We investigated the effect of phospholipase A2 (PLA2) inhibitors on PLA2 activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [3H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA2 inhibitors. PLA2 activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m- chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA2 activity was also suppressed (58% of control) by trifluoperazine (50 μM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 μM) suppressed PLA2 activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA2 activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA2 activity. To study the effects of PLA2 inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37°C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA2 inhibitors blocked PLA2 activity, there was no suppression of LDH release. PLA2 may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes. (C) 2000 Academic Press.
- Phospholipase A
- Preservation injury