Effect of fumonisin B1 on phosphatidylethanolamine biosynthesis in Chinese hamster ovary cells

BIOCHIMICA ET BIOPHYSICA ACTA

2~ : f ' I

ELSEVIER

Biochimica et Biophysica Acta 1304 (1996) 190-196

8aN

Effect of fumonisin B 1 on phosphatidylethanolamine biosynthesis in Chinese hamster ovary cells K e t a n Badiani, D a v i d M. Byers, H a r o l d W. C o o k , N e a l e D. R i d g w a y ': Departments of Pediatrics and Biochemistry and the Atlantic Research Centre, Dalhousie UniL'ersity, 5849 University A venue, Halifax, Nova Scotia, Canada B3H 4H7

Received 21 March 1996; revised 7 June 1996; accepted 24 July 1996

Abstract

Fumonisin B 1 has been shown to inhibit dihydroceramide synthesis and elevate cellular sphinganine levels in several cultured cell lines. In Chinese hamster ovary (CHO)-K1 cells, 20 #xM fumonisin Bl inhibited sphingomyelin synthesis by 75% after 5 h, but stimulated [3H]serine incorporation into PtdEtn by 5- to 7-fold. Fumonisin caused a 10-20% increase in [3H]serine labelling of PtdSer. While fumonisin (20 #zM) caused sustained inhibition of sphingomyelin synthesis, PtdEtn labelling peaked at 7-fold above controls at 12 h and declined to 4-fold by 24 h. Fumonisin treatment for 12 h increased the in vitro activity of PtdSer synthase by 62% and inhibited PtdSer decarboxylase by 35%, suggesting that increased PtdEtn labelling by [3H]serine is not by this pathway. An ethanolamine 'trap' experiment was performed to assess the contribution of phosphoethanolamine from sphinganine degradation for PtdEtn labelling. Stimulation of [3H]serine incorporation into PtdEtn by fumonisin could be reduced by 60% with the inclusion of 50/xM unlabelled ethanolamine in the culture medium. The ethanolamine-mediated reduction in [3H]serine incorporation into PtdEtn was accompanied by 4-fold increase in cellular [3H]phosphoethanolamine. In control cells labelled with [3H]serine, 50 /xM ethanolamine did not cause [3H]phosphoethanolamine to accumulate. Consistent with elevated phosphoethanolamine production in fumonisin-treated cells, [3H]ethanolamine incorporation into PtdEtn was inhibited by 75% after 12 h. The degradation of endogenous long-chain bases to phosphoethanolamine and entry into the CDP-ethanolamine pathway appears to be a major pathway for PtdEtn synthesis in fumonisin-treated CHO-K1 cells. Keywords: Phosphatidylethanolamine; Fumonisin; CHO-K1 cell; Ovary; (Chinese hamster)

1. Introduction

Sphingomyelin constitutes 5-10% of the total phospholipid of mammalian cell membranes [1].

Abbreviations: CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdCho, phosphatidylcholine; SM, sphingomyelin. * Corresponding author. Fax: + 1 (902) 4941394.

Originally regarded as a structural component of membranes, sphingolipids such as sphingomyelin are now regarded as reservoirs for second messengers such as sphingosine, ceramide and sphingosine 1phosphate [2]. Ceramide can be generated by agonist induced hydrolysis of sphingomyelin and is thought to be a mediator of cell proliferation, differentiation and apoptosis [3]. Long-chain bases such as sphingosine and sphinganine are inhibitors of protein kinase C [4]. As well, long-chain bases inhibit non-protein kinase C-dependent phosphorylation, activate phos-

0005-2760/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0005-2760(96)00119-1

K. Badiani et al./ Biochimica et Biophysica Acta 1304 (1996) 190-196

phatidylinositol metabolism, promote calcium mobilization, and activate phospholipase D [5-8]. An approach to determining the effects of longchain bases on cell function is to elevate intracellular concentrations by inhibition of N-acylation to dihydroceramide using the mycotoxin fumonisin B 1. Fumonisin B~ is structurally similar to sphinganine and is a competitive inhibitor of sphinganine N-acyltransferase [9]. Treatment of a number of cultured cell and animal models with fumonisin B 1 resulted in inhibition of sphingolipid biosynthesis [10-14] and a concomitant increase in the levels of sphinganine, but not sphingosine [13,14]. The metabolic fate of this endogenous sphinganine has not been extensively studied. Exogenous long chain bases are known to be degraded to phosphoethanolamine and incorporated into PtdEtn [15,16]. More recent studies using cultured cells have shown that fumonisin B 1 caused a 30-60% increase in [3H]serine incorporation into PtdEtn [13,16] and inhibited ethanolamine labelling of PtdEtn by a similar degree [16]. In mouse macrophage J774A.1 cells, exogenous [13H]sphingosine was rapidly metabolized to aqueous metabolites and [3H]PtdEtn was the major product in the organic extract [16]. These studies showed that long-chain base catabolism is an important route for supplying ethanolamine for PtdEtn synthesis. In this study we report the influence of fumonisin B~ on PtdEtn synthesis by the CDP-ethanolamine and PtdSer decarboxylase pathways in CHO-K1 cells. Results indicate that fumonisin stimulated [3H]serine labelling of PtdEtn via increased production of phosphoethanolamine from long-chain base catabolism and not as a result of increased PtdSer decarboxylation. CHO-K1 cells appear to be unique in that the extent of PtdEtn labelling from [3H]serine during fumonisin treatment is 10-fold greater than previously reported in hepatocytes [13] and macrophages [161. 2. Materials and methods

191

ethanolamine and ethanolamine were obtained from the Sigma. PtdEtn, sphingomyelin, PtdSer and PtdCho were purchased from Serdary Research Laboratories. Phosphatidyl-[3-t4C]serine (53 mCi/mmol) was a product of Amersham. Thin-layer chromatography plates were from Merck (silica gel 60) or Fisher (silica gel G). All tissue culture media was purchased from Gibco/BRL. 2.2. Cell culture

CHO-K1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum and proline (34 /zg/ml) in 60 mm dishes at 37°C in an atmosphere of 5% CO 2. Cells were seeded at a density of 175 000 cells per dish in 3 ml of medium and experiments were done on day 3 or 4. 2.3. Cell labelling and phospholipid analysis

For [3H]serine labelling of phospholipids, medium was replaced with 2 ml of serine-free DMEM with 5% fetal calf serum and proline (34 /zg/ml). Cells were labelled with 15 /zCi/ml [3H]serine for 1 h, harvested by scraping in phosphate-buffered saline (PBS, 10 mM potassium phosphate (pH 7.4) and 150 mM NaC1) and lipids were extracted as described previously [17]. For experiments using [14C]ethanolamine, the procedure followed was as described above except that all dishes received 1 /zCi/ml of radiolabel. Phospholipids were separated by thin-layer chromatography in a s o l v e n t system of chloroform/methanol/acetic acid/water ( 6 0 / 4 0 / 4 / 1 , by vol.). After iodine staining, the radioactivity associated with phospholipids was determined by scintillation counting and phospholipid phosphorus was quantitated using the procedure of Rouser et al. [18]. [3H]Sphinganine was quantitated after base hydrolysis of glycerolipids and thin-layer chromatography in a solvent system of chloroform/methanol/water ( 6 5 / 2 5 / 4 , v / v / v ) [17].

2.1. Materials

[1-14C]Ethanolamine h y d r o c h l o r i d e (3.9 mCi/mmol) and [3H-(G)]serine (21.7 Ci/mmol) were purchased from Du Pont-New England Nuclear. Fumonisin B~, L-serine, phosphoethanolamine, CDP-

2.4. Isolation and analysis of aqueous phase metabolites

[3H]Serine incorporation into aqueous phase metabolites of the CDP-ethanolamine pathway was

192

K. Badiani et al. / Biochimica et Biophysica Acta 1304 (1996) 190-196

quantitated using a modification of the lipid extraction procedure described above. Cells were scraped in 4 ml of methanol/water (5/4, v/v), an equivalent volume of chloroform was added, samples were vortexed and the phases separated by centrifugation. Aliquots of the upper phase were dried under a stream of nitrogen and separated by thin-layer chromatography on silica gel G plates in a solvent system of ethanol/2% ammonia (1/2, v/v). Authentic standards were included as cartier. Metabolites were detected by spraying the plate with 0.2% ninhydrin in ethanol and heating for 15 min at l l0°C. CDPethanolamine could not be accurately quantitated due to co-migration with [3H]serine. Bands corresponding to the metabolites were scraped into vials and the radioactivity determined by scintillation counting.

2.6. PtdSer decarboxylase assay PtdSer decarboxylase activity in CHO-K1 cell homogenates was measured by determining the rate of formation of radiolabelled PtdEtn from phosphatidyl[3-14C]serine [20,21]. Briefly, [14C]PtdSer (0.8 mM) was dispersed in water by sonication with a probe tip sonicator for 40 seconds. Assays (250 /zl) contained 0.1 M KH2PO 4 buffer (pH 6.8), 10 mM EDTA, 0.5 m g / m l Triton X-100 and 75 /zg cell homogenate protein. Assays was initiated by the addition of [HC]PtdSer (102 /xM, 0.33 Ci/mol) and terminated after 30 min at 37°C by 3 ml chloroform/methanol (2/1, v / v ) followed by 1 ml 0.9% KC1. The reaction products were isolated and quantitated as described above for the PtdSer synthase assay.

2.5. PtdSer synthase assay 3. Results

Enzyme assays were performed on cell homogenates prepared at 4°C in the following manner. Cells were washed twice with 2 ml PBS, scraped in 1 ml PBS and sedimented by centrifugation at 3000 × g for 10 min. Cells were resuspended in 2 ml of cold 0.25 M sucrose, 10 mM Hepes buffer (pH 7.4) and lysed by 12-14 passages through a 21 gauge needle. Protein content of cell homogenates was determined by the method of Lowry et al. [19]. PtdSer synthase base exchange activity was determined by measuring the formation of [3H]PtdSer from [3H]serine with egg yolk PtdCho as exogenous donor lipid [20]. PtdCho was dried under nitrogen, solubilized in 0.1% (w/v) Triton X-100, dispersed by vortexing and sonicated for 30 sec using a probe tip sonicator. The assay was performed in a total volume of 500 /xl containing 10 mM CaC12, 4 mM hydroxylamine, 25 mM Hepes (pH 7.4), 0.8 mM PtdCho, 0.12 m g / m l Triton X-100 and 0.4 mM [3H]serine (8 /zCi//zmol). Assays were started by the addition of 100 /zg cell homogenate protein, incubated for 20 min at 37°C and terminated by the addition of 3 ml chloroform/methanol (2/1, v / v ) and 1 ml 0.9% KC1. Phases were separated by centrifugation and [3H]PtdSer in the organic phase were resolved by thin-layer chromatography in a solvent system of chloroform/methanol/acetic acid/water ( 6 0 / 4 0 / 4 / 1 , by vol.).

The incorporation of [3H]serine was used to monitor de novo biosynthesis of sphingomyelin, and synthesis of PtdSer and PtdEtn via the PtdSer synthase and decarboxylase reactions, respectively. When CHO-K1 cells were treated with varying amounts of fumonisin B 1 for 5 h, there was a concentration-dependent inhibition of [3H]serine incorporation into sphingomyelin that was maximal (75% inhibition relative to control) with 20 /xM fumonisin (Fig. 1). Over the same concentration range, there was a marked stimulation of [3H]serine incorporation into PtdEtn that reached a maximum of 7-fold over controis at 20/zM fumonisin. Fumonisin had little effect on label accumulation in PtdSer, suggesting that either labelled PtdSer was rapidly converted to PtdEtn or [3H]serine was entering PtdEtn by a pathway not involving PtdSer. The effect of time of cell exposure to fumonisin on inhibition of sphingomyelin biosynthesis and stimulation of PtdEtn biosynthesis is shown in Fig. 2. Using 20 /zM fumonisin, [3H]serine incorporation into sphingomyelin was maximally inhibited by 6 h of treatment and this level of inhibition was maintained for 24 h. Reduction of sphingomyelin labelling was accompanied by increased [3H]sphinganine. Persistent inhibition of sphingolipid biosynthesis with fumonisin B l has been observed previously [13]. The reason

K. Badiani et al./ Biochimica et Biophysica Acta 1304 (1996) 190-196 800 0

600 L_ Q.

A

SM

• •

PtdEtn PtdSer

400 j

.= "5

~

co'5 200 E 00--

5

10 15 20 Fumonisin B~ (~M)

25

Fig. 1. Effect of fumonisin B t concentration on [3H]serine labelling of sphingomyelin, phosphatidylethanolamine and phosphatidylserine. CHO-K1 cells were cultured in serine-free DMEM with 5% FCS and the indicated concentrations of fumonisin B 1 for 5 h. One hour prior to harvest, cells were pulse-labelled with [3H]serine, and lipids were extracted and analysed by thin-layer chromatography. Values are the means and standard error of three experiments each performed in duplicate. Abbreviation: SM, sphingomyelin.

for prolonged inhibition is not known, but it is thought to be due to the inability of cells to catabolize fumonisins [13,14]. Stimulation of [3H]serine labelling of PtdEtn was coincident with inhibition of

400 F rO

o t-

!

O.

!

•

SM PtdSer PtdEtn Spa

•

o 300 0

Q_

e-

--

200 /

/

~E v

0 6 --

5

10

15

20

25

Time (h)

Fig. 2. Time-course for fumonisin B 1 effects on [3H]serine labelling of phospholipids. CHO-KI cells were cultured in serine-free DMEM with 5% FCS medium and fumonisin (20 /xM) was added at staggered times so that all cells were harvested at once. Cells were labelled with [3H]serine for 1 h prior to the end of fumonisin treatment. Thus all cells were cultured in serine-free medium for 24 h compared to 6 h in Fig. 1. Values are the means and standard error of four experiments each performed in duplicate. Values for sphinganine are the means and range of two experiments done in duplicate. Abbreviations: SM, sphingomyelin; Spa, sphinganine.

193

sphingomyelin s y n t h e s i s and i n c r e a s e d [3H]sphinganine. The main increase in labelling of PtdEtn was observed by 4 h and was maximum by 12 h. The decline in PtdEtn labelling from 12 to 24 h does not appear to be the result of cytotoxicity since PtdSer synthesis was elevated by 20-50% over the entire time course and there was no evidence of cell death or changes in cellular morphology. Experiments shown in Fig. 2 were performed in serine free-medium, but a similar magnitute increase in PtdEtn labelling was observed if cells were maintained in medium containing serine prior to addition of [3H]serine. Also, it should be noted that the incorporation of [3H]serine into lipids was reduced by one-half in Fig. 2 compared to experiments shown in Fig. 1, but the magnitude increase in labelling of PtdEtn was similar. This is the result of the culture and labelling conditions for these experiments; in Fig. 1, cells were in serine-free medium for 6 h compared to 24 h in Fig. 2. (refer to figure legends for details). Fumonisin (20 /xM) treatment for 12 h resulted in a 50-60% reduction in sphingomyelin mass (results not shown). However, even with the large increase in PtdEtn synthesis as measured by [3H]serine labelling in fumonisin-treated cells, the mass of this phospholipid was unchanged (12.1 + 0.1% vs. 13.7 + 2.1% of total lipid phosphorus in control and fumonisintreated cells, respectively). Fumonisin B 1 perturbs sphingomyelin synthesis, but this agent has little effect on the incorporation of [3H]serine into PtdSer, and PtdEtn labelling was increased. Since enhanced labelling of PtdEtn could represent increased flux through the PtdSer decarboxylation pathway, we measured the activity of PtdSer synthase and PtdSer decarboxylase in fumonisin treated cells. While there was a 65% increase in PtdSer synthase activity in fumonisin-treated cell homogenates (Table 1), PtdSer decarboxylase activity was inhibited in homogenates prepared from cells treated with 20/xM fumonisin for 6 and 12 h. Hence, the 7-fold increase in [3H]serine incorporation into PtdEtn that we had observed in the presence of 20 /zM fumonisin cannot be explained by decarboxylation of newly synthesized PtdSer. The possibility that sphinganine degradation could be providing phosphoethanolamine for the CDPethanolamine pathway was investigated. It is well established that exogenous sphingosine and sphinga-

194

K. Badiani et al. / Biochimica et Biophysica Acta 1304 (1996) 190-196

Table 1 Effect of fumonisin B j on phosphatidylserine synthase and phosphatidylserine decarboxylase activities Activity (% of control) Time of fumonisin B 1 treatment (h)

Phosphatidylserine synthase

Phosphatidylserine decarboxylase

None 6 12

100% " 122+9 162+7

100% b 89+5 64+2

Cells were incubated with vehicle (PBS) or fumonisin (20 /xM) for the indicated times. Cell homogenates were prepared and enzyme activities were assayed as described in Section 2. All values are the mean and standard error of three experiments performed in duplicate. " Control values for phosphatidylserine synthase activity were 4 + 1 pmol PtdSer/min per mg protein. b Control values for phosphatidylserine decarboxylase activity were 13 + 2 nmol PtdEtn/h per mg protein. nine are degraded to phosphoethanolamine [15,16], but the extent of catabolism of endogenously synthesized sphinganine is not certain. Long-chain bases are degraded by a two step process. First, sphingosine kinase phosphorylates the 1-position of sphingosine to form sphingosine-l-phosphate which is subsequently hydrolysed by sphingosine phosphate lyase to produce phosphoethanolamine and a long-chain fatty aldehyde [22,23]. In cells labelled with [3H]serine, fumonisin B 1 elevates the levels of [3H]sphinganine which would be degraded to phosphoethanolamine and enter the PtdEtn biosynthetic pathway. To determine whether this was occurring in fumonisin-treated CHO-K1 cells, an experiment was carried out wherein cells were treated with 2 0 / z M fumonisin in a medium supplemented with and without ethanolamine (50 /~M) for 6 h. The rationale was that catabolism of [3H]sphinganine in fumonisin-treated cells would result in production of [3H]phosphoethanolamine that, in cells cultured in medium with excess ethanolamine, should accumulate due to isotope dilution in the expanded, intracellular pool. As shown in Table 2, there was 4 - f o l d increase in [ 3 H ] p h o s phoethanolamine in fumonisin-treated cells cultured with ethanolamine compared to cells grown without ethanolamine supplementation. The increase in phosphoethanolamine was paralleled by a 60% reduction in [3H]serine-labelled PtdEtn in fumonisin-treated cells. With the exception of fumonisin/ethanolamine-treated cells, which displayed slight elevation

Table 2 Effect of fumonisin B~ on incorporation of [3H]serine into lipid and water-soluble metabolites in the presence of ethanolamine Addition

Etn P-Etn PtdEtn (dpm/nmol lipid phosphorus)

PtdSer

None Fumonisin None + Etn Fumonisin+Etn

119+7 126+28 124+ 12 166+ 17 115_+11 152+26 159+ 12 650+48

211+20 242+ 17 257+30 219+ 12

96+13 480+24 114+10 181+18

Cells were grown for 6 h in the presence or absence of fumonisin B 1(20 /xM) in serine-free DMEM medium plus 5% FCS with or without ethanolamine (50 ~M). Cells were labelled with [3H]serine and lipids extracted as described in Section 2. Each value is the mean and standard error of three experiments each performed in duplicate. Abbreviations: Etn, ethanolamine; P-Etn, phosphoethanolamine. in [3H]ethanolamine, there was no change in labelling of the ethanolamine pool under any conditions. These results suggest that phosphoethanolamine produced from endogenous sphinganine degradation in fumonisin-treated cells directly enters the CDP-ethanolamine pathway. Results shown in Table 2 suggested that enhanced production of phosphoethanolamine in fumonisin treated cells would inhibit ethanolamine labelling of PtdEtn. To determine if this was the case, CHO-K1 cells were treated with fumonisin for up to 12 h and pulsed with [14C]ethanolamine for the last hour of each time point (Fig. 3). There was a steady decline 1201

oo -

40p

20[ JO

14

Time (h)

Fig. 3. Inhibition by fumonisin B~ of [~4C]ethanolamine incorporation into phosphatidylethanolamine. Ceils were treated with 20 /zM fumonisin for the indicated times. One hour prior to the end of each time period, cells were pulse labelled with [14C]ethanolamine (1 /zCi/ml). Values are expressed as a percentage of radiolabel incorporated compared to vehicle-treated cells (2138 dpm/nmol lipid phosphorus). Values are the means and range of 2 experiments each performed in duplicate.

K. Badiani et al. / Biochimica et Biophysica Acta 1304 (1996) 190-196

in radiolabel incorporation into PtdEtn with only 25% of initial activity remaining at 12 h.

4. Discussion Although it has been shown that fumonisin treatment of cells contributes to synthesis of PtdEtn via breakdown of long-chain bases [16], the magnitude of this contribution to PtdEtn biosynthesis is uncertain. This is a difficult problem since [3H]serine used to label sphingolipids and long-chain bases can also enter PtdEtn via base exchange and PtdSer decarboxylation. A study of the effects of fumonisin on sphingolipid metabolism in hepatocytes reported a 50% increase in PtdEtn labelling with [3H]serine [13]. A similar 30-50% increase in [3H]serine-labelled PtdEtn was reported in J774.A.1 cells and fumonisin treatment also caused a 30% reduction in [~4C]ethanolamine incorporation into PtdEtn [16]. In CHO-K1 cells, fumonisin inhibited [14C]ethanolamine labelling by 75% and produced a 5- to 7-fold increase in [3H]serine incorporation into PtdEtn. The magnitude of fumonisin-stimulated PtdEtn labelling in CHO-K1 cells is unusually large and could indicate a greater capacity to degrade endogenous longchain bases or more efficient utilization of phosphoethanolamine derived from long-chain base catabolism. Several pieces of evidence presented here support the conclusion that endogenous long-chain base-derived phosphoethanolamine is incorporated into PtdEtn in fumonisin treated CHO-K1 cells. First, the PtdSer synthase and decarboxylation pathway cannot account for the large increase in serine-labelled PtdEtn. PtdSer base exchange activity is stimulated by fumonisin, consistent with a 10-40% increase in [3H]serine labelling of PtdSer, but in vitro PtdSer decarboxylase activity was inhibited. Second, [3H]phosphoethanolamine was identified as the precursor for [3H]PtdEtn synthesis in fumonisin treated cells. An excess of cold ethanolamine in the medium did not completely inhibit fumonisin-stimulated PtdEtn synthesis; however, this method clearly demonstrated that phosphoethanolamine, and not ethanolamine, was the precursor for PtdEtn labelling. Third, inhibition of [~4C]ethanolamine labelling of PtdEtn by fumonisin is consistent with phosphoethanolamine

195

from long-chain base catabolism and de novo synthesis entering a common pool for CDP-ethanolamine synthesis, as was shown in cultured macrophages [16]. The potential for incorporation of [3H]serine from long-chain base degradation illustrates an important limitation when measuring PtdEtn biosynthesis; the base exchange and PtdSer decarboxylase pathway may not be the only route for serine incorporation into PtdEtn. In untreated CHO-K1 cells, ethanolamine and phosphoethanolamine pools were radiolabelled suggesting that a portion of PtdEtn labelling by [3H]serine could be via the CDP-ethanolamine pathway (Table 2). However, radioactivity in these two precursors was not 'trapped' by inclusion of ethanolamine in the medium, as was the case in fumonisin-treated CHO-K1 cells. This suggests that the contribution from sphinganine catabolism to the CDP-ethanolamine pathway in cultured cells occurs primarily under conditions of long-chain base accumulation such as inhibition of N-acylation or addition of exogenous long-chain bases [15,16]. However, ethanolamine did not completely inhibit fumonisinstimulated PtdEtn labelling. The same could be true for control conditions, thus giving an underestimation of the contribution of label from long-chain base catabolism. Other factors such as ethanolamine availability and PtdSer decarboxylase activity could influence the proportion of label in PtdEtn from long-chain base catabolism. The accumulation of phosphoethanolamine in fumonisin-treated cells would not be due to stimulation of a PE-specific phospholipase C. This futile cycle of rapid synthesis of PtdEtn, subsequent hydrolysis by phospholipase C and reutilization of phosphoethanolamine would not result in net accumulation of label in PtdEtn as observed in this study. In light of these findings, care should be taken when interpreting the results of [3H]serine labelling experiments in CHO-K1 and other cultured cells. In CHO-K1 and BHK-21 cells the main pathway for synthesis of PtdEtn is the PtdSer decarboxylase pathway [24]. Reports also have been published suggesting that the decarboxylation pathway is less significant than the CDP-ethanolamine pathway [25]. The CDP-ethanolamine pathway is a preferred route for the synthesis of ethanolamine plasmalogens [26], although this may not be true for all cells [27]. In fumonisin-treated cells, and under other conditions of

196

K. Badiani et al. / Biochimica et Biophysica Acta 1304 (1996) 190-196

long-chain base accumulation, the CDP-ethanolamine pathway could also have an important function in removal of excess phosphoethanolamine. Phosphoethanolamine from sphinganine catabolism does not result in a net increase in cellular PtdEtn mass indicating relatively minor input or compensatory down regulation of other PtdEtn biosynthetic routes. Conversion of PtdSer to PtdEtn could be reduced in fumonisin-treated cells because decarboxylase activity measured in vitro was inhibited by 35%. Inhibition of PtdSer decarboxylase in treated cells indicates that phosphoethanolamine or increased synthesis of PtdEtn may reciprocally regulate both the decarboxylase and de novo pathways of PtdEtn biosynthesis. It has been shown in human keratinocytes that phosphoethanolamine and PtdEtn can inhibit the activity of the PtdSer decarboxylase [21]. In summary, this study shows that endogenous long-chain bases contribute to PtdEtn biosynthesis in CHO-K1 cells, and the entry point into PtdEtn biosynthesis is phosphoethanolamine. As shown in other cultured cells [15,16], this pathway appears to function primarily when endogenous long-chain bases are increased. However, the rapid catabolism of exogenous [1- 3H]sphingosine to [ 3H]PtdEtn [ 16] suggests that the pathway may be active under normal conditions and its contribution to PtdEtn synthesis masked by synthesis via the PtdSer decarboxylase pathway. Acknowledgements Thanks to Robert Zwicker and Gladys Keddy for their assistance with cell culture. This work was supported by a program grant from the Medical Research Council of Canada (PG-11476) and a Scholarship to N.D.R.K.B. is a Heart and Stroke Foundation of Canada Research Fellow. References [1] White, D.A. (1973) in Form and Function of Phospholipids (Ansell, G.B., Dawson, R.M.C. and Hawthorne, J.N. eds.), pp. 441-482, Elsevier, Amsterdam.

[2] Merrill, A.H., Jr., Hannun, Y.A. and Bell, R.M. (1993) Adv. Lipid Res. 25, 1-24. [3] Hannun, Y. (1994) J. Biol. Chem. 269, 3125-3128. [4] Hannun, Y. and Bell, R.M. (1989) Science 243, 500-507. [5] Ghosh, T.K., Bian, J. and Gill, D.L. (1990) Science 248, 1658-1656. [6] Chao, C.P., Laulederkind, S.J.F. and Ballou, L. (1994) J. Biol. Chem. 269, 5849-5856. [7] Lavie, Y. and Liscovitch, M. (1990) J. Biol. Chem. 264, 7617-7623. [8] Bluztajn, J.K., Hudson, P.L., Slack, B.E. (1994) in SignalActivated Phospholipases (Liscovitch, M., ed.), pp. 213 -230, R.G. Landes Company, Austin, TX. [9] Merrill, A.H., Jr., Van Echten, G., Wang, E. and Sandhoff, K. (1993) J. Biol. Chem. 268, 27299-27306. [10] Schroeder, J.J., Crane, H.M., Xia, J., Liotta, D.C. and Merrill, A.H., Jr. (1994) J. Biol. Chem. 269, 3475-3481. [ll] Yoo, H.-S., Norred, W.P., Wang, E., Merrill, A.H., Jr. and Riley, R.T. (1992) Toxicol. Appl. Pharmacol. 114, 9-15. [12] Wu, W.-I., McDonough, V.M., Nickels, J.T., Jr., Ko, J., Fischl, A.S., Vales, T.R., Merrill, A.H., Jr. and Carman, G.M. (1995) J. Biol. Chem. 270, 13171-13178. [13] Wang, E., Norred, W.P., Bacon, C.W., Riley, R.T. and Merrill, A.H., Jr. (1991) J. Biol. Chem. 266, 14486-14490. [14] Merrill, A.H., Jr., Wang, E., Gilchrist, D.G. and Riley, R.T. (1993) Adv. Lipid Res. 26, 215-234. [15] Stoffel, W. (1973) Mol. Cell. Biochem. l, 147-155. [16] Smith, E.R. and Merrill, A.H., Jr. (1995) J. Biol. Chem. 270, 18749-18758. [17] Ridgway, N.D. and Merriam, D.L. (1995) Biochim. Biophys. Acta 1256, 57-70. [18] Rouser, G., Siakatos, A.N. and Fleisher, S. (1966) Lipids l, 85-86. [19] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [20] Voelker, D.R. and Frazier, J.L. (1986) J. Biol. Chem. 261, 1002-1008. [21] Arthur, G. and Lu, X. (1993) Biochem. J. 293, 125-130. [22] Buehrer, B.M. and Bell, R.M. (1993) Adv. Lipid. Res. 26, 59-67. [23] Van Veldhoven, P.P. and Mannaerts, G.P. (1993) Adv. Lipid Res. 26, 69-98. [24] Voelker, D.R. (1984) Proc. Natl. Acad. Sci. USA 81, 26692673, [25] Tijburg, L.B.M., Gelen, M.J.H. and Van Golde, L.M.G. (1989) Biochim. Biophys. Acta 1004, 1-19. [26] Miller, M.A. and Kent, C. (1986) J. Biol, Chem. 261, 1002-1008. [27] Xu, Z.L., Byers, D.M., Palmer, F.B.St.C., Spence, M.W. and Cook, H.W. (1991) J. Biol. Chem. 266, 2143-2150.