Isoform-selective activation of protein kinase c by nitric oxide in the heart of conscious rabbits - A signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning

Although isoform-selective translocation of protein kinase C (PKC) epsilon appears to play an important role in the late phase of ischemic preconditio ning (PC), the mechanism(s) responsible for such translocation remains uncl ear. Furthermore, the signaling pathway that leads to the development of la te PC after exogenous administration of NO in the absence of ischemia (NO d onor-induced late PC) is unknown. In the present study we tested the hypoth esis that NO activates PKC and that this is the mechanism for the developme nt of both ischemia-induced and NO donor-induced late PC. A total of 95 chr onically instrumented, conscious rabbits were used. In rabbits subjected to ischemic PC (six 4-minute occlusion/4-minute reperfusion cycles), administ ration of the NO synthase inhibitor N-omega-nitro-L-arginine (group III), a t doses previously shown to block the development of late PC, completely bl ocked the ischemic PC-induced translocation of PKC epsilon but not of PKC e ta, indicating that increased formation of NO is an essential mechanism whe reby brief ischemia activates the epsilon isoform of PKC, Conversely, a tra nslocation of PKC epsilon and -eta quantitatively similar to that induced b y ischemic PC could be reproduced pharmacologically with the administration of 2 structurally unrelated NO donors, diethylenetriamine/NO (DETA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), at doses previously shown to elici t a late PC effect. The particulate fraction of PKC epsilon increased from 35+/-2% of total in the control group (group I) to 60+/-1% after ischemic P C (group II) (P<0.05), to 54+/-2% after SNAP (group IV) (P<0.05) and to 52/-2% after DETA/NO (group V) (P<0.05), The particulate fraction of PKC eta rose from 66+/-5% in the control group to 86+/-3% after ischemic PC (P<0.05 ), to 88+/-2% after SNAP (P<0.05) and to 85+/-1% after DETA/NO (P<0.05). Ne ither ischemic PC nor NO donors had any appreciable effect on the subcellul ar distribution of PKC alpha, -beta 1, -beta 2, -gamma, -delta, -mu, or -t/ lambda; on total PKC activity; or on the subcellular distribution of total PKC activity. Thus, the effects of SNAP and DETA/NO on PKC closely resemble d those of ischemic PC. The DETA/NO-induced translocation of PKC epsilon (b ut not that of PKC eta) was completely prevented by the administration of t he PKC inhibitor chelerythrine at a dose of 5 mg/kg (group VI) (particulate fraction of PKC epsilon, 38+/-4% of total, P<0.05 versus group V; particul ate fraction of PKC eta, 79+/-2% of total). The same dose of chelerythrine completely prevented the DETA/NO-induced late PC effect against both myocar dial stunning (groups VII through X) and myocardial infarction (groups XI t hrough XV), indicating that NO donors induce late PC by activating PKC and that among the 10 isozymes of PKC expressed in the rabbit heart, the epsilo n isotype is specifically involved in the development of this form of pharm acological PC. In all groups examined (groups I through VI), the changes in the subcellular distribution of PKC epsilon protein were associated with p arallel changes in PKC epsilon isoform-selective activity, whereas total PK C activity was not significantly altered. Taken together, the results provi de direct evidence that isoform-selective activation of PKC epsilon is a cr itical step in the signaling pathway whereby NO initiates the development o f a late PC effect both after an ischemic stimulus (endogenous NO) and afte r treatment with NO-releasing agents (exogenous NO). To our knowledge, this is also the first report that NO can activate PKC in the heart, The finding that NO can promote isoform-specific activation of PKC identifies a new biological function of this radical and a new mechanis m in the signaling cascade of ischemic PC and may also have important impli cations for other pathophysiological conditions in which NO is involved and for nitrate therapy.