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Papers In Press, published online ahead of print October 1, 2009
J. Lipid Res., doi:10.1194/jlr.M800367-JLR200

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Journal of Lipid Research, Vol. 50, 1986-1995, October 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Chain length specificity for activation of cPLA₂ by C1P: use of the dodecane delivery system to determine lipid-specific effects

Dayanjan S. Wijesinghe^*, Preeti Subramanian^*, Nadia F. Lamour^*, Luciana B. Gentile^*, Maria H. Granado, Alicja Bielawska, Zdzislaw Szulc, Antonio Gomez-Munoz and Charles E. Chalfant^1,***,

^* Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA
Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country
Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC
^*** Research and Development, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, VA
Research and Development, The Massey Cancer Center, Richmond, VA

¹ To whom correspondence should be addressed. e-mail: cechalfant{at}vcu.edu

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A₂ (cPLA₂) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of 6 carbons efficiently activated cPLA₂ 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 cPLA₂, 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 C_18:1 C1P. Here, we demonstrate that C1P requires 6 carbon acyl-chain to activate cPLA₂. 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 cPLA₂ by C1P: use of the dodecane delivery system to determine lipid-specific effects.

Supplementary key words ceramide-1-phosphate • ceramide kinase • prostaglandins • phospholipase A₂ • inflammation • arachidonic acid • dodecane • eicosanoids

Abbreviations: AA, arachidonic acid; CERK, ceramide kinase; C1P, ceramide-1-phosphate; cPLA₂, group IVA cytosolic phospholipase A₂; EtOH, ethanol; GFP, green fluorescent protein; PA, phosphatidic acid; PAPC, 1-palmitoyl-2-arachidonoylphosphotidylcholine; PC, phosphatidylcholine; PDI, protein disulfide isomerase; PGE₂, prostaglandin E₂; SMase, sphingomyelinase

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