MODULATION OF CA2-MUSCLE BY GENISTEIN AND PROTEIN-TYROSINE PHOSPHORYLATION( SENSITIVITY IN SMOOTH)

Genistein, a potent tyrosine kinase inhibitor, inhibits contraction of several types of smooth muscle, suggesting that protein tyrosine phos phorylation may be an important regulatory mechanism for smooth muscle contraction. We suspected that one site between activation of smooth muscle and contraction which might be modulated by protein tyrosine ph osphorylation involved mechanisms for control of Ca2+ sensitivity. Sin ce smooth muscle permeabilized with staphylococcal alpha-toxin permits direct assessment of agonist-induced Ca2+ sensitivity, we studied the effects of genistein on potential coupling between tyrosine phosphory lation and Ca2+ sensitivity in permeabilized ileal smooth muscle. Resu lts show that contraction of intact preparations with carbachol is mar kedly and reversibly inhibited by 40% at 4 mu g genistein/ml and by 60 % at 20 mu g genistein/ml. Permeabilized preparations that are contrac ted with a submaximal [Ca2+] in the presence of GTP relax when geniste in is added to the medium. Genistein also reversibly inhibits contract ions induced in permeabilized muscle with either a submaximal or maxim al [Ca2+] in the presence of GTP, as well as receptor-coupled activati on of Ca2+ sensitization with 10 mu M carbachol/10 mu M GTP. Activatio n of permeabilized preparations at pCa 4.6 in the presence of 100 mu M GTP promotes time-dependent tyrosine phosphorylation of several subst rates. Both phosphorylation and force are inhibited by genistein. Howe ver, relatively high levels of myosin light chain phosphorylation pers ist during genistein-induced inhibition of Ca2+ sensitivity. In contra st, genistein has no effect on Ca2+-activated contraction in Triton-sk inned preparations in either the presence or the absence of GTP. This shows that it does not directly inhibit actin-myosin interaction and s uggests that its target(s) may be a cytosolic or membrane-bound regula tory protein(s) that is leached from the preparations during Triton-sk inning. Taken together, these new data suggest that (a) tyrosine phosp horylation of one or more substrates may be coupled to mechanisms whic h regulate Ca2+ sensitivity and (b) the inhibitory effects of genistei n are probably due to inhibition of agonist-induced Ca2+ sensitivity. (C) 1995 Academic Press, Inc.