Abstract
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-γ1. In contrast, with ET-1 and bombesin, phosphorylated phospholipase C-γ1 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.
Footnotes
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Send reprint requests to: Dr. Denis Leiber, Laboratoire de Signalisation et Régulations Cellulaires, CNRS, EP 1088, Bâtiment 430, Université Paris-Sud, 91405 Orsay Cedex, France.
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↵1 This work was supported by grants from the Centre National de la Recherche Scientifique (ERS 0570 and EP1088) and by a contribution from the Association de la Recherche contre le Cancer (1355).
- Abbreviations:
- ET-1
- endothelin-1
- InsP3
- inositol trisphosphate
- InsP2
- inositol bisphosphate
- InsP1
- inositol monophosphate
- PIP2
- phosphatidylinositol-4,5-bisphosphate
- PTK
- protein tyrosine kinase
- PTP
- protein tyrosine phosphatase
- PLC
- phospholipase C
- Tyr
- tyrphostin
- Fura-2/AM
- acetoxy methyl ester (AM) form of fura-2
- Received May 14, 1998.
- Accepted January 13, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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