Chain length specificity for activation of cPLA2alpha 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 >or=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 (EC(50) = 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 >or=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 deliver exogenous phospholipids to cells for the examination of specific biological effects.
Wijesinghe
Dayanjan S
DS
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
Subramanian
Preeti
P
Lamour
Nadia F
NF
Gentile
Luciana B
LB
Granado
Maria H
MH
Bielawska
Alicja
A
Szulc
Zdzislaw
Z
Gomez-Munoz
Antonio
A
Chalfant
Charles E
CE
D000473:Alkanes / Q000737:chemistry; D045744:Cell Line, Tumor; D002518:Ceramides / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D054513:Group IV Phospholipases A2 / Q000378:metabolism*; D006801:Humans; D013329:Structure-Activity Relationship
D000473:Alkanes; D002518:Ceramides; C065576:ceramide 1-phosphate; C007548:n-dodecane; D054513:Group IV Phospholipases A2