Tyrosine phosphorylation as a convergent pathway of heterotrimeric G protein- and rho protein-mediated Ca2+ sensitization of smooth muscle of rabbit mesenteric artery.
1. The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery. 2. In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [32P]-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody. Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (> or = 30 microM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment. 3. The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction. However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization. 4. The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction. The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S. The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors. 5. These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein. However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.
Sasaki
M
M
Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
Hattori
Y
Y
Tomita
F
F
Moriishi
K
K
Kanno
M
M
Kohya
T
T
Oguma
K
K
Kitabatake
A
A
D036002:ADP Ribose Transferases / Q000378:metabolism / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins; D002118:Calcium / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D019833:Genistein / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects* / Q000502:physiology; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects* / Q000502:physiology; D009281:Naphthalenes / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011817:Rabbits; D012266:Ribose / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D020741:rho GTP-Binding Proteins
D002273:Carcinogens; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D020032:Tyrphostins; D014662:Vasoconstrictor Agents; C079836:tyrphostin 25; D010656:Phenylephrine; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D000244:Adenosine Diphosphate; D012266:Ribose; D006632:Histamine; D006160:Guanosine Triphosphate; D019833:Genistein; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D019311:Staurosporine; C058819:calphostin C; D002118:Calcium