Vol. 289, Issue 2, 1022-1030, May 1999

A Tyrosine Kinase Signaling Pathway, Regulated by Calcium Entry and Dissociated from Tyrosine Phosphorylation of Phospholipase C-1, Is Involved in Inositol Phosphate Production by Activated G Protein-Coupled Receptors in Myometrium¹

Bruno Palmier, Monique Vacher, Simone Harbon and Denis Leiber

Signalisation et Régulations Cellulaires, Centre National de la Recherche Scientifique, Université Paris-Sud, Orsay Cedex, France

Our experiments were conducted to evaluate, in rat myometrium, the potential contribution of a protein tyrosine kinase (PTK) pathway in the hydrolysis of phosphatidylinositol-4,5-bisphosphate mediated by bombesin, endothelin-1 (ET-1), and carbachol. The production of inositol phosphates (InsP) by agonists and AlF₄ was partly inhibited (35-40%) by genistein and tyrphostins, two PTK inhibitors. Genistein attenuated uterine contractions elicited by the stimulation of muscarinic and bombesin receptors, whereas pervanadate, a protein tyrosine phosphatase inhibitor, potentiated receptor-mediated contraction. Tyrosine-phosphorylated proteins were detected in detergent extracts from agonist- and pervanadate-stimulated myometrium. The amount of InsP produced in response to pervanadate was related to the tyrosine phosphorylation status of phospholipase C-1. In contrast, with ET-1 and bombesin, phosphorylated phospholipase C-1 made a minor contribution. Additional findings were rather consistent with a role for Ca²⁺. In fura-2-loaded cells, genistein partly decreased both the transient and sustained intracellular Ca²⁺ concentration phases induced by bombesin. The removal of extracellular Ca²⁺ or the addition of nifedipine inhibited (35%) InsP production due to bombesin and ET-1. The inhibitory effects of genistein and tyrphostins were abolished in Ca²⁺-depleted medium, were not additive with that of nifedipine, and (as for nifedipine) were counteracted by the Ca²⁺ channel agonist Bay K 8644. The data are consistent with a PTK-mediated process in the activation of the voltage-gated Ca²⁺ influx that is involved in the production of InsP by stimulated G protein-coupled receptors.