Ischemic preconditioning reduces Na+ accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia

Short periods of ischemia followed up by reperfusion are known to protect t he heart against injury caused by a subsequent sustained ischemia. This phe nomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknow n. By using isolated hepatocytes as an in vitro model of liver precondition ing, we have investigated the possible effect of preconditioning on intrace llular pH and Na+ homeostasis. Freshly isolated rat hepatocytes were precon ditioned by 10 minutes of incubation under hypoxic conditions followed up b y 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hyp oxia. Although preconditioning did not ameliorate adenosine triphosphate (A TP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Naaccumulation developing during hypoxia were appreciably reduced in precondi tioned cells. The effects of preconditioning on intracellular pH, Na+ homeo stasis, and citotoxicity were mimicked by stimulating protein kinase C (PKC ) with 4 beta-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glyce rol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking va cuolar proton ATPase (V-ATPase) with bafilomycin A(1) abolished the protect ion given by preconditioning or by PMA treatment on hypoxic acidosis, Na+ o verload, and hepatocyte killing. Similarly, the addition of Na+ ionophore m onensin also reverted the cytoprotection exerted by preconditioning. This i ndicated that ischemic preconditioning of isolated hepatocytes decreased ce ll 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 an d Na+ overload promoted by Na+-dependent acid buffering systems.