title: Thrombin-induced Ca2+ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein. Evidence for the involvement of a G protein in inositol trisphosphate-dependent Ca2+ release.
abstract: The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca2+ mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the G alpha i2, G alpha i3, G alpha s, G beta 35, and G beta 36 protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca2+ mobilization, we investigated the effect of PT on Ca2+ responses using fluorescence spectroscopy and the Ca2+ indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 microgram/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca2+ from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca2+ release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca2+ response was inhibited by approximately 72%, suggesting that the major fraction of the Ca2+ response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the beta-subunit of G proteins because quantitative immunoblots showed that levels of the beta-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca2+ pool because Ca2+ release induced by agents that release Ca2+ directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [3H]inositol trisphosphate (IP3) formation because the level of inhibition of IP3 formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT-sensitive species is the major G protein pathway that couples thrombin-receptor activation to Ca2+ mobilization. This G protein appears to be involved not in the mechanisms that generate IP3 but rather possibly in coupling at the level of the intracellular Ca2+ store.
authors.affiliation: Baker Medical Research Institute, Alfred Hospital, Prahran, Victoria, Australia.
authors.forename: C B
authors.initials: CB
authors.lastname: Neylon
authors.forename: A
authors.initials: A
authors.lastname: Nickashin
authors.forename: P J
authors.initials: PJ
authors.lastname: Little
authors.forename: V A
authors.initials: VA
authors.lastname: Tkachuk
authors.forename: A
authors.initials: A
authors.lastname: Bobik
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000975:Antioxidants; D001345:Autoradiography; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006873:Hydroquinones / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007811:Lanthanum / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D051381:Rats; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D006873:Hydroquinones; D013729:Terpenes; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; C019359:2,5-di-tert-butylhydroquinone; D004533:Egtazic Acid; D019284:Thapsigargin; D007811:Lanthanum; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D002118:Calcium