No evidence for protective erythropoietin alpha signalling in rat hepatocytes.

BACKGROUND: Recombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. METHODS: Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4 degrees C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2-100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. RESULTS: In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. CONCLUSION: Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.

Enhanced oxidation of NAD(P)H by oxidants in the presence of dehydrogenases but no evidence for a superoxide-propagated chain oxidation of the bound coenzymes.

Recently we demonstrated that lactate dehydrogenase (LDH)-bound NADH is oxidized by O2, H2O2, HNO2 and peroxynitrite predominantly via a chain radical mechanism which is propagated by superoxide. Here we studied both whether other dehydrogenases also increase their coenzymes' reactivity towards these oxidants and whether a chain radical mechanism is operating. Almost all dehydrogenases increased the oxidation of their physiological coenzymes by at least one of the oxidants. The oxidation of NADH or NADPH depended both on the binding dehydrogenase and the applied oxidant and in some cases the reactions were remarkably fast. The highest rate constant (k = 370 M-1 s-1) was found for the reaction of HNO2 with NADH bound to alcohol dehydrogenase. Regardless of the applied oxidant, superoxide dismutase failed to inhibit the oxidation of protein-bound NADH and NADPH. We therefore conclude that several dehydrogenases increase the oxidation of NADH and/or NADPH by the employed set of oxidants in bimolecular reactions, but, unlike LDH, do not mediate a O2*(-) dependent chain radical mechanism.

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes.

Heme catalases are considered to degrade two molecules of H(2)O(2) to two molecules of H(2)O and one molecule of O(2) employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H(2)O(2) (relative to catalase concentration), adjusted by H(2)O(2)-generating systems. At a ratio of a H(2)O(2) flux (given in microM/min(- 1)) to catalase concentration (given in microM) of 10 min(- 1) and above, H(2)O(2) degradation occurred via the catalatic cycle. At lower ratios, however, H(2)O(2) degradation proceeded with increasingly diminished production of O(2). At a ratio of 1 min(- 1), O(2) formation could no longer be observed, although the enzyme still degraded H(2)O(2). These results strongly suggest that at low physiological H(2)O(2) fluxes H(2)O(2) is preferentially metabolised reductively to H(2)O, without release of O(2). The pathways involved in the reductive metabolism of H(2)O(2) are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H(2)O(2) fluxes but kinetically outcompete the reaction of compound I with H(2)O(2) at low H(2)O(2) production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H(2)O(2) are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.

Initiation of a superoxide-dependent chain oxidation of lactate dehydrogenase-bound NADH by oxidants of low and high reactivity.

In cells, NADH and NADPH are mainly bound to dehydrogenases such as lactate dehydrogenase (LDH). In cell-free systems, the binary LDH-NADH complex has been demonstrated to produce reactive oxygen species via a chain oxidation of NADH initiated and propagated by superoxide. We studied here whether this chain radical reaction can be initiated by oxidants other than LDH largely increased the oxidation of NADH (but not of NADPH) by O(2), H(2)O(2) and during the intermediacy of HNO(2). LDH also increased the oxidation of NADH by peroxynitrite. The increases in NADH oxidation were completely prevented by superoxide dismutase (SOD). In contrast, the nitrogen dioxide-dependent oxidation of NADH and NADPH was decreased by LDH in a SOD-independent manner. These experimental data strongly indicate that oxidation of LDH-bound NADH can be induced from reaction of either weak oxidants with LDH-bound NADH or of strong oxidants with free NADH thus yielding which is highly effective to propagate the chain. Our results underline the importance of SOD in terminating superoxide-dependent chain reactions in cells under oxidative stress.