Fig. 6.

Effect of scyphostatin on C₆-NBD-Cer formation. A, Rat embryonic cerebral cortex was homogenized in TK buffer, pH 7.4 (with or without 10 mm MgCl₂) or in 10 mm MES buffer, pH 4.7, for analysis of SMase activities. Homogenates (20 μg of protein) were incubated for 15 min with scyphostatin (dissolved in ethanol; corresponding amounts of ethanol were added to control samples), followed by a 30 min incubation with 10 μmC₆-NBD-SM. After terminating the reaction, C₆-NBD-Cer formation was analyzed. Results are means ± SEM of three independent experiments. The slope of A-SMase activity is significantly different from that of N-SMase activity (with or without Mg) (p < 0.0001).B, Top, Coverslips containing neurons were placed in dishes that did not contain a glial monolayer and incubated for 1 hr with or without scyphostatin (1 μm), followed by an additional 1 hr incubation after addition of 1.5 μm C₆-NBD-SM. NGF (100 ng/ml) was then added and, after a further 1 hr, the amount of C₆-NBD-Cer formed was analyzed. Data are means ± SEM for three independent experiments. Bottom, Neuronal morphology was analyzed 18 hr after adding reagents. Results are means of the number of cells in stage 3 as a percent of the control ± SEM for three different cultures in which 50 cells per coverslip were analyzed for four coverslips per treatment (bottom). *p < 0.01, statistically significant differences; ANOVA.