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Angiotensin II signal transduction pathways in chick cardiomyocytes: involvement of tyrosine phosphorylation

University of British Columbia

The general subject of these studies is the cellular action of angiotensin II (All) on cardiac myocytes. The present work was based on the hypothesis that several intracellular messengers or processes mediated by both AT] and AT2 receptors, underlie the response of cardiomyocytes to All and that protein tyrosine phosphorylation plays an important role in All-mediated signal transduction in the cardiomyocyte. The aim of this work was to elucidate some aspects of the effect of All on cardiac myocytes. Cardiomyocytes from 7 day old chick embryo were selected for investigations. The ability of All to induce several different and important second messengers, namely hydrolysis of phosphatidylinositol 4,5- biphosphate and phosphatidylcholine were examined The involvement of protein tyrosine phosphorylation in signal transduction activated by All in chick cardiomyocytes as well as the effect of All on Ptdlns 3-kinase were also assessed The role of AT] and AT2 subtypes of All receptors in complex signaling mechanisms following All stimulation of chick cardiomyocytes was investigated using relatively selective ATj and AT2 receptor antagonists. All stimulation of cardiac cells produced phosphoinositide hydrolysis and accumulation of inositol phosphates, mainly InsP^. The kinetics of InsP$ production, a hallmark of PtdInsP2~PLC activation, suggest that PtdIns2~PLC activation by All in chick cardiomyocytes occurs within I minute. The All-stimulated production oflnsP], InsP2, and InsP^ was partially but not completely blocked by the AT] receptor antagonist. Forty percent of the increase in InsP^ after All stimulation was blocked by AT2 receptor antagonism while both AT] and AT2 receptor blockade completely prevented IP3 formation. Thus, activation of PLC by All in chick cardiac myocytes is not solely mediated by AT] receptors, as has been previously proposed, but rather occurs through both receptor subtypes. The hypothesis that protein tyrosine phosphorylation induced by All might play an important role in early activation of PtdInsP2 hydrolysis and formation oflnsP^ in cardiac cells was tested. Genistein, a tyrosine kinase inhibitor, partially reduced All-induced accumulation oflnsP^. Consequently, not only PLC-/3 is activated in response to All cell stimulation but PLC-y is also likely activated. In chick cardiomyocytes, All produced phosphatidylcholine (PC) hydrolysis. However, the maximal effect, in contrast to PtdInsP2 hydrolysis, was late as it was observed at 30 minutes and remained increased over the I hour observation period One minute exposure to All induced tyrosine phosphorylation of proteins in chick cardiac myocytes. The most prominent of these proteins were proteins with approximate molecular weights 70 kDa and 195 kDa. All-induced protein tyrosine phosphorylation was almost completely blocked by pretreatment of cells with Lavendustin A, a tyrosine kinase inhibitor. Both AT] and AT2 are involved, to different extent, in Allinduced tyrosine phosphorylation in chick cardiac myocytes. Ptdlns 3-kinase was activated in response to AH stimulation of chick cardiac myocytes and was blocked by tyrosine kinase inhibitor. This suggests that Ptdlns 3-kinase might play an important role in All-induced signal transduction in cardiomyocytes. In summary, All produces immediate, within 1 minute, activation of PLC to produce InsP$ through AT} and AT2 receptors. All produces immediate activation of Ptdlns 3-kinase while PC hydrolysis is a late event. These data also show that tyrosine phosphorylation induced by All plays an important role in InsP$ formation and Ptdlns 3-kinase activation in the heart.

Thesis/Dissertation

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2009-03-05

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http://hdl.handle.net/2429/5572

Experimental Medicine

University of British Columbia

UBCV

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