METABOLISM OF SHORT-CHAIN CERAMIDE AND DIHYDROCERAMIDE ANALOGS IN CHINESE-HAMSTER OVARY (CHO) CELLS

A series of radiolabelled ceramides (D-erythro and L-threo) and dihydr oceramides (DL-erythro and DL-threo) with 2, 4 or 6 carbon N-acyl grou ps were synthesized. These analogues were incubated with cultured CHO cells and radioactive products isolated and analyzed. In addition to s ynthesis of short-chain sphingomyelin and glucosylceramide, radiolabel led sphingosine and sphinganine were released from short-chain ceramid es and dihydroceramides and subsequently utilized for synthesis of lon g-chain ceramide and sphingolipids. Substrate preference for short-cha in sphingomyelin synthesis in cells was D-erythro-ceramides > L-threo- ceramides > DL-erythro-dihydroceramides > DL-threo-dihydroceramides, a nd C-4-and C-6-analogues were preferred over the C-2-analogue. Kinetic constants for conversion of short-chain (dihydro)ceramides to short-c hain sphingomyelin were determined using CHO cell membranes and found to correlate with substrate preference in cultured cells. D-erythro-C- 6-Ceramide was the preferred substrate for short-chain glucosylceramid e synthesis. D-erythro-C-2-ceramide inhibited incorporation of [H-3]se rine into sphingomyelin, glucosylceramide and ceramide rapidly (2 h) a nd in a dose-dependent manner. Over a similar time period, [H-3]cholin e-labelling of sphingomyelin was not affected. Inhibition of [H-3]seri ne-Labelling of sphingolipids appeared to correlate with release of [H -3]long-chain bases from short-chain ceramides and dihydroceramides an d synthesis of long-chain sphingolipids. However, some discrepancies b etween DL-erythro-C-4- and C-6-dihydroceramides, and D-erythro-C-2-cer amide suggested that short-chain dihydroceramides were less efficient in suppressing de novo synthesis from [H-3]serine, while contributing substantially to endogenous sphingolipid synthesis. Inhibition of de n ovo sphingolipid synthesis by short-chain ceramides and dihydroceramid es could not be related to inhibition of serine palmitoyltransferase a ctivity in vitro.