Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells

The cellular mechanisms by which ischemic preconditioning increases liver tolerance to ischemia/reperfusion injury are still poorly understood. This study investigated the role of the hypoxia-inducible factor-1 (HIF-1) in the protection associated with the late phase of liver preconditioning. Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 minute) hypoxic stress or by 15 minutes incubation with the adenosine A _2A receptors agonist CGS21680 24 hours before exposure to 90 minutes of hypoxia in a serum-free medium. Late preconditioning induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a HIF-1-regulated transmembrane enzyme that catalyzes bicarbonate production. Such effects were associated with prevention of hepatocyte killing by hypoxia and the amelioration of intracellular acidosis and Na⁺ accumulation. The inhibition of PKC-mediated and PI3-kinase-mediated signals with, respectively, chelerythrine and wortmannin abolished HIF-1 activation and blocked both CAIX expression and the protective action of late preconditioning. CAIX expression was also prevented by interfering with the transcriptional activity of HIF-1 using a dominant negative HIF-1β subunit. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4-acetamido- 4′-isothiocyanato-stilben-2,2′-disulfonate also reverted the protective effects of late preconditioning on intracellular acidosis and Na ⁺ accumulation. Conclusion: The stimulation of adenosine A _2A receptors induced late preconditioning in liver cells through the activation of HIF-1. HIF-1-induced expression of CAIX increases hepatocyte tolerance to ischemia by maintaining intracellular Na⁺ homeostasis. These observations along with the importance of HIF-1 in regulating cell survival indicates HIF-1 activation as a possible key event in liver protection by late preconditioning.