Phosphorylation vanadate-induced contraction

Because earlier studies in this and other laboratories showed that inhibitors of tyrosine kinase activity suppressed contraction of smooth muscle (Section II), we suspected that vanadate-induced contraction might be due to its efficacy as an inhibitor of tyrosine phosphatase activity. Our experiments showed that treatment of taenia coli with 1.5 mM vanadate induced a tonic contraction that was associated with enhanced tyrosine phosphorylation of at least three substrates (M 85,000, 116,000, and 205,000 Figs. 2A and 2B). Maximal force attained in response to vanadate was about one-fourth to one-third of the maximal force attained during the phasic contraction elicited by stimu-... 

In this section we review data showing that activation of smooth muscle preparations induces tyrosine phosphorylation of several substrates. As shown in Fig. 7, a similar set of substrates (M, 42,000-205,000) are tyrosine phosphorylated during (1) activation of muscarinic receptors in intact taenia coli, (2) vanadate-induced contraction of the same preparation, (3) activation of aj-adrenergic receptors in VSMC cultured from canine femoral artery, and (4) Ca +-activated contraction of permeabilized ileal longitudinal smooth muscle. It is likely that one or more of these substrates... 

Di Salvo J, Semenchuk LA, Lauer, J (1993a) Vanadate-induced contraction of smooth muscle and enhanced protein tyrosine phosphorylation. Arch Biochem Biophys 304 386-391... 

Tyrosine kinases have been implicated in Ca +-sensitization largely as the result of the inhibitory effects of tyrosine kinase inhibitors, genistein and vanadate, and the correlation between tyrosine phosphorylation and vanadate- or agonist-induced contractions (Di Salvo et al. 1993a,b, 1994, 1997 Steusloff et al. 1995). However, the tyrosine-phosphorylated proteins have yet to be directly linked to a known contractile regulatory mechanism i.e., MLCK, SMPP-IM) and evaluation of the relationships between tyrosine phosphorylation and modulation of Ca +-sensitization of contraction in intact muscle is somewhat complicated by concurrent changes in cytosolic Ca (Di Salvo et al. 1994,1997), possibly as the result of activation of PLC-y (Marrero et al. 1994) or Gaq.n (Umemori et al. 1997). 

Accordingly, the maintenance of tyrosine phosphorylation during relaxation induced by chelation of extracellular Ca + with EGTA probably reflects persistent inhibition of tyrosine phosphatase activity by the continued presence of vanadate. The tyrosine-phos-phorylated substrates may be poised to promote Ca + entry so that replacement of the medium with fresh Ca2+-PSS allows for rapid entry of Ca2+ and a large spontaneous transient contraction. In contrast, no spontaneous contraction occurred in the experiment with La3+ and vanadate because La3+ blockade of the Ca + channels persists when the medium is replaced with fresh Ca2+-PSS (Fig. 2D). Based on this interpretation of the data, further studies were performed to more directly test the hypothesis that protein tyrosine phosphorylation is an important mechanism for regulating [Ca +Jj in smooth muscle cells. 

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