Modulation of Ca 2+ sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation

Abstract

Genistein, a potent tyrosine kinase inhibitor, inhibits contraction of several types of smooth muscle, suggesting that protein tyrosine phosphorylation 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 phosphorylation involved mechanisms for control of Ca 2+ sensitivity. Since smooth muscle permeabilized with staphylococcal α-toxin permits direct assessment of agonist-induced Ca 2+ sensitivity, we studied the effects of genistein on potential coupling between tyrosine phosphorylation and Ca 2+ sensitivity in permeabilized ileal smooth muscle. Results show that contraction of intact preparations with carbachol is markedly and reversibly inhibited by 40% at 4 μg genistein/ml and by 60% at 20 μg genistein/ml. Permeabilized preparations that are contracted with a submaximal [Ca 2+] in the presence of GTP relax when genistein is added to the medium. Genistein also reversibly inhibits contractions induced in permeabilized muscle with either a submaximal or maximal [Ca 2+] in the presence of GTP, as well as receptor-coupled activation of Ca 2+ sensitization with 10 μ m carbachol/10 μ m GTP. Activation of permeabilized preparations at pCa 4.6 in the presence of 100 μ m GTP promotes time-dependent tyrosine phosphorylation of several substrates. Both phosphorylation and force are inhibited by genistein. However, relatively high levels of myosin light chain phosphorylation persist during genistein-induced inhibition of Ca 2+ sensitivity. In contrast, genistein has no effect on Ca 2+-activated contraction in Triton-skinned preparations in either the presence or the absence of GTP. This shows that it does not directly inhibit actin-myosin interaction and suggests that its target(s) may be a cytosolic or membrane-bound regulatory protein(s) that is leached from the preparations during Triton-skinning. Taken together, these new data suggest that (a) tyrosine phosphorylation of one or more substrates may be coupled to mechanisms which regulate Ca 2+ sensitivity and (b) the inhibitory effects of genistein are probably due to inhibition of agonist-induced Ca 2+ sensitivity.