Liver
ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic
ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the
purine metabolites, such as
adenosine and
inosine, and studied the mode of their pharmacological actions. The human
hepatocellular carcinoma-derived cell line HepG2 was subjected to combined
oxygen-
glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h).
Adenosine or
inosine (300-1,000 µM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and
lactate dehydrogenase methods, respectively. The results showed that both
adenosine and
inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective
adenosine receptor antagonists. On the other hand, the
adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)
adenine hydrochloride (
EHNA, 10 µM) markedly and almost fully reversed the protective effect of
adenosine during COGD, while it did not influence the cytoprotective effect of
inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of
adenosine also involve intracellular conversion to
inosine. The likely interpretation of these findings is that
inosine serves as an alternative source of energy to produce
ATP during hypoxic conditions. The protective effects are also partially dependent on
adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6‑morpholino-pyridin-3-yl)pyrido[2,3-d]
pyrimidine, 2HCl (
ABT 702, 30 µM) significantly reversed the protective effect of both
adenosine and
inosine during
hypoxia and re-oxygenation. Collectively, the current results support the view that during
hypoxia,
adenosine and
inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of
inosine for protection against various forms of warm and cold liver
ischemia is relevant.