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

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of >= 6 carbons efficiently activated cPLA(2)alpha in vitro, whereas C-2-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA(2)alpha, AA release, and PGE(2) 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, C-2-C1P was shown to be ineffective in the induction of AA release as compared with C-6-C1P, C-16-C1P, and C-18:1 C1P. Here, we demonstrate that C1P requires = 6 carbon acyl-chain to activate cPLA(2)alpha. Thus, published reports on the biological activity of C-2-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently de-liver 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 cPLA(2)alpha by C1P: use of the dodecane delivery system to determine lipid-specific effects. J. Lipid Res. 2009. 50: 1986-1995.