Postconditioning (PostC) modifies early post-ischemic pH, redox-environment and activity of enzymes. We hypothesized that early acidosis in PostC may affect superoxide-dismutase (SOD) and catalase(CAT) activities, may reduce 3-nitrotyrosine(3-NT)-and may increase S-nitrosylated (SNO)-protein levels, thus deploying its protective effects. To verify this hypothesis, we studied early (7 th min) and late (120 th min) phases of reperfusion a) endogenous-SOD and -CAT activities, and b) 3-NT-and SNO-protein levels. Isolated rat hearts underwent 30-min ischemia/120-min reperfusion(I/R) or PostC (5 cycles of 10-s I/R at the beginning of 120-min reperfusion) either with or without exogenous-CAT or -SOD infused during the initial 3-min of reperfusion. The effects of early reperfusion with acidic-buffer(AB, pH 6.8) on endogenous antioxidant-enzymes were also tested. Pressure, infarct-size and lactate-dehydrogenase release were also measured. At the 7 th min, PostC induced a significant decrease in SOD-activity with no major change in both Mn-and Cu/Zn-SOD levels, and in CAT-activity and level. PostC also reduced 3-NT and increased SNO levels. Exogenous-SOD, but not -CAT abolished PostCcardioprotection. In late reperfusion(120-min), I/R increased SOD-activity, but decreased CATactivity and Cu/Zn-SOD levels; these effects were reversed by PostC; 3-NT was not affected, but SNO was increased by PostC. AB reproduced PostC effects on antioxidant-enzymes.Conclusions: PostC downregulates endogenous-SOD and preserves -CAT activity, thus increasing SNO and reducing 3-NT levels. These effects are triggered by early post-ischemic acidosis. Yet acidosis-induced SOD-downregulation may limit denitrosylation; thus contributing to PostC-triggering. Hence, exogenous-SOD, but not -CAT, interferes with PostC-triggering.Persistent SOD-downregulation and SNO-increase may contribute to PostC and AB beneficial effects.

The Frank -Starling mechanism is a fundamental property of the vertebrate heart, which allows the myocardium to respond to increased filling pressure with a more vigorous contraction of its lengthened fibres. In mammals, myocardial stretch increases cardiac nitric oxide (NO) release from both vascular endothelium and cardiomyocytes. This facilitates myocardial relaxation and ventricular diastolic distensibility, thus influencing the Frank -Starling mechanism.In the in vitro working heart of the eel Anguilla anguilla, we previously showed that an endogenous NO release affects the Frank -Starling response making the heart more sensitive to preload. Using the same bioassay, we now demonstrate that this effect is confirmed in the presence of the exogenous NO donor S-nitroso-N-acetyl penicillamine, is independent from endocardial endothelium and guanylate cyclase/ cGMP/protein kinase G and cAMP/protein kinase A pathways, involves a PI(3)kinase-mediated activation of endothelial NO synthase and a modulation of the SR-CA 2þ ATPase (SERCA2a) pumps. Furthermore, we show that NO influences cardiac response to preload through S-nitrosylation of phospholamban and consequent activation of SERCA2a. This suggests that in the fish heart NO modulates the Frank -Starling response through a beat-to-beat regulation of calcium reuptake and thus of myocardial relaxation.We propose that this mechanism represents an important evolutionary step for the stretch-induced intrinsic regulation of the vertebrate heart, providing, at the same time, a stimulus for mammalian-oriented studies.

Being the largest form of intravascular and tissue storage of nitric oxide (NO) and a signalling molecule itself, the nitrite anion (NO(2)(-)) has emerged as a key player in many biological processes. Since the heart is under an important NO-mediated autocrine-paracrine control, in mammals the cardiac effects of nitrite are under intensive investigation. In contrast, nothing is known in non-mammalian vertebrates. We evaluated nitrite influence on cardiac performance in the perfused beating heart of three different cold-blooded vertebrates, i.e. two teleost fishes, the temperate red-blooded Anguilla anguilla, the Antarctic stenotherm, hemoglobinless Chionodraco hamatus (icefish), and the frog Rana esculenta. We showed that, under basal conditions, in all animals nitrite influences cardiac mechanical performance, inducing negative inotropism in eel and frog, while being a positive inotrope in C. hamatus. In all species, these responses parallel the inotropic effects of authentic NO. We also demonstrated that the nitrite-dependent inotropic effects are i) dependent from NO synthase (NOS) activity in fish; ii) sensitive to NO scavenging in frog; iii) cGMP/PKG-dependent in both eel and frog. Results suggest that nitrite is an integral physiological source of NO and acts as a signalling molecule in lower vertebrate hearts, exerting relevant inotropic actions through different species-specific mechanisms.

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Nitrite, a physiological nitric oxide (NO) storage form and an alternative way for NO generation, affects numerous biological processes through NO-dependent and independent pathways, including the S-nitrosylation of thiol-containing proteins. Mechanisms underlying these phenomena are not fully understood. The purpose of this study was to analyse in the rat heart (as prototype of mammalian heart) whether nitrite affects S-nitrosylation of cardiac proteins and the potential targets for S-nitrosylation. Rat hearts, perfused according to Langendorff, were exposed to nitrite. By Biotin Switch Method, we showed that nitrite treatment increased the degree of S-nitrosylation of a broad range of membrane proteins. Further analysis, conducted on subfractioned proteins, allowed us to identify a high level of nitrosylation in a small range of plasmalemmal proteins characterized by using an anti-Kir2.1 rabbit polyclonal antibody. We also verified that this effect of nitrite is preserved in the presence of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide). Our results suggest, for the first time, that nitrite represents a direct S-nitrosylating agent in cardiac tissues and that inward-rectifier potassium ion channels (Kir2.1) are one of the targets. These observations are of relevance since they support the growing evidence that nitrite is not only a NO reserve but also a direct modulator of important functional cardiac proteins.

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