Sphingosine reverses growth inhibition caused by activation of protein kinase c in vascular smooth muscle cells

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1, 2‐dioctanoyl‐snglycerol (DiC8), when added 2 h after α‐thrombin, reverses by 95% the induction of DNA synthesis in VSM cells by α‐thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N‐acetylsphingosine and C₁₁‐sphingosine were used to investigate this phenomenon further. Neither phorbol 12‐myristate 13‐acetate (PMA;200 ng/ml) nor sphingosine (up to 10 μM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by α‐thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose‐dependent manner with complete reversal at 10 μM. Neither N‐acetylsphingosine nor C₁₁‐sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III‐S phosphorylation. Protein kinase C activity was inhibited 50% by 300 μM sphingosine, but 15% by similar concentrations of N‐acetylsphingosine and C₁₁‐sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [³H]phorbol dibutyrate binding. Sphingosine (at > 5 μM), but not N‐acetylsphingosine or C₁₁‐sphingosine, blocked [³H]phorbol dibutyrate binding in a dose‐ and time‐dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.