Figure 3

S1P induces cellular S1P-production and -secretion through G _i protein coupled receptors. A. HeLa cells were exposed to either vehicle or 3 μM S1P for the indicated period of times in the presence of [³H]sphingosine. The amount of produced [³H]S1P is expressed as percent of that in vehicle-treated cells. B. Secreted [³H]S1P was extracted from the cell culture medium following a 10 minute stimulation with vehicle or 3 μM S1P, and was analyzed using thin layer chromatography and scintillation counting. C. The cells were treated with 50 ng/ml pertussis toxin or vehicle for 16 h. Cellular lipids were extracted following a 10 minute stimulation with 3 μM S1P or vehicle in the presence of [³H]sphingosine. The formed [³H]S1P was assayed using thin layer chromatography and scintillation counting. D. Concentration-response curve for S1P-induced S1P production. HeLa cells were stimulated with varying concentrations of S1P or vehicle together with [³H]sphingosine for 10 minutes. Cellular lipids were extracted and separated by HPTLC. The results are expressed as percent increased [³H]S1P production in S1P stimulated cells compared with control. The data points and bars in panels A-D denote the mean ± SEM of at least three independent experiments (*, p < 0.05).