Studies during the initial funding period have demonstrated that activation of the phosphatidylinositol signaling pathway (PSP) results in cytosolic calcium oscillation (CCO) driven phasic myometrial contractions. The repetitive cytosolic calcium transients are produced by the release of stored intracellular calcium, along with the influx of extracellular calcium. Additional studies have demonstrated that several isoforms of the signal transduction proteins involved in the PSP are expressed in rat myometrial tissue, including five isoforms for phospholipase C (PLC). The physiologic significance of the simultaneous expression of these multiple PLC isoforms in myometrial tissue is yet to be completely defined; however, it appears that different isoforms are necessary to mediate PSP activation via different membrane receptor classes (i.e. G-protein coupled v. tyrosine kinase coupled). The overall goal of the studies being proposed in this revised Competing Continuation Application is to test the hypothesis that one (or both) of the PLC-gamma isoforms expressed in myometrium are required for the generation of phasic contractions in response to activators of tyrosine kinase (TK), including thrombin and platelet activating factor (PAF).
The specific aims are to test the following hypotheses: 1) that pervanadate (a tyrosine phosphatase inhibitor) produces increased PLC- gamma phosphorylation, inositol phosphate (IP) production, the generation of CCOs, and phasic myometrial contractions, 2) that myometrial contractions in response to thrombin are mediated by TK activation, increased PLC-gamma phosphorylation, IP production, and the generation of CCOs, 3) that PAF produces phasic myometrial contractions mediated by TK activation, increased PLC-gamma phosphorylation, IP production, and the generation of CCOs and 4) that one (or both) of the PLC-gamma isoforms are essential for the pervanadate, thrombin and PAF stimulated activation of the PSP and the generation of cytosolic calcium transients. The latter studies will be performed utilizing antisense oligonucleotides for PLC-gamma1 and PLC-gamma2 with primary uterine myocytes cultures. These proposed studies will continue to improve our understanding of the molecular mechanisms underlying myometrial contractions, and ultimately improve our ability to more effectively treat clinically important disturbances of uterine contractile activity, especially premature labor which contributes substantially to the excessive preterm delivery rate in the U.S.
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