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) ε appears to play an important role in the late phase of ischemic preconditioning (PC), the mechanism(s) responsible for such translocation remains unclear. Furthermore, the signaling pathway that leads to the development of late PC after exogenous administration of NO in the absence of ischemia (NO donor-induced late PC) is unknown. In the present study we tested the hypothesis that NO activates PKC and that this is the mechanism for the development of both ischemia-induced and NO donor-induced late PC. A total of 95 chronically instrumented, conscious rabbits were used. In rabbits subjected to ischemic PC (six 4-minute occlusion/4-minute reperfusion cycles), administration of the NO synthase inhibitor N(ω)-nitro-L-arginine (group III), at doses previously shown to block the development of late PC, completely blocked the ischemic PC-induced translocation of PKCε but not of PKCη, indicating that increased formation of NO is an essential mechanism whereby brief ischemia activates the ε isoform of PKC. Conversely, a translocation of PKCε and -η quantitatively similar to that induced by 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 elicit a late PC effect. The particulate fraction of PKCε increased from 35 ± 2% of total in the control group (group I) to 60 ± 1% after ischemic PC (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 PKCη 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). Neither ischemic PC nor NO donors had any appreciable effect on the subcellular distribution of PKCα, -β1, -β2, -γ, -δ, -μ, or -ι/λ; on total PKC activity; or on the subcellular distribution of total PKC activity. Thus, the effects of SNAP and DETA/NO on PKC closely resembled those of ischemic PC. The DETA/NO-induced translocation of PKCε (but not that of PKCη) was completely prevented by the administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg (group VI) (particulate fraction of PKCε, 38 ± 4% of total, P < 0.05 versus group V; particulate fraction of PKCη, 79 ± 2% of total). The same dose of chelerythrine completely prevented the DETA/NO-induced late PC effect against both myocardial stunning (groups VII through X) and myocardial infarction (groups XI through 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 ε isotype is specifically involved in the development of this form of pharmacological PC. In all groups examined (groups I through VI), the changes in the subcellular distribution of PKCε protein were associated with parallel changes in PKCε isoform-selective activity, whereas total PKC activity was not significantly altered. Taken together, the results provide direct evidence that isoform-selective activation of PKCε is a critical step in the signaling pathway whereby NO initiates the development of a late PC effect both after an ischemic stimulus (endogenous NO) and after 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 mechanism in the signaling cascade of ischemic PC and may also have important implications for other pathophysiological conditions in which NO is involved and for nitrate therapy.