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 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 [Ca²⁺]. Chelation of free cytosolic Ca²⁺ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosome-plasma 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.