Contribution of pH-dependent group II phospholipase A₂ to chemical hypoxic injury in rat hepatocytes

Numerous studies have suggested that enhanced membrane phospholipid degradation contributes to hypoxic and ischemic injury. Recently, acidosis has been found to potently protect against hypoxic and ischemic injury. To investigate the interrelationships of these two events in hypoxic injury, we studied the role of a pH-dependent group II phospholipase A₂ (PLA₂, E.C. 3.1.1.4) in chemical hypoxic injury in rat hepatocytes. Northern blot analysis of RNA extracted from normoxic and hypoxic rat hepatocytes with group II rat liver PLA₂-specific oligonucleotide cDNA probes revealed a 0.9 kb transcript whose abundance was significantly increased in rat hepatocytes within 15 min after initiation of chemical hypoxia and remained high until cells lost viability. Immunofluorescence staining of hepatocytes with polyclonal antibodies that recognize group II PLA₂ demonstrated a substantial increase in the level of PLA₂ protein in hypoxic rat hepatocytes within 30 min of initiation of hypoxic injury. Treatment of hepatocytes with group II PLA₂-specific anti-sense DNA oligonucleotides: 1) abolished accumulation of PLA₂ protein in hypoxic rat hepatocytes as assessed by immunofluorescence staining with anti-PLA₂ antibodies; 2) decreased the enzymatic activity of PLA₂ manifested as decreased arachidonic acid release in hypoxic hepatocytes; and 3) significantly delayed cell death evoked by chemical hypoxia as indicated by a decrease in propidium iodide uptake. These findings suggest that pH-dependent group II PLA₂ plays an important role in chemical hypoxic injury of rat hepatocytes.