JaumaI a/NeurochemistryRaven Press, Ltd., New York@ 1989 International Society for Neurochemistry

Uptake of Exogenous Phosphatidylserine by HumanNeuroblastoma Cells Stimulates the Incorporation of

[methyl-14C]Cholineinto Phosphatidylcholine

B. E. Slack, M. Liscovitch, J. K. Blusztajn, and R. J. Wurtman

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

Abstract: The phosphatidylserine (PtdSer) content of humancholinergic neuroblastoma (LA-N-2) cells was manipulatedby exposing the cells to exogenous PtdSer, and the effectsonphospholipid content, membrane composition, and incor-poration of choline into phosphatidylcholine (PtdCho) wereinvestigated. The presence of liposomes containing PtdSer(l0-130 p.M)in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, andsmaller and slower increases in the contents of other mem-brane phospholipids. The PtdSer levels in plasma membraneand mitochondrial fractions prepared by discontinuous su-crose density gradient centrifugation increased by 50 and100%, respectively, above those in control cells after 24 h ofexposure to PtdSer (130 p.M). PtdSer caused a concomitant,concentration-dependent increase of up to twofold in the in-corporation of [methyp4C]choline chloride into PtdCho ata choline concentration (8.5 p.M) compatible with activationof the CDP-choline pathway, suggesting that the levels ofPtdSer in membranes may serve as a stimulus to regulateoverall membrane composition. PtdSer caused a mean in-

crease of 41% in PtdCho labeling, but the phorbol ester,phorbol 12-myristate 13-acetate (PMA), which stimulatesPtdCho synthesis in a number of cell lines, increased[14C]PtdCho levels by only 14% in LA-N-2 cells, at a con-centration (100 oM) which caused complete translocation ofthe calcium- and phospholipid-dependent enzyme proteinkinase C to the membrane. The translocation was inhibitedby prior exposure of the cells to PtdSer. Treatment with PMAfor 24 h diminished protein kinase C activity by 80%, butincreased the labeling of PtdCho in both untreated andPtdSer-treated cells. These data suggest that uptake ofPtdSerby LA-N-2 cells alters both the phospholipid composition ofthe membrane and synthesis of the major membrane phos-pholipid PtdCho; the latter effect does not involve activationof protein kinase C. Key Words: Phospholipids-Phospha-tidylserine-Phorbol esters-Phosphatidylcholine-Proteinkinase C-Neuroblastoma. Slack B. E. et al. Uptake of ex-ogenous phosphatidylserine by human neuroblastoma cellsstimulates the incorporation of [methyl-14C]choline intophosphatidylcholine. J. Neurochem. 53,472-481 (1989).

Phosphatidyiserine (PtdSer), a major negatively-charged phospholipid component of mammalian cellmembranes, influences fluidity and fusion capacity ofmembrane lipid bilayers (Papahadjopoulos et aI., 1973)and modulates the activity of various enzymes, in-cluding cytidyltransferase (CTP: cholinephosphate cy-tidyltransferase; Choy and Vance, 1978) and proteinkinase C (Takai et aI., 1979a). A variety of techniqueshave been used to study the regulation and functionof PtdSer in the membranes of intact cultured mam-malian cells, including the development of mutantsdeficient in PtdSer synthesis (Kuge et aI., 1985) and

the exposure of cells to exogenous PtdSer added to themedium (Nishijima et aI., 1986). Using a human cho-linergic neuroblastoma (LA-N-2) cell line (Seeger etaI., 1977),we have attempted to manipulate the PtdSercomposition of the membrane in intact cells, and toanalyze the effects of such alterations on the metabo-lism of phosphatidylcholine (PtdCho), the major phos-pholipid component of cell membranes.

The rate limiting step in the synthesis of PtdCho iscatalyzed by cytidyltransferase (Pelech and Vance,1984), an enzyme of the Kennedy pathway, which isthe major route of PtdCho synthesis in most cells

Received November 29, 1988;revised manuscript receivedJanuary9, 1989; accepted January 10, 1989.

Address correspondence and reprint requests to Dr. B. E. Slack atthe Department of Brain and Cognitive Sciences, Massachusetts In-stitute of Technology, Cambridge, MA 02139, U.S.A.

The present address of Dr. M. Liscovitch is Department of Hor-mone Research, Weizmann Institute of Science, Rehovot 76100, Is-rael.

Abbreviations used: AcCho, acetylcholine; GroPCho, giycero-phosphocholine; LysoPtdSer, lysophosphatidylserine; PtdCho, phos-phatidylcholine; PCho, phosphocholine; PtdEtn, phosphatidyletha-nolamine; Ptdlns, phosphatidylinositol; PMA, phorbol12-myristate13-acetate; PMSF, phenylmethylsulfonyl fluoride; PtdSer, phospha-tidylserine; CerPCho, sphingomyelin.

472

--- . - -- -. - -. - ._- -.-

ENDOGENOUS PtdSer INCREASES PtdCho LABELING

(Kennedy and Weiss, 1956). In a number of differentcell lines, activators of the calcium- and phospholipid-dependent enzyme protein kinase C (Nishizuka,I984a), such as the phorbol esters and diacylglycerols,stimulate PtdCho synthesis (Kreibich et aI., 1971; Susset aI., 1971; Paddon and Vance, 1980; Warden andFriedkin, 1984; Kreutter et aI., 1985; Liscovitch et aI.,1986, 1987b; Kolesnick and Paley, 1987). In this report,we show that exposure of LA-N-2 cells to liposomalsuspensions of PtdSer alters membrane phospholipidcomposition and stimulates the incorporation of[14C]choline into PtdCho. In in vitro mixed micellarsystems, the binding of protein kinase C is dependenton the presence of PtdSer and calcium, whereas itsactivation requires addition of diacylglycerol (Hannunet aI., 1985; Ganong et al., 1986). We investigated thepossibility that the stimulation of PtdCho labeling inLA-N-2 cells by exogenous PtdSer might be due tomodulation of protein kinase C activity, perhaps be-cause of alterations in its distribution between the cy-toplasm (where it is inactive) and the membrane (whereit is activated in the presence of PtdSer and diacyl-glycerol) (Nishizuka, 1984a). To test this hypothesis,we compared the effects of PtdSer and phorbol 12-myristate 13-acetate (PMA), a potent activator of pro-tein kinase C in many cell types, on PtdCho synthesisand on protein kinase C activity in these cells.

MATERIALS AND METHODS

MaterialsLA-N-2 cells (passage 69) were kindly provided by Dr. R.

C. Seeger (Department of Pediatrics, UCLA Medical School,Los Angeles, CA, U.S.A.). Plasticware for tissue culture wasfrom Falcon Plastics (Los Angeles, CA, U.S.A.)or from CostarCo. (Cambridge, MA, U.S.A.). Leibovitz L-15 nutrient me-dium, F-12, Dulbecco's modified Eagle's medium, and serawere from GIBCO Laboratories (Grand Island, NY, U.S.A.).Serum-free N2 medium was made according to the methodof Bottenstein and Sato (1979) and routinely supplementedwith 100 p.M choline. When cells were labeled for short pe-riods of time with [14C]choline, nonradioactive choline wasomitted from the medium. PMA, leupeptin, phenylmethyl-sulfonyl fluoride (PMSF), PtdSer, Nonidet P-40, and histonetype III-S were from Sigma Chemical Co. (St. Louis, MO,U.S.A.). Lysophosphatidylserine (LysoPtdSer) was kindlysupplied by Fidia (Abano Terme, Italy). h-32P]Adenosinetriphosphate ([32p]ATP; to Ci/mmol) was from Amersham(Arlington Heights, IL, U.S.A.) and [methyl-'4C]cholinechloride (58.5 mCi/mmol) was from New England Nuclear(Boston, MA, U.S.A.). Solutions ofPMA (10 mM in dimethylsulfoxide) were kept frozen at -20°e.

Cell cultureLA-N-2 cells (passage 73-105) were maintained in LI5

medium, supplemented with 10% fetal calf serum and 100p.M choline in humidified room air at 37°C. Medium waschanged once or twice weekly. Cells were subcultured at sub-confluent density by brief (I min) exposure to a solution of0.1% Viokase (a crude extract of pancreatic enzymes; ViobinCo., Monticello, IL, U.S.A.) in phosphate-buffered saline,and plated onto 60- or 35-mm dishes. At 24 h prior to treat-

473

ments, the medium bathing the cells was replaced with N2medium supplemented with 100 p.M choline.

Phospholipid assayCells were exposed for various periods of time to liposomal

suspensions ofPtdSer in N2 medium, orto N2 medium alone.To make the liposomes, PtdSer was dissolved in chloroform,dried under nitrogen, resuspended in phosphate-buffered sa-line, and sonicated on ice for 5 min. The suspension wasboiled for 5 min to sterilize it, and diluted with N2 mediumto the desired concentration. Stock concentrations of PtdSerwere determined by extracting and assaying for phospholipidcontent. Stocks contained pure PtdSer as determined by one-dimensional thin-layer chromatography. Following exposureto PtdSer, cells were washed twice with saline, harvested withViokase, and suspended in N2 medium. An aliquot wascounted to determine cell number. The cell suspension wascentrifuged briefly to pellet the cells, and the medium wasdiscarded. The pellet was extracted in chloroform-methanolaccording to the method ofFolch et al. (1957), and the phos-pholipids were separated by one-dimensional thin-layerchromatography on preadsorbent silica gelG plates (Analtech,Newark, DE, U.S.A.) using a mobile phase containing chlo-roform/ethanol/water/triethylamine (30:34:8:30 by volume).Under these conditions, phosphatidic acid cochromatographswith phosphatidylinositol (PtdIns), but because of the smallamount normally present in the cells, its presence was ne-glected in our calculations. In some experiments, a differentmobile phase composed of chloroform/methanol/ammo-nium hydroxide (65:25:5 by volume) was used in order toseparate PtdCho from LysoPtdSer. The phospholipids weremeasured by phosphate assay (Svanborg and Svennerholm,1961).

Labeling of cells with (14Qcholine and measurementof lipid- and water-soluble metabolites

Following pretreatment with PtdSer or PMA, the mediumwas removed, and the cells were rinsed with N2 medium andthen incubated for 2 h at 37°C with [methyl-14C]cholinechloride (0.5 p.Ci/rol; 8.5 p.M) in choline-free N2 medium.The medium was removed, the dishes were rinsed once withchilled N2 medium, and I ml of ice-cold methanol was addedto each dish. [The addition of methanol to cultured cellsprior to scraping has been shown to prevent cellular metab-olism of lipids as effectively as immediate extraction of cellswith chloroform and methanol (Van Veldhoven and Bell,1988).] The cells were scraped from the plates and two vol-umes of chloroform were added. The mixture was vortexed,I ml of water was added, and the mixture was vortexed again.The upper phase containing the water-soluble labeled me-tabolites of choline [acetylcholine (AcCho), phosphocholine(PCho), and glycerophosphocholine (GroPCho)] and thelower phase containing phospholipids were separated by briefcentrifugation. Both phases were dried under vacuum. Theorganic phase was redissolved in chloroform/methanol (I: I)and an aliquot counted by liquid scintillation spectrometry.As determined by thin layer chromatography, 90% of thelabel incorporated into [14C]choline-labeled phospholipidswas located in PtdCho. Labeled water-soluble choline me-tabolites were analyzed by a modification of a high perfor-mance liquid chromatographic assay described previously(Liscovitch et aI., 1985, 1987b). Proteins were determinedby the method of Lowry et al. (1951).Protein kinase C assay

Cells previously exposed to N2 medium, or to N2 con-taining PtdSer, were incubated in fresh N2 medium contain-

J. Nl!Urochl!m..Vol. 53. No.2. 1989

474 B. E. SLACK ET AL.

ing various concentrations of PMA for I h, at which timethe effect of PMA in these cells was found to be maximal.The cells were scraped from the dishes in 2 ml of Buffer A(20 mM Tris-HCI, 2 mM sodium EDTA, 0.5 mM EGTA,and 350 Itg/ml of PMSF at pH 7.5; Kraft and Anderson,1983),to which had been added 0.33 M sucrose, and sonicatedwith 10 I-see pulses. The lysate was centrifuged at 100,000g for I h, and the resulting supernatant fluid kept on ice. ThepeUetwas solubilized by homogenizing in Buffer A containing0.1% Nonidet P-40 and centrifuged at 100,000 g for I h. Thesupernatant fluid containing the detergent-solubilized en-zyme, and the supernatant fluid from the first centrifugationstep containing the soluble enzyme, were applied to identicaldiethylaminoethyl (DEAE) cellulose columns equilibratedwith Buffer A. The enzyme was eluted with 0.5 ml of 0.15M NaG in the same buffer. The assay tubes contained, in avolume of 0.25 ml, the following: Tris-HCI (25 mM), CaCh(I mM), MgCh (10 mM), PMSF (0.4 mM), histone (500 Itg/ml), ATP (0.1 mM), 106dpm [32p]ATP,and 1-6 Itg of theenzyme preparation. Each sample was assayed in triplicate,in both the presence and absence of PtdSer (20 Itg/ml). Theassay tubes were incubated for 5 min at 37°C, and the phos-phorylated histone was precipitated with 20% trichloraceticacid and filtered using 0.45 Itm filters (Millipore Corp., Bed-ford, MA, U.S.A.). The amount of radioactivity on the filterswas determined by liquid scintillation counting. Protein ki-nase C activity was calculated as the difference between theradioactivity incorporated into trichloracetic acid-insolublematerial in the presence and absence of PtdSer. The in vitroproperties of protein kinase C extracted from LA-N-2 cellswere similar to those reported previously for NG I08-15 cells(Liscovitch et aI., 1987b); in the presence of 20 Itg/ml ofPtdSer and low concentrations of calcium, protein kinaseactivity was slightly above background, but was increased tonear maximal levels by the addition of 20 oM PMA. Proteinwas assayed by the method of Lowry et al. (1951).

Subcellular fractionation procedureCells were harvested with Viokase, resuspended in serum-

containing L-15 medium, and pelleted as described above.The cells were resuspended in five volumes of lysis buffer(0.5 mM dithiothreitol, 1 mM EGTA, 0.5 mM sodium bi-carbonate, and 10 mM HEPES at pH 7.9; Imai and Ger-shengorn, 1987) and incubated at 37°C for 5 min. Ten vol-umes of ice-cold lysis buffer were added, and the cell sus-

pension was homogenized (five strokes with a teflon pestlein a glass homogenizer) and centrifuged for 20 min at 1,000g to sediment the nuclei. The nuclear pellet was designatedfraction no. 6. The supernatant fraction was centrifuged for1 h at 100,000 g, and the pellet was resuspended in lysisbuffer, layered on top of a discontinuous sucrose density gra-dient consisting of fractions of 1.2, 1.0, 0.8, 0.6, 0.4, and 0.2M sucrose in lysis buffer, and centrifuged for 20 min at188,000 g in a Beckman SW41Ti rotor. The resulting bands(designated fractions no. 1-5 from top to bottom of the gra-dient) were diluted in lysis buffer, sedimented at 70,000 g,and frozen at -70°C for later analysis. ATPase activity wasdetermined by the method of Post and Sen (1967), and suc-cinate dehydrogenase activity was determined by measuringtetrazolium reductase activity (Pennington, 1961). Phospho-lipids were extracted and assayed as described above.

StatisticsThe statistical significance of differences was estimated by

Student's t test or by two-way analysis of variance. Valuesquoted in the text are expressed as means :t SEM unlessotherwise stated.

RESULTS

LA-N-2 cells take up PtdSer and incorporateit into membranes

Exposure of LA-N-2 cells to PtdSer-containing li-posomes suspended in N2 medium led to time- andconcentration-dependent increases in cell PtdSer con-tent (Table I; see Fig. 2b). At low (30 ILM)PtdSer con-centrations, uptake occurred progressively over a periodof days and was accompanied by small but significantincreases in PtdCho, PtdIns, phosphatidylethanol-amine (PtdEtn), and sphingomyelin (CerPCho) con-tents (Table 1). After 4 days of exposure to PtdSer,when cell PtdSer contents were increased sevenfold,cell contents of both PtdCho and PtdEtn were -40%higher than those of untreated time-matched controls.No further increases were observed after 6 days. Levelsof PtdIns and CerPCho were approximately doubledafter 2 days in PtdSer, and remained so until day 6(Table I). LA-N-2 cells, once transferred to N2 me-

CeUs were treated with N2 medium, or N2 supplemented with 30 p.M PtdSer for 1-6 days, then harvestedand assayed for phospholipid content as described in Materials and Methods. Values represent meansI SEM. The number of individual determinations is indicated in parentheses..Values are significantly different from time-matched controls, p < 0.05.

J. Neurochem.. Vol. 53. No.2, /989

TABLE 1. Phospholipid content of cells exposed to PtdSer (30 ItM) for 1-6 days

Phospholipid content (pmol/IO,OOOceUs)

Day Treatment PtdSer PtdCho Ptdlns PtdEtn CerPCho

Control 57 I 7 (4) 137 I 26 (7) 32 I 4 (4) 49 I 12 (3) 19I4 (4)PtdSer 143 I 19 (4)- 131 I 17 (7) 36 I 5 (4) 45 I 2 (3) 30 I 9 (4)

2 Control 42 I 5 (7) 151 I 17 (7) 27 I 6 (6) 73 I II (6) 22 I II (5)PtdSer 161 I 19 (7)- 167 I 17 (7) 52 I 10 (6)- 78 I 10 (6) 49 I 8 (5)-

4 Control 44 I 3 (7) 152 I 12 (7) 17 I 2 (7) 54 I 4 (7) 17 I 3 (6)PtdSer 331 I 49 (7)- 211 I 13 (7)- 47 I6 (7)- 78 I 3 (7)- 33 I 3 (6)-

6 Control 58 I 8 (3) 175 I I (3) 38 I 2 (3) 63 I 5 (3) 29 I 3 (3)PtdSer 471 I 80 (3)- 242 I 1 (3)- 70 I 9 (3)- 86 I 5 (3)- 54 I 7 (3)-

ENDOGENOUS PtdSer INCREASES PtdCho LABELING

AG. 1. PtdSer content and characterization of subcellular mem-brane fractions from LA-N-2 cells. Cells were exposed to N2 me-dium, or N2 supplemented with PtdSer (130 pM) for 24 h. Thecells were lysed and the membranes were prepared by centrifu-gation on a sucrose density gradient (see Materials and Methods).The fractions were collected and assayed for phospholipid content,Na+,K+-ATPase activity, and succinate dehydrogenase activity. a:PtdSer content of fractions 1-5 from the sucrose density gradient,and of the low-speed nuclear pellet ("nuc", fraction 6) from control(filledbars) and PtdSer-treated (unfilledbars) cells. The graph showsvalues derived from two experiments. b: Na+,K+-ATPase (circles)and succinate dehydrogenase (squares) activity in subcellularmembrane fractions from control (filledsymbols) and PtdSer-treated(unfilled symbols) cells.

dium, undergo only one or two divisions before be-coming quiescent. The cell number per dish after 4-6days was not affected by the presence of 30 ILMPtdSerin the N2 medium.

Overnight treatment with higher concentrations ofPtdSer also increased the total PtdCho content of thecells (Fig. 2b). The increase in cell PtdSer content (withI00 ~ PtdSer in the medium) was rapid, approachinghalf-saturation after 3 h (not shown). In four of fiveexperiments, PtdCho content was elevated (to 143:t 6% of control) by 24-h treatment with 100 ILMPtdSer. Concomitant increases in PtdEtn, Ptdlns, andCerPCho to 164 :t 7, 173 :t 39, and 144 :t 32% ofcontrol, respectively, were also observed. The mostconsistently affected phospholipids, other than PtdSeritself, were PtdCho and PtdEtn. The increase in PtdChocontent was observed irrespective of the thin-layerchromatographic system used for its purification (seeMaterials and Methods). Incubation of cells in thepresence of elevated serine concentrations (up to 50mM) or in 10 ILM LysoPtdSer (higher concentrationswere toxic to the cells) did not increase their PtdSercontent, suggesting that uptake of PtdSer did not re-quire its prior metabolism.

- - -

475

Subcellular membrane fractions prepared from cul-tures of LA-N-2 cells exposed to PtdSer (130 ILM) for24 h contained increased amounts of PtdSer (expressedas a percentage of total phospholipid content) (Fig. Ia).The observed increases ranged from approximately30% in fraction 3, and 50% in fraction 2, to twofoldincreases (on average) in fractions 4 and 5. A threefoldelevation of PtdSer was observed in the crude nuclearpellet (which should also contain unbroken cells andlarge cell fragments) (Fig. la). Fraction 2 was enrichedwith the plasma membrane marker Na+,K+-ATPase,and fractions 4 and 5 contained the mitochondrialmarker succinate dehydrogenase (Fig. Ib). (No attemptwas made to determine the extent to which the mito-chondrial fraction was contaminated with microsomalmembranes.) The results are consistent with the pos-sibility that the contents ofPtdSer in both intracellularand plasma membranes were elevated by pretreatmentwith PtdSer. Small elevations in PtdEtn and PtdChocontent (nanomoles per milligram of protein) also oc-curred in some fractions (data not shown). The activ-ities of Na+,K+-ATPase and succinate dehydrogenaseand their distribution among various membrane frac-tions were similar in PtdSer-treated and untreated cells(Fig. Ib).

Exposure to PtdSer stimulates incorporation of(14qcholine into PtdCho

LA-N-2 cells treated overnight with various concen-trations of PtdSer (0-130 ~) and then labeled for 2h with [14C]cholineshowed a concentration-dependentstimulation ofPtdCho labeling by PtdSer of up to two-fold (Fig. 2a). The degree of stimulation was correlatedwith the amount of PtdSer taken up by the cells (Fig.2b) and paralleled the increase in PtdCho content (Fig.2b). PtdCho labeling was also stimulated by PtdSer inP4C]choline-prelabeled cells (not shown), suggestingthat this effect was not due to a change in the specificactivity of the precursor pool. The mean increase of[14C]choline incorporation into PtdCho by 100 ILMPtdSer in II experiments was 141 :t 6%(mean :t SEM);no significant change in the protein content per dishwas observed (control, 0.523 :t 0.06; PtdSer, 0.525:t 0.056 mg per dish). Table 2 shows a typical experi-ment in which the increase in [14C]PtdCho (after 2-hlabeling with ['4C]choline) elicited by 24-h pretreat-ment with 100 ILMPtdSer was accompanied by a de-crease in [14C]PCholevels. When the results from threesuch experiments were combined and expressed as apercentage of control values, cells pretreated withPtdSer showed a mean significant increase in[14C]PtdCho to 144 :t 9% of control, and a mean de-crease in [14C]PChoto 90 :t 4% of control. (The averagedecrease did not achieve significance.) ['4C]AcCho lev-els did not change (10 I :t 9% of control in PtdSer-treated cells) (Table 2), again suggesting that PtdSerdid not affect choline specific activity. In two of threeexperiments, the amount of label lost from [14C]PChowas greater than that transferred to p4C]PtdCho (Ta-ble 2).

J. Neurochem., Vol. 53, No.2, /989

25, (0)

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B. E. SLACK ET AL.

FIG. 2. PtdSer causes concentration-dependent increases InPtdCho labeling and content. Cells were treated overnight withvarious concentrations of PtdSer in N2 medium. a: Cells werewashed once, and labeled for 2 h with [1'C]choline. The incorpo-ration of radioactivity into PtdCho was determined as describedin Materials and Methods. Values represent means :t SEM of rep-rlCatedeterminationsfrom two experiments.b: Cells were extractedand assayed for PtdSer (filled circles) and PtdCho (filled triangles).Values represent means :t SEM from a representative experiment.

The effects of PtdSer on [14C]PtdCho degradationwere examined in pulse-chase experiments in whichcells were prelabeled for 24 h in the absence ofPtdSer,then exposed to various concentrations of PtdSer forthe first 24 h of a 72-h chase period in N2 mediumsupplemented with 100 p.M choline (Fig. 3). The rel-atively long chase period was chosen in order to allow

TABLE 2. Effect of PtdSer on labeling of PtdCho,AcCho, and PCho

Radioactivity (dpmfmg of protein)

Treatment AcCho PChoPtdCho

ControlPtdSer

3,560 :t 485,151 :t 202"

64,024 :t 2,88966,010:t 2,601

93,421 :t 2,69678,798:t 1,515"

Cells were treated for 24 h with lOO,.M PtdSer or N2 mediumalone, then washed and labeled with [1'C]choline for 2 h. The cellswere extracted and the metabolites in the aqueous and lipid-solublephaseswere measured asdescribed in Materials and Methods. Valuesrepresent means :t SEM of triplicate determinations from a repre-sentative experiment which was repeated three times.

" Values are significantly different from controls, p < 0.0 I by Stu-dent's t test.

J. Neurochem., Vol. 53, No.2, 1989

+-t PtdCho+ r t

+-+~hO .~~

TA. ,

GroPCho .a._~--.A-+

o 20 40 60 BO 100 120 140

PtdSer (jJt.A)

FIG.3. PtdSer stimulates PtdCho degradation in prelabeled cells.Cells were labeled overnight with [1'C]choIine in N2 medium c0n-taining 5% dialyzed fetal calf serum, then chased with N2 containing100 pM choline and various concentrations of PtdSer. After 24 h,the medium was replaced with control N2 medium. After an ad-ditional48 h, the cells were extracted and assayed for [1'C]PtdCho(upper panel), and for [1'C]PCho (filled circles) and [1'C]GroPCho(filled triangles) (lower panel). Values represent means :t SO ofreplicate determinations from a representative experiment that wasrepeated twice. .p < 0.05; values are significantly different fromcorresponding control values.

detectable amounts of the degradation productP4C]GroPCho to accumulate. PtdSer added during thechase period resulted in a concentration-dependentdecrease in [14C]PCho and a concomitant significantincrease in the production of [14C]GroPCho (Fig. 3),the product of sequential degradation of PtdCho byphospholipase A and lysophospholipase. The levels of[14C]choline and [14C]AcChoin these cells were verylow (the two compounds combined contained <5,000dpm/mg of protein), and were not altered by PtdSertreatment. Treatment of cells for 24 h with variousconcentrations of PtdCho did not alter the incorpo-ration of [14C]choline into PtdCho in cells in whichPtdSer treatment increased labeling by 50% (Fig. 4).

PMA activates protein kinase C but has little effecton PtdCho labeling

It is known that phorbol esters stimulate choline in-corporation into PtdCho in many cell lines, an effectwhich may be partially attributable to activation ofprotein kinase C (Liscovitch et aI., 1987b).Because thePtdSer concentration can influence the binding of pro-tein kinase C in vitro to mixed micelles composed ofTriton X-lOO, phospholipids, and diacylglycerols(Hannun et aI., 1985; Ganong et aI., 1986), we hy-pothesized that increased levels of PtdSer in LA-N-2cell membranes might stimulate PtdCho labeling by

476

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ENDOGENOUS PtdSer INCREASES PtdCho LABELING

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RG. 4. PtdSer, but not PtdCho, stimulates [14C]PtdCho synthesis.Cells were treated for 24 h with various concentrations of PtdSer(filled cirdes) or PtdCho (unfilled cirdes) in N2 medium. The cellswere then washed once, and labeled for 2 h with [14C]choline.Incorporation of radioactivity into PtdCho was determined as de-scribed in Materials and Methods. Values represent means :t SEMof triplicate determinations from a single experiment.

enhancing the binding of protein kinase C to mem-branes, thereby stimulating its activity. Therefore, theeffectsof PtdSer and the phorbol ester PMA on PtdChosynthesis in LA-N-2 cells were compared. In experi-ments in which cells were treated with 100 ILMPtdSeror N2 medium alone for 24 h, then washed and labeledfor 2 h in the presence of vehicle (dimethyl sulfoxide)or PMA (100 nM), respectively, PtdSer increased[J4C]PtdCho synthesis to 140 j: 8% of control values(mean j: SEM of six experiments), whereas, in the sameexperiments, PMA increased labeling to only 114j: 4% of control values (data not shown). However, inseparate experiments, this concentration of PMAcaused maximal activation of protein kinase C [seenas the translocation of the enzyme from the cytosol tothe membrane (Fig. 5)] within 1 h of administrationin LA-N-2 cells. Prolonged treatment with phorbol es-

6000

100010PMA(nM)

RG. 5. Translocation of protein kinase C activity from the cyto-plasm to the membrane of LA-N-2 cells by PMA. Cells were pre-treated with N2 medium for 24 h, then exposed to various con-centrations of PMA in N2 medium for 1 h. Cells were sonicatedand separated into soluble and particulate fractions which wereassayed for protein kinase C activity as described in Materials andMethods. Filled symbols represent cytosolic fraction; unfilled sym-bols represent membrane fraction.

477

TABLE 3. Effect of24-h exposure to PMA on basal andPtdSer-stimulated accumulation offuC]PtdCho

PretreatmentRadioactivity in PtdCho

(dpm/mg of protein)

None

PMA (24 h)PtdSer (24 h)PtdSer + PMA (24 h)

7,390 :t 24814,750:t 951"14,687 :t 826"19,558 :t 2,230"

Cells were treated overnight with PtdSer (100 p.M), PMA (100oM), both PtdSer and PMA, or N2 medium alone. The cells werewashed once with N2, labelled for 2 hours with [l4C]choline, andextracted as described in Materials and Methods, and the radioactivityin the lipid fraction was determined. Values represent means:t SEMof triplicate determinations from a representative experiment.

" Values are significantly different from control, p < 0.05.

ters has been reported to desensitize cells to subsequenttreatment with these agents (Solanki and Siaga, 1981;.Collins and Rozengurt, 1982), probably by depletingcells of protein kinase C (Rodriguez-Pena and Rozen-gurt, 1984). In LA-N-2 cells, 24 h of exposure to 100nM PMA reduced protein kinase C activity by 80%(from 1,316 to 252 pmoljmg of protein in one exper-iment). However, following this treatment, incorpo-ration of [J4C]choline into [J4C]PtdCho was signifi-cantly increased (Table 3), suggesting that in LA-N-2cells, protein kinase C may actually inhibit PtdChoaccumulation. Stimulation of PtdCho labeling byPtdSer was not inhibited by coincubation for 24 h withPMA (Table 3); in fact, the effect of PtdSer appearedto be further increased (although not significantly) un-der these conditions.

PtdSer inhibits translocation of protein kinase C tomembranes by PMA

The tumor-promoting phorbol ester PMA stimu-lated in a concentration-dependent fashion the redis-tribution of protein kinase C from the cytosol to themembrane in intact LA-N-2 cells (Fig. 5), and increasedthe activity of the enzyme in vitro in the presence ofsuboptimal concentrations of calcium (see Materialsand Methods); both actions have been observed inmany cell types. PtdSer alone caused no significantchange in the distribution of protein kinase C in theabsence of PMA (Fig. 6). However, treatment for 3 hor longer with PtdSer (130 ILM)shifted the PMA dose-response curve to the right (Fig. 6). This shift was max-imal after 3 h of exposure to PtdSer, at which time thePtdSer content of the cells was approximately half thatachieved at saturation. At this time, the ECsofor PMAwas increased from about 20 nM in controls to 140nM in treated cells (Fig. 6b). A shift of similar mag-nitude was seen after 24 h of PtdSer treatment.

DISCUSSION

A number of cell lines have the ability to accumulateexogenous phospholipids. Chinese hamster fibroblasts

J. Neurochem., Vol. 53. No.2, 1989

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FIG. 6. Inhibition of PMA-induced translocation of protein kinaseC by prior treatment with PtdSer. Cells were treated with N2 me-dium (filled symbols) or 130 ,.M PtdSer in N2 medium (unfilledsymbols) for (8) 15 min, (b) 3 h, or (c) 24 h. The medium wasreplaced with N2 containing various concentrations of PMA for 1h. Then the cells were collected, extracted, and assayed for proteinkinase C activity (see Materials and Methods). Data were normal-ized to express protein kinase C activity as a percentage of themaximum membrane-associated activity observed in the presenceof PMA. Values represent means :t SEM of at least three separateexperiments.

incubated with liposomes containing a fluorescent an-alog of PtdSer were able to incorporate the analog intothe plasma membrane and various intracellular mem-branes (Martin and Pagano, 1987). In Chinese hamsterovary cells treated with exogenous PtdSer, synthesis ofPtdSer was depressed and total PtdSer content was un-changed; addition of PtdEtn to the medium, however,did not suppress PtdEtn synthesis, and PtdEtn contentwas increased (Nishijima et aI., 1986).

Our data show that human LA-N-2 neuroblastomacells take up PtdSer from the medium in a time- andconcentration-dependent manner (Fig. 2b and Table1).Measurement of phospholipids followingsubcellular

J. Nrorochem., Vol. 53, No.2, /989

fractionation of PtdSer-treated cells showed increasedlevels of PtdSer in intracellular and plasma membranes(Fig. I). The increased PtdSer content was accompa-nied by an increase in cell content of other phospho-lipids and by a concentration-dependent increase inthe incorporation of [14C]choline into [14C]PtdCho, aresponse which does not appear to be related to acti-vation of protein kinase C. [Although high concentra-tions (100 p.M) of PtdSer were found to be somewhattoxic to mutant Chinese hamster ovary cells (Voelkerand Frazier, 1986), we found no significant alterationsin protein content or in AcCho content in cells exposedto 100 or 130 p.M PtdSer for 24 h, whereas alterationsin protein kinase C activation were detectable within3 h and increases in PtdCho labeling within 24 h. Fur-thermore, no alteration in cell number relative to con-trols was elicited by exposure to 30 p.M PtdSer for upto 6 days (data not shown).]

Although the cell content ofPtdSer increased several-fold after 24 h of exposure to 130 p.M PtdSer, the ratioof PtdSer to total phospholipids in subcellular mem-brane fractions was increased by 50-140% above con-trol, indicating that not all of the PtdSer taken up bythe cells ultimately became associated with membranes.The balance presumably remained in the cytoplasm,or may have been taken up by lysosomes. The distri-bution of Na+,K+-ATPase and succinate dehydroge-nase among the various membrane fractions obtainedfrom the sucrose density gradient was not altered bytreatment of the cells with PtdSer (Fig. 2), suggestingthat the observed increase in PtdSer associated withplasma or mitochondrial membranes did not affectATPase activity, and did not interfere with the sepa-ration of plasma membrane fragments on the gradient.

The subcellular fractionation data, although sugges-tive, do not demonstrate unequivocally that exogenousPtdSer is actually incorporated into the cell mem-branes. However, this possibility seems more likely inview of the evidence that the amounts of other mem-brane phospholipid species also increased. PtdSer mayserve as a precursor for both PtdEtn and PtdCho viadecarboxylation (Borkenhagen et aI., 1961) to PtdEtnand subsequent stepwise methylation to PtdCho (Bre-mer et aI., 1960).The latter synthetic pathway is presentin LA-N-2 cells (Blusztajn et al., 1987) and, coupledwith decarboxylation of excess PtdSer, could lead toenhanced levels of PtdEtn and PtdCho content inPtdSer-treated cells. Since the enzymes catalyzing thesereactions are membrane-bound, exogenous PtdSermust become rather closely associated with the mem-brane in order to act as a substrate. (It is worth notingthat ethanolamine is not present in the medium; there-fore, decarboxylation of PtdSer is the only route ofPdtEtn synthesis available to these cells.) Alternatively,metabolism of excess PtdSer might release diglyceridemetabolites, which could increase the synthesis of otherphospholipid species by acting as precursors or as reg-ulators of synthetic enzyme activity (e.g., cytidyltrans-ferase; Choy et al., 1979). Although the mechanism of

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ENDOGENOUS PtdSer INCREASES PtdCho LABELING

the PtdSer-related elevations in other phospholipids inLA-N-2 cells is as yet unclear, this process partiallyoffsets the increase in PtdSer relative to other mem-brane phospholipids that results from PtdSer uptake.

The uptake of PtdSer by LA-N-2 cells was associatedwith a concentration-dependent increase in the incor-poration of[14C]choline into P4C]PtdCho. The lack ofeffect of PtdSer on formation of [14C]AcCho(Table 2)suggests that the specific activity of the choline poolwas unchanged by this treatment, and supports theconclusion that PtdCho synthesis was increased byPtdSer. This interpretation is further supported by theobservation that PtdSer enhanced PtdCho labelingwithin I h in cells prelabeled with [14C]choline (notshown). Concomitant with the increase in PtdCho la-beling was a variable decrease in the radioactivity ofthe PCho pool in PtdSer-treated cells (Table 2), con-sistent with increased conversion of PCho to PtdCho,again suggesting stimulation of the synthetic pathway.The loss of label from PCho was greater than the in-crease in PtdCho, possibly reflecting increased degra-dation of PtdCho in PtdSer-treated cells. [Activationof phospholipase D in NG I08-15 cells (Liscovitch etaI., 1987a) released choline into the medium; however,in the present study, released metabolites were notquantitated.] Pulse-chase experiments (in which cellswere exposed to PtdSer during a 72-h chase period)show that the formation of the PtdCho metabolite[14C]GroPCho (which was not detectable after shortlabeling periods) was increased in cells exposed toPtdSer (Fig. 3). This, together with the observed de-crease in [14C]PCho, suggests that both synthesis anddegradation of PtdCho may be increased by PtdSer,and that the enhancement of PtdCho labeling is notdue to decreased degradation. The levels of labeledcholine and AcCho were very low in these cells, andwere not affected by PtdSer treatment, indicating thatthe decrease in PCho was probably not due to accel-erated dephosphorylation. The unchanged levels ofP4C]PtdCho after a prolonged chase period (Fig. 3)may reflect a compensatory increase in PtdCho deg-radation with increasing concentrations ofPtdSer. Thestability of the labeled PtdCho pool in these experi-ments contrasts with the net increase in labeling ofPtdCho seen after short (2 h) labeling periods in PtdSer-treated cells (Fig. 2), and may indicate that recentlysynthesized PtdCho in these cells is not subject to deg-radation.

Under control conditions, the amount of radioac-tivity located in PCho in LA-N-2 cells following shortlabeling periods was 10-30 times greater than that inPtdCho; in contrast, about threefold (Liscovitch et aI.,1986) and fivefold (Warden and Friedkin, 1984)greateramounts were observed in NG 108-15 neuroblastomacells and 3T3 fibroblasts, respectively. This suggeststhat the rate of conversion of PCho to PtdCho in LA-N-2 cells is relatively slow, and may account for thelong doubling time (about 56 h) of these cells and thedelayed appearance of the metabolite [14C]GroPCho.

479

In many cell lines, the incorporation of [14C]cholineinto [14C]PtdChois stimulated by the tumor promotorPMA (Kreibich et aI., 1971; Suss et aI., 1971; Paddonand Vance, 1980; Warden and Friedkin, 1984; Lis-covitch et aI., 1986, 1987b; Kolesnick and Paley, 1987)and by diacylglycerol (Kreutter et aI., 1985; Muir andMurray, 1986; Kolesnick and Paley, 1987; Liscovitchet aI., 1987b). Both compounds can activate cytidyl-transferase (Choy et aI., 1979; Paddon and Vance,1980; Pelech et aI., 1984b), the rate limiting enzymein the PtdCho synthetic pathway, as well as proteinkinase C (Nishizuka, 1984a,b). There is evidence thatin NG 108-15 cells the stimulation of cytidyltransferaseactivity by PMA is due to activation of protein kinaseC (Liscovitch et aI., 1987b). However, PtdCho synthesisalso can be regulated via protein kinase C-independentmechanisms (Kolesnick and Paley, 1987; Liscovitchet aI., 1987b).

Because protein kinase C may regulate PtdCho syn-thesis in some cell lines, and because this enzyme isdependent on phospholipids (particularly PtdSer) foractivation, we assessed the involvement of protein ki-nase C in the stimulation of PtdCho labeling in cellsexposed to PtdSer. Three observations suggest thatprotein kinase C does not mediate this response toPtdSer in LA-N-2 cells. First, the effect of PtdSer wasnot mimicked by concentrations of PMA that maxi-mally activated protein kinase C. Acute exposure toPMA increased PtdCho synthesis in LA-N-2 cells byonly 14%, although it caused a complete, concentra-tion-dependent translocation of protein kinase C tothe membrane (Fig. 5). [Activation of protein kinaseC by hormones and neurotransmitters that generatediacylglycerol (Fearon and Tashjian, 1985; Hirota etaI., 1985; Sugden et aI., 1985), or by the action of di-acylglycerol analogs such as the phorbol ester PMA(Kraft and Anderson, 1983), is accompanied by trans-location of the enzyme's activity from the cytosol tocell membranes, and an increase in the apparent affinityof the enzyme for both PtdSer and calcium (Takai etaI., 1979b; Kishimoto et aI., 1980).] Second, the effectof PtdSer was not inhibited by prolonged treatmentwith PMA. Although in NG 108-15 cells (Liscovitch etaI., 1987b) and in GH3 pituitary cells (Kolesnick andPaley, 1987) prolonged treatment with PMA reducedprotein kinase C levels and abolished the stimulationof PtdCho synthesis by acute administration of PMA,prolonged treatment of LA-N-2 cells with PMA (whichreduced protein kinase C activity by 80%) increasedbasal synthesis of PtdCho and did not inhibit the actionof PtdSer (Table 3). Third, PtdSer treatment alone didnot affect the distribution of protein kinase C, and in-hibited translocation of this enzyme to the membranein LA-N-2 cells by PMA. The increase in [14C]PtdCholevels following prolonged exposure to PMA (Table 3)may indicate that protein kinase C inhibits PtdChosynthesis, or stimulates its degradation in LA-N-2 cells.If this is the case, then depletion of the enzyme mightbe expected to result in accumulation of PtdCho.

J. Neurochem.. Vol. 53. No.2. 1989

480 B. E. SLACK ET AL.

Alternative mechanisms which might underlie ac-tivation of PtdCho synthesis by PtdSer include directactivation of cytidyltransferase by the phospholipid(Choy and Vance, 1978), by its diglyceride metabolites(Choy et aI., 1979), or by its fatty acid constituents(Pelech et aI., 19840). The levels of the latter might beincreased by metabolism of some of the PtdSer accu-mulated by LA-N-2 cells. Hence, although PMA stim-ulates PtdCho synthesis via activation of protein kinaseC in some cell lines, there is evidence that an alter-native, protein kinase C-independent pathway, whichis stimulated by diacylgycerol, also exists (Kolesnickand Paley, 1987; Liscovitch et aI., 1987b).

PtdSer treatment caused a rightward shift in the dose-response curve to PMA, but did not alter the total pro-tein kinase activity associated with the membrane inthe presence of supramaximal PMA concentrations(Fig. 6). This is consistent with a decreased affinity ofprotein kinase C for the membrane-phorbol estercomplex to which it is believed to bind. It is significantthat the inhibitory effectofPtdSer observed in our studywas seen after 3 h of exposure to PtdSer, but not after15 min (Fig. 6), suggesting that this effect requires in-corporation of PtdSer into the cell membrane. It hasbeen shown that the ratio of PtdEtn to PtdSer influ-ences protein kinase C activity in vitro (Kaibuchi etaI., 1981); lowering this ratio by elevating membranePtdSer in LA-N-2 cells might inhibit binding of theenzyme to the membrane. Cell-specific variations inPtdSer composition might partially explain the widerange of ECso values for PMA which have been ob-served in various cell types. Alternatively, we have notruled out the possibility that PtdSer accumulation inthe cell cytoplasm may impede translocation of proteinkinase C to the membrane by PMA.

Elsewhere, it has been shown that the CerPCho pre-cursor and metabolite, sphingosine, inhibited phorbolester binding and protein kinase C activity in platelets(Hannun et al., 1986), whereas sphinganine inhibitedthe differentiation of human promyelocytic leukemiccells by phorbol esters (Merrill et al., 1986). In contrast,alterations in the phospholipid head group compositionof human promyelocytic leukemic cells enhancedbinding of phorbol esters (Cabot, 1983). It remains tobe determined whether other phospholipids will alsomodify protein kinase C activity in intact LA-N-2 cells.

The data presented in this report suggestthat changesin the PtdSer composition of cell membranes maymodify the synthesis and content ofPtdCho, the majorphospholipid component of mammalian cell mem-branes. It will be of interest to determine whether thiseffect is confined to PtdSer, or shared by other phos-pholipids. The ease with which the phospholipid com-position of LA-N-2 neuroblastoma cells can be ma-nipulated to produce measurable physiological con-sequences demonstrates the potential value of thissystem in elucidating relationships between cell mem-brane phospholipid composition, membrane synthesis,and neuronal cell function.

J. Neurochem., Vol. 53. No.2, /989

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Acknowledgment: These studies were supported in part bygrants from the National Institute of Mental Health (MH-28783) and from FIOlA, Abano Terme, Italy. We wish tothank Ms. Asma Ahmed for expert technical assistance.

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