Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A2α and inducer of cell toxicity

Ceramide-1-phosphate (C1P) regulates cellular functions including arachidonic acid (AA) metabolism and modulates cell fate. The mechanism by which C1P is taken up is unclear, and the development of lipophilic analogs may be useful for regulating C1P's actions. We synthesized new mono- and di-methyl-ester (MM and DM, respectively) analogs of C1P with N-acyl chains of different lengths, and examined their effects on AA release and cell toxicity. Short-N-acyl-DM-C1P analogs including C5- and C6-DM-C1P, but not long-N-acyl-DM-C1P analogs, inhibited the release of AA mediated by α type cytosolic phospholipase A2 (cPLA2α) in Chinese hamster ovary (CHO) cells and the enzymatic activity. Short-N-acyl-DM-C1P analogs including C6-DM-C1P caused morphological changes with cell toxicity 24 h after the treatment in three cells lines (CHO, L929, and RLC-18 cells), although the role of AA in the toxicity was not clear. Neither long-N-acyl-DM-C1P analogs nor MM-C1P analogs including C6-MM-C1P affected cPLA2α activity and cell toxicity. Similar to C6-ceramide having a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) group on a C6-N-acyl chain (NBD-C6-ceramide), NBD-C6-DM-C1P and C6-DM-C1P-NBD (with a C6-N-acyl chain and an NBD-labeled C14-alkyl chain) were accumulated in the Golgi complex, although less C6-DM-C1P-NBD than NBD-C6-DM-C1P was taken up. NBD-C6-ceramide was converted to various metabolites including NBD-C6-sphingomyelin, but both NBD-C6-DM-C1P and C6-DM-C1P-NBD were stable in cells within 2 h. The short-N-acyl-DM-C1P analogs acted directly as an inhibitor of cPLA2α and an inducer of cell toxicity, and may be useful for the regulation of ceramide/C1P-regulated responses.

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