Chain length specificity for activation of cPLA2α by C1P: use of the dodecane delivery system to determine lipid-specific effects

Abstract

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A2 (cPLA2α) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of ≥6 carbons efficiently activated cPLA2α in vitro, whereas C2-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA2α, AA release, and PGE2 synthesis (EC50 = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C2-C1P was shown to be ineffective in the induction of AA release as compared with C6-C1P, C16-C1P, and C18:1 C1P. Here, we demonstrate that C1P requires ≥6 carbon acyl-chain to activate cPLA2α. Thus, published reports on the biological activity of C2-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.—Wijesinghe, D. S., P. Subramanian, N. F. Lamour, L. B. Gentile, M. H. Granado, A. Bielawska, Z. Szulc, A. Gomez-Munoz, and C. E. Chalfant. Chain length specificity for activation of cPLA2α by C1P: use of the dodecane delivery system to determine lipid-specific effects.

Keywords

• ceramide-1-phosphate
• ceramide kinase
• prostaglandins
• phospholipase A2
• inflammation
• arachidonic acid