Utilization of Caged Ceramide and Ceramide 1-Phosphate Analogs for Monitoring Cellular Events after Photoactivation

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Caged compounds possess a photolabile covalent bond. This chapter reviews studies of caged derivatives of two important sphingolipid signaling molecules, ceramide and ceramide 1-phosphate. Biophysical studies were carried out after a 6-bromo-7-hydroxycoumarinyl-ceramide conjugate was inserted into model bilayer membranes. Uncaging with long-wavelength UV light liberated N-palmitoylceramide, and reorganization of lipid domains in the bilayer was monitored. Two derivatives of N-palmitoyl-ceramide 1-phosphate in which the phosphate group was esterified to a caging group were investigated in macrophages; in one derivative the cage is 7-(N,N-diethylamino)coumarin (DECM-C1P)while in the other it is a 4-bromo-5-hydroxy-2-nitrobenzhydryl moiety (BHNB-C1P). The caged derivatives were delivered to macrophages in aqueous solution. The photolytic uncaging process then released ceramide 1-phosphate in the cytosol of macrophages, which was accompanied by stimulation of macrophage proliferation, reactive oxygen species production, and other intracellular signaling events. A distinction can thus be made in some cells between extracellular events evoked by ceramide 1-phosphate, as for example by its interaction with a putative cell-surface receptor, from its intracellular bioactivities. These studies show that elevation of ceramide or ceramide 1-phosphate levels by uncaging of their inactive caged forms enable investigations of a wide variety of biophysical and biochemical processes.