Phospholipase A2 enzymes differently impact PUFA release and oxylipin formation ex vivo in rat hearts

Phospholipase A₂ (PLA₂) enzymes cleave cell membrane phospholipids and release polyunsaturated fatty acids (PUFA), which can be converted into oxylipins. However, little is known about PLA₂ preference for PUFA, and even less is known about how this further impacts oxylipin formation. Therefore, we investigated the role of different PLA₂ groups in PUFA release and oxylipin formation in rat hearts. Sprague-Dawley rat heart homogenates were incubated without or with varespladib (VAR), methyl arachidonyl fluorophosphonate (MAFP) or EDTA. Free PUFA and oxylipins were determined by HPLC-MS/MS, and isoform expressions by RT-qPCR. Inhibition of sPLA₂ IIA and/or V by VAR reduced the release of ARA and DHA, but only DHA oxylipins were inhibited. MAFP reduced the release of ARA, DHA, ALA, and EPA, and the formation of ARA, LA, DGLA, DHA, ALA, and EPA oxylipins. Interestingly, cyclooxygenase and 12-lipoxygenase oxylipins were not inhibited. mRNA expression levels of sPLA₂ and iPLA₂ isoforms were highest whereas levels of cPLA₂ were low, consistent with activity. In conclusion, sPLA₂ enzymes lead to the formation of DHA oxylipins, while iPLA₂ is likely responsible for the formation of most other oxylipins in healthy rat hearts. Oxylipin formation cannot be implied from PUFA release, thus, both should be evaluated in PLA₂ activity studies.