Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells

Btl

ELSEVIER

Biochimica et Biophysica Acta 1256 (1995) 57-70

Biochi~ic~a et BiophysicaA~ta

Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells Neale D. Ridgway *, Deborah L. Merriam Department of Pediatrics and Biochemistry and the Atlantic Research Centre, Dalhousie University, 5849 University Acenue, Halifax, Nova Scotia, Canada B3H 4H7 Received 19 May 1994; revised 17 October 1994; accepted 23 December 1994

Abstract

A series of radiolabelled ceramides (D-erythro and L-threo) and dihydroceramides (DL-erythro and DL-threo) with 2, 4 or 6 carbon N-acyl groups were synthesized. These analogues were incubated with cultured CHO cells and radioactive products isolated and analyzed. In addition to synthesis of short-chain sphingomyelin and glucosylceramide, radiolabelled sphingosine and sphinganine were released from short-chain ceramides and dihydroceramides and subsequently utilized for synthesis of long-chain ceramide and sphingolipids. Substrate preference for short-chain sphingomyelin synthesis in cells was o-erythro-ceramides> L-threo-ceramides > DL-erythrodihydroceramides > OL-threo-dihydroceramides, and C4-and C6-analogues were preferred over the C2-analogue. Kinetic constants for conversion of short-chain (dihydro)ceramides to short-chain sphingomyelin were determined using CHO cell membranes and found to correlate with substrate preference in cultured cells. D-erythro-C6-Ceramidewas the preferred substrate for short-chain glucosylceramide synthesis. D-erythro-C2-ceramide inhibited incorporation of [3H]serine into sphingomyelin, glucosylceramide and ceramide rapidly (2 h) and in a dose-dependent manner. Over a similar time period, [3H]choline-labelling of sphingomyelin was not affected. Inhibition of [3H]serine-labelling of sphingolipids appeared to correlate with release of [3H]long-chain bases from short-chain ceramides and dihydroceramides and synthesis of long-chain sphingolipids. However, some discrepancies between DL-erythro-C 4- and C6-dihydroceramides, and D-erythro-C2-ceramide suggested that short-chain dihydroceramides were less efficient in suppressing de novo synthesis from [3H]serine, while contributing substantially to endogenous sphingolipid synthesis. Inhibition of de novo sphingolipid synthesis by short-chain ceramides and dihydroceramides could not be related to inhibition of serine palmitoyltransferase activity in vitro. Keywords: Short-chain ceramide; Short-chain dihydroceramide; Sphingomyelin synthesis; CHO cell

I. Introduction

Sphingolipids, which include sphingomyelin and glycosphingolipids, are important constituents of all eukaryotic cell membranes. Sphingolipids are structurally diverse, but have a common hydrophobic component, generically

Abbreviations: BSA, bovine serum albumin; C8-APPD, L-threo-2(N-octanoyl-amino)-l-phenyl-l,3-propanodiol; C2-ceramide, N-acetylsphingosine; C2-dihydroceramide, N-acetyl-sphinganine; C4-ceramide, N-butyl-sphingosine; C4-dihydroceramide, N-butyl-sphinganine; C 6ceramide, N-hexanoyl-sphingosine; C6-dihydroceramide, N-hexanoylsphinganine; L-MAPP, L-threo-2-(N-myristoyl-amino)-l-phenyl-l-propanol; D-MAPP, D-erythro-2-(N-myristoyl-amino)-l-phenyl-l-propanol; NBD, 7-nitro-2,1,3-benzoxadiazol-4-yl; PtdSer, phosphatidylserine; PtdCho, phosphatidylcholine; PtdEm, phosphatidylethanolamine; SPT, serine palmitoyltransferase. * Corresponding author. Fax: + 1 (902) 4941394 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 0 1 0 - 0

referred to as ceramide, composed of a long-chain base (i.e., sphingosine or sphinganine) and N-acylated fatty acid [1]. In addition to its role as a structural component of cell membranes, sphingomyelin can be regarded as a reservoir for the lipid derived second messengers ceramide and sphingosine. Sphingosine and other long-chain bases are proposed regulators of protein kinase C, while ceramide has been shown to mediate intracellular signalling events fundamental to cell proliferation, differentiation and cell death (apoptosis) [2,3]. Ceramide can be produced either de novo or by catabolism of endogenous membrane sphingolipids. The enzymes involved in dihydroceramide biosynthesis have been localized to the endoplasmic reticulum [4], but identity and localization of the enzyme that introduces the 4-trans double bond in ceramide is not known. Next, ceramide is delivered to glycosyltransferases in the Golgi apparatus [5] and sphingomyelin synthase, which resides

58

N.D. Ridgway,D.L. Merriam/ Biochimica et BiophysicaActa 1256 (1995)57-70

predominantly in Golgi and plasma membrane [6,7], for the synthesis of glycosphingolipids and sphingomyelin, respectively. Ceramide can also be produced by sphingomyelinase hydrolysis of the phosphocholine head-group. Acid sphingomyelinase degrades sphingomyelin in the lysosomes and is probably involved in lipoprotein catabolism and membrane turnover [8]. In addition to the lysosomai enzyme, there are several different forms of sphingomyelinase distinguished by cation requirements and activity at neutral pH [8]. One of these neutral sphingomyelinases appears to be important in signal transduction and has been proposed to generate ceramide in response to external stimuli [2,3,9]. Due to limited solubility of long-chain ceramides in aqueous environments, the study of ceramide metabolism in cultured cells or in vitro is difficult. This problem can be circumvented by substitution of the N-acyl group with a shorter acyl chain. Short-chain fluorescent and radiolabelled cell-permeable ceramide analogues have been useful in demonstrating the intracellular pathways involved in ceramide and sphingolipid metabolism and trafficking [10-12]. Short-chain ceramides have also been used extensively to mimic cellular responses elicited by endogenous ceramide generated in signal transduction processes [2,3]. Relatively little is known about metabolism of short-chain (dihydro)ceramides 1 in cultured cells. The influence of stereochemical configuration on metabolism of C6-NBDceramides has been addressed [11], but the effect of altering N-acyl chain length was not examined. This has important implications when using these compounds as biologically active metabolites like sphingosine and sphinganine could be released from short-chain (dihydro)ceramides in cultured cells. In the present study, we have investigated the metabolism in CHO cells and in vitro of radiolabelled short-chain (dihydro)ceramide analogues with 2-, 4- or 6-carbon N-acyl groups. Metabolism of the analogues was specified by the chain-length, stereochemistry and longchain base component. The influence of short-chain (dihydro)ceramides on endogenous sphingolipid synthesis was also investigated and related to the degree of metabolism of the analogues.

2. Materials and methods

2.1. Materials D-erythro-Sphingosine, DL-erythro-sphinganine, DLthreo-sphinganine, fatty acid-free BSA and sphingomyelinase (Staphylococcus aureus) were purchased from Sigma Chemical, St. Louis, MO. [U-14C]Serine, NaB[3H]4,

i Refers to both cerarnidesand dihydroceramides.

[3H(G)]serine, [methyl-3H]choline and En3Hance spray were purchased from DuPont-New England Nuclear. Thin-layer chromatography plates (silica gel 60) were from Merck. Autoradiography film (Hyperfilm-MP) was from Amersham Corp. D-MAPP, L-MAPP and C8-APPD were gifts from Dr. Y u s u f H a n n u n , Division of Hematology/Oncology, Duke University Medical Center, Durham NC. Fetal calf serum was adjusted to 1.21 g / m l with NaBr and delipidated by ultracentrifugation, followed by dialysis against phosphate buffered saline (150 mM NaC1, 10 mM sodium phosphate, pH 7.4). 2.2. Tissue culture Chinese hamster ovary (CHO) K-1 cells were cultured in Dulbecco's modified Eagles medium containing 5% fetal calf serum (medium A) and proline (34 /zg/ml) at 37°C in an atmosphere of 5% CO 2, For experiments, 175000 cells were seeded in 60 mm dishes in 3 ml of medium A and used 4 days later when approx. 70% confluent. In some experiments, cells were switched into Dulbecco's modified Eagles medium containing 5% delipidated fetal calf serum (medium B) and proline (34 p,g/ml) 18-24 h prior to the addition of (dihydro)ceramides. Short-chain (dihydro)ceramides were added to cells as a 1:1 (mol/moi) complex with BSA [11]. The complex was prepared by adding an appropriate amount of analogue, dissolved in 1 vol. ethanol, to 10 vol. of 1 mM BSA in 20 mM Tris-HC1 (pH 7.4). The complex was vortexed briefly and incubated at 37° C for 10 rain prior to use. 2.3. Preparation of labelled short-chain (dihydro)ceramides D-erythro-Sphingosine and DL-erythro-sphinganine (1015 mg) were acylated with acetic, butyric or hexanoic anhydride, and the N-acetyl ceramide and dihydroceramide products recovered as previously described [13]. C 4- and C6-(dihydro)ceramides were recovered by extraction into chloroform/methanol (1:1, v / v ) , washed 5 to 6 times with an ideal upper phase [14] and purified by thin-layer chromatography using chloroform/methanol (93:7, v / v ) as the developing solvent. Subsequently, the 3-hydroxyl group was oxidized by treatment with 10 mg chromic anhydride in 1.5 ml pyridine and 1.5 ml benzene for 3 h, followed by extraction of the 3-keto (dihydro)ceramides and purification by thin-layer chromatography [13]. (Dihydro)ceramides were radiolabelled by reduction of the 3-keto group with a 5-fold molar excess of NaB[3H]4 (468 mCi/mmol) in methanolic 0.05 M NaOH for 30 rain at 4°C [15,16]. The labelled analogues were extracted with 4 ml of chloroform and residual NaB[3H]4 removed by 3 washes with 1 vol. of distilled water. The racemic mixture of erythro and threo isomers was resolved by thin-layer chromatography in diethyl ether/methanol (9:1 ( v / v ) for C 2- and C4-analogues and 31:1 ( v / v ) for Ca-analogues). Thin-layer chro-

N.D. Ridgway, D.L. Merriam/ Biochimica et Biophysica Acta 1256 (1995) 57-70

matography plates were exposed to film at - 7 0 ° C for 6-10 h, [3-3H](dihydro)ceramides identified using the autoradiogram as a template, scraped and eluted in chloroform/methanol (2:1, v / v ) . The erythro and threo isomers of all six short-chain ceramides were purified by one additional thin-layer chromatography step. The identity and purity of (dihydro)ceramide analogues was routinely checked by analysis of the long-chain component following acid hydrolysis in acetonitrile/water (9:1, v / v ) , 0.5 M HC1 for 1 h at 75°C [17] and co-chromatography with authentic standards in chloroform/methanol/2 M NH4OH (40:10:1, v / v ) . Specific activity of (dihydro)ceramides (200 dpm/pmol) was calculated from the specific activity of NaB[3H]4: one tritium atom is introduced per molecule of (dihydro)ceramide making the specific activity onequarter that of the hydride donor. This was confirmed by measuring specific activities of several labelled C4- and C6-ceramides and dihydroceramides based on mass measurements using the diacylglyerol kinase assay [18] and unlabelled standards. 2.4. Incubation of CHO cells with radiolabelled ceramides and dihydroceramides

CHO cells were washed once with 2 ml of warm phosphate-buffered saline, and 1 ml of serum-free medium A was added to each dish followed by 5 nmol of ceramide/BSA complex. Cells were incubated at 37°C in a 5% CO 2 atmosphere for up to 1 h, medium removed and 1 ml of methanol/water (5:4, v / v ) was added to each dish. Cells were scraped into glass extraction tubes and 5 ml chloroform/methanol (1:1, v / v ) was added to give a single phase and extracted by the method of Folch et al. [14]. Unless otherwise indicated, total CHO cell lipids were separated by thin-layer chromatography in a solvent system of chloroform/methanol/15 mM CaC12 (65:35:8, v / v ) , sprayed with En3Hance and exposed to film at - 7 0 ° C for 1-4 days. Using the fluorogram as a guide, bands were scraped from the plate and the associated radioactivity quantitated by liquid scintillation counting. The identity of [3H]-labelled sphingomyelin, sphingosine, sphinganine, glucosylceramide and ceramide was confirmed by co-migration with unlabelled authentic standards and [14C]serine.labelled sphingolipids prepared from CHO cells. Short-chain sphingomyelin was identified by digestion of total lipid extracts with sphingomyelinase (Staphylococcus aureus) according to the manufacturer or comparison to R r values for C6-sphingomyelin [6]. 2.5. Labelling,

extraction and analysis of [3H]serine-

labelled lipids

Unless otherwise indicated, monolayers of CHO cells were pulse-labelled for 1 h with 7.5 /zCi [3H(G)]serine in 1 ml of serine-free medium A (with this short-labelling protocol less then 8% of the total label in the lipid fraction

59

was incorporated into PtdCho and neutral lipid, and 95% of the label in sphingolipids was in the long-chain base faction). At the end of a 1 h pulse, medium was removed, cells rinsed with 1 ml of cold phosphate-buffered saline, scraped in l ml of methanol/water (5:4, v / v ) and transferred to a screw-cap tube. The culture dish was rinsed with 1 ml of methanol/water, extracts combined and 5 ml of chloroform/methanol (1:2, v / v ) added. Extracts were shaken vigorously, 4 ml of 0.58% NaC1 added and the phases separated by centrifugation at 2000 X g X 5 min. The organic phase was extracted twice with 2 ml of methanol/0.58% NaCl/chloroform (45:47:3, v / v ) and dried over a small column of anhydrous sodium sulphate. Total phospholipids in the organic extract were separated by thin-layer chromatography in a solvent system of chloroform/methanol/acetic acid/water (60:40:4:1, v / v ) and visualized with iodine vapour. Bands corresponding to phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and sphingomyelin were scraped, and radioactivity quantitated by liquid scintillation counting. Glycerolipids in an aliquot of the organic extract were subjected to alkaline methanolysis in 0.1 M KOH in methanol/water (95:5, v / v ) for 2 h at 37°C and sphingolipids extracted following neutralization with HC1. Sphingolipids and ceramide were separated by thin-layer chrom atography on silica-gel G plates in chloroform/methanol/water (65:25:4, v / v ) , identified by fluorography and radioactivity quantitated by scintillation counting. Acid hydrolysis of total lipid extracts and isolation of sphingosine and sphinganine was performed as described in a previous section. Total lipid phosphorus was determined by the method of Rouser et al. [19] and used to calculate the specific activity of [3H]serine-labelled lipids. 2.6. Sphingomyelin synthase and SPT assays

CHO cells were homogenized in 10 mM EDTA, 20 mM Tris-HC1 (pH 7.4) by 10 passages through a 22 gauge needle. Total cell membranes were prepared by centrifugation of CHO cell homogenates at 100 000 X g for 1 h and resuspension of the membrane pellet in homogenization buffer using a glass dounce homogenizer. Sphingomyelin synthase was assayed as previously described [6] in a final volume of 500 /zl containing 25 mM KCI, 0.5 mM EDTA and 50 mM Tris-HC1 (pH 7.4) and 100 /xg of protein. The reaction was initiated by the addition of [3-3 H]ceramide or [3-3H]dihydroceramide (200 d p m / p m o l ) as a 1:1 (mol/mol) complex with BSA, incubated at 37°C for 15 min, terminated by the addition of 4 ml of chloroform/methanol (1:1, v / v ) and extracted. Lipid extracts were resolved by thin-layer chromatography in chloroform/methanol/15 mM CaC12 (65:35:8, v / v ) and subjected to fluorography for 18-48 h. The reaction gave a single product corresponding to short-chain sphingomyelin, which was scraped and quantitated by liquid scintillation counting. Synthesis of short-chain sphingomyelin

60

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

was linear for 20 min with up to 200 /xg protein in the assay. SPT was assayed as previously described [20]. CHO cell membranes ( 2 5 - 5 0 mg), isolated as described above, were incubated with 100 mM HEPES (pH 8.0), 2.5 mM EDTA, 50 p,M pyridoxyl phosphate, 5 mM DTT, 2 0 0 / x M palmitoyl CoA and 1 mM [3H-(G)]serine in a final volume of 100 /zl. 3-Ketosphinganine was extracted from the reaction mixture, separated by thin-layer chromatography, and identified by fluorography and comparison to an authentic standard. Membrane protein was measured by the method of Lowry et al. [21] using BSA as a standard.

3. Results Short-chain radiolabelled (dihydro)ceramide analogues with 2-, 4- or 6-carbon N-acyl groups were synthesized and their metabolism investigated in CHO cells. An exam-

pie of the spectrum of metabolites from o-erythro-C4-[33H]ceramide and DL-erythro-C4-[3-3H]dihydroceramide following a 1 h incubation with CHO cells is shown in Fig. 1. Both compounds were converted to C4-sphingomyelin and C4-glucosylceramide, with D-erythro-C4-ceramide being a substantially better substrate for these reactions. Interestingly, another set of metabolites was formed that co-migrated in two different solvent systems with standards for long-chain sphingomyelin, ceramide and glucosylceramide. In addition, sphingosine was produced by metabolism of both C4-compounds, while sphinganine was generated in cells receiving DL-erythro-C4-[3-3H]dihydro ceramide. Results from these fluorograms suggest that short-chain ceramides undergo a series of complex reactions that involve (1) addition of a phosphocholine or glucose headgroup, (2) hydrolysis of the N-butyl group (or other acyl group) to yield labelled long-chain bases, (3) conversion of [3H]long-chain bases to ceramide and ultimately sphingomyelin and glucosylceramide and (4)

B

....

]C4-Cer]C4-DHCI Std I

iiiii

Q

A

SC-SM" Fig. 1. Metabolism of D-erythro-C4-[3-3H]ceramide and DL-erythro-C4-[3-3H]dihydroceramide by CHO cells. CHO cells (two 60 mm dishes) were incubated with 20 nmol each of ceramide (C4-Cer) or dihydroceramide (C4-DHC) in serum-free medium for 1 h. Cells were harvested, lipids extracted and separated by thin-layer chromatographyin (A) chloroform/methanol/2 M NHaOH (40:10:1, v/v) or (B) chloroform/methanol/15 mM CaC1z (60:35:8, v/v). Thin-layer chromatography plates were subject to fluorography for 11 (panel A) or 4 days (panel B). The standard lanes (Std) are sphingolipids prepared by base hydrolysis of [14C]serine-labelled CHO cells lipids. Abbreviations are: LC-SM, long-chain sphingomyelin; SC-SM, short-chain sphingomyelin; LC-GIcCer, long-chain glucosylceramide; SC-GlcCer, short-chain glucosylceramide; LC-Cer, long-chain ceramide; SC-Cer, short-chain ceramide (or dihydroceramide); Spo, sphingosine; Spa, sphinganine.

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

catabolism of radiolabelled long- and short-chain sphingolipids and ceramides to long-chain bases or other intermediates. A comprehensive analysis of the cellular products of radiolabelled short-chain (dihydro)ceramide metabolism is shown in Table 1. The most extensively metabolized ceramides and dihydroceramides were those with N-butyl and N-hexanoyl groups (Table 1). D-erythro-C 4- and oerythro-C6-[3-3H]Ceramide were taken up by cells and converted to short-chain sphingomyelin to a similar extent. However, D-erythro-C6-ceramidewas converted to C6-glycosylceramide 4-times more efficiently compared to both the D-erythro-C2- and D-erythro-C4-ceramides. Similarly, 3-fold less D-erythro-C2-ceramidewas converted to shortchain sphingomyelin compared to the D-erythro-C 4 and C6-ceramides. Thus, sphingomyelin synthesis prefers an N-acyl group of 4 carbons or more, while glucosylceramide synthesis increases to a level comparable to sphingomyelin synthesis when the N-acyl group is 6 carbons. Differences in the specificity of sphingomyelin and glucosylceramide synthesis were observed for ceramide and dihydroceramide isomers. Short-chain sphingomyelin synthesis was reduced 2- to 6-fold with the L-threo isomer of ceramide. This is in contrast to results for short-chain glucosylceramide synthesis where the L-threo isomer was utilized 5- to l 1-fold less efficiently then the D-erythro isomer (the greatest difference was with the two C6-ceramide isomers). Overall, dihydroceramides were poorer substrates for sphingomyelin and glucosylceramide synthesis then their ceramide counterparts. Part of this may be explained by the relatively poor uptake of the DL-erythrodihydroceramides (as reported previously for DL-erythroC:-dihydroceramide [22]). For example, approximately 3fold more of the C6-ceramides and DL-threo-C6-dihydro-

61

ceramide were recovered in cells compared to DL-erythroC6-dihydroceramide. If synthesis of short-chain sphingomyelin and glucosylceramide is normalized by a factor of three, DL-erythro-C6-dihydroceramide is still a poor substrate compared to D-erythro-C6-ceramide, but substantially better then DL-threo-dihydroceramide. Of the C2-derivatives studied only D-erythro-C2-ceramide generated significant amounts of short-chain sphingomyelin and glucosylceramide, but this was 3- to 4-fold less then that produced from 4 and 6 carbon N-acyl ceramides. Also included in Table 1 is the distribution of radioactivity in long-chain sphingomyelin and glucosylceramide, and the long-chain bases, sphingosine and sphinganine. Metabolism of radiolabelled D-erythro-Ca-ceramide and D-erythro-C6-ceramide resulted in production of sphingosine, which could enter the endogenous sphingolipid synthetic pathway. Results in Fig. 1 and Table 1 show that after a 1 h incubation with these two analogues, radiolabelled long-chain ceramide was produced, along with approximately equal amounts of sphingomyelin and glucosylceramide. Interestingly, the L-threo isomer of long-chain ceramide (made from the C4-ceramide) was less efficiently converted to sphingomyelin and glucosylceramide (1.6% and 7.1%, respectively, of label in long-chain ceramide) compared to the D-erythro isomer (21.1% and 31.4%, respectively, of label in long-chain ceramide). Thus, shortand long-chain ceramides with the L-threo configuration are poor substrates for sphingomyelin and glycosphingolipid synthesis compared to their D-erythro counterparts. Conversion of long-chain ceramides, derived from radiolabelled DL-erythro-C4- and C6-dihydroceramide, to sphingomyelin and glucosylceramide was similar to Derythro analogues (although less long-chain ceramide was made from dihydroceramides). This is not surprising since

Table 1 Distribution of ceramide and dihydroceramide metabolites in CHO cells Ceramide

~erythro-C2-Cer e-threo-C2-Cer DL-erythro-C2-DHC DL-threo-C2-DHC D-erythro-C4-Cer L-threo-C4-Cer DL-erythro-C4-DHC DL-threo-C4-DHC D-erythro-C6-Cer L-threo-C6-Cer DL-erythro-C6-DHC DL-threo-C6-DHC

Medium products ( p m o l / m g protein) SC-SM

SC-GIcCer

SC-Cer

Spo/Spa

LC-SM

LC-GIcCer

LC-Cer

38.4 5.8 9.3 2.1 126.7 43.4 34.1 6.2 139.2 82.8 21.1 14.2

25.9 4.6 3.8 2.4 30.1 6.9 4.0 5.8 118.4 10.0 5.9 6.3

3140.5 3454.3 2598.1 3549.9 2253.9 3100.2 1193.2 3524.4 2128.5 2134.3 637.4 2226.3

2.9 1.3 1.7 0.8 41.2 7.3 10.8 4.5 12.6 3.0 4.3 0.9

6.0 1.0

6.9 4.6

26.1 1.4 8.9 1.0 19.3 1.8 3.9 1.2

39.0 5.9 10.7 6.3 21.7 3.9 9.2 3.1

19.8 11.8 18.0 9~2 124.2 82.9 36.9 15.6 127.2 61.3 18. 9.0

CHO cells were cultured in serum-free medium for 1 h in the presence of 5 nmol [3-3H]ceramide or [3-3H]dihydroceramide presented as a 1:1 (mol/mol) complex with BSA. Lipid products were extracted, resolved by thin-layer chromatography and radioactivity measured as described in Section 2. Results are the means of three separate experiments (range was no greater than 15% of the mean). Abbreviations are as follows: SC-SM, short-chain sphingomyelin; SC-GIcCer, short-chain glucosylceramide; SC-Cer, short-chain ceramide; LC-SM, long-chain sphingomyelin, LC-GIcCer, long-chain glucosylceramide; LC-Cer, long-chain ceramide; Cer, ceramide; DHC, dihydroceramide.

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

62

Table 2 Distribution of ceramide and dihydroceramide metabolites in medium from CHO cells Ceramide

Medium products ( p m o l / m g protein) SC-SM

SC-GIcCer

SC-Cer

D-erythro-C2-Cer L-threo-C2-Cer DL-erythro-C2-DHC DL-threo-C2-DHC D-erythro-C4-Cer L-threo-C4-Cer Dt,-erythro-C4-DHC DL-threo-C4-DHC D-erythro-C6-Cer L-threo-C6-Cer DL-erythro-C6-DHC DL-threo-C6-DHC

4.6 0.6

1.5 0.8

28.7 5.8 6.4 2.2 41.8 14.7 8.3 3.5

4.4 1.6 3. I 1.6 12.2 2.1 3.3 1.4

1000.8 997.2 1925.7 941.2 995.5 1019.0 3437.7 1219.0 905.7 1203.3 4329.3 1948.3

CHO cells were cultured and incubated with short-chain (dihydro)ceramide analogues as described in Table 1. Lipid products were extracted from culture medium, resolved by thin-layer chromatography and radioactivity measured as described in Section 2. Results are the mean of two or three separate experiments. Abbreviations are described in Table 1.

CELLS

MEDIUM

LC-Cer SC-Cer

LC-GlcCer [ SC-GlcCer-Q

SpolSpa

--

LC-SM

-

........

-

sc-sM-

w

Origin -Fig. 2. C6-[3-3H]ceramide and C6-[3-3H]dihydroceramide metabolites in CHO cells and culture medium. CHO cells (two 60 mm dishes) were incubated with 5 nmol of each ceramide or dihydroceramide for 1 h. Cells and medium were harvested, lipids isolated and separated by thin-layer chromatography as described in Section 2. Thin-layer chromatography plates was subjected to fluorography for 4 days at - 70 ° C. o-erythro-C6-ceramide (lanes 1 and 5), D-threo-C6-ceramide (lanes 2 and 6), DL-erythro-C6-dihydroceramide (lanes 3 and 7), DL-threo-C6-dihydroceramide (lanes 4 and 8). Abbreviations are the same as Fig. 1.

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

the ceramides should be identical (except the dihydroceramides are an enantomeric mixture) once the 4-trans double bond is introduced after acylation [23]. Bioactive sphingolipid derivatives other than sphingosine have recently been identified and implicated in cell signalling processes (reviewed in [2]). One of these, sphingosine-l-phosphate, could be responsible for some of the biological activity of sphingosine [24]. In preliminary studies, we were unable to identify sphingosine-l-phosphate or ceramide-l-phosphate synthesis from any of the analogues tested here. Short-chain SM and glycosylceramide synthesized in intact cells are known to be released into the medium of cultured cells, probably by absorption to acceptors like albumin or lipoproteins [11,25]. All the ceramide analogues tested here produced short-chain sphingomyelin and glucosylceramide that were released into the medium in amounts proportional to cellular synthesis (Table 2). Generally, between 8% and 23% of total short-chain sphingolipids were recovered in the medium. All the OLerythro-isomers of dihydroceramide were poorly taken up by cells compared to other analogues, as evidenced by excess of these isomers in the medium (Table 2) vs. the cell (Table 1). Radiolabelled long-chain sphingolipids, sphingosine or sphinganine were not detected in the medium. A representative fluorogram showing the distribution of products for the four radiolabelled C6-(dihydro)ceramide analogues in cells and medium is shown in Fig. 2. We consistently noted that short-chain sphingomyelin derived from DL-erythro-C6-dihydroceramide, but not the threo isomer, migrated as doublet; the lower band having the mobility of C6-sphingomyelin derived from D - e r y t h r o - C 6ceramide. Although less clearly visible, the same result is seen for oL-erythro-C4-dihydroceramide (Fig. 1). This result suggests that the desaturase that introduces the 4-trans double bond in ceramide will use short-chain dihydroceramides or short-chain sphingomyelin as substrates. To confirm this, the long-chain base composition of sphingolipids derived from oL-erythro-C4-dihydroceramide was examined (Table 3). Sphingosine comprised 23.6% of the long-chain base composition of C4-sphingomyelin indicating a desaturase is active on oL-erythro-C4-dihydroceramide or the short-chain sphingomyelin. A similar distribution of sphingosine and sphinganine was seen for C 4 - g l u cosylceramide. Sphingosine was also present (3.2%) in the long-chain bases recovered from cellular DL-erythro-C4-dihydroceramide, but we cannot be sure if this resulted from dihydroceramide desaturation or catabolism of sphingolipids. [1-14C]C8-dihydroceramide was recently shown to be desaturated in neuroblastoma cells [26], further supporting the concept of desaturation of intact dihydroceramides and not free sphinganine. Results in Table 1 clearly show that sphingomyelin synthase has a preference for substrates with N-acyl chains longer than 4 carbons, a 4-trans double bond and the naturally occurring D-erythro configuration. To determine

63

Table 3 Long-chain base composition of [3H]sphingolipids derived from DLerythro_C 4_[3 - 3H]dihydroceramide Percent distribution of:

Spo Spa

SC-SM

LC-SM

SC-GIuCer

LC-GluCer

SC-Cer

LC-Cer

23.6 76.4

61.0 39.0

35.0 65.0

36.3 63.7

3.2 96.8

48.1 51.9

Percent distribution. CHO cells were cultured and incubated with C4-dihydroceramide for 1 h as described in Table 1. Lipids were extracted from cells, separated by thin-layer chromatography and visualized by autoradiography or exposure to iodine vapours. Bands corresponding to the sphingolipid fractions listed below were eluted from the silica-gel and the distribution of label in sphingosine and sphinganine determined following acid hydrolysis as described in Section 2. Sphingosine accounted for 0.2% of the long-chain bases composition of DL-erythro-C 4[3-3H]dihydroceramide that was not incubated with cells. Results are the means of 3 or 4 separate analysis. Abbreviations are the same as described in Fig. 1.

if this preference is maintained in vitro, K m and Vmax constants for sphingomyelin synthase were measured with short-chain (dihydro)ceramides using CHO cell membranes as a source of enzyme. These short-chain analogues

35

I

I

I

I

A

30 ca25 E c 20 E E

"10

1

0

20

I

2 3 Cs-Ceramide (gM) I

I

I

4

5

I

I

O 16 E

"212 o E

8 4 0

0

I

I

I

I

I

I

5

10

15

20

25

30

Cs-Dihydrocoramido (~M)

35

Fig. 3. Substrate-velocity curves for sphingomyelin synthesis in CHO cell membranes using C6-[3- 3H]ceramides and C 6-[3- 3H]dihydroceramides as substrates. Synthesis of N-hexanoyl sphingomyelin was assayed as described in Section 2. Results for (A) D-erythro-C6-ceramide ( 0 ) and DL-threo-C6-ceramide (0), and (B) DL-erythro-C6-dihydroceramides ( 0 ) and DL-threo-C6-dihydroceramides ( © ) are the means of duplicate assays from a representative experiment.

N.D. Ridgway, D.L. Merriam/ Biochimica et Biophysica Acta 1256 (1995) 57-70

64

are more soluble than natural ceramides and when added to the enzyme assay as a 1:1 ( m o l / m o l ) complex with B S A rapidly exchange into membranes. Substrate velocity curves with C6-ceramide and C6-dihydroceramide isomers were hyperbolic (Fig. 3) and gave linear double reciprocal plots from which K m and V,,~× constants were determined (Table 3). Sphingomyelin synthase kinetics, reported previously for C6-ceramide, are independent of substrate concentration in the aqueous phase and subject to surface dilution-like kinetics when excess lipids are included in the assay [6,27]. To avoid potential variability between assays related to substrate partitioning into excess membranes, the amount of membrane protein assayed and assay volume was constant for all assays. Because catalysis is occurring in the membrane phase of the assay, it would be more appropriate to express substrate concentration as a mol%. We did not employ a detergent mixed micellar enzyme assay, and the mol% of substrate in the membrane phase cannot be accurately measured, so K m is expressed in molarity and comparisons are made on that basis. Kinetic constants for (dihydro)ceramide analogues are shown in Table 4. No activity was observed in vitro for both C2-dihydroceramides or L-threo-C2-ceramide. Similar to results for intact CHO cells, D-erythro ceramide analogues proved to be the best substrates; K m values for L-threo ceramide isomers were increased 3- to 6-fold and Vmax were reduced by 2-fold relative to the corresponding D-erythro ceramide. A similar pattern was seen for the series of dihydroceramides. Sphingomyelin synthase activity was also dependent on the length of the N-acyl group. There was a 10-fold increase in Vmax and 2-fold decrease in Kr, as the N-acyl chain increased from 2 to 6 carbons for the o-erythro-ceramides. In general, Wmax values for sphingomyelin synthesis using the various ceramides and dihydroceramides corresponded to results obtained with CHO cells (Table 1), indicating that these substrates are

Table 4 Kinetic constants for sphingomyelin synthase using short-chain ceramide and dihydroceramide substrates Substrate

D-erythro-C2-Cer D-erythro-C4-Cer L-threo-C4-Cer DL-erythro-C4-DHC DL-threo-C4-DHC o-erythro-C6-Cer L-threo-C6-Cer DL-erythro-C6-DHC DL-threo-C6-DHC

K m

Vma x

(/xM)

(pmol/min per rag)

4.0 1.1 6.9 5.1 28.6 1.7 4.2 9.9 12.8

3.5 14.0 7.4 8.0 3.7 34.8 19.9 11.6 4.5

Increasing concentrations of 1:1 (mol/mol) complexes of (dihydro)ceramides and BSA were incubated with 100 /xg of CHO cell membranes. K m and Vm,~ constants were derived from double-reciprocal plots of initial velocity data. Abbreviations are described in Table 1. All values represent the means of two experiments.

140 A

120

~.

loo

,9 ° "

80

¢.-

{TJ

60 O

E e~

40 [3

20

10

0

120

20

30 40 Time (min)

~

1

I

t

50

60

T

r

70

~'e--~_. lOO

6o8°

o

40 20 0

0

10

20

30 40 Time (min)

Fig. 4. Time course for metabolism of

50

60

70

D-erythro-C4-[3-3H]ceramide in

CHO cells. Cells were incubated with 5 nmol of ceramide in 1 ml of serum-free medium. At the indicated times cells were harvested, lipids extracted and separated by thin-layer chromatography in chloroform/methanol/15 mM CaCI2 (60:35:8, v/v), and labelled shortchain sphingolipids (A) and long-chain bases and sphingolipids (B) scraped and quantitated. Results are the means of duplicatc determinations from a representative experiment. Abbreviations are the same as Fig. I or as follows: SC-SM, N-butyl sphingomyelin; SC-GIcCer, N-butyl glucosylceramide; GlcCer, glucosylceramide; Cer, ceramide.

freely accessible to intracellular compartments where sphingomyelin synthase resides. Long-chain bases and ceramides are intermediates in complex sphingolipid synthesis, but the p r e c u r s o r / p r o d u c t relationship and source of radiolabel in these compounds was not obvious in results for 1 h incubations with shortchain (dihydro)ceramides (Tables 1 and 2). To clearly define how short-chain (dihydro)ceramide serve as precursors for synthesis of long-chain sphingolipids in CHO cells, the time course for metabolism of radiolabelled D-erythr o-C 4-ceramide and DL-erythro-C 4-dihydroceramide was investigated. Intracellular synthesis of C 4sphingomyelin and C4-glucosylceramide is linear for 20 min and ceases by 60 min (Fig. 4A). This can be partially accounted for by equilibrium between intracellular synthesis and secretion into the medium, which accounts for about 20% of the total C4-sphingomyelin synthesized at 1 h (Table 2). Long-chain sphingolipids seem to be derived

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

2000

I

I

I

I

I

40

i

,-- 1600

c ,0,>

9

m.1200

2

E

F:

E

O

% 8oo

E O.

4OO

T

1

A

30 25 20 15 10 5

I

0

r

35

O

O~

65

10

I

I

I

I

I

20

30

40

50

60

Time (rain)

0 70

0

Fig. 5. Uptake of D-erythro-C4-[3-3H]ceramide and DL-erythro-C4-[33H]dihydroceramide by CHO cells. The amount of C4-ceramide ( O ) and C4-dihydroceramide ( O ) taken up by CHO cells was determined as described in Fig. 4.

from [3H]sphingosine since it is a major radiolabelled product at 10 min, and there is a distinct lag in ceramide, sphingomyelin and glucosylceramide synthesis relative to sphingosine indicative of a precursor/product relationship (Fig. 4B). The rapid release of [3H]sphingosine suggests it derived from hydrolysis of D-erythro-C4-ceramide and rapidly equilibrated with the pool of long-chain bases destined for acylation to ceramide. The time course for DL-erythro-C4-dihydroceramide metabolism is complex due to relatively poor uptake and metabolism of this substrate compared to C4-ceramide. Whereas D-erythro-Ca-ceramide uptake was almost saturated at 10 min, oL-erythro-Ca-dihydroceramide entered CHO cells slowly and at a constant rate for 1 h (Fig. 5). The rate of uptake of the C4-dihydroceramide substrate clearly influences its metabolism as shown in Fig. 6A and

40 35

10

30 40 50 Time (rain)

~

._c 30 ,-,° 25 c~ 20 E o

20

60

T //~

t

[] GIcCerI = ~

70

B

/

15

[lO 5 0

0

10

20

30

40

Time (min)

50

60

70

Fig. 6. Time course for metabolism of DL-erythro-C4-[3-3H]dihydroceramide in CHO cells. The short-chain sphingolipid (A) and long-chain sphingolipid (B) cellular products of DL-erythro-Ca-dihydroceramide were determined as described in Fig. 4.

B. Short-chain SM and glucosylceramide synthesis from

DL-erythro-C4-[3-3H]dihydroceramide (Fig.

6A) is almost linear for 1 h and lags slightly if secretion into the medium

Table 5 Effect of ceramide and dihydroceramide analogues on [3H]serine incorporation into sphingolipids, phosphatidylserine and phosphatidylethanolamine Addition

NA" Spo Spa

~erythro-C2-Cer L-threo-C2-Cer DL-erythro-C2-DHC DL-threo-C2-DHC D-erythro-C4-Cer DL-erythro-Ca-DHC D-erythro-C6-Cer DL-erythro-C6-DHC L-MAPP D-MAPP C8-APPD

Incorporation of ( d p m / n m o l lipid phosphorus): SM

GluCer

Ceramide

PtdSer

PtdEtn

112.0 ± 24.6 (100) b 13.8 ± 8.7 (12.3) 17.3 ± 10.6 (15.4) 10.4 ± 4.3 (9.2) 106.5 ± 27.3 (95.0) 71.2 ± 10.6 (63.5) 117.0 ± 19.7 (104.4) 3.0 + 1.1 (2.6) 28.7 + 9.4 (25.0) 4.8 ± 2.7 (4.0) 46.4 ± 8.8 (41.4) 47.1 ± 15.8 (42.0) 119.6 ± 18.9 (106.2) 122.3 + 44.6 (108.9)

39.3 ± 5.1 (100) 5.2 ± 3.3 (13.2) 5.8 ± 4.7 (14.7) 4.9 ± 1.5 (12.4) 30.6 (77.8) 34.3 (87.2) 37.5 (95.4) 3.5 + 1.6 (8.9) 11.4 + 2.8 (29.0) 3.2 ± 0.7 (8.1) 16.8 ± 5.9 (42.7) 17.5 + 2.3 (44.5) 31.4 ± 11 (79.8) 32.9 + 4.4 (83.7)

132.5 ± 19.6 (100) 56.0 _+ 18.1 (42.2) 40.0 _+ 7.5 (30.1) 54.1 _+ 7.0 (40.8) 114.1 (86.1) 88.7 (66.9) 112.0 (84.5) 48.5 ± 4.4 (36.6) 52.8 ± 11 (39.5) 45.7 ± 7.0 (34.4) 75.3 ± 16.0 (56.8) 113.1 ± 7.3 (85.3) 124.3 ± 11.8 (93.8) 129.6 ± 8.5 (97.8)

394.3 308.2 553.2 352.5 335.6 332.1 343.2 252.1 502.1 373.8 523.4 392.5 301.1 344.3

98.2 ± 18.8 (100) 74.5 ± 10.9 (75.8) 97.1 ± 11.4 (98,8) 81.8 ± 16.4 (83,2) 88.4 (90.0) 87.6 (89.2) 132.0 (134.4) 84.1 + 2.4 (85.6) 100.7 + 3.9 (102.5) 88.5 ± 2.8 (90.1) 108.1 ± 4.7 (110.8) 91.2 ± 11.8 (92.8) 101.1 ± 14.5 (102.9) 92.0 ± 27.0 (93.6)

_+ 62.3 (100) ± 25.6 (78.2) ± 74.1 (140.2) ± 76.4 (89.3) (85.1) (84.2) (87.0) -t- 14.5 (63.9) + 32.7 (127.3) ± 21 (94.8) ± 25.0 (132.7) + 75.8 (99.5) _+ 60.5 (76.3) ± 66.9 (87.3)

CHO cells (in medium B) were treated for 2 h with each analogue (10 p.M dissolved in ethanol or as BSA complex). Cells were pulsed with 7.5 /.~Ci/ml [3H]serine for the last hour, harvested and lipids analyzed. Results are the means of 2 to 9 separate experiments + S.D. Abbreviations are described in Table 1. No addition. Cells received BSA or ethanol alone. h Percent [3H]serine incorporation relative to control.

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

66

is taken into account (11% of total C4-sphingomyelin secreted at 1 h, refer to Table 2). There is a pronounced lag in production of long-chain sphingolipids and longchain bases (Fig. 6B). Sphingosine was found to account for 4 0 - 5 0 % of the labelled long-chain bases as all time points. Unlike data for D-erythro-C4-ceramide (Fig. 4B), there is no clear p r e c u r s o r / p r o d u c t relationship between either long-chain base and the long-chain sphingolipids. Also, it is uncertain whether [3H]ceramide is synthesized exclusively from sphingosine, sphinganine or both. Results similar to that shown in Figs. 3, 4 and 5 were found for C6-ceramide and C6-dihydroceramide metabolism in CHO cells. It was previously reported that CHO cells incubated with C2-ceramide for 2 4 - 4 8 h showed an almost complete cessation of de novo sphingolipid synthesis, as measured by [3H]serine incorporation, and no effect on incorporation of [3H]choline or [3H]galactose into sphingomyelin or

120

I

I

I

I

--eI []

100

A

SM GIcCer

80 E O

O

60

"6

40 20 I

0

2

I

I

I

4 6 8 C2-Ceramide (gM)

T

10

100 80 E

- - T

r

1

1

r

4

A 1001

O O

0

0 [] ¢

60 40

GIcCer Ceramlde

20 -----0

0

120

J

L

1

2

3

4

5

I

I

I

I

I

Time (h)

0

2

4 6 8 C2-Ceramide (pM)

10

12

Fig. 8. Dose-dependent inhibition of [3H]serine incorporation into sphingolipids by C2-ceramide. Cells reccived medium B containing increasing concentrations of C2-ceramide (dissolved in ethanol) for 3 h at 37° C. In the last hour of the incubation, cells were pulsed with [3H]serine (7.5 /zCi/ml) and incorporation into alkali-stable sphingolipids (panel A) and phospholipids (panel B) was measured. Results are the means of duplicate determinations from a representative experiment. Control values and abbreviations are the same or similar to Fig. 7.

#

0

~

20

130 co

dSe

-6

120

B 1 O0

~

80

O

o 60 "6 .~ 4.0 •~ 0 0

0

I

I

1

2

I

3 Time (h)

I

I

4

5

6

Fig. 7. Time course for inhibition of [3H]serine incorporation into sphingolipids by C2-ceramide. Cells received 10 /xM C2-ceramide (dissolved in ethanol) in medium B for the indicated times. In the last hour of each incubation, cells were pulsed with [3H]serine (7.5 /xCi/ml) and harvested. Incorporation of [3H]serine into alkali-stable sphing01ipids(panel A) and phospholipids (panel B) was measured as described in Section 2. Results are the means of duplicate measurements from a representative experiment. Control values for SM, glucosylceramide, ceramide, PtdSer and PtdEtn were 116.5, 40.6, 111.4, 343.0 and 95.7 dpm/nmol lipid phosphorus, respectively (each determination had on average 140 nmol lipid phosphorus). Abbreviations used are described in Fig. 4.

glycosphingolipids, respectively [28]. We found that 10 /zM C2-ceramide inhibited [3H]serine incorporation into sphingomyelin and glucosylceramide by 8 0 - 9 0 % after only 2 h (Fig. 7A). Label incorporation into total sphingolipid long-chain bases (primarily sphingosine) was also inhibited by 80% (results not shown). Interestingly, [3H]serine incorporation into ceramide was reduced by only 60%. Dose response curves for [3H]serine-labelling of CHO cells treated with C2-ceramide for 4 h show specific inhibition of sphingomyelin and glucosylceramide synthesis, and a lesser effect on ceramide-labelling, at C2-ceramide concentrations as low as 1 /xM (Fig. 8A). Inhibition of sphingolipid-labelling is not the result of decreased serine uptake since PtdSer and PtdEtn synthesis was effected by < 15% relative to controls (Fig. 7B and 8B). The results from Fig. 7 and 8 suggest direct inhibition of sphingolipid synthesis by C2-ceramide or an indirect effect due to utilization of sphingosine released from C 2ceramide [28]. If inhibition is related to production of sphingosine or sphinganine, then short-chain

N.D. Ridgway, D.L. Merriam/Biochimica et Biophysica Acta 1256 (1995) 57-70

67

Table 6 Short-chain ceramides and dihydroceramides do not inhibit [3H]choline-labellingof phosphatidylcholineor sphingomyelin Analogue Sphingomyelin Phosphatidylcholine (dpm/nmol lipid phosphorus) (dpm/nmol lipid phosphorus) No addition o-erythro-C2-Cer 1 ~M 10/.xM D-erythro-C6 Cer 1 /xM 10/xM DL-erythro-C6-DHC 1 /xM 10/.tM DL-threo-C6-DHC 1 p,M 10/xM

150.6 + 8.5

11495 + 949

141.0_+ 26.2 121.2 _+28.5

12916 + 1666 12497 + 449

153.7 _+18.9 145.2 _+15.4

12538 _+806 12365 + 1204

160.8 _+16.2 142.2 _+16.0

11528 _+1218 10918 _+490

143.1 + 3.8 147.0 + 18.9

10256 + 542 11295 + 489

CHO cells were cultured in choline-free medium A containing the indicated amount of ceramide or dihydroceramide for l h and pulsed with [3H]choline (2.5 p~Ci/ml) for an additional 2 h. Cells were harvested and radioactivity in PtdCho and SM determined as described in Section 2. Results are the means + S.D. of three experiments. Abbreviations are described in Table 1.

(dihydro)ceramides should have widely different effects on [3H]serine incorporation into sphingolipids based on the amount of long-chain base released in the cell by (dihydro)ceramide degradation. We tested the ability of the various analogues to inhibit [3H]serine-labelling of sphingolipids, ceramide, PtdEtn and PtdSer over a 2 h period (Table 5). t)-Erythro-ceramides proved to be the most potent inhibitors of [3H]serine incorporation into sphingomyelin and glucosylceramide. Consistent with the degree of catabolism of the analogues to sphingosine and longchain sphingolipids (Table 1), C 4- and C6-ceramides were slightly better inhibitors then C2-ceramide. While [3H]serine incorporation into sphingomyelin and glucosylceramide was inhibited by greater than 90%, labelling of ceramide in the presence of short-chain o-erythro-ceramides was reduced by only 6 0 - 7 0 % . Inhibition of sphingolipid-labelling in the presence of dihydroceramide analogues was again proportional to the metabolism of these compounds to sphingosine, sphinganine and long-chain sphingolipids (Table 1), and followed the hierarchy C4-dihydroceramide > C6-dihydroceramide > C2-dihydroceramide. However, inhibition of [3H]serine incorporation into sphingomyelin and glucosylceramide was 75% for C4-dihydroceramide and 60% for C6-dihydroceramide even though these two compounds produced as much or more long-chain bases and long-chain sphingolipids as C2-ceramide (Table 1). Interestingly, L-MAPP inhibited [3H]serine incorporation into sphingomyelin and glucosylceramide by 60%, with a lesser effect on ceramide, but the D-erythro isomer (which has the same stereochemical configuration as natural ceramide) was without effect. Addition of 10 p,M sphingosine or sphinganine to CHO cells inhibited labelling to a similar degree as 10 /zM of the o-erythro short-chain ceramides. The threo isomer of C2-ceramide and dihydroceramide did not inhibit [3H]serine-labelling of sphingolipids. [3H]Serine-labelling of PtdSer and PtdEtn

was unaffected by these analogues with the exception of sphinganine, D-erythro-C 4- and C6-dihydroceramide, which produced a 2 5 - 4 0 % increase in PtdSer-labelling. Since (dihydro)ceramide analogues are metabolized to sphingosine and sphinganine which are used for sphingolipid synthesis, sphinganine synthesis could be inhibited without any effect on overall sphingomyelin synthesis. This was tested by measuring [3H]choline transfer from PtdCho to sphingomyelin in the presence of selected ceramide and dihydroceramide analogues (Table 6). Derythro-C 2- and C6-ceramide inhibited [aH]serine-labelling of sphingomyelin by 90% but had no effect on [3H]choline incorporation into this phospholipid. Erythro and threo isomers of C6-dihydroceramide were also without effect. Radioactivity in PtdCho, the phosphocholine donor, was not effected by any of the analogues. Results from Table 5 suggest, with some exceptions, inhibition of [3H]serine incorporation into sphingolipids is the result of long-chain base release and subsequent utilization for sphingolipid synthesis. To test whether inhibition of de novo synthesis was the result of direct effects on biosynthetic enzymes, cells were treated with 10 /.tM C:-ceramide for 2 h and SPT and sphingomyelin synthase activity assayed in isolated membranes. SPT activity in treated membranes (38.8 ___5.0 p m o l / m i n per mg protein, n = 3) was not significantly different than untreated membranes (42.5 + 6.8 p m o l / m i n per mg protein, n = 3). Sphingomyelin synthase activity was slightly reduced in treated membranes (20.9 + 1.4 p m o l / m i n per mg protein, n = 3) vs. controls (27.0 + 3.9 p m o l / m i n per mg protein, n = 3). This inhibition could be due to residual C2-ceramide in membranes which would then compete with C6-[3a H]ceramide used to assay enzyme activity. This type of inhibition seems likely since in CHO membranes the apparent K i for C2-ceramide inhibition of C6-[3-aH]cera mide conversion to C6-[3-3H]sphingomyelin is 16.6 /zM.

68

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

Incubation of CHO membranes with 10 /xM C2-ceramide for 5 rain prior to the initiation of the enzyme assay did not significantly inhibit SPT activity (23.0 _+ 5.5 vs. 16.9 _+ 6.9 p m o l / m i n per mg protein for control and C2-ceramide-treated, respectively, n = 3). Preincubation with C 2ceramide (10 /xM) at 20 ° C for up to 1 h had no effect on SPT activity.

4. Discussion

The enhanced solubility and uptake of short-chain (dihydro)ceramides by cultured cells makes them useful substrates to identify the intracellular fate of ceramide and specificity of enzymes involved in ceramide metabolism. In this study a variety of radiolabelled (dihydro)ceramide analogues were tested for metabolism in cultured CHO cells with the intent of (1) identifying the intracellular and secreted products of short-chain (dihydro)ceramide metabolism, (2) assessing the stereochemical and structural specificity of enzymes which utilize ceramide as a substrate, and (3) assessing the effect analogues have on de novo sphingolipid synthesis as measured by radiolabelled precursor incorporation. Short-chain analogues were synthesized from I> erythro-sphingosine and DI,-erythro-sphinganine precursors, and the 3-position tritiated by reduction of 3-keto (dihydro)ceramides with NaB[3H]4 . This method has the advantage of labelling both sphingosine and sphinganine backbones, and generating substrates with 3R or 3S configurations that can be easily separated and studied. One potential disadvantage is displacement of the tritium group due to the allylic nature of the 3-position in sphingosine. However, displacement occurs under acidic conditions and was not a problem in this study since we routinely recovered 80-90% of the poorly metabolized ceramides (threoCz-, C 4- and C6-ceramides) and 60-70% of the more extensively metabolized ceramides (erythro-C 4 and -C 6 ceramide) after a 1 h incubation in cultured cells. The fate of the 10-30% of unrecovered material is not known but is assumed to be lost during extraction of labelled sphingolipids and oxidation of the sphingosine degradation product palmitaldehyde. Unlike studies where short-chain ceramides were modified or radiolabelled in the N-acyl group [11,22], we are able to determine the fate of long-chain bases liberated from ceramides and dihydroceramides during short-term incubations with CHO cells. Other studies have used C sceramide [25] and C2-dihydroceramide [28] labelled in the long-chain base moiety and demonstrated long-chain sphingomyelin-labelling after 24 or 48 h incubations. Within minutes of addition to CHO cells, (dihydro)ceramides (with the exception of the C~-compounds) generated significant amounts of labelled long-chain bases (5-40 p m o l / m g protein) that entered the endogenous pathway for ceramide and sphingolipid synthesis. We cannot be

sure if the release of long-chain bases from ceramide analogues resulted in a net increase in the mass of longchain bases. Other studies using HL-60 cells have shown that little or no sphingosine is formed from labelled C 2ceramide [29]. Similar findings are shown here; however, C2-ceramide is relatively inert compared to C 4- and C 6ceramide. [3H]Sphingosine generated from C 4- and C 6ceramide (41.2 and 12.6 p m o l / m g protein for approximately 106 cells, respectively) would be expected to expand the cellular pool given the range of long-chain base content of cells (10 to 100 pmol/106 cells) [30]. Indeed, Goldkorn et al. [31] have shown that ceramide and sphingosine mass increased 2-fold in A431 cells incubated for 15 min with 10 /xM Cs-ceramide. Incubation of MDCK cells with Cs-ceramide for 24 h elevated sphingosine by 10-fold [25]. Based on mass and isotope studies it appears that sphingosine increases rapidly after addition of shortchain ceramides due primarily to deacylation of these analogues. [3H]Sphingosine and [3H]sphinganine released from short-chain (dihydro)ceramides is effectively incorporated in ceramide, sphingomyelin and glucosylceramide. One interesting feature of long-chain base release in cells was the rapid steady-state equilibrium that was reached: this indicates the endogenous pool of long-chain bases is small relative to that from exogenous sources, and as a result there is little or no isotope dilution and rapid labelling of sphingolipids. This could also result from rapid inhibition of endogenous sphinganine synthesis by short-chain (dihydro)ceramides. Direct deacylation-reacylation of shortchain ceramide seems unlikely to be involved in long-chain ceramide synthesis since free sphingosine is released, and there is a clear precursor/product relationship between sphingosine, ceramide and other sphingolipids. Ceramidase has been reported to catalyze deacylation/reacylation, but this reaction does not involve release of long-chain base [32]. Deacylation/reacylation of short-chain sphingomyelin or glucosylceramide could account for some labelling of long-chain counterparts. Although this process cannot be ruled out, it is likely to be a minor pathway compared to synthesis from radiolabelled long-chain bases. The remodelling of sphingomyelin acyl chains by deacylation/reacylation has been shown to account for actin antibody [33] and retinoic acid [34] stimulated labelling of sphingomyelin by fatty acids in cultured cells. The naturally occurring I>erythro (2S,3R) conformation of sphingosine is conferred by the stereochemical specificity of the first two enzymes in sphingolipid synthesis [35]. Other isomers of long-chain bases have been detected, but appear to be an artefact of harsh acid or base hydrolysis used to liberate long-chain bases from sphingolipids [36]. There is some disagreement as to the utilization of non-natural isomers of long-chain bases and ceramides for sphingolipid synthesis. A report showed that D-erythro and IAhreo isomers of sphinganine, intravenously injected into rats, were preferred for sphingo-

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

myelin synthesis in the liver [37]. Others have shown preferred conversion of oL-threo-sphingosine to sphingomyelin in rat brain [38]. A study using C6-NBD-ceramides and dihydroceramides concluded there was little preference for configuration at the 2 and 3 position for sphingomyelin synthesis, but the D- or L-erythro-sphingosine isomers of C6-NBD-ceramide were preferred for glycosylceramide synthesis [12]. However, in these studies either intact ceramides were not used, metabolism was studied in whole animals or product formation was difficult to quantitative and only qualitative estimates of substrate utilization were presented. In our study, D-erythro- and L-threoceramides were utilized for synthesis of short-chain sphingomyelin in CHO cells and in vitro, but the former was a substantially better substrate. Glucosyltransferase displayed a narrow substrate specificity compared to sphingomyelin synthase and used D-erythro-C6-ceramide almost exclusively for glucosylceramide synthesis. We found that short-chain dihydroceramides are poor substrates for glucosylceramide synthesis in CHO cells compared to Derythro-C6-ceramide, a finding confirmed in studies with C6-NBD dihydroceramides in cultured fibroblasts [12]. The stereochemical specificity for short-chain ceramide utilization does not seem to be an artefact of the solubility properties of these compounds since long-chain ceramide isomers made from threo-sphingosine or threo-sphinganine were converted less efficiently to sphingomyelin and glycosylceramide compared to their counterparts derived from D-erythro long-chain bases. The relatively p o o r m e t a b o l i s m of s o m e (dihydro)ceramide analogues, and the effects they have on long-chain base and ceramide levels in other cell types, suggests that these compounds may actually inhibit endogenous sphingolipid synthesis or catabolism. This has been suggested for inhibition of sphingomyelin synthase by C8-ceramide, which reduced the incorporation of [3H]choline into sphingomyelin in MDCK cells by about 40% after a 24 h incubation [25]. Similarly, C2-ceramide treatment of CHO cells for 24-48 h was shown to inhibit synthesis of sphingolipids, as measured by precursor-labelling with [3H]serine [28]. However, a lack of effect of C2-ceramide on [3H]choline incorporation into sphingomyelin suggested that enough sphingosine was released from C2-ceramide and converted to sphingomyelin to support cellular requirements [28]. We extended previous studies by examining the short-term effect of other shortchain (dihydro)ceramides on sphingolipid synthesis and correlated this to the contribution of these radiolabelled analogues to sphingolipid synthesis. A correlation between release of long-chain bases, and their conversion to longchain sphingolipids, and inhibition of de novo sphingolipid synthesis was observed. The exception to this finding was that dihydroceramides were less effective inhibitors relative to their metabolism to long-chain sphingolipids. If inhibition of [3H]serine incorporation into sphingolipids by C2-ceramide was the result of isotopic dilution

69

of endogenous [3H]serine-labelled long-chain bases, these compounds should have accumulated. However, [3H]sphinganine and [3H]sphingosine do not accumulate in short-chain ceramide-treated CHO cells and, as shown previously [28], free [3H]long-chain base synthesis was reduced by long-term C2-ceramide treatment of CHO cells. Suppression of de novo sphingolipid synthesis by sphingosine-mediated inhibition of SPT is possible during longterm ( > 24 h) incubation with C2-ceramide but seems unlikely for the short time courses presented in this study. This conclusion is based on studies using mouse cerebellar cells where 4 - 8 h treatment with 50 /zM sphingosine was required to reduce SPT activity by 80% [39]. Down-regulation of SPT or sphingomyelin synthase activity was not evident in membranes from CHO cells treated for 2 h treatment with C2-ceramide. As well, C2-ceramide added directly to SPT assays had no effect on activity. These results indicate that long-chain bases (or other unidentified sphingolipid metabolite) derived from short-chain ceramides inhibit sphinganine synthesis rapidly and by a mechanism different than that reported for exogenous long-chain bases. It is difficult to discriminate between inhibition of de novo sphingolipid synthesis by short-chain ceramides or an unidentified metabolite of these compounds since all the inhibitory (dihydro)ceramides generate long-chain bases and sphingolipids. However, L-MAPP, an aminophenyl propanol analogue of ceramide [40], was found to inhibit [3H]serine-labelling of sphingomyelin and glucosylceramide by 58% during a 2 h treatment. This compound, if deacylated, does not produce sphingosine or contribute to sphingolipid synthesis. It is possible that L-MAPP mimics the effect of ceramide by directly inhibiting a component of the sphingolipid biosynthetic pathway. Further work with this and other ceramide analogues may prove useful in determining a regulatory role for ceramide in sphingolipid synthesis. Depending on the stereochemistry of the 2 and 3 positions and acyl-chain length, short-chain (dihydro)ceramides undergo metabolism to short-chain sphingolipids and long-chain bases, which are then utilized for endogenous ceramide and sphingolipid synthesis. Short-chain (dihydro)ceramides have a complex inhibitory effect on endogenous sphingolipid synthesis and, as shown in an accompanying paper, cholesterol esterification [41]. Since (dihydro)ceramide analogues are metabolized to biologically active compounds and are known to affect other metabolic processes, the extent of these effects should be measured and controlled for appropriately.

Acknowledgements Thanks to Tom Lagace for excellent technical assistance and Ketan Badiani for critical review of the manuscript. This work was supported by a grant and

70

N.D. Ridgway, D.L. Merriam / Biochimica et Biophysica Acta 1256 (1995) 57-70

s c h o l a r s h i p to N D R f r o m the M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a , a n d an e s t a b l i s h m e n t g r a n t f r o m I z a a k W a l t o n K i l l i a m C h i l d r e n ' s Hospital.

References [1] Merrill, A.H., Jr. and Jones, D.D. (1990) Biochim. Biophys. Acta 1044, 1-12. [2] Hannun, Y.A. and Linardic, C.M. (1993) Biochim. Biophys. Acta 1154, 223-236. [3] Hannun, Y.A. (1994) J. Biol. Chem. 269, 3125-3128. [4] Mandon, E.C., Ehses, l., Rother, J., Van Echten, G. and Sandhoff, K. (1992) J. Biol. Chem. 267, 11144-11148. [5] Van Echten, G. and Sandhof, K. (1993) J. Biol. Chem. 268, 53415344. [6] Futerman, A.H., Stieger, B., Hubbard, A.L. and Pagano, R.E. (1990) J. Biol. Chem. 265, 8650-8657. [7] Volker, D.R. and Kennedy, E.P. (1982) Biochemistry 21, 27532759. [8] Spence, M.W. (1989) in Phosphatidylcholine Metabolism (Vance, D.E., ed.), pp. 186-196, CRC Press, Boca Raton, FL. [9] Okazaki, O., Bielawska, A., Domae, N., Bell, R.M. and Hannun, Y.A. (1994) J. Biol. Chem. 269, 4070-4077. [10] Lipsky, N.G. and Pagano, R.E. (1985) J. Cell Biol. 100, 27-34. [11] Pagano, R.E. and Martin, O.C. (1988) Biochemistry 27, 4439-4445. [12] Karrenbauer, A., Jeckel, D., Just, W., Birk, R., Schmidt, R.R., Rothman, J.E. and Wieland, F.T. (1990) Cell 63, 259-267. [13] Gaver, R.C. and Sweeley, C.C (1966) J. Am. Chem. Soc. 88, 3643-3647. [14] Folch, J., Lees, M., Sloane-Stanley, G.H. and Randall, R.J. (1951) J. Biol. Chem. 226, 497-50. [15] lwamori, M., Moser, H.W. and Kishimoto, Y. (1975) J. Lipid Res. 16, 332-336. [16] Van Veldhoven, P.P., Bishop, W.R. and Bell, R.M. (1989) Anal. Biochem. 183, 177-189. [17] Kadowaki, H., Bremer, E.G., Evans, J.E., Jungalwala, F.B. and McCluer, R.H. (1983) J. Lipid Res. 24, 1389-1397. [18] Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E. and Bell, R.M. (1966) J. Biol. Chem. 261, 8597-8600. [19] Rouser, G., Siakatos, A.N. and Fleisher, S. (1966) Lipids 1, 85-86.

[20] Williams, R.D., Wang, E. and Merrill, A.H. Jr. (1984) Arch. Biochem. Biophys. 228, 282-291. [21] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [22] Bielawska, A., Crane, H.M., Liotta, D., Obeid, L.M. and Hannun, Y.A. (1993) J. Biol. Chem. 268, 26226-26232. [23] Merrill, A.H., Jr. and Wang, E. (1986) J. Biol. Chem. 261, 37643769. [24] Desai, N.N., Zhang, H., Olivara, A., Mattie, M.E. and Spiegel, S. (1992) J. Biol. Chem. 267, 23122-23128. [25] Abe, A., Wu, D., Shayman, J.A. and Radin, N.S. (1992) Eur. J. Biochem. 210, 765-773. [26] Rother, J., Van Echten, G., Schwarzmann, G. and Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14-20. [27] Futerman, A.H. and Pagano, R.E. (1992) Methods Enzymol. 209, 437-446. [28] Ladenson, R.C., Monsey, J.D., Allin, J. and Silbert, D.F. (1993) J. Biol. Chem. 268, 7650-7695. [29] Okazaki, T., Bielawski, A., Bell, R.M. and Hannun, Y.A. (1990) J. Biol. Chem. 265, 15823-15831. [30] Kolesnick, R.N. (1991) Prog. Lipid Res. 30, 1-38. [3A] Goldkorn, T., Dressier, K.A., Muindi, J., Radin, N.S., Mendelsohn, J., Menaldino, D., Liotta, D. and Kolesnick, R.N. (1991) J. Biol. Chem. 266, 16092-16097. [32] Okabe, H. and Kishimoto, Y. (1977) J. Biol. Chem. 252, 7068-7073. [33] Ulrich, R.G. and Shearer, W.T. (1984) Biochem. Biophys. Res. Commun. 121,605-611. [34] Anders, A., Borebardt, R.A. and Bell, R.M. (1992) Biochim. Biophys. Acta 1125, 90-96. [35] Sweeley, C.C. (1991) in Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D.E. and Vance, J., eds.), pp. 327-361, Elsevier Science Publishers, Amsterdam, The Netherlands. [36] Christie, W.W. (1987) in High-Performance Liquid Chromatography and Lipids (Christie, W.W., ed.), pp. 211-232, Pergamon Press, Oxford, GB. [37] Stoffel, W. and Bister, K. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 169-181. [38] Kanfer, J.N. and Gal, A.E. (1966) Biochem. Biophys. Res. Commun. 22, 442-451. [39] Mandon, E.C., Van Echten, G., Birk, R., Schmidt, R.R. and Sandboff, K. (1991) Eur. J. Biochem. 198, 667-674. [40] Bielawska, A., Linardic, C.M. and Hannun, Y.A. (1992) J. Biol. Chem. 267, 18493-18497. [41] Ridgway, N.D. (1994) Biochim. Biophys. Acta, in press.