The Influence of Phospholipase A 2 (PLA 2 ) Enzymes on Fatty Acid Release and Oxylipin Formation in Rat Hearts

Abstract

Polyunsaturated fatty acids (PUFA) released by PLA2 are converted into diverse bioactive lipid mediators called oxylipins. However, little is known about the preference of PLA2 isoforms for PUFA release, and even less is known about how this further impacts the formation of oxylipins. Further, oxylipin levels in tissues do not always mimic the levels of their precursor PUFA, so conclusions about oxylipins cannot be drawn from PUFA content. Therefore, we aimed to determine the impact of PLA2 isoforms on PUFA release and oxylipin formation. We hypothesized that different PLA2 isoforms would impact PUFA release and oxylipin formation differently, and that oxylipin formation would not necessarily reflect changes seen in PUFA. Rat heart homogenates were incubated at 37C for 20 min with or without inhibitors, as the increase of both free PUFA and oxylipins was linear at 20 min incubation. Free PUFA and oxylipins were analyzed by HPLC-MS/MS. Two inhibitors were used: Varespladib (VAR) targeting secreted (sPLA2) isoforms IIA, V and X, and methyl arachidonyl fluorophosphonate (MAFP) targeting cytosolic (cPLA2) and calcium-independent (iPLA2) isoforms. Arachidonic Acid (ARA) release was similarly inhibited by both VAR and MAFP, however, while 15 out of 29 ARA oxylipins were inhibited with MAFP, only 3 out of 29 were inhibited with VAR. Docosahexaenoic acid (DHA) release was more strongly inhibited by VAR than by MAFP, which corresponded with 7 out of 12 DHA oxylipins being more strongly inhibited by VAR than by MAFP. Both alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA) were only inhibited by MAFP, which also inhibited 3 out of 5 ALA and 1 out of 3 EPA oxylipins. Further, 9 out of 10 LA oxylipins were inhibited by MAFP while none were affected by VAR. In conclusion, in the rat heart ARA is released by sPLA2 IIA, V, and/or X as well as by cPLA2 and/or iPLA2, DHA is released more by sPLA2 IIA, V, and/or X than by cPLA2 and/or iPLA2, and EPA and ALA are released primarily by cPLA2 and/or iPLA2. Effects on oxylipin formation largely reflected those on precursor PUFA release, with an exception, as inhibition of sPLA2 IIA, V and X by VAR mostly did not affect ARA oxylipin formation even though it inhibited ARA release. Therefore, careful evaluation of the effects on the oxylipin profile is needed when considering PLA2 inhibitors for potential treatments of diseases.