Journal of NeurochemistryRaven Press, Ltd., New York

@ 1992 International Society for Neurochemistry

Enhancement by Cytidine of MembranePhospholipid Synthesis

Ignacio Lopez G.-Coviella and Richard J. Wurtman

Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A.

Abstract: Cytidine, as cytidine 5'-diphosphate choline, is amajor precursor in the synthesis of phosphatidylcholine incell membranes. In the present study, we examined the rela-tionships between extracellular levels of cytidine, the con-version of (14C]cholineto (14C]phosphatidylcholine, and thenet syntheses of phosphatidylcholine and phosphatidyleth-anolamine by PCl2 cells. The rate at which cytidine (as(3H]cytidine) was incorporated into the PCl2 cells followednormal Michaelis-Menten kinetics (Km = 5p.M;Vmax = 12X 10-3 mmol/mg of protein/min) when the cytidine con-centrations in the medium were below 50 p.M; at higherconcentrations, intracellular (3H]cytidine nucleotide levelsincreased linearly. Once inside the cell, cytidine was con-verted mainly into cytidine triphosphate. In pulse-chase ex-periments, addition of cytidine to the medium caused a

time- and dose-<lependent increase (by up to 30%) in theincorporation of (14C]choline into membrane (14C]_phosphatidylcholine. When the PCl2 cells were supple-mented with both cytidine and choline for 14 h, small butsignificant elevations (p < 0.05) were observed in their abso-lute contents of membrane phosphatidylcholine, phospha-tidylethanolamine, and phosphatidylserine, all increasingby 10-15% relative to their levels in cells incubated withcholine alone. Exogenous cytidine, acting via cytidine tri-phosphate, can thus affect the synthesis and levels of cellmembrane phospholipids. Key Words: Acetylcholine-Choline-Cytidine-PC 12 cells-Phosphatidylcholine-Phospholipids. LOpezG.-Coviella I. and Wurtman R. J. En-hancement by cytidine of membrane phospholipid synthe-sis. J. Neurochem. 59, 338-343 (1992).

Most of the phosphatidylcholine (PtdCho) synthe-sized by cells is formed via a biosynthetic pathwaythat utilizes cytidine in the form of 5'-cytidine diphos-phate choline (CDP-choline) as an intermediate(Kennedy and Weiss, 1956). This pathway involves,sequentially, the phosphorylation of choline, the com-bination of the resulting phosphorylcholine with CTPto generate CDP-choline, and the addition of diacyl-glycerol to this compound to yield PtdCho. We previ-ously showed that administration of CDP-choline toanimals increased plasma cytidine and choline levels(Lopez G.-Coviella et aI., 1987). Moreover, measure-ments of the arteriovenous differences in plasma cyti-dine concentrations occurring during a constant intra-venous cytidine infusion suggested that more than30% of the circulating cytidine enters the brain with asingle pass (unpublished observations). The fate of ex-ogenous cytidine in neural tissues, however, was notexplored. The present study examined the fate of exog-enous cytidine in PCl2 cells, a neuron-related line,and sPecifically its conversion to CTP and CDP-cho-line and its effects on intracellular PtdCho levels.

MATERIAlS AND METHODS

Cell culturePCI2 cells were routinely grown in 250-ml flasks at 37°C

in Dulbecco's modified Eagle's medium (DMEM) contain-ing 5%fetal bovine serum and 10%horse serum in a humidi-fied atmosphere of 10% CO;z/90% air. Cells were plated in35-mm dishes at least 72 h prior to each experiment. Whenthe medium was changed in the course of an experiment,the cells were washed three times.with cold (4°C) phos-phate-buffered saline (PBS) before addition of the new me-dium. To harvest the cells, their medium was aspirated andthe cells washed and scraped off the dishes with cold metha-nol (I ml). Cell counts and assays of protein (Lowry et aI.,1951)and DNA (Labarca and Paigen, 1991) were done rou-tinely in each experiment.

Incorporation of radiolabeled cytidine into PC12 cellsPCl2 cellswere incubated in Hanks' balanced salt solu-

tion supplemented with 5, 10, 15,20, 50, 100, or 200 p.M(3H]cytidine(l Ci/mol) at 37°C. Aftera 20-min incubationperiod,the medium wasremovedand the cellswerewashedwith 5 ml of PBSand scraped from the dish in 1M formicacid(pH 2).Duplicatesampleswereanalyzedfor the radio-

Received September 10, 1991; revised manuscript received Jan-uary 7, 1992; accepted January 7, 1992.

Address correspondence and reprint requests to Dr. R. J. Wurt-man at Department of Brain and Cognitive Sciences, Massachu-setts Institute of Technology, Cambridge, MA 02139, U.S.A.

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Abbreviations used: CDP-choline, 5'-cytidine diphosphate cho-line; DMEM, Dulbecco's modified Eagle's medium; PBS, phos-phate-buffered saline; PtdCho, phosphatidylcholine; PtdEtn, phos-phatidylethanolamine; PtdSer, phosphatidylserine.

338

... ..- ....

CYTIDINE AND MEMBRANE PHOSPHOLIPIDS

activity in total cell material and in acid-soluble and -insolu-ble materials. To measure total radioactivity, an aliquot ofthe cell suspension was dried under vacuum, redissolved in200 IIIof 0.5 Mtrichloroacetic acid, heated at 70°C for 30min, mixed with scintillator-solvent mixture, and countedin a liquid scintillation spectrophotometer (Beckman In-struments, Berkeley, CA, U.S.A.). To measure the radioac-tivity associated with the acid-insoluble material, an aliquotwas centrifuged at 2,000 g for 10 min; the resulting pelletwas then washed twice with cold 0.5 M perchloric acid and0.5 M trichloroacetic acid, and its radioactivity was countedafter heating at 70°C for 30 min in 0.5 M trichloroaceticacid. To measure acid-insoluble radioactivity, the superna-tant fluid was centrifuged again at 5,000 g for 15 min (4°C),and an aliquot was taken for counting.

Ribonucleotide extraction and analysisCells were harvested with I M formic acid, as above. The

formic acid was collected and centrifuged at 4°C to removecells and debris, and the supernatant fluid was lyophilized.Nucleotides were purified by boronate-affinity chromatogra-phy (Affi-gel 601, Bio-Rad Laboratories, Richmond, CA,U.S.A.) prior to analysis by HPLC. To separate the ribonu-cleotide fractions, dried residues of the samples were redis-solved in I M ammonium acetate (pH 8.8) and applied at4°C to gel columns (1.0 X 1.5 em) which had been equili-brated with I M ammonium acetate (pH 8.8). The columnswere washed twice with 5 ml of the ammonium acetatesolution and the ribonucleotides eluted with 5 ml of 0.1 Mformic acid. Fractions containing ribonucleotides weredried under vacuum and redissolved in water (100 Ill) be-fore analysis by HPLC.

Nucleotides were resolved by ion-pair chromatography,using a reverse-phase column (Dynamax, 250 X 4.6 mm,ODS 3 Ilm) and the following gradient system: solvent A,S'mM tetrabutylammonium phosphate, 30 mM KH2P04,pH 6; solvent B, 50% acetonitrile in solvent A; 35-min con-cave gradient (no. 5) to 55% of solvent B; at 1.4 ml/min,with a 23-min equilibration delay.

Choline and acetylcholine assaysCholine was assayed by a modification of the method of

Goldberg and McCaman (1973). Aliquots of cell extractswere diluted with an equal volume of sodium phosphatebuffer (10 mM, pH 6.7), and the quaternary amines wereextracted into a solution of tetraphenylboron dissolved inheptanone (5 mg/rol), reextracted into 0.4 M HCl, anddried. The residues were then incubated for IS min at 38°Cwith 30 ml of a buffer solution [50 mM sodium phosphate,5 mMMg02, I mM ATP, 0.5 unit/ml choline kinase (EC2.7.1.32) from Saccharomyces cerevisiae, pH 8] containing[32P]ATP (sp. act. 30 Ci/mmol). The resulting [32p]_phosphocholine was isolated using an ion-exchange resin(Dowex AG-l X 8; Bio-Rad Corp.), eluted with ammoniumacetate buffer (75 roM, pH 10), and quantitated by liquidscintillation spectrophotometry. Acetylcholine levels in ali-quots of sample extracts previously incubated with acetyl-cholinesterase were determined using the same method.

Extraction and separation of cell phospholipidsCell phospholipids were extracted according to the

method of Folch et al. (1957). Tissues were sonicated inmethanol, brought to 20%(vol/vol) with chloroform/metha-nol (2: I), and mixed with two volumes of a 50% solution ofmethanol/water. After centrifugation, both the organicphase (locus of the phospholipids) and the aqueous phase

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339

(locus of the water-soluble choline-containing metabolites)were dried under vacuum. The residue from the organicphase was reconstituted in chloroform/methanol (I: I).

An aliquot (50 ml) of the phospholipid extract was puri-fied subsequently by TLC on silica gel G plates (LK-6D orLK-5D plates, Whatman) using a system consisting of chlo-roform/ethanol/triethylamine/water (30:34:30:8, by vol-ume) as the mobile phase. Phospholipid standards wereused to identify the corresponding bands under UV lightafter spraying the plates with 0.1% diphenylhexatriene inpetroleum ether. The radioactivities associated with the dif-ferent phospholipid bands were counted by liquid scintilla-tion spectrometry, and the total amounts of each of thephospholipids were determined by phosphate assay (Svan-borg and Svennerholm, 1961).

Determination of water-soluble choline-containingmetabolites from cultured cells

The water-soluble choline-containing metabolites werepurified by HPLC, using a normal phase silica column (3-Ilm spherical particles, 10 X 4.6 mm) and a gradient elutionsystem based on increasing polarity and ionic strength (Li-scovitch et aI., 1985). The mobile phase consisted of twosolvents: solvent A, acetonitrile/water/ethanol/acetic acid/0.83 M ammonium acetate (800:127:68:2:3, by volume);and solvent B, same components as solvent A(400:400:68:53:79, by volume; pH 3.6). A linear gradientfrom 0 to 100% of solvent B was started 7 min after theinjection (100 Ill). The flow rate was 1.5 ml/min and thetemperature 55°C.

Phosphocholine in the aqueous extracts was quantitatedby collecting the corresponding HPLC fraction, hydrolyz-ing it with alkaline phosphatase, and measuring the cholinederived from it. The purified phosphocholine fraction wasdried under vacuum and reconstituted in 200 IIIof a mix-ture (pH 10.7) of 0.1 Mglycylglycine, I mMMg02, 2 roMzinc sulfate, and 2 units of alkaline phosphatase [orthophos-phoric-monoester phosphohydrolase (alkaline optimum),EC 3.1.3.1] (Sigma Type III, St. Louis, MO, U.S.A.). Thismixture was incubated at 37°C and the reaction stoppedafter 4 h by adding 250 ml of chilled tetraphenylboron dis-solved in heptanone (5 mg/ml). Subsequently, choline wasdetermined by radioenzymatic assay.

Data analysisStatistical analysis of the data was performed using t tests

for comparisons between two groups, and by one-way ortwo-way analysis of variance, followed by multiple compari-son tests, when more than two groups were compared.Transport kinetics and regression analyses of the data weredone following linear and nonlinear models.

RESULTS

Kinetics of cytidine incorporation into PC12 cellsTo measure the rate of incorporation of cytidine,

PC 12 cells were incubated with various concentra-tions of (3H]cytidine (1 Ci/mol), and the radioactivi-ties associated with total cell extract and with acid-soluble and -insoluble materials were measured. Therate of incorporation of cytidine into PC 12 cells wasfound to follow normal Michaelis-Menten kinetics(Km = 5 ILM)when the cytidine concentrations in themedium were below 50 ILM (Fig. 1). However, when

J. Neurochem.. Vol. 59. No. /. /992

340 I. LOPEZ G.-COVIELLA AND R. J. WURTMAN

cytidine concentrations in the medium were in-creased above this level, the amounts of intracellularPH]cytidine nucleotides increased linearly, suggest-ing a simple diffusion mechanism or a low-affinitytransport system. At all concentrations of cytidinetested, its rate of incorporation into the acid-solublepool was more than four times that of its incorpora-tion into acid-insoluble material. Theoretical VMax val-ues for cytidine incorporation into acid-soluble and-insoluble material were estimated as 9 X 10-3 and 2X 10-3 mmol/mg of protein/min.

Nucleotide levels in PCl2 cells supplemented withcytidine

When PC 12 cells were incubated for 2 h in the pres-ence of various concentrations of cytidine, theyshowed a dose-dependent increase in the intracellularlevels of cytidine nucleotides (Fig. 2A). IntracellularCDP and CTP levels increased by 200 and 287%, re-spectively, when cells were incubated with 25 p,M cy-tidine. Further elevations in the cytidine concentra-tion of the medium led to comparatively smaller in-creases. CMP levels apparently did not change withany of the concentrations of cytidine used in thisstudy, and CDP-choline was found only in traceamounts in both control and supplemented cells,making its quantification difficult. No significantchanges in any of the other ribonucleotides were ob-served as a consequence of supplementing PC 12 cellswith cytidine.

PC 12 cells incubated with 100 p,M cytidine for pe-riods of 0.5-8 h (Fig. 2B) showed maximal increasesin CTP and CDP levels (by more than five and threetimes, respectively) at the earliest time tested; thesepersisted for at least 8 h. The uridine nucleotide poolswere also increased, cellular UTP rising graduallyfrom 3.66 to a maximum of 5.86 nmol/mg of proteinafter 4 h of incubation, and UDP and UMP peakingat 8 h (from 3.20 to 5.38 and from 1.09 to 1.84 nmol/mg of protein, respectively).

Effect of cytidine on [I4QPtdCho synthesisin PC12 cells

To determine if the cytidine-induced increase in in-tracellular CTP levels could affect PtdCho biosynthe-

J. Neurochem.. Vol. 59, No.1, 1992

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sis, PC12 cells, incubated in serum-containingDMEM with and without cytidine (100 p,M), weregiven a I-h pulse with radio labeled choline (34 p,M,7.4 mCi/mmol) and "chased" every 30 min thereafter(for a total of2.5 h). The rate of [l4C]choline incorpo-ration into PtdCho was found to be significantlyhigher in cytidine-supplemented cells (by 30%; p< 0.01), and was maintained for a longer period oftime (Fig. 3). The observed change in radioactivePtdCho was not due to an isotope dilution effect, be-cause the amounts of radioactive choline and the spe-cific activity of the phosphocholine, measured at theend of the pulse and at 30, 60, and 150 min thereafter,did not differ between cytidine-supplemented and un-supplemented cells. We were not able to quantify[l4C]CDP-choline, because only trace amounts of thiscompound could be detected (see Discussion).

In other experiments, cells incubated for 15 minwith 0, 10, 25, or 100 p,M cytidine showed dose-de-

FIG. 2. Dose- and time-dependent increase in cytidine nucleo-tides in PC12 cells. A: Nucleotide content as a function of cytidineconcentration in the medium. PC12 cells were incubated in N2medium supplemented with 0, 25, 50, 100, 200, and 1,000 pMcytidine, and harvested 2 h thereafter. B: Time course of the ef-fect of cytidine supplementation on nucleotide levels in PC12cells. PC12 cells were incubated in N2 medium supplementedwith 100 pM cytidine, and their nucleotide contents measured at30 min, and at 1, 2, 4, and 8 h. At appropriate times, media wereremoved and the cells washed with PSS and scraped from thedish with 3 ml of ice-cold 1 M formic acid. Cell extracts werecentrifuged and the nucleotides in the supernatant (acid-soluble)fraction measured by boronate-affinity chromatography and re-verse-phase ion-pair HPLC with UV detection (254 nm). Data rep-resent the means :t SEM of three experiments.

- -

.r 20 //

I

200' . Total J KID - .4.098jIJi BFIG. 1. Incorporation of cytidine into & Acid-soluble fracUonPC12 cells as a function of the concen- N 16 . Acid-InsolublefracUon

tration of cytidine in the medium. PC12 .!'! /& I 160.:: /

cells were incubated in Hanks' balanced 0 /

salt solution supplemented with 5, 10, !;. 12 Vi15,20,50,100, and 200 pM [3H]cytidine If

/ I 1/V 120. /(1 mCi/mmol) at 37°C. After a 20-min in- N 8cubation period, the medium was re- ..

moved and the cells were washed with N I T, I 80PSS and scraped from the dish into 1 M '0 4

formic acid. Duplicate samples were ana- j ..: . . .Iyzed for the radioag.tivities in total cell ma- > , . I 40terial and in the acid-soluble and -insolu- o . I I ./-:,/I ,

ble fractions. a 8 16 24 60 120 180 0.00 0.06 0.12 0.18 0.24

5 <fH]CyUdlne concentraUon, jIJi) 1/5

10A I .CliP

8 t . CDPc . CTP'ije 6Q.'"E 4"-"0

? V t-t------..--_41EC

E-----------/.. --- - -e- - - . - - ....o' , , ,,'," , . -. - -8- - - - - __

I , , .0 4 8 12 50 100 0 2 4 6 8 10

Cytidineconcentrotion x 10-1) Time (hours)

CYTIDINE AND MEMBRANE PHOSPHOLIPIDS

FIG. 3. Effects of cytidine on [14C]PtdCho synthesis in PC12cells. Cells were incubated in serum-containing DMEM in the pres-ence of [14C]ch0Iine (34 p.M, 7.4 mCi/mmol), with and withoutcytidine (100 pM). After 60 min, the medium was removed, andthe cells were washed twice with PBS and further incubated withunlabeled medium. At 3O-min intervals (for 2.5 h), the media werediscarded, and the cells were washed with PBS, scraped from thedish, and collected in 1 mt of ice-cold methanol. Two volumes ofchloroform were added to the cell suspension, and the mixturewas washed with 1 ml of water. Phospholipids in the organicextract were purified by TLC and the radioactivity correspondingto PtdCho counted. The aqueous phase was analyzed for cOO-line-containing metabolites by HPLC at the end of the pulse and at30, 60, and 150 min thereafter. Data represent means :t SEM ofthree experiments.

pendent increases in the incorporation of[l4C]cholineinto PtdCho (Table I). Increasing the concentrationof cytidine over 100 pM did not result in further in-creases in radiolabeled PtdCho.

Levels of phospholipid and choline metabolites incytidine-supplemented PC12 cells

To determine if the accelerated rate ofPtdCho syn-thesis in cytidine-supplemented cells produced a netincrease oftotal PtdCho content, PC12 cells were in-cubated with or without choline (l00 p.M) for 48 hand then supplemented with choline (50 p.M), cyti-dine (50 pM), or both. Cellular levels of the phospho-lipids PtdCho, phosphatidylethanolamine (PtdEtn),and phosphatidylserine (PtdSer), and of choline andacetylcholine, were measured 14 h thereafter (Table2). Cells incubated in the absence of choline for 48and 14 h had lower choline and acetylcholine levelsthan those supplemented with choline in all groupsstudied. Incubation with cytidine for 14 h did not af-fect the levels of either compound, whereas incuba-tion with choline significantly increased both (p< 0.01).

Total PtdCho concentration was increased slightly,but significantly, in cells incubated with both cholineand cytidine, compared with its levels in control cellsor in cells supplemented with choline alone (p < 0.05;Table 2). Choline supplementation for 14 h signifi-cantly elevated PtdCho levels only in cells which hadbeen incubated previously without choline for 48 h.Cytidine-supplemented cells also exhibited increasedlevels of PtdEtn and PtdSer, whether or not the me-dium also contained choline (p < 0.05; Table 2). Cyti-

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341

dine supplementation failed to affect cellular sphingo-myelin content (data not shown).

DISCUSSION

These data show that cytidine is taken up into PC 12cells and converted to CTP, and that the exogenouspyrimidine increases both the rate of synthesis and thetotal contents of PtdCho, PtdEtn, and PtdSer in thecells.

When cytidine concentrations in the medium arelow, cytidine incorporation follows normal Michae-lis-Menten kinetics, and its incorporation into theacid-soluble pool is greater than that into nucleicacids. Similar observations have been made for theincorporation of uridine and other nucleosides in awide variety of cells (Jacquez, 1962; Scholtissek,1968; Plagemann, 1971c); this suggests that at con-centrations below 50 p.M, cytidine is taken up byPC 12 cells mainly by a transport system, perhaps in-volving facilitated diffusion, whereas at concentra-tions above this level, simple diffusion or a low-affin-ity uptake system becomes the predominant mode ofentry.

Chromatographic analysis of acid extracts fromPC 12 cells showed that the total content of pyrimi-dine nucleotides in these cells is about one-fourth thatof purine nucleotides (3.85 versus 11.32 nmol/106cells). The estimated values in the present study arevery close to those reported by Plagemann (1972) forNovikoff rat hepatoma cells, lower than those foundby Khym et al. (1978) for some cell types, and in thesame range as those reported by Brenton et al. (1977)for lymphoid cells and by Weber and Edlin (1971) for3T3 cells. Considering that the radius of PC 12 cells isapproximately 5 p.m (Watanabe et aI., 1983), and onthe basis of a total volume of 2.5 p.1/106cells (Wardand Plagemann, 1969), the average overall intracellu-lar concentration of cytidine nucleotides is 0.17 mM,and at least 55% of this may be present as CTP.

The expansion of the cytidine nucleotide pool inthe present study, when PCI2 cells were supple-

TABLE 1. Dose-dependentincrease in [uClPtdCho inPCl2 cellssupplementedwith cytidine

PCI2 cells were incubated in serum-containing DMEM in thepresence of [14C]choline(34 p.M, 7.4 mCi/mmol), with 0, 10,25, or100 pM cytidine. After 15 min, the medium was removed and thecells were washed with PBS, scraped from the dish, and collected inI ml of ice-cold methanol. Phospholipids were extracted, separatedby TLC, and their radioactivity counted as described in Materialsand Methods. Values represent means :t SEM of four experiments..p < 0.0 I versus preceding value.

J. Neurochem., Vol. 59, No.1. 1992

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JOO

o Cytidine (-)0 250 i . Cytidine (+)Ec 200"'"

Ea. 150"0 100or;U"

ITiL

5: .0 40 80 120 160 200 240

Time (min)

Cytidine ['4C]PtdCho(pM) (dpm x 1O-3/mg) % increase

0 2.20 :t 0.0410 2.35 :t 0.04" 725 2.81 :t 0.03" 22

100 3.19 :t 0.20" 32

342 /. LOPEZ G.-COV/ELLA AND R. J. WURTMAN

TABLE 2. Choline, acetylcholine. and phospholipid contents (nmol/mg of protein) in PCl2 cells

Intracellular choline (Ch) and acetylcholine (ACh) levels and membrane phospholipid levels of PC12 cells supplemented with cytidine(Cyd). PC 12 cells were incubated with and without choline (100 p.M) for 48 h, and then supplemented with choline (50 p.M),cytidine (50 p.M),or both during the following 14 h. Values represent means:t SEM of three experiments.

"p < 0.01 versus control.bp < 0.05 versus choline alone.<p < 0.05 versuscontrol.

mented with cytidine, was fast and dose-dependent(Fig. 2), and due primarily to increased levels ofCTP.The presence of 25 p,M cytidine in the medium wassufficient to cause a two- to threefold increase in CTPlevels after 2 h of incubation. In spite of the markedincrease in cytidine nucleotide levels, the total con-centration of nucleotides in the cells was affected onlylittle (74.6 versus 60.7 nmol/mg of protein in unsup-plemented cells). A slight increase in uridine nucleo-tides was apparent, with time, in cells supplementedwith 100 #LMcytidine, but cytidine had practically noeffect on the levels of other nucleotides. This observa-tion agrees with previous reports by Plagemann(1971a,b, 1972) on Novikoff cells: uridine and cyti-dine nucleotide levels fell when these cells were incu-bated with high concentrations of guanosine, inosine,or adenosine, but levels of other nucleotides failed tochange when cells were incubated with cytidine oruridine.

It is generally assumed that the formation ofCDP-choline is a rate-limiting step in the synthesis ofPtdCho (Vance et al., 1980). Thus, it would not besurprising for intracellular levels of this intermediateto be low. Despite the increase in CTP levels in cyti-dine-supplemented cells, we could not measure CDP-choline levels in these cells or in unsupplementedcells. However, we have found that PC12 cells incu-bated with [l4C]choline (8.6 #LM,0.5 #LCi,58.5 mCi/mmol, 20 min) and subsequently "chased" in the pres-ence of depolarizing concentrations of potassium (50mM) exhibited higher radiolabeled CDP-choline lev-els than cells incubated without potassium. In thesecircumstances, addition of cytidine to the medium(100 p,M) produces a two- to threefold increase in[14C]CDP-choline (data not shown). The effect of po-tassium may be explained by activation of cholinekinase by this ion (Ando et al., 1987), which may in-crease the availability of radiolabeled phosphocholinethat reacts with CTP to form CDP-choline.

The enhancement by cytidine of the incorporationof radiolabeled choline into PtdCho in PC 12cells (Ta-ble 1) may reflect increased synthesis of this phospho-

J. Neurochem., Vol. 59, No.1. 1992

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lipid, secondary to elevations in intracellular CTP lev-els. These findings resemble previous observations inpoliovirus-infected HeLa cells, in which a two- tothreefold increase in the concentration of CTP in thecytosolic compartment stimulated the incorporationof [methyPH]choline into PtdCho and produced atwofold increase in cytidylyltransferase activity (pre-sumably the rate-limiting step in PtdCho synthesis)(Vance et al., 1980). That CTP could be a critical regu-lator in PtdCho synthesis is also suggested in experi-ments in BHK-21 cells incubated with Semliki Forestvirus: a 70% depletion in this nucleotide was asso-ciated with inhibition of PtdCho formation (White-head et al., 1981). However, the activity of micro-somal cytidylyltransferase in PC 12cells is not known.In HeLa cells, the apparent Km for CTP ofthe cytidy-lyltransferase is 0.2 mM (Choy et al., 1980), and in rathepatocytes 0.54 mM or more (Pelech and Vance,1984). If one assumes that the Km for CTP of the en-zyme is within this range in PC 12 cells, then the con-centration of CTP in these cells ( 90 p,M) would bewell below this value. Thus, by providing cytidine, wecould increase intracellular CTP levels and the rate ofsynthesis of PtdCho, and explain the observed in-crease in the incorporation of radiolabeled cholineinto PtdCho.

If this hypothesis is true, one might expect to findan absolute increase in the total PtdCho levels in cyti-dine-supplemented cells. A slight, but significant, in-crease of this phospholipid was indeed found in cellssupplemented with cytidine when choline was alsopresent in the medium. Interestingly, this increasewas also accompanied by increased levels of two othermajor phospholipid constituents of cell membranes,PtdEtn and PtdSer. These data suggest that the rela-tive composition of cell membrane phospholipids ismaintained within certain limits, and that an increasein the synthesis of one phospholipid constituent canlead to increased synthesis (or decreased breakdown)of some of the others. Similar conclusions have beendrawn from studies on LA-N-2 cells incubated withPtdSer liposomes (Slack et aI., 1989); the increase in

-- - ---

Preincubationin the absenceof Ch Preincubation in the presenceof Ch

Ch Cyd Ch + Cyd - Ch Cyd Ch + Cyd

Ch 0.09 :t 0.01 0.53 :t 0.04" 0.07 :t 0.01 0.58 :t 0.02" 0.20 :t 0.02 0.71 :t 0.07" 0.02 :t 0.01 0.65 :t 0.02"ACh 0.02 :t 0.01 0.27 :t 0.03" 0.04 :t 0.01 0.27 :t 0.03" 0.06 :t 0.01 0.29 :t 0.03" 0.07 :t 0.01 0.23 :t 0.01 a

PtdCho 39.2:t 1.7 66.3 :t 2.7 44.0 :t 1.4 73.1 :t 0.9".b 73.6 :t 1.2 75.7 :t 1.9 76.1 :t 2.8 82.2 :t l.ijb.<PtdEtn 27.4 :t 1.0 28.8 :t 1.5 33.0 :t 1.1b.< 31.9:t 0.7< 29.9:t 0.7 33.6 :t 1.7 35.4 :t 1.8< 34.5 :t 0.4<PtdSer 8.1 :t 0.2 9.6:t0.1< 9.4 :t 0.3< 10.7 :t 0.5" 12.3 :t 0.7 11.3 :t 0.7 13.0:t 0.6 13.2 :t 0.7

.-.............. ......-...........-...

CYTIDINE AND MEMBRANE PHOSPHOLIPIDS

membrane PtdSer was followed by activation of theincorporation of choline into PtdCho and of ethanol-amine into PtdEtn. Thus, cytidine, by increasing in-tracellular CfP, and as a consequence membranePtdCho levels, may stimulate the overall formation ofcell membranes or slow the degradation of other phos-pholipids. Because, in humans cytidine plasma levelsrange between 1 and 2 p.M (Lopez G.-Coviella et at.,1987), it would not be surprising if, in some circum-stances (i.e., cell membrane deterioration and neuro-nal cell loss), this substrate may limit the synthesis ofmembrane phospholipids.

Acknowledgment:This work was supported by grantsfrom the National Institute of Mental Health (MH-28783)and the Center for Brain Sciencesand MetabolismCharita-ble Trust.

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Brenton D. P., Astrin K. H., Cruikshank M. K., and Seegmiller J. E.(1977) Measurement of free nucleotides in cultured humanlymphoid cells using high pressure liquid chromatography.Biochern. Med. 17,231-247.

Choy P. C, Paddon H. E., and Vance D. E. (1980) An increase incytoplasmic CTP accelerates the reaction catalyzed byCTP:phosphocholine cytidyltransferase in poliovirus-infectedHeLa cells. J. Bioi. Chern. 255,1070-1073.

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