Effect of phospholipase A(2) inhibitors on the release of arachidonic acidand cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA (2) activity and cell viability in cold-stored rat hepatocytes. The cells w ere radiolabeled with [H-3] arachidonic acid (AA) and cold stored in the Un iversity of Wisconsin (UW) solution containing various PLA(2) inhibitors. P LA(2) activity was determined by measuring the total free (cellular + super natant) AA by thin-layer chromatography after inhibiting reacylation of fre e XA with inhibitors of energy production (carbonyl cyanide m-chlorophenylh ydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h c old storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 mu M), but its effe ct was limited to only 24 h, In contrast, pretreatment of the cells prior t o hypothermic preservation with trifluoperazine (10 to 100 mu M) suppressed PLA(2) activity during 48 h storage, Inclusion of calmodulin antagonist W- 7 did not affect PLA(2) activity. Thus, the inhibitory activity of these ag ents appears unrelated to Ca-calmodulin-phospholipid interaction but to hav e an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) i nhibitors on cell viability, lactate dehydrogenase (LDH) release was measur ed in the presence or absence of inhibitors upon rewarming cold-stored cell s in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) i nhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-st ored hepatocytes. (C) 2000 Academic Press.