PERVANADATE MEDIATED AN INCREASED GENERATION OF INOSITOL PHOSPHATES AND TENSION IN RAT MYOMETRIUM - ACTIVATION AND PHOSPHORYLATION OF PHOSPHOLIPASE C-GAMMA-1

Stimulation of [H-3]inositol-labeled rat myometrial strips with pervan adate, formed by mixing orthovanadate and H2O2, induced a dose-depende nt accumulation of [H-3]inositol phosphates. Orthovanadate or H2O2 add ed alone had no effect. Pretreatment of myometrium with two tyrosine k inase inhibitors, namely genistein and tyrphostin 47 (at 100 mu M), re duced pervanadate-stimulated inositol phosphate formation by 50%. Perv anadate induced a time-sequential formation of inositol phosphates in the order inositol trisphosphate, inositol bisphosphate, and inositol monophosphate. The inhibitory effect of genistein was observed at the level of the three inositol phosphates. Pervanadate induced contractio n of the myometrium; the response was dose-dependent. H2O2 or orthovan adate was without effect. Pervanadate-mediated contraction was inhibit ed (50%) by genistein and tyrphostin 47 (100 mu M). Western blot analy sis, using anti-phosphotyrosine antibodies, revealed that phosphorylat ed proteins were present in detergent extracts from pervanadate-stimul ated myometrium. Tyrosine phosphorylation was reduced by a preincubati on with 100 mu M genistein or tyrphostin 47. Phospholipase C-gamma 1 w as immunodetected in myometrial extracts and was identified as one of the substrates subject to tyrosine phosphorylation following pervanada te treatment. The results demonstrate that in myometrium, protein tyro sine kinase/phosphatase activities controlled both phosphorylation and activation of phospholipase C-gamma 1, contributing to the modulation of the generation of inositol phosphates and tension.