Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (ϩ119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (ϩ103% versus control) and at 30 minutes (ϩ89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the ⑀ isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (ϩ72%) and at 30 minutes (ϩ67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKC⑀, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKC⑀ but also that of Src and Lck, indicating that Src and Lck are downstream of PKC⑀ in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction. (Circ Res. 1999;85:542-550.)
Activation of protein kinase C (PKC) ⑀ by nitric oxide (NO) has been implicated in the development of cardioprotection. However, the cellular mechanisms underlying the activation of PKC⑀ by NO remain largely unknown. Nitration of protein tyrosine residues has been shown to alter functions of a variety of proteins, and NO-derived peroxynitrite is known as a strong nitrating agent. In this investigation, we demonstrate that NO donors promote translocation and activation of PKC⑀ in an NO-and peroxynitrite-dependent fashion. NO induces peroxynitrite-mediated tyrosine nitration of PKC⑀ in rabbit cardiomyocytes in vitro, and nitrotyrosine residues were also detected on PKC⑀ in vivo in the rabbit myocardium preconditioned with NO donors. Furthermore, coimmunoprecipitation of PKC⑀ and its receptor for activated C kinase, RACK2, illustrated a peroxynitrite-dependent increase in PKC⑀-RACK2 interactions in NO donor-treated cardiomyocytes. Moreover, using an enzyme-linked immunosorbent assaybased protein-protein interaction assay, PKC⑀ proteins treated with the peroxynitrite donor SIN-1 exhibited enhanced binding to RACK2 in an acellular environment. Our data demonstrate that post-translational modification of PKC⑀ by NO donors, namely nitration of PKC⑀, facilitates its interaction with RACK2 and promotes translocation and activation of PKC⑀. These findings offer a plausible novel mechanism by which NO activates the PKC signaling pathway. Protein kinase C (PKC)1 is a family of serine-threonine kinases that participate in numerous biological processes (1, 2). In the heart, activation of PKC reduces the myocardial ischemic injury, whereas inhibition of PKC abolishes ischemic preconditioning (3-5). Recently, it has been shown that this cardioprotective effect can be fully mimicked by modulating the activity of a single isozyme of this family, the ⑀ isoform of PKC (6 -9). Multiple molecular events have been shown to have an activating effect on this enzyme, among which, of particular interest, is nitric oxide (NO). Although the effects of NO on PKC depend on its biological functions and on the cell types (10 -14), NO-induced activation of PKC is well documented in the heart (15, 16). Several investigations have demonstrated that at doses that produce a cardioprotective effect, exogenous NO (released by NO donors) activates PKC⑀ in an isoformspecific manner (17-20). Furthermore, activation of this isozyme has been demonstrated to play an essential role in orchestrating the signal transduction events during NO-induced cardioprotection against ischemic injury (15, 21). However, the exact molecular mechanism(s) whereby NO activates PKC⑀ in the heart remain largely unknown.As a relatively stable hydrophobic free radical gas, NO can readily diffuse through cell membranes (22). Within cells, NO itself and NO-derived reactive nitrogen species are capable of reacting with various molecular targets that include complex biological molecules, such as proteins, lipids, and DNA, as well as low molecular weight compounds (23). One of the impo...
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