N,N-DIMETHYLSPHINGOSINE INHIBITION OF SPHINGOSINE KINASE AND SPHINGOSINE 1-PHOSPHATE ACTIVITY IN HUMAN PLATELETS

Potential sphingosine (Sph) metabolites include phosphorylated, N-acyl ated, and N-methylated derivatives. Phosphorylated Sph, i.e., sphingos ine l-phosphate (Sph-1-P), may act as an autocrine stimulator of blood platelets, as it is abundantly stored in platelets and released extra cellularly and its exogenous addition induces platelet activation. In this study, we evaluated Sph-1-P formation and its effects in human pl atelets in the presence of other Sph metabolites. On addition of [H-3] Sph to intact platelets, the label was rapidly converted to Sph-1-P. T his conversion into [3H]Sph-1-P was inhibited by N,N-dimethylsphingosi ne (DMS) in a dose-dependent manner, but not by other structurally rel ated Sph derivatives, including ceramide. The inhibition of Sph-1-P fo rmation by DMS was reproduced using a cell-free system (Sph kinase obt ained from platelet cytosolic fractions) and much stronger than that b y DL-threo-dihydrosphingosine, which had been considered to be the str ongest inhibitor of Sph kinase. Administration of DMS to intact platel ets resulted in a decrease in Sph-1-P mass and an increase in Sph mass . Furthermore, DMS inhibited the release of Sph-1-P from platelets sti mulated with 12-O-tetradecanoylphorbol 13-acetate and inhibited platel et aggregation induced by exogenous addition of Sph-1-P. Collectively, our results indicate that DMS is useful as a Sph kinase inhibitor and that Sph-1-P actions as an autocrine stimulator of platelets are inhi bited by DMS.