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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Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na ؉ homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na ؉ accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na ؉ homeostasis, and citotoxicity were mimicked by stimulating protein kinase C (PKC) with 4␤-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A 1 abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na ؉ overload, and hepatocyte killing. Similarly, the addition of Na ؉ ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na ؉ accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na ؉ overload promoted by Na ؉ -dependent acid buffering systems. (HEPATOLOGY 2000;31:166-172.)In 1986, Murry et al. 1 reported that a short period of ischemia led to an unexpected resistance of the myocardium to a subsequent prolonged ischemia. Since then, the resistance to ischemic injury acquired after 1 or more brief periods of ischemia followed up by reperfusion has been termed ischemic preconditioning. 1,2 In the myocardium, 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) that begins 12 to 24 hours from the transient ischemia and lasts for 3 to 4 days. 3 Besides the heart, ischemic preconditioning has been shown in several organs including the brain, the skeletal muscles, and the small intestine. 3 Recently, the development of preconditioning has also been observed in livers exposed to brief interruptions of blood perfusion. [4][5][6] Hepatic preconditioning prevents hepatocellular damage caused by both warm and cold ischemia and improves liver transplantation in rats. [4][5][6] Extensive studies in the myocardium have s...

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Atrial natriuretic peptide (ANP) reduces ischemia and/or reperfusion damage in several organs, but the mechanisms involved are largely unknown. We used freshly isolated rat hepatocytes to investigate the mechanisms by which ANP enhances hepatocyte resistance to hypoxia. The addition of ANP (1 mol/L) reduced the killing of hypoxic hepatocytes by interfering with intracellular Na ؉ accumulation without ameliorating adenosine triphosphate (ATP) depletion and pH decrease caused by hypoxia. The effects of ANP were mimicked by 8-bromo-guanosine 3 , 5 -cyclic monophosphate (cGMP) and were associated with the activation of cGMP-dependent kinase (cGK), suggesting the involvement of guanylate cyclase-coupled natriuretic peptide receptor ( A trial natriuretic peptide (ANP) belongs to the natriuretic peptide (NP) family, which includes a number of peptides acting as neurotransmitters or hormones. 1 Like other NPs, ANP is produced in response to stress conditions such as cardiac hypoxia and atrial stretch and has potent vasodilating, hypotensive, and natriuretic activities. 2,3 In addition, evidence indicates that ANP modulates cell proliferation and cardiomyocyte hypertrophy 4 and exerts cytoprotective functions. Indeed, ANP has been shown to efficiently reduce kidney damage by both warm and cold hypoxia 5,6 to prevent reperfusion injury in perfused hearts. 7 The mechanisms responsible for the cytoprotective action of ANP have not been entirely elucidated. In the kidney, Shaw et al. 5 have shown that the cytoprotective activity of ANP is associated with the block of catecholamine-mediated renal vasoconstriction, resulting in an increased glomerular filtration rate and in the prevention of intratubular obstruction by protein casts. Conversely, in the heart, ANP decreases Pselectin expression by coronary endothelial cells and reduces neutrophil infiltration during reperfusion. 7 Recently, ANP has been shown to reduce liver ischemia/ reperfusion injury 8,9 and improve graft survival after liver transplantation. 10 These effects have been ascribed to a guanosine 3Ј, 5Ј-cyclic monophosphate (cGMP)-medi-

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A short period of hypoxia reduces the cytotoxicity produced by a subsequent prolonged hypoxia in isolated hepatocytes. This phenomenon, termed hypoxic preconditioning, is mediated by the activation of adenosine A2A-receptor and is associated with the attenuation of cellular acidosis and Na + overload normally occurring during hypoxia. Bafilomycin, an inhibitor of the vacuolar H + /ATPase, reverts the latter effects and abrogates the preconditioning-induced cytoprotection. Here we provide evidence that the acquisition of preconditioning-induced cytoprotection requires the fusion with plasma membrane and exocytosis of endosomal-lysosomal organelles. Poisons of the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actin cytoskeleton, prevented lysosome exocytosis and also abolished the preconditioning-associated protection from acidosis and necrosis provoked by hypoxia. Preconditioning was associated with the phosphatydilinositol 3-kinase-dependent increase of cytosolic [ [Ca 2+ ] ]. Chelation of free cytosolic Ca 2+ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosomeplasma membrane fusion is the mechanism through which hypoxic preconditioning allows hepatocytes to preserve the intracellular pH and survive hypoxic stress. This process is under the control of phosphatydilinositol 3-kinase and requires the integrity of the cytoskeleton and the rise of intracellular free calcium ions.

Ischemic preconditioning has been shown to improve liver resistance to hypoxia/reperfusion damage. A signal pathway involving A 2A -adenosine receptor, G i -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 A 2A -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 A 1 . SB203580 and bafilomycin A 1 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. ß