1 The aim of this study was to determine whether dierent signal transduction mechanisms underlie the Ca 2+ sensitizing eects of guanosine 5'-O-(3-thiotriphosphate) (GTP g S) and receptor agonists on b-escin-skinned smooth muscle of rabbit mesenteric artery. 2 In the homogenate of the b-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the [ 32 P]-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 g S-induced Ca 2+ sensitization, although the eect of GTP g S at higher concentrations (530 mM) was not completely blocked by this treatment. In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca sensitizing eects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors. 5 These results suggest that rho p21 predominantly mediates GTP g S-induced Ca 2+ sensitization of b-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca 2+ 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 Ca 2+ sensitization mechanisms.
This study was undertaken to explore possible signal-transduction mechanisms involved in the Ca2+-sensitizing effects of carbachol and endothelin-1 (ET-1) by using beta-escin-skinned smooth muscle of porcine coronary artery. Pretreatment with C3 exoenzyme of Clostridium botulinum, which selectively inactivates rho p21 by adenosine diphosphate (ADP) ribosylation, resulted in a significant inhibition of ET-1-induced Ca2+ sensitization, but had no effect on carbachol-induced Ca2+ sensitization. Whereas the protein kinase C (PKC) inhibitors calphostin C and staurosporine did not affect the Ca2+-sensitizing effect of carbachol, the tyrosine kinase inhibitors genistein and tyrphostin 25 greatly but incompletely suppressed it. In contrast, the Ca2+-sensitizing effect of ET-1 was significantly inhibited by either calphostin C or genistein. Although the inhibitory effect of calphostin C on ET-1-induced Ca2+ sensitization was less than that of genistein, the effects of calphostin C and genistein were additive. The genistein-sensitive component of ET-1-induced Ca2+ sensitization appeared to include the C3-sensitive one. However, a substantial enhancement by ET-1 of the Ca2+-induced contraction was observed even in the presence of the two inhibitors. In beta-escin-skinned smooth muscle of rabbit mesenteric artery, ET-1-induced Ca2+ sensitization was marginally affected by C3 pretreatment, calphostin C, and genistein. We conclude that, although PKC activation and rho p21 protein-dependent and -independent tyrosine phosphorylation each plays an important role in an increase in myofilament Ca2+ sensitivity, the contributions of these signaling pathways to Ca2+ sensitization are different depending on receptor agonists and tissues used. Furthermore, these data suggest the existence of an as yet undefined signal-transduction mechanism involved in Ca2+ sensitization caused by receptor agonists.
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