CONVERSION OF DIACYLGLYCEROL TO PHOSPHATIDYLCHOLINE ON THE BASOLATERAL SURFACE OF EPITHELIAL (MADIN-DARBY CANINE KIDNEY) CELLS - EVIDENCE FOR THE REVERSE ACTION OF A SPHINGOMYELIN SYNTHASE

When ,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-P A) is inserted into the plasma membrane of fibroblasts, it is metaboli zed by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -pho sphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034 -2040). In Madin-Darby canine kidney (MDCK) cells incubated at 10-degr ees-C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no Co-NBD-PE could be depleted from the basolateral cell surface by t he addition of bovine serum albumin to the medium. Preincubation of th e cells with [H-3]choline for 2 h at 37-degrees-C prior to C6-NBD-PA a ddition at 10-degrees-C labeled non-depletable C6-NBD-PC with a specif ic activity of >10 times that of the depletable C6-NBD-PC on the basol ateral cell surface, indicating that the latter had not been synthesiz ed by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-P A as substrate for both intracellular and surface C6-NBD-PC synthesis. In addition, C6-NBD-PC synthesis on the cell surface was independent of the location of the C6-NBD-chain on the 1- or 2-position, indicatin g that the reaction occurred by transfer of phosphorylcholine. Using C 6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovere d on the basolateral but not on the apical cell surface. The conversio n of PC plus ceramide to DG and SM on the basolateral MDCK cell surfac e suggests that,the synthesis of C6-NBD-PC on this surface occurred vi a the reverse reaction of a SM synthase. Indeed, the surface C6-NBD-PC synthesis was reduced to 40-50% by addition of C6-NBD-ceramide or hyd rolysis of cell surface SM by exogenous neutral sphingomyelinase. Sinc e DG activates protein kinase C and ceramide indirectly inhibits,this kinase but activates other kinase(s) and phosphatase(s), the phosphoch oline transferase at the cell surface may have a regulatory role in si gnal transduction.