CELL-PERMEABLE CERAMIDES INHIBIT THE STIMULATION OF DNA-SYNTHESIS ANDPHOSPHOLIPASE-D ACTIVITY BY PHOSPHATIDATE AND LYSOPHOSPHATIDATE IN RAT FIBROBLASTS

The interactions of ceramides with phosphatidate and lysophosphatidate in the regulation of signal transduction in rat fibroblasts were exam ined. C-2- and C-6-ceramides (N-acetylsphingosine and N-hexanoylsphing osine, respectively) at 10 mu M inhibited the stimulation of DNA synth esis that was produced by 50-100 mu M phosphatidate, or lysophosphatid ate, or by exogenous phospholipase D. Sphingosine (10 mu M) had the op posite effect to the ceramides on DNA synthesis. C-2- or C-6-ceramides failed to inhibit the stimulation of DNA synthesis by insulin or seru m. The ceramides did not modify the actions of phosphatidate, or lysop hosphatidate, in decreasing the forskolin-induced increase in cAMP. C- 2- and C-6-ceramides inhibited the stimulation of phospholipase D acti vity by: (a) phosphatidate, lysophosphatidate, phorbol ester, thrombin , or serum in intact fibroblasts and (b) phorbol ester or guanosine 5' -3-O-(thio)triphosphate in permeabilized fibroblasts. The ceramides ca n therefore modify cell signaling via phospholipase D, but this effect alone could not explain the decreased DNA synthesis. Incubation of fi broblasts with C-2- or Cs-ceramides or sphingomyelinase inhibited the interaction of exogenous phosphatidate or lysophosphatidate with the f ibroblasts by 42 and 53%, respectively. Furthermore, a greater proport ion of the phosphatidate, or lysophosphatidate, that was associated wi th the fibroblasts was metabolized further when the cells were pretrea ted with ceramides or sphingomyelinase. This effect was accompanied by an increased activity of N-ethylmaleimide-insensitive phosphatidate p hosphohydrolase. Ceramides may therefore produce part of their growth inhibitory effects by blocking some of the signal transducing effects of phosphatidate and lysophosphatidate.