Ischemic preconditioning improves liver resistance to hypoxia and reduces reperfusion injury following transplantation. However, the intracellular signals that mediate the development of liver hypoxic preconditioning are largely unknown. We have investigated the signal pathway leading to preconditioning in freshly isolated rat hepatocytes. Hepatocytes were preconditioned by 10-minute incubation under hypoxic conditions followed by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Preconditioning reduced hepatocyte killing by hypoxia by about 35%. A similar protection was also obtained by preincubation with chloro-adenosine or with A 2A -adenosine receptor agonist CGS21680, whereas A 1 -adenosine receptor agonist N-phenyl-isopropyladenosine (R-PIA) was inactive. Conversely, the development of preconditioning was blocked by A 2 -receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), but not by A 1 -receptor antagonist 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX). In either preconditioned or CGS21680-treated hepatocytes a selective activation of ␦ and protein kinase C (PKC) isoforms was also evident. Inhibition of heterotrimeric G i protein or of phospholypase C by, respectively, pertussis toxin or U73122, prevented PKC activation as well as the development of preconditioning. MEK inhibitor PD98509 did not interfere with preconditioning that was instead blocked by p38 MAP kinase inhibitor SB203580. The direct activation of p38 MAPK by anisomycin A mimicked the protection against hypoxic injury given by preconditioning. Consistently, an increased phosphorylation of p38 MAPK was observed in preconditioned or CGS21680-treated hepatocytes, and this effect was abolished by PKC-blocker, chelerythrine. We propose that a signal pathway involving A 2A -adenosine receptors, G i -proteins, phospholypase C, ␦-and -PKCs, and p38 MAPK, is responsible for the deve- The term ischemic preconditioning refers to the resistance to ischemic injury acquired by tissue following one or more brief periods of ischemia followed by reperfusion. 1,2 Ischemic preconditioning was first described in the myocardium, 1 but has been shown in several other organs, including the brain, the skeletal muscles, and the small intestine. 3 In the heart, ischemic preconditioning occurs in 2 phases: an early phase (early preconditioning) that immediately follows the transient hypoxia and lasts 2 to 3 hours and a late phase (late preconditioning), which begins 12 to 24 hours from the transient ischemia and lasts for about 3 to 4 days. 3 Recent studies have shown that the same phenomenon could also be observed in the liver. [4][5][6][7][8][9][10] In particular, 10-minute interruption of liver blood supply in anesthetized rats followed by 10 minutes of reperfusion reduces transaminases released during a subsequent 90-minute period of ischemia and 90-minute reoxygenation. 5 A similar effect has also been observed in steatosic livers following heat shock preconditioning. 7 Furthermore, ischemic preconditioning before cold preserva...

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A major aldehydic end product of the peroxidation of arachidonic acid, 4-hydroxy-2,3-nonenal (HNE), has recently been considered for its potential involvement in a variety of cell functions. Here we report on the differential regulation of rat hepatocyte protein kinase C (PKC) isoforms by concentrations of HNE actually detectable in specific biological fluids or tissues. PKC ␤I and, to a much greater extent, PKC ␤II activities were markedly increased by 0.1 mol/L HNE (final concentration in cell medium) whereas they were unaffected or even inhibited by 1 to 10 mol/L HNE. On the contrary, the calcium independent PKC ␦ activity was inhibited by 0.1 mol/L and increased by 1 and 10 mol/L. The carbonyl compound 4-hydroxy-2,3-nonenal (HNE) is one of the major products of the oxidative breakdown of -6 fatty acids, the featuring process of membrane lipid peroxidation. 1 This aldehyde, being strongly electrophylic, readily reacts at neutral or slightly alkaline pH with sulphydryl and amino groups. HNE production was quantified several years ago in rat liver microsomes and isolated hepatocytes undergoing various experimental conditions of enhanced lipid peroxidation. 2,3 In more recent in vivo experimental reproduction of liver 4 and arterial 5,6 fibrosclerotic lesions, the detectable steady-state HNE concentration reached the low-micromolar range, up to 5 to 10 µmol/L.Until rather recently, in vitro biochemical studies were oriented towards seeking evidence of the many biochemical effects of HNE, generally using concentrations above 10 µmol/L; these were normally performed by adding the aldehyde to cell cultures or suspensions or cellular subfractions. 1 Such an approach was adopted to counteract the high aldehyde metabolism shown by the majority of the cell types tested 7 ; however, it directed the investigation to quantities of HNE of rather unlikely biological interest. Further, the overall negative effects exerted by relatively high HNE dosage contributed to the short-sighted opinion that this and related lipid peroxidation products were simply detrimental and acutely toxic to cells and tissues and consequently were of limited interest in pathophysiology.This state of affairs was rectified in particular by extensive research on the role of oxidatively modified low density lipoproteins in the pathogenesis of atherosclerosis 8 and the detection in the human plasma of autoantibodies against HNE-modified low-density lipoproteins 9 or of HNE-modified proteins, 10 and a role of HNE in pathophysiology became more favorably considered.At present, because of the use of specific monoclonal antibodies raised against tissue HNE-protein adducts, the number of human diseases with detectable accumulation of HNE is rapidly growing: increased HNE levels have been recognized in the liver of patients with alcohol-induced Abbreviations: HNE, 4-hydroxy-2,3-nonenal; PKC, protein kinase C; CD, cathepsin D; DAG, diacylglycerol.From the

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