Protein kinase C and platelet inhibition by D-erythro-sphingosine: comparison with N,N-dimethylsphingosine and commercial preparation.
Sphingosine has been shown to be a potent and specific inhibitor of protein kinase C in vitro and in cell systems including human platelets. Questions have been raised as to the validity of commercial sphingosine as a protein kinase C inhibitor and whether sphingosine or N,N-dimethylsphingosine is the active species. In the present study, we compared the effects of synthetic D-erythro-sphingosine, N,N-dimethylsphingosine and commercial sphingosine on purified protein kinase C in vitro and washed human platelets. These three compounds were found to be of high purity and well-defined structure based on [1H]NMR, FAB-mass Spectrometry, and TLC analysis. Both synthetic D-erythro-sphingosine and commercial sphingosine inhibited protein kinase C in vitro using vesicle as well as mixed micellar assays. N,N-dimethylsphingosine also significantly inhibited purified protein kinase C in vitro. Both preparations of sphingosine inhibited phosphorylation for 40 kD protein, a known substrate of protein kinase C in platelets. Similarly both sphingosine preparations inhibited aggregation and secretion of human platelets induced by 8 nM gamma-thrombin. These results indicate that sphingosine from commercial source, synthetic sphingosine and N,N-dimethylsphingosine are equipotent in inhibiting protein kinase C. These studies also validate the utility of sphingosine as a phamarcologic inhibitor of protein kinase C in vitro and in cell systems.
Khan
W A
WA
Department of Medicine, Duke University Medical Center, Durham, N. C. 27710.
Dobrowsky
R
R
el Touny
S
S
Hannun
Y A
YA
D001792:Blood Platelets / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011493:Protein Kinase C / Q000097:blood*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology*
D007202:Indicators and Reagents; D010975:Platelet Aggregation Inhibitors; D011493:Protein Kinase C; C061800:N,N-dimethylsphingosine; D013110:Sphingosine