Nitric oxide (NO) improves liver resistance to hypoxia/reperfusion injury acting as a mediator of hepatic preconditioning. However, the mechanisms involved are still poorly understood. In this study, we have investigated the mechanisms by which short-term exposure to the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])-diazen-1-ium-1,2-diolate (NOC-9) increases hepatocyte tolerance to hypoxic injury. Isolated rat hepatocytes preincubated 15 min with NOC-9 (0.250 mM) became resistant to the killing caused by hypoxia. NOC-9 cytoprotection did not involve the activation of protein kinase C, but was instead blocked by inhibiting soluble guanylate cyclase with 1H-(1,2,4)-oxadiazolo-(4,3) quinoxalin-1-one (ODQ) (50 microM) or cGMP-dependent kinase (cGK) with KT 5823 (5 microM). Conversely, cGMP analogue, 8Br-cGMP (50 microM) mimicked the effect of NOC-9. Western blot analysis revealed that hepatocyte treatment with NOC-9 or 8Br-cGMP significantly increased dual phosphorylation of p38 MAPK. The activation of p38 MAPK was abolished by inhibiting guanylate cyclase or cGK. Pretreatment with NO significantly reduced intracellular Na(+) accumulation in hypoxic hepatocytes. This effect was reverted by KT 5823 as well as by the p38 MAPK inhibitor SB203580. SB203580 also reverted NOC-9 protection against hypoxic injury. Altogether, these results demonstrated that NO can induce hepatic preconditioning by activating p38 MAPK through a guanylate cyclase/cGK-mediated pathway.