Abstract
When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (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 degrees C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no C6-NBD-PE could be depleted from the basolateral cell surface by the addition of bovine serum albumin to the medium. Preincubation of the cells with [3H]choline for 2 h at 37 degrees C prior to C6-NBD-PA addition at 10 degrees C labeled non-depletable C6-NBD-PC with a specific activity of > 10 times that of the depletable C6-NBD-PC on the basolateral cell surface, indicating that the latter had not been synthesized by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-PA 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, indicating that the reaction occurred by transfer of phosphorylcholine. Using C6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovered on the basolateral but not on the apical cell surface. The conversion of PC plus ceramide to DG and SM on the basolateral MDCK cell surface suggests that the synthesis of C6-NBD-PC on this surface occurred via 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 hydrolysis of cell surface SM by exogenous neutral sphingomyelinase. Since DG activates protein kinase C and ceramide indirectly inhibits this kinase but activates other kinase(s) and phosphatase(s), the phosphocholine transferase at the cell surface may have a regulatory role in signal transduction.
Highlights
From the Department of Cell Bwbgy, Medical School,AZU H02.314, University of Utrecht, Heidelberglaan 100,3584 CX Utrecht, The Netherlands and TDepartment of Anatomy, College of Medicine, University of Iowa, Iowa City, Iowa 52242
Preincubation of the cells with [SH]cholinefor ogeneous distribution is presented by the plasma membrane 2 h at 37 O C prior to Ce-NBD-phosphatidic acid (PA) addition at 10 OC of epithelial cells of kidney and intestine, where the apical labeled non-depletable Ca-NBD-PCwith a specific ac- domain, lining the body cavities, has a PC contentht at isfour tivity of >10 times that of the depletable Ce-NBD-PC times lower than that of the basolateral domain, which faces on the basolateralcell surface, indicatingthat thelat- the neighboring cells andthe underlying tissue [1]
Presence of Newly Synthesized c6-NBD-PC on the Cell,S'urfuce"In a first series of experiments, C,NBD-PA was added to Madin-Darby canine kidney (MDCK) cells for 3 h at 10 "C as described under "Experimental Procedures." This resulted in thesynthesis of 23 f 7 pmol of Cs-NBD-PC/107cells ( n = 13)
Summary
Ardy van Helvoort, Wouter van’t Hof$, Tita Ritsemaj, Alex Sandra(, and Gerrit vaMneer 11. Preincubation of the cells with [SH]cholinefor ogeneous distribution is presented by the plasma membrane 2 h at 37 O C prior to Ce-NBD-PA addition at 10 OC of epithelial cells of kidney and intestine, where the apical labeled non-depletable Ca-NBD-PCwith a specific ac- domain, lining the body cavities, has a PC contentht at isfour tivity of >10 times that of the depletable Ce-NBD-PC times lower than that of the basolateral domain, which faces on the basolateralcell surface, indicatingthat thelat- the neighboring cells andthe underlying tissue [1] This ter had not been synthesized by the CDP-choline path- difference in lipid composition may be caused by local syncpNcwsPrUsneyueeloCBasuallnbtillycsDnstssos.thyctgiuu-nreConecCarrsrtnhefftthiaeahea-saome-NcceiNcsanfeeBicioalBdsosusprD.ewonuDriIb-rcgant-wDetaoghcsodaliGtaeeDenhdsrscGda1dtcbesidme-ioynlatpitluoisithneodrlcrstaddnarneuo2tacd,s,vrS-neeuCfepMtaClnsbhrsolcfteus-easoeeNdto-slinr.iNatyftBiTorunotoBtnhDhthntfaeDheee,-ntepPh-ifdlsnsbhcoCoeipodaorcssshsnbisuaCyociopvatrnfnaailhfseogat-taCoorihNnotcsnlereemiaeyBgor-osCtaNflyieDntclsshterMBh--haoolaNPoDilenDftnlABbi-CctPnPtuhDaeh(KCeCtesS--e.
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