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Effect of Cytidine on Membrane Phospholipid Synthesis in Rat Striatal Slices

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Effect of Cytidine on Membrane Phospholipid Synthesis in Rat Striatal Slices Journal of Neurochemi,enry Raven Press, Ltd ., New York © 1995 International Society for Neurochemistry Effect of Cytidine on Membrane Phospholipid Synthesis in Rat Striatal Slices Vahide Savci and Richard J . Wurtman Delzcartrnent of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, U .S .A . Abstract : Using rat striatal slices, we examined the effect cell culture systems and in vivo experiments, exoge- of cytidine on the conversion of [ 3H]choline to [ 3H]- nous cytidine has been shown to be taken up into cells phosphatidylcholine ([ 3 H]PC), and on net syntheses of and converted sequentially to CMP, CDP, and CTP PC, phosphatidylethanolamine (PE), and phosphatidyl- (Plagemann, 1971a,b ; Trovarelli et al ., 1982 ; 1984 ; serine, when media did or did not also contain choline, Lopez G.-Coviella and Wurtman, 1992) . CTP is re- ethanolamine, or serine . Incubation of striatal slices with quired to form key intermediates in the biosynthesis cytidine (50-500 NM) caused dose-dependent increases in intracellular cytidine and cytidine triphosphate (CTP) of both phosphatidylcholine (PC) and phosphati- levels and in the rate of incorporation of [ 3H]choline into dylethanolamine (PE) (quantitatively the most sig- 3 membrane [ H] PC . In pulse-chase experiments, cytidine nificant phospholipids of eukaryotic cells) via the Ken- (200 N,M) also increased significantly the conversion of nedy pathway (Kennedy and Weiss, 1956 ; Pelech and [ 3H]choline to [ 3 H]PC during the chase period . When Vance, 1984 ; Vance, 1985) and also in the biosynthe- slices were incubated with this concentration of cytidine sis of phosphatidylinositol (Vance, 1985 ; Majerus, for 1 h, small (7%) but significant elevations were ob- 1992 ; Pike, 1992) . This pathway involves, sequen- served in the absolute contents (nmol/mg of protein) of tially, the phosphorylation of choline or ethanolamine, membrane PC and PE (p < 0 .05), but not phosphatidyl- combination of the resulting phosphocholine or phos- serine, the synthesis of which is independent of cytidine- CTP generate 5'-cytidinedi- containing CTP . Concurrent exposure to cytidine phoethanolamine with to (200 or 5'-cytidinediphos- NM) and choline (10 pM) caused an additional significant phosphocholine (CDP-choline) increase (p < 0.05) in tissue PC levels beyond that pro- phoethanolamine (CDP-ethanolamine), and, finally, duced by cytidine alone . Exposure to choline alone at a their combination with diacylglycerol (DAG) to yield higher concentration (40 N,M) increased the levels of all PC or PE (Kennedy and Weiss, 1956 ; Pelech and three membrane phospholipids (p < 0 .01) ; the addition Vance, 1984) . The rate of PC biosynthesis apparently of cytidine, however, did not cause further increases . depends upon the activity of CTP :phosphocholine cyti- Concurrent exposure to cytidine (200 ^ and ethanol- dylyltransferase, which catalyzes the formation of amine (20 ^ also caused significantly greater eleva- CDP-choline from phosphocholine and CTP (Pelech tions < in PE than those caused by (p 0.05) tissue levels and Vance, 1984) . Brain CTP levels are low (Mandel cytidine alone . In contrast, the addition of serine (500 and Edel-Harth, 1966 ; Abe et al ., 1987) and below did not enhance cytidine's effects on any membrane phospholipid . Exposure to serine alone, however, like ex- this enzyme's apparent K , for the nucleotide (Pelech posure to sufficient choline, increased levels of all three and Vance, 1984 ; Mages et al ., 1988) . Hence, we sus- membrane phospholipids significantly (p < 0.01) . These pected that exogenous cytidine might, by increasing data show that exogenous cytidine, probably acting via intracellular CTP levels, affect brain phospholipid syn- CTP and the Kennedy cycle, can increase the synthesis thesis . This hypothesis was previously confirmed in a and levels of membrane PC and PE in brain cells . Key stable cell line of PC 12 cells (Lopez G.-Coviella and Words: Cytidine-Choline-Phosphatidylcholine-Phos- Wurtman, 1992) but had not yet been examined in pholipid-Ethanolamine-Serine-Striatum . animal tissue . J. Neurochem. 64, 378-384 (1995) . Received December 22, 1993 ; final revised manuscript received May 23, 1994 ; accepted May 23, 1994 . Address correspondence and reprint requests to Dr . R . J . Wurtman The nucleoside cytidine, once it is phosphorylated at Department of Brain and Cognitive Sciences, E25-604, Massachu- (Santos et al ., 1968) to form the nucleotides cytidine setts Institute of Technology, Cambridge, MA 02139, U .S .A . triphosphate (CTP), cytidine diphosphate (CDP), and Abbreviations used: CDP, cytidine diphosphate ; CDP-choline, 5'- cytidinediphosphocholine ; CDP-ethanolamine, 5'-cytidinediphos- cytidine monophosphate (CMP), exerts important ef- phoethanolamine ; CMP, cytidine monophosphate ; CTP, cytidine tri- fects on a variety of brain functions (Geiger and Yama- phospate ; DAG, diacylglycerol ; PC, phosphatidylcholine; PE, phos- saki, 1956 ; Roberts, 1973 ; Drago et al ., 1990) . In both phatidylethanolamine ; PS, phosphatidylserine . 378 CYTIDINE PHOSPHOLIPID SYNTHESIS IN RAT BRAIN 379 In this study, we investigated the effects of cytidine KH 2 PO 4 , 4% acetonitrile, pH 6 : solvent B, 50% acetonitrile on the synthesis and levels of PC, PE, and phosphati- in solvent A; 35-min concave gradient (no . 3) to 50% of dylserine (PS) in striatal slices, as well as its interac- solvent B ; at 1 .5 ml/min, with a 25-min equilibration delay . tions with choline, ethanolamine, and serine in affect- Extraction and separation of phospholipids and ing phospholipid production . water-soluble choline-containing metabolites After incubation, slices were rinsed several times with ice-cold Krebs solution and homogenized in 1 ml of distilled MATERIALS AND METHODS water containing eserine (20 pM) . Three milliliters of meth- Slice preparation and experimental procedure anol/chloroform (1 :2 ; vol/vol) was then added . After cen- Male Sprague-Dawley rats (250-280 g) (Charles River, trifugation, both the organic phase ( 1,800 pl ) (locus of phos- Cambridge, MA, U.S .A .) were killed by decapitation and pholipids) and the aqueous phase (1,800 p.l) (locus of water- brains were placed in ice-cold Krebs buffer (mM : NaCl 120, soluble choline-containing metabolites) were dried under KCl 3 .5, CaC1 2 1 .3, MgS0 4 1 .2, NaH2PO 4 1 .2, NaHC0 3 25, vacuum . glucose 10, eserine salicylate 0.02) . The buffer was gassed Aliquots of the lower phase were used for determination with a mixture of 95% 02 + 5% C02 for at least 30 min of phosphorus and for separation of individual classes on before use . Striata were rapidly excised, and slices (0 .3 mm) of phospholipids . These classes were purified by TLC were prepared using a McIlwain tissue chopper (Mickle Lab- silica gel G plates (LK-6D plates, Whatman), using a sys- oratory Engineering Co ., Gomstall, U.K .) . The slices were tem consisting of chloroform/ethanol/ triethylamine/water et washed several times with Krebs buffer solution and trans- (30:34:30:8, by volume) as a mobile phase (Touchstone ferred to incubation tubes (five to seven slices per tube) ; al ., 1980) . Bands containing the individual phospholipids these were placed in a Dubnoff shaker and incubated at 37°C were scraped off the plates and their radioactivities counted for the periods indicated. During incubation periods media by liquid scintillation spectrophotometry ; the total amount were replaced every 10 min with fresh Krebs buffer continu- of each phospholipid was determined by phosphate assay ously bubbled with 95% 02 + 5% COZ . After a 60-min (Svanborg and Svennerholm, 1961) . equilibration period, incubation media were discharged ; Aliquots of the aqueous phase (containing water-soluble, slices were then incubated for 1 h with l ml of Krebs buffer choline-containing metabolites) were separated by TLC on containing 5 p,Ci of [ ;H]choline (86.7 Ci/mmol, Du Pont- silica gel G plates (LK-5D plates, Whatman) . The following New England Nuclear) (final concentration of choline per composition was used as a mobile phase : acetonitrile, etha- tube was 65 nM) and different concentrations of cytidine nol, 30% NH 4 0H, 8.3°h NaCl (40:8 :3 :24, by volume) (Slack (50-500 pM) . et ad ., 1991 ) . Corresponding hands containing choline, ace- To determine the effect of incubation of slices with exoge- tylcholine, and phosphocholine were scraped and their radio- nous cytidine on tissue cytidine and nucleotides, slices were activities counted . incubated with different concentrations of cytidine (50-200 Measurement of choline and acetylcholine pM) for 1 h . For time-course experiments, incubations were During the extraction of lipids, the upper aqueous phase of 10-, 30-, or 60-min duration. Two different sets of experi- (1,800 pl) was separated for determination of choline and ments were done for cytidine and nucleotide measurements . acetylcholine and dried under vacuum. Aliquots of dried In some experiments, unlabeled choline (10, 40 pM), aqueous phases were resuspended in water and subjected to ethanolamine (20 pM), or serine (500 pM) was added to HPLC analysis using polymeric reverse-phase column incubation media, alone or together with cytidine (200 pM), (BAS, West Lafayette, IN, U.S .A .) with a mobile phase of before a 1-h incubation . 50 mM phosphate (pH 8.5) containing 0.005% Kathon CG (Rohm and Hass, Philadelphia, PA, U .S .A .) as a bactericide . Extraction and analysis of cytidine and Once separated, acetylcholine and choline were converted nucleotides
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