Maria Grazia De Cesaris

Signal pathway responsible for hepatocyte preconditioning by nitric oxide

Nitric oxide (NO) improves liver resistance to hypoxia/reperfusion injury acting as a mediator of hepatic preconditioning. However, the mechanisms involved are still poorly understood. In this study, we have investigated the mechanisms by which short-term exposure to the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium-1,2-diolate (NOC-9) increases hepatocyte tolerance to hypoxic injury. Isolated rat hepatocytes preincubated 15 min with NOC-9 (0.250 mM) became resistant to the killing caused by hypoxia. NOC-9 cytoprotection did not involve the activation of protein kinase C, but was instead blocked by inhibiting soluble guanylate cyclase with 1H-(1,2,4)-oxadiazolo-(4,3) quinoxalin-1-one (ODQ) (50 microM) or cGMP-dependent kinase (cGK) with KT 5823 (5 microM). Conversely, cGMP analogue, 8Br-cGMP (50 microM) mimicked the effect of NOC-9. Western blot analysis revealed that hepatocyte treatment with NOC-9 or 8Br-cGMP significantly increased dual phosphorylation of p38 MAPK. The activation of p38 MAPK was abolished by inhibiting guanylate cyclase or cGK. Pretreatment with NO significantly reduced intracellular Na(+) accumulation in hypoxic hepatocytes. This effect was reverted by KT 5823 as well as by the p38 MAPK inhibitor SB203580. SB203580 also reverted NOC-9 protection against hypoxic injury. Altogether, these results demonstrated that NO can induce hepatic preconditioning by activating p38 MAPK through a guanylate cyclase/cGK-mediated pathway.

Alterations of Na+ homeostasis in hepatocyte reoxygenation injury

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na(+) distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na(+) content that was associated with cytotoxicity. Na(+) accumulation and hepatocyte death were prevented by the omission of Na(+) from the incubation medium, but not by the addition of antioxidants. Blocking Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO(3)(-) from the reoxygenation medium significantly decreased Na(+) overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na(+) accumulation and cell death during reoxygenation. A significant protection against Na(+)-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na(+) influx with the Na(+)/H(+) ionophore monensin. Altogether, these results suggest that Na(+) accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.

Stimulation of p38 MAP kinase reduces acidosis and Na(+) overload in preconditioned hepatocytes.

Ischemic preconditioning has been shown to improve liver resistance to hypoxia/reperfusion damage. A signal pathway involving A2A‐adenosine receptor, Gi‐proteins, protein kinase C and p38 MAP kinase is responsible for the development of hypoxic preconditioning in hepatocytes. However, the coupling of this signal pathway with the mechanisms responsible for cytoprotection is still unknown. We have observed that stimulation of A2A‐adenosine receptors or of p38 MAPK by CGS21680 or anisomycin, respectively, appreciably reduced intracellular acidosis and Na+ accumulation developing during hypoxia. These effects were reverted by p38 MAPK inhibitor SB203580 as well as by blocking vacuolar proton ATPase with bafilomycin A1. SB203580 and bafilomycin A1 also abolished the cytoprotective action exerted by both CGS21680 and anisomycin. We propose that the stimulation of p38 MAPK by preconditioning might increase hepatocyte resistance to hypoxia by activating proton extrusion through vacuolar proton ATPase, thus limiting Na+ overload promoted by Na+‐dependent acid buffering systems.

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.