Preconditioning-induced cytoprotection in hepatocytes requires Ca2+-dependent exocytosis of lysosomes.

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 A_2A-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 3methyladenine, which inhibit phosphatydilinositol 3kinase, 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 [Ca²⁺]. Chelation of free cytosolic Ca²⁺ 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. [ABSTRACT FROM AUTHOR]