteins could be detected in vascular smooth muscle cells.