Regulation of L-type Ca2+ channels in rabbit portal vein by G protein alphas and betagamma subunits.
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.
Zhong
J
J
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Dessauer
C W
CW
Keef
K D
KD
Hume
J R
JR
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015220:Calcium Channels / Q000145:classification / Q000187:drug effects* / Q000378:metabolism*; D002227:Carbazoles; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011169:Portal Vein / Q000378:metabolism; D011487:Protein Conformation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011758:Pyrroles / Q000494:pharmacology; D011817:Rabbits; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
C105593:8-bromoadenosine-3',5'-cyclic monophosphorothioate; D015220:Calcium Channels; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D009281:Naphthalenes; D011758:Pyrroles; D013873:Thionucleotides; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015240:Phorbol 12,13-Dibutyrate; C057416:KT 5720; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; C058819:calphostin C; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate