N-acetylsphingosine stimulates phosphatidylglycerolphosphate synthase activity in H9c2 cardiac cells

Cardiolipin and phosphatidylglycerol biosynthesis were examined in H9c2 cells incubated with short-chain ceramides. Incubation of cells with N-acetylsphingosine or N-hexanoyl-sphingosine stimulated [1,3-H-3]glycerol incorporation into phosphatidylglycerol and cardiolipin, with N-acetylsphingosine having the greater effect, The mechanism for the ceramide-mediated stimulation of de novo phosphatidylglycerol and cardiolipin biosynthesis appeared to be an increase in the activity of phosphatidylglycerolphosphate synthase, the committed step of phosphatidylglycerol and cardiolipin biosynthesis. The presence of the potent protein phosphatase inhibitors calyculin A or okadaic acid attenuated the N-acetylsphingosine-mediated stimulation of phosphatidylglycerolphosphate synthase activity and of phosphatidylglycerol and cardiolipin biosynthesis, indicating the involvement of a ceramide-activated protein phosphatase(s). The presence of 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated enzyme activity and [1,3-H-3]glycerol incorporation into phosphatidylglycerol and cardiolipin. The effects of CPT-cAMP and N-acetylsphingosine on phosphatidylglycerol and cardiolipin bio synthesis and on phosphatidylglycerolphosphate synthase activity were additive. Phosphatidylglycerol biosynthesis from sn-[C-14]glycerol 3-phosphate in permeabilized H9c2 cells was stimulated by preincubation with N-acetylsphingosine, and this was attenuated by okadaic acid. N-Acetylsphingosine treatment of cells elevated mitochondrial phospholipase A(2) activity. Since the pool sizes of phosphatidylglycerol and cardiolipin were unaltered in these cells, the observed increase in phosphatidylglycerolphosphate synthase activity may be a compensatory mechanism for the N-acetylsphingosine-mediated elevation of mitochondrial phospholipase A(2) activity. Finally, addition of tumour necrosis factor alpha to H9c2 cells resulted in an elevation of both phosphatidylglycerolphosphate synthase and phospholipase A(2) activities. The results suggest that phosphatidylglycerol and cardiolipin metabolism in H9c2 cells may be regulated by intracellular ceramide signalling.