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

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Abstract
1. The effect of purified G protein subunits alphas and betagamma 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 Galphas or Gbetagamma exhibited significant increases in peak Ba2+ current (IBa) density (148 % and 131 %, respectively) compared with control cells. The combination of Galphas and Gbetagamma further increased peak IBa density (181 %). Inactive Galphas and Gbetagamma did not have any effect on Ca2+ channels. 3. The stimulatory effect of Galphas on peak IBa 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 Gbetagamma 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 Gbetagamma on peak IBa. In addition, acute application of phorbol 12,13-dibutyrate enhanced peak IBa density in control cells, which could be entirely blocked by calphostin C. 4. These data indicate that enhancement of Ba2+ currents by Galphas and Gbetagamma 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 Gs proteins could be detected in vascular smooth muscle cells.