Vanadate-Induced Contraction of Smooth Muscle and Enhanced Protein Tyrosine Phosphorylation

This study was guided by the hypothesis that protein tyrosine phosphorylation may participate in signal transduction that is associated with contraction of smooth muscle. Accordingly, because sodium vanadate stimulates contraction of smooth muscle and inhibits protein tyrosine phosphatases, we sought to determine if vanadate-induced contraction was linked to enhanced protein tyrosine phosphorylation. Pronounced and time-dependent increases (5- to 20-fold) occurred in tyrosine phosphorylation during vanadate-induced contraction of guinea pig taenia coli. Three substrates of 86, 116, and 205 kDa were tyrosine phosphorylated as detected by immunoblotting with phosphotyrosine monoclonal antibody. Vanadate-induced contraction and enhanced protein tyrosine phosphorylation were both reduced in the presence of 2.5-5 μg/ml genistein, a potent tyrosine kinase inhibitor. In the continued presence of vanadate, chelation of extracellular Ca2+ with 5 mM ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) caused relaxation. However, pronounced phosphorylation of the substrates persisted even though isometric force decreased to control level. In this setting, replacement of the muscle bathing medium with fresh vanadate and EGTA-free medium caused a prompt spontaneous contraction which was immediately followed by relaxation and dephosphorylation of the substrates. Similarly, blockade of Ca2+-entry sites with 10 mM LaCl3 also relaxed smooth muscle strips that had been contracted with 1.5 mM vanadate. However, unlike EGTA-induced relaxation, replacement of the medium with fresh vanadate and LaCl3-free medium did not cause a spontaneous contraction. Taken together, these results show that vanadate-induced contraction of smooth muscle is probably coupled to enhanced protein tyrosine phosphorylation. The results also suggest that tyrosine phosphorylation may participate in Ca2+-dependent signalling mechanisms which regulate contraction of smooth muscle.