©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Roles of de Novo Sphingolipid Biosynthesis and Turnover in the Burst of Free Sphingosine and Sphinganine, and Their 1-Phosphates and N-Acyl-Derivatives, That Occurs upon Changing the Medium of Cells in Culture (*)

(Received for publication, December 1, 1994; and in revised form, May 30, 1995)

Elizabeth R. Smith Alfred H. Merrill , Jr. (§)

From theDepartment of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUDING REMARKS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Long-chain (sphingoid) bases are highly bioactive intermediates of sphingolipid metabolism, yet relatively little is known about how the amounts of these compounds are regulated. This study used J774A.1 cells to characterize the ``burst'' of sphinganine and sphingosine, or the transient increase of up to 10-fold in long-chain base mass, that occurs when cells in culture are changed to fresh medium. The increase in sphinganine was attributable to de novo sphingolipid biosynthesis because: 1) there is increased incorporation of [^3H]serine and [^3H]palmitate into sphinganine; 2) the incorporation of [^3H]serine was equivalent to the increase in sphinganine mass; 3) beta-F-alanine, an inhibitor of serine palmitoyltransferase, blocked the sphinganine burst; 4) the magnitude of the burst depended on the concentration of serine in the medium, which is known to affect long-chain base biosynthesis; and 5) the appearance of sphinganine was relatively unaffected by lyso-osmotrophic agents (NH(4)Cl and chloroquine) that blocked sphingolipid hydrolysis in these cells. In contrast, the sphingosine burst arose mainly from turnover of complex sphingolipids because no incorporation of [^3H]serine or [^3H]palmitate into sphingosine was detected; sphingosine mass was not affected by beta-F-alanine or the serine concentration; and, the burst could be followed by the release of sphingosine and ceramide from complex sphingolipids (especially sphingomyelin) in a process that was inhibited by NH(4)Cl and chloroquine. Additionally, the fate of these long-chain bases differed: sphinganine was mostly (80-85%) acylated and incorporated into dihydroceramide and complex sphingolipids, whereas most of the sphingosine (70%) was phosphorylated and degraded, with incorporation of the resulting ethanolamine phosphate into phosphatidylethanolamine. Sphinganine, however, could be diverted toward degradation by adding an inhibitor of N-acylation (fumonisin B(1)). In accounting for the elevation in sphingosine and sphinganine after cells are changed to new medium, these studies have provided fundamental information about long-chain base metabolism. The existence of differential changes in sphinganine and sphingosine, as well as their 1-phosphates and N-acyl-derivatives, should be considered when evaluating the roles of sphingolipid metabolites in cell regulation.


INTRODUCTION

Long-chain (sphingoid) bases have long been known to serve as the backbones of sphingolipids(1) ; however, only recently have they gained attention as bioactive compounds. Since the discovery that sphingosine acts as a potent inhibitor of protein kinase C(2) , many other systems have been found to be affected by long-chain bases, including the Na,K-ATPase(3) , phosphatidic acid phosphatase(4, 5, 6, 7) , phospholipases (including phospholipase D)(8, 9) , retinoblastoma protein phosphorylation(10) , and sphingosine-activated protein kinase(s)(11) . Furthermore, the N-acyl- (i.e. ceramide) (12, 13, 14) and 1-phosphate(15, 16, 17) derivatives of sphingosine are highly bioactive and are emerging as mediators of various cell signaling pathways (for recent reviews, see (12, 13, 14) , 18, 19).

Relatively little is known about the metabolism of long-chain bases as intermediates of de novo biosynthesis and the turnover of more complex sphingolipids. The amounts of free long-chain bases in cells are generally low (20, 21, 22) but can be affected by various factors (13, 20, 23) and toxins(24) . For example, treatment of growth arrested fibroblasts with platelet-derived growth factor increases sphingosine and sphingosine 1-phosphate, with the latter appearing to act as a mediator in the release of intracellular calcium (25) and activation of AP1(26) .

We have discovered that a common laboratory procedure, the changing of cells in culture to fresh medium, induces a transient ``burst'' of sphinganine and sphingosine in J774A.1 cells (27, 73, 74, 75) and Swiss 3T3 cells(28) . A similar observation has been made with several other cell types(29) , which suggests that this may be a common occurrence. The cellular levels of sphingosine that are achieved by adding fresh culture medium (approximately 0.5 nmol/mg protein) are similar to the amounts that affect cell function when sphingosine is added exogenously (18, 28, 30) . Therefore, this burst warrants further characterization to determine its potential relevance to the regulation of cell behavior by sphingolipids.

In this study, we establish that the free sphinganine of the burst arises from increased de novo sphingolipid biosynthesis, whereas the sphingosine is derived from sphingolipid turnover. The fate of the long-chain bases differed: although both can be converted to the 1-phosphates, sphinganine is mainly acylated, whereas sphingosine is degraded by J774A.1 cells. As far as we are aware, this study is the first to identify conclusively the origins and fates of these endogenous compounds in intact cells.


EXPERIMENTAL PROCEDURES

Materials

The J774A.1 cells (no. TIB 67), a murine macrophage-like cell line, was obtained from the American Type Culture Center (Rockville, MD), FBS (^1)was purchased from BioCell (Rancho Dominguez, CA), and DMEM was from Life Technologies, Inc. All other cell culture reagents were obtained from Sigma.

The D-erythro-sphingosine, DL-threo-sphingosine, ceramides, sphingomyelin, and gangliosides were from Sigma; DL-erythro-sphinganine was from United States Biochemical Corp. (Cleveland, OH); 3-ketosphinganine, D-threo-sphingosine and C-sphinganine, and [1-^3H]sphingosine were synthesized according to Di Mari et al.(31) , Nimkar et al.(32) , Sarmientos et al.(33) , and Cooke et al.(34) , respectively.

The [3-^3H]serine (30 mCi/mmol), [14-^3H]palmitate (54.4 Ci/mmol), and [2-^14C]ethanolamine-HCl (54.4 mCi/mmol) were from Amersham Corp. The beta-fluoro-L-alanine (beta-F-alanine) was donated by Merck (Rahway, NJ); FB(1) was purchased from Sigma or from the Division of Food Sciences and Technology, Council for Scientific and Industrial Research (Pretoria, South Africa).

Cell Culture

J774A.1 cells were grown in suspension at 37 °C in a spinner flask (Corning; Corning, NY) in DMEM, 10% FBS, 100 units/ml of penicillin-G, 100 µg/ml of streptomycin sulfate, and sodium bicarbonate (3.7 g/liter). Cells were passaged every 2-3 days by a 1:4 dilution with fresh medium to a density of approximately 2.5 10^5 cells/ml. Unless indicated differently, cells from the spinner flask were collected by gentle centrifugation, resuspended in new medium, and allowed to adhere to 60-mm tissue culture dishes (Corning) at 5-7.5 10^5 cells/2 ml of medium. These cells were incubated at 37 °C, 5% CO(2), for 3 days before beginning the experiment by removing the conditioned medium and addition of fresh medium.

Analysis of Sphingosine and Sphinganine Mass

After incubation of the cells under the conditions described in the text, the medium was removed, 0.5 ml of ice-cold methanol was added, and the cells were scraped from the dishes with a rubber spatula. The plates were scraped again with 0.4 ml of deionized water, followed by 0.4 ml of methanol. Cells and washes were pooled in 13 100-mm test tubes, 200-300 pmol of C-sphinganine was added as an internal standard, and the long-chain bases were extracted and analyzed by HPLC (21) .

[^3H]Serine and [^3H]Palmitate Labeling of Cellular Sphingolipids

In experiments to evaluate the de novo biosynthesis of sphingolipids, the medium was removed from the cells and replaced with 1 ml of new DMEM containing 10-100 µCi of [^3H]serine or 10 µCi of [^3H]palmitate as described previously(35) . [^3H]Palmitate was prepared as a 20 µM palmitatebulletBSA (1:1) complex of unlabeled- and [^3H]palmitate in a molar ratio of 9.9:1. The cells were incubated in a 37 °C, 5% CO(2) tissue culture incubator; for experiments involving incubations of less than 2 h, cells were kept in 10 mM HEPES-buffered DMEM, pH 7.4, at 37 °C and atmospheric CO(2).

In experiments to evaluate the turnover of labeled sphingolipids, cells were incubated with 0.1 mCi of [^3H]serine/ml medium for 3 days, then the medium was removed, the cells were washed twice with ice-cold PBS, and incubated in new DMEM with 10% FBS (or HEPES-buffered DMEM for short term experiments).

Labeling of Cell Lipids with [1-^3H]Sphingosine

[1-^3H]sphingosine was prepared from commercial D-erythro-sphingosine by the reduction of the 1-aldehyde derivative with Na[^3H]BH(4)(33, 34) . Beginning with 25 mg of D-erythro-sphingosine, the synthesis involved: 1) protection of the NH(2) group using methyl dichloracetate; 2) blocking the 1-OH group to form N-dichloroacetyl-1-O-trityl-sphingosine; 3) blocking the 3-OH group by treating with benzoyl chloride; 4) deprotection of the 1-OH group by HBr in acetic acid; 5) oxidation of the 1-OH group with DESS-Martin periodinane(34) ; 6) reduction of the aldehyde group with Na[^3H]BH(4); 7) deprotection of the 3-OH and NH(2) groups; and 8) purification of the [1-^3H]sphingosine by reverse-phase HPLC (C18-Waters Radial Pak; Milford, MA) using methanol, 5 mM potassium phosphate, pH 7.0 (90:10 v/v) as the mobile phase. The final specific activity was determined by analytical HPLC to be 45,000 dpm/nmol.

Lipid Extraction and Analysis

For analyses of phospholipids, long-chain base degradation products, and the long-chain base composition of total cellular sphingolipids, cells were extracted with 1.5 ml of chloroform/methanol (1:2 v/v) for 1 h at 37 °C. Following centrifugation to remove precipitated cell protein and debris, the chloroform/methanol extract was dried en vacuo and redissolved in chloroform. The chloroform-soluble material was used for lipid analysis. For determination of the long-chain base backbone of cellular sphingolipids, an aliquot of the chloroform-soluble material was acid hydrolyzed(36) , and sphingosine and sphinganine were quantitated by HPLC or separated by thin layer chromatography. In experiments where total and complex sphingolipids had been labeled with [^3H]serine, the sphingolipids were extracted using a LiChroprep RP18 column as described by van Echten et al.(37) . To aid in extraction and chromatographic detection of radiolabeled lipids, approximately 20 µg of each lipid of interest (i.e. sphingosine, ceramide, sphingomyelin, etc.) was added as a carrier.

To determine the sphingoid base 1-phosphate mass, the cell lipids were extracted by the one-phase method described for total sphingolipids and cleavage products (above), but C-sphinganine was included as an internal standard. The dried fraction was resuspended in 40 µl of methanol, followed by 0.1 ml of 0.1 M Tris, pH 8.8, 0.2 ml of 0.2 M glycine buffer pH 9.0, 25 µl of H(2)0, 25 µl of 100 mM MgCl(2), briefly sonicated(39) , and 25 units of alkaline phosphatase (from bovine intestinal mucosa (Sigma), a nonspecific phosphatase that has been shown to hydrolyze phosphorylated farnesylated derivatives)(38) , was added (pilot experiments showed that approximately 70-80% of a sphingosine 1-phosphate standard was cleaved to sphingosine under these conditions) (data not shown). The tubes were capped under nitrogen and incubated at room temperature for 6-8 h, then the long-chain bases were extracted and quantitated by HPLC as described above.

To resolve individual lipid classes, aliquots of the lipid extracts were applied to Silica Gel 60 plates (Merck) and the chromatograms developed as follows: ceramide (diethyl ether/methanol, 99:1 v/v); long-chain bases (chloroform/methanol, 2 N ammonium hydroxide, 40:10:1.5 v/v); phosphatidylethanolamine and sphingoid bases (chloroform/methanol, 28% ammonium hydroxide, 13:7:1 v/v); complex sphingolipids, specifically gangliosides (chloroform/methanol, 0.22% calcium chloride, 60:35:8 v/v)(40) ; long-chain base cleavage products (hexane/diethyl ether/acetic acid, 70:30:1 v/v). Spots were generally detected by iodine; ninhydrin was used to visualize free long-chain bases. For autoradiography, TLC plates were sprayed with Fluoro-Hance (Research Products Intl., Mount Prospect, IL) and exposed to film for 5-7 days. In some experiments, radiolabeled lipids were detected on a Bioscan System 200 Imaging Scanner. Following identification with authentic standards, the radiolabeled lipids were scraped from the plate into scintillation vials, 0.2 ml of water and 4 ml of scintillation mixture were added, and the samples were counted for 10 min. Measurements are normalized for protein and expressed as disintegrations/min per mg protein, unless otherwise stated in the text. Protein was determined by the method of Lowry and co-workers (41) using BSA as standard. Phospholipid mass was determined by the measurement of inorganic phosphate content(42) .

In Vitro Coupled Serine Palmitoyltransferase and Ceramide Synthase Assays

To determine if J774A.1 cells have both the capability to synthesize and to hydrolyze newly formed dihydroceramides, N-acyl-[^3H]sphinganine was synthesized in situ utilizing the precursor substrates and an NADPH-regenerating system as described in Braun et al.(43) and Williams et al.(44) . [^3H]Serine was purified by cation-exchange chromatography(44) , and unlabeled serine was added to give a specific activity of approximately 1 10^5 dpm/nmol. Macrophages (>10^8/experiment) were removed from the spinner flask, washed twice with ice-cold PBS containing 5 mM EDTA, and resuspended in 5 ml of ice-cold 50 mM potassium phosphate with 5 mM EDTA, 10 mM DTT, and 0.25 M sucrose (pH 7.4). Cells were disrupted by sonication in a water bath sonicator (kept cold with ice), the sonicate was centrifuged for 20 min at 18,000 g, and the supernatant centrifuged at 217,000 g for 1 h in a Beckman TL100 airfuge centrifuge. The high speed membrane pellet was resuspended in 50 mM potassium phosphate with 5 mM EDTA and 10 mM DTT (pH 7.4) and used for activity assays within 1-1.5 h following isolation. Each assay tube contained 50 µl of 2 mM [^3H]serine, 0.2 mM pyridoxal 5-phosphate, 50 mM potassium phosphate (pH 7.5), 8 mM glucose 6-phosphate, 4 units of glucose-6-phosphate dehydrogenase (Sigma Type VII from Bakers' yeast), 0.2 mM palmitoyl-CoA, 0.2 mM stearoyl-CoA, 0.4 mM MgCl(2), 2 mM NADP, and 0.2 mM ATP (magnesium salt). To this mixture, 50 µl (approximately 50 µg of protein) of the resuspended pellet was added for a final volume of 100 µl and the tube incubated in a 37 °C shaking water bath. To optimize for sphinganine conversion to dihydroceramide, stearoyl-CoA was included in the initial reaction mixture and additional stearoyl-CoA (0.2 mM) was added at 20 min to replace substrate removed by a highly active acyl-CoA hydrolytic system(44, 45) . Terminations of reactions and extraction of [^3H]products were performed as described (46) . Radioactivity in tubes that did not contain acyl-CoAs was subtracted as background from sample tubes and was generally <10% of the total distintegrations/minute.

[^14C]Ethanolamine Labeling

To determine the effect of FB(1) on the biosynthesis and turnover of phosphatidylethanolamine, cells were grown for 48 h in medium containing 0.25 µCi/ml [^14C]ethanolamine, washed, and changed to fresh medium. To dilute the endogenous [^14C]ethanolamine and prevent reutilization of the ^14C-labeled headgroup released during hydrolysis, 2 mM cold ethanolamine was added to cells in conditioned medium for 1 h and included in fresh DMEM during the chase period(47) . FB(1) was added 5 min before, as well as during, the chase.


RESULTS

Fig.1illustrates the changes in sphingosine and sphinganine mass that occurred when conditioned culture medium was removed from J774.A1 cells and fresh DMEM was added. Free sphingosine increased 6.4-fold within 15 min and reached a maximum (approximately 300 pmol/mg protein or an 8-fold increase) by 30 min. Sphinganine mass also rose, increasing approximately 10-fold in 15-30 min. The sum of these two long-chain bases was approximately 0.5 nmol/mg protein. This time course and the fold changes varied somewhat among experiments, but the maxima always occurred within an hour after changing the medium. Long-chain base levels typically decreased to about half of the maximum by 120 min (Fig.1) and returned to the initial level found in conditioned medium within 4 h (not shown).


Figure 1: Free sphingoid base mass after removal of conditioned culture medium. J774 cells were cultured for 3 days, then the conditioned culture medium was removed and replaced with fresh medium (without FBS) for various times. Sphingosine (open circles) and sphinganine (filled circles) were analyzed as described under ``Experimental Procedures.'' Results are the mean ± range (pmol/mg protein) from duplicate samples of a representative experiment.



Changes in Long-chain Base Labeling by

Serine and [^3H]Palmitate

Elevations in free sphingosine and sphinganine could arise from de novo synthesis and (or) turnover of sphingolipids. To discriminate between these two possibilities, radiolabeled precursors of long-chain base biosynthesis ([^3H]serine and [^3H]palmitate) were included in the fresh culture medium and the incorporation of radiolabel into long-chain bases was determined. [^3H]Serine (Fig.2A) was rapidly incorporated into free sphinganine, with the greatest incorporation found at 15 and 30 min, a reduction to about half this amount by 1 h, and little label in either free long-chain base after 2 h. This pattern of labeling resembled the time-dependent changes in sphinganine mass (Fig.1). Based on the specific activity of the added [^3H]serine (30 mCi/mmol), the mass of [^3H]sphinganine present at 15 and 30 min would be geq200 pmol/mg cell protein, and greater amounts could be present if the [^3H]serine is diluted with endogenous, unlabeled serine. This amount was very close to the elevation in sphinganine mass (200 pmol/mg protein) (Fig.1). Sphinganine mass and labeling differed only at 2 h, which might be caused by depletion of the labeled precursors (35) and/or to dilution of the [^3H]serine (see below). Sphinganine synthesis from [^3H]palmitate also increased (Fig.2B), but based on the specific activity of the starting [^3H]palmitate, only 1 pmol of [^3H]sphinganine/mg cell protein was produced, which is less than 1% of the sphinganine mass. This difference is probably due to dilution of the [^3H]palmitate with unlabeled, endogenous palmitate.


Figure 2: [^3H]Serine and [^3H]palmitate incorporation into free sphingosine and sphinganine. J774 cells were cultured for 3 days, then the medium was removed and replaced with 1 ml of fresh DMEM containing 0.1 mCi [^3H]serine (A) or 1 µM palmitate spiked with 10 µCi of [^3H]palmitate (B). [^3H]Sphingosine (open circles) and sphinganine (closed circles) were extracted, separated by TLC, and quantitated by scintillation counting. Results are the mean ± S.D. (dpm/mg protein; n = 3) for A, and mean ± half range (dpm/mg protein; n = 2) for B.



There was little incorporation of [^3H]serine and [^3H]palmitate into sphingosine (Fig.2, A and B, open circles). Thus, the labeling time courses and changes in mass indicate that the burst of sphinganine is due to increased de novo synthesis; sphingosine, however, arises from another source. These findings are consistent with the pathway for sphingosine synthesis, wherein the addition of the 4-trans double bond to sphinganine occurs after N-acylation(35, 48, 60) .

beta-Fluoro-alanine Inhibition of de Novo Sphingolipid Biosynthesis

beta-Haloalanines act as mechanism-based irreversible inhibitors of serine palmitoyltransferase, the enzyme that catalyzes the first step of sphingolipid biosynthesis(49) . J774A.1 cells were treated for 20 min with beta-F-alanine before the change to fresh medium (Fig.3): 1 mM beta-F-alanine did not alter free sphingosine mass (Fig.3A), whereas 0.1 mM (Fig. 3B, closed squares) and 1.0 mM beta-F-alanine (Fig. 3B, closed triangles) reduced the increase in sphinganine mass by 38 and 74%, respectively, at the 30-min time point. Treatment of cells with 1 mM beta-F-alanine for 5 min in conditioned medium, followed by addition of new DMEM containing beta-F-alanine and [^3H]palmitate, also eliminated [^3H]palmitate labeling of free sphinganine (not shown). These findings confirm that de novo biosynthesis is required for the sphinganine burst but not for the changes in sphingosine.


Figure 3: Effect of beta-F-alanine inhibition of de novo sphingolipid biosynthesis on sphingosine (A) and sphinganine (B) masses following change to new medium. J774 cells were cultured for 3 days, then beta-F-Alanine (0 mM, open circles; 0.1 mM, filled squares; 1.0 mM, filled triangles) was added 20 min prior to the removal of the conditioned medium and addition of new DMEM containing beta-F-alanine at the same concentrations. The long-chain bases were quantitated after incubation for the indicated times. Results are the average of duplicate samples ± half range (pmol/mg protein).



Role of Serine Concentration in the Sphinganine Burst

Another way to alter the rate of de novo sphingolipid biosynthesis is to vary the amount of serine in the medium(50, 51) . Fig.4shows the increases in sphinganine and sphingosine in cells changed to new medium (both were elevated 8-15-fold), conditioned medium (no change from time 0), and conditioned medium supplemented with 0.4 mM serine (the concentration in new DMEM), which restored some of the increase in sphinganine (i.e. 5-fold), but had no effect on sphingosine. Analyses of the amounts of free amino acids in the cells (some of which are shown in Fig.5) confirmed that there was a transient, 4-fold increase in cellular serine after changing the medium. The amounts of other amino acids (shown in Fig.5for Asx and Glx) decreased over this time course, which establishes that the change in serine concentration was not an artifact of incomplete removal of the medium.


Figure 4: Serine stimulation of de novo sphingolipid synthesis. J774 cells were cultured for 3 days, then changed to the indicated media, incubated for 15 min, and analyzed for long-chain base mass. Results are expressed as the mean ± S.D. (pmol/10^6 cells/dish, n = 3). Symbols: sphingosine, solid bars; sphinganine, hatched bars. The incubation media were as follows: NM, new HEPES-buffered DMEM; CM, conditioned medium taken from the same or similarly treated cells (both give the same result); CM + Ser, the same conditioned medium supplemented with 0.4 mML-serine; PBS, 10 mM HEPES-buffered phosphate-buffered saline; PBS + Ser, PBS supplemented with 0.4 mML-serine.




Figure 5: Cellular amounts of serine and other amino acids in cells incubated in new medium. J774 cells were incubated in fresh DMEM (containing 10% FBS) for the given times, washed twice to remove the medium, and extracted for analyses of amino acids of the aqueous phase of the lipid extract as described under ``Experimental Procedures.'' The range for the data are within 10% of the mean for all measurements.



When cells were changed to PBS, the increase in sphingosine was comparable to new medium (Fig.4). Sphinganine increased 6-fold, which is 49% of the stimulation by new medium. Addition of 0.4 mM serine to PBS increased sphinganine by 40-fold over conditioned medium (6-fold over PBS alone) and had no effect on sphingosine. These results indicate that addition of exogenous serine enhances the sphinganine burst but is not necessary for some increase in sphinganine, presumably because cells can produce this amino acid precursor, and may release some sphinganine by hydrolysis of sphinganine-containing sphingolipids.

Chloroquine and Ammonium Chloride Inhibition of Sphingoid Base Generation

The results described above indicate that the sphingosine arises from the turnover of complex sphingolipids rather than de novo synthesis. Most sphingolipids are located in the plasma membrane, endosomes, lysosomes, and the Golgi apparatus(52) ; therefore, if the sphingosine burst involves the turnover of complex sphingolipids, it may involve hydrolases in acidic compartments such as lysosomes or endosomes(52, 53) . Chloroquine, a lyso-osmotrophic agent, inhibited the sphingosine burst with an IC of approximately 11 µM (Fig.6A, open circles), but had little or no effect on the sphinganine burst (Fig.6A, closed circles). Ammonium chloride, a less specific lyso-osmotrophic agent, also inhibited the sphingosine burst, but at higher concentrations (IC = 2 mM) (Fig.6B, open circles), and caused some inhibition of sphinganine formation (35% at 10 mM) (Fig.6B, closed circles). It is not clear why ammonium ion causes this reduction in sphinganine, but ammonium ion can also increase cytosolic pH. One of the reasons that condition medium inhibits the sphingosine burst may be that ammonium ion accumulates in cell culture medium from amino acid metabolism and the non-enzymatic deamidation of glutamine. (^2)


Figure 6: Chloroquine (A) and ammonium chloride (B) suppression of sphingoid base mass levels 1 h after change to new culture medium. Conditioned culture medium was removed, and cells were incubated in fresh DMEM containing either chloroquine or ammonium chloride for 1 h, after which sphingosine (open circles) and sphinganine (filled circles) were extracted and quantitated. Values represent the mean of triplicate determinations and the S.D. lie within 10% of the mean for all values except the 5 µM chloroquine data where the S.D. for sphingosine are ± 19% of the mean and sphinganine ± 16% of the mean. The sphingosine and sphinganine mass in control cells were 252 ± 23 and 164 ± 5.6 pmol/mg cell protein (mean ± S.D.), respectively, for the chloroquine experiment. In control cells for the ammonium chloride experiment, the sphingosine mass was 377 ± 59 pmol/mg protein, and sphinganine was 381 ± 44 pmol/mg protein.



Analyses of Long Chain Bases of More Complex Sphingolipids Formed (and Turned Over) during the Sphinganine/Sphingosine Burst

These were also analyzed as follows.

De Novo Sphingolipid Biosynthesis

To determine the fate of the sphinganine synthesized after the change to new medium, the mass and labeling of the long-chain base backbone of both ceramide(s) and total sphingolipids were determined (Fig.7). No detectable change occurred in the mass of sphingosine in total sphingolipids following addition of new medium (Fig.7C, open circles); however, the mass of sphinganine-containing sphingolipids increased by 2-3-fold (approximately 1 nmol/mg protein/h) (Fig.7C, closed circles). About 10% of the sphinganine-containing sphingolipids were N-acyl-sphinganines (i.e. dihydroceramides), which increased 6.3-fold over 2 h (Fig.7A, closed circles).


Figure 7: Changes in sphingoid base composition of ceramide and total sphingolipids following the change to new culture medium. After incubation for various times in new DMEM, J774 cells were extracted and the sphingosine (open circles) and sphinganine (filled circles) content of ceramides (A) and total sphingolipids (C) determined as described under ``Experimental Procedures.'' The incorporation of [^3H]serine (0.1 mCi/ml, added at t(0)) was determined by acid hydrolysis of the ceramide (B) and total sphingolipids (D) to sphingoid bases, which were separated by TLC and quantitated by scintillation counting. Results are the mean ± S.D. (dpm/mg protein) of triplicate samples.



The amount of ceramide increased 3-fold (Fig.7A, closed circles) within 30 min and remained elevated at approximately 200 pmol/mg protein for 120 min. Ammonium chloride (10 mM) completely abrogated the increases in ceramide, but not in dihydroceramide (data not shown). The lack of an observable decrease in the mass of complex sphingolipids (Fig.7C, open circles) is probably due to the small amounts of ceramide and free sphingosine that are produced (400-500 pmol) compared to the total sphingolipid mass (>6 nmol).

Analyses of the labeled long-chain bases (Fig.7, B and D) established that over this time course, [^3H]serine was incorporated almost exclusively into the sphinganine backbone of dihydroceramide (Fig.7B, closed circles), with little conversion to ceramide (Fig.7B, open circles), which further substantiates the hypothesis that ceramide is derived mainly from the turnover of unlabeled sphingolipids rather than from the conversion of dihydroceramide to ceramide. The labeling of sphinganine and sphingosine in total sphingolipids resembles a precursor-product relationship (Fig.7D): the [^3H]sphinganine in complex sphingolipids rose steadily in the first hour and thereafter leveled off, whereas [^3H]sphingosine increased more gradually and did not equal [^3H]sphinganine until 2-4 h (Fig.7D, openversusclosed circles). Only a portion (23% after 30 min and 11% after 4 h) of this label was in dihydroceramide per se, with the remainder in complex sphingolipids. These data reveal that a substantial amount of dihydroceramide can be incorporated into complex sphingolipids and raise the possibility that the 4,5-trans-double bond might be added to complex (dihydro)sphingolipids, as well as to dihydroceramides(60) .

Complex Sphingolipid Turnover

Cells were incubated with 0.1 mCi of [^3H]serine for 3 days (to incorporate [^3H]serine into sphingolipids and ``condition'' the medium), washed with cold PBS, and incubated in fresh DMEM for 1 h with or without NH(4)Cl (Fig.8) or chloroquine. Sphingomyelin (145,000 ± 36,000 dpm/mg protein) accounted for approximately 60% of the [^3H]sphingosine in cellular sphingolipids at time 0 and appeared to decrease about 5% when incubated in fresh medium. Although this change was not statistically significant due to large variations in the amounts of label in sphingomyelin at time 0, in cells treated with 10 mM NH(4)Cl, a significantly greater amount of radiolabel remained in sphingomyelin. Therefore, it appears that between 10,000 and 25,000 dpm of [^3H-sphingosine]sphingomyelin is lost during the 1-h incubation in fresh medium. The hydrolysis of sphingomyelin was also demonstrated by labeling cells with[^14C]choline (0.5 µC/dish) for 2 days followed by removal of the medium and incubation with either conditioned medium with choline or fresh DMEM. The distintegrations/minute in sphingomyelin decreased by approximately 20% in 30 min, and conditioned medium, ammonium chloride, or chloroquine blocked the decrease (data not shown). The remainder of the radiolabel was distributed among several glycolipid bands on the TLC plate, the largest being lactosylceramide (Fig.8, left panel). However, the distintegrations/minute in these species increased rather than decreased, and NH(4)Cl blocked this change. Our interpretation of these findings is that sphingomyelin could be a source of the free [^3H]sphingosine and [^3H]sphingosine-containing ceramide (which constituted only 1-2% of the total disintegrations/minute in sphingomyelin) and that, once released, some of these intermediates may be recycled into glycolipids.


Figure 8: Turnover of [^3H]serine-labeled sphingolipids. J774 cells were plated at a density of 5 10^5 cells/2 ml in 60-mm dishes in DMEM containing 10% FBS and 0.1 mCi/ml [^3H]serine. After 3 days, the conditioned medium was removed, and the cells were washed with PBS and incubated in fresh DMEM with or without 2 or 10 mM ammonium chloride (NH(4)Cl). After 1 h, the lipids were extracted and analyzed as described under ``Experimental Procedures.'' Free sphingosine (Free So) was determined by HPLC, and the long-chain bases of sphingomyelin (SM), lactosylceramide (LacCer), and ceramide (Cer) were quantitated after acid hydrolysis as described in the text. Results are expressed as dpm/µg protein ± S.D. (n = 3).



Evaluation of the Turnover of Newly Made Dihydroceramides as a Possible Source of Some of the Free Sphinganine during the Burst

Although de novo sphingolipid synthesis is necessary for the sphinganine burst, some sphinganine might arise also from hydrolysis of newly made dihydroceramides, explaining the reduction in sphinganine when ammonium chloride was added to the cells (Fig.6). To test this hypothesis, dihydroceramides were synthesized de novo from [^3H]serine by incubating microsomes with palmitoyl-CoA, an NADPH-regenerating system, and stearoyl-CoA, followed by a chase with a 10-fold excess of serine and beta-fluoroalanine (to end further synthesis of 3-ketosphinganine), then addition of fumonisin B(1) (FB(1)) (54) to block the reacylation of sphinganine.

During the first 20 min, label appeared in 3-ketosphinganine (Fig.9, triangles), sphinganine (circles), and N-acyl-sphinganines (squares). The chase reduced, but did not abolish, the labeling of sphinganine and N-acyl-sphinganine; nonetheless, there was a significant reduction in N-acyl[^3H]sphinganine after addition of FB(1) and an equivalent increase in [^3H]sphinganine and 3-keto-[^3H]sphinganine compared to both the control and the 40-min time point. Thus, at least in this system, sphinganine (and possibly 3-ketosphinganine) can be acylated as well as released from newly made dihydroceramides.


Figure 9: Incorporation of [^3H]serine into newly synthesized long-chain bases and N-acyl-sphinganine (dihydroceramide) in vitro. A cell membrane fraction was incubated with the precursors for N-acyl-sphinganine as described in the text. After 20 min, 1 mM cold serine, 1 mM beta-F-alanine, and 0.1 mM stearoyl-CoA were added (indicated as the Chase); and after an additional 20 min (at FB(1)), 25 µM FB(1) was added to the groups designated by the striped line. At each time, the amount of radiolabel in sphinganine (closed circles), dihydroceramide (open squares), and 3-ketosphinganine (closed triangles) were determined. Results are expressed as the mean ± S.D. (pmol/mg protein, n = 3), and represents one of two experiments that gave this profile.



Disappearance of Free Long-chain Bases after the Burst

Since free sphinganine and sphingosine begin to decline after 30-60 min (Fig.1), they must be undergoing acylation to (dihydro)ceramides and/or phosphorylation and cleavage to a long-chain fatty aldehyde and ethanolamine phosphate (reviewed in Refs. 52 and 55). At time 0, the cells contained <10 pmol of the 1-phosphates (Fig.10); upon changing the cells to new medium, both sphinganine- and sphingosine 1-phosphate (calculated as the difference between long-chain base mass in lipid extracts treated with or without alkaline phosphatase) increased rapidly to 50-70 pmol/mg protein (each) for the first hour, which was approximately one-third of the free long-chain bases. By 120 min, the long-chain bases had returned to nearly the same level as at time 0. Therefore, the burst involves a transient increase in not only sphingosine and sphinganine, but also in the 1-phosphates.


Figure 10: Determination of phosphorylated sphingoid bases. Free sphingosine (filled circles) and sphinganine (open circles) were analyzed directly by HPLC, and the 1-phosphates were determined by incubation of the lipids with 20 units of alkaline phosphatase for 6-8 h, followed by analysis of sphingosine (filled squares) and sphinganine (open squares) by HPLC. Results are expressed as pmol/mg cell protein (mean ± S.D., n = 4).



The data in Fig.10suggest that newly made sphinganine, as well as sphingosine, are accessible to both ceramide synthase and sphingosine kinase. If so, inhibition of acylation by FB(1) should divert more of the long-chain base toward phosphorylation. Incubation of the cells with 50 µM FB(1) caused sphinganine to increase from 6.3 ± 1.9 pmol/dish at time 0 to 169 ± 9.9 pmol/dish after 30 min (compared to 77 ± 6 pmol/dish for cells that were not treated with FB(1)). A comparable increase was found in sphinganine 1-phosphate (Fig.11), with the greatest effect of the FB(1) seen at 120 min. In contrast, FB(1) had little effect on the amounts of sphingosine (i.e. 88 ± 7 versus 81 ± 15 pmol/dish at 30 min with and without FB(1), respectively) or on sphingosine 1-phosphate (Fig.11). Therefore, FB(1) had little effect on sphingosine accumulation or on its phosphorylation.


Figure 11: Long-chain base 1-phosphate concentrations in the presence of FB(1). At the time of changing the cells to fresh medium, 50 µM FB(1) was added and sphingosine 1-phosphate (open bar) and sphinganine 1-phosphate (solid bar) were determined as described under ``Experimental Procedures'' and the legend for Fig.10. Results are expressed as pmol/mg cell protein (mean ± S.D., n = 4).



Degradation of Sphinganine and Utilization of the Ethanolamine Moiety for Phosphatidylethanolamine Synthesis

The ethanolamine phosphate derived from the lytic cleavage of the sphingosine (or sphinganine) 1-phosphate has been reported to be incorporated into phosphatidylethanolamine (PE), although this has only been shown with exogenously added long-chain bases(56, 57) . Conditions that increase sphinganine- and sphingosine 1-phosphate, such as changing the medium and/or addition of FB(1), should lead to increased synthesis of ethanolamine phosphate. This is a difficult hypothesis to prove because radiolabeled serine is incorporated into both long-chain bases and phosphatidylserine, and the latter is known to be decarboxylated to PE.

When J774 cells were labeled with 10 µCi/ml [^3H]serine for 1 or 12 h in the presence and absence of 50 µM FB(1) (Table1), FB(1) reduced the amount of [^3H]sphingosine in ceramide, sphingomyelin, and total sphingolipids, but did not alter the incorporation of radiolabel into the total lipid extract, PC, or PS. In contrast, FB(1) increased the amount of radiolabel in PE by 48 and 31% after 1 and 12 h, respectively. Incubation of J774A.1 cells with 50 µM FB(1) also did not alter the mass of PE significantly (36.3 ± 3.9 nmol/mg protein for FB(1)-treated and 40.5 ± 0.9 nmol/mg protein for control cells, or approximately 25% of the total cellular phospholipid, at 12 h), whereas the sphingomyelin mass decreased by 46% (from 18.4 ± 1.0 nmol/mg protein to 9.9 ± 1.2 nmol/mg protein). These findings are consistent with FB(1) diverting newly made [^3H]sphinganine 1-phosphate to [^3H]ethanolamine phosphate for use in [^3H]PE synthesis, and raises the possibility that sphinganine serves not only as an intermediate of sphingolipid biosynthesis, but also as a source of ethanolamine phosphate in some cells. A similar observation was made, but not explained, in studies of the effects of FB(1) on hepatocytes(24) .



Ethanolamine phosphate synthesized via the long-chain base phosphates should also compete with the incorporation of exogenously added [^14C]ethanolamine into PE. When J774 cells were incubated with 0.25 µCi of [^14C]ethanolamine in the fresh culture medium, FB(1) reduced the amount of label incorporated into [^14C]PE (Fig.12). The inhibition (30% at 12 h) was comparable to the increase in incorporation of [^3H]serine into PE (31%, Table1).


Figure 12: Effect of FB(1) on the incorporation of [^14C]ethanolamine into PE. Cells were cultured for 3 days, and new medium containing [^14C]ethanolamine (0.25 µCi) with (closed circles) or without (open circles) 50 µM FB(1) was added and the amount of radiolabel in PE determined. Results are expressed as dpm/nmol PE/mg cell protein (mean ± S.D., n = 3).



Since long-chain bases (58) and the 1-phosphates (59) have been reported to activate a PE-specific phospholipase D, FB(1) might induce turnover and reutilization of the ethanolamine-derived headgroup. This possibility was eliminated because, when J774 cells were cultured for 48 h in medium containing 0.25 µCi/ml [^14C]ethanolamine and subsequently changed to new DMEM containing 2 mM cold ethanolamine with or without FB(1), the rate of release of label from [^14C]phosphatidylethanolamine was 4.2 ± 1.0 pmol/min/mg protein for both groups (data not shown).

Utilization of Exogenously Added

Sphingosine for the Synthesis of Ceramide, Sphingomyelin, and Phosphatidylethanolamine

Utilization of long-chain bases for PE biosynthesis was demonstrated directly by synthesizing [1-^3H]sphingosine and following the fate of the radiolabel upon incubation with the cells (Table2). After 3 h, about half of the radiolabel was recovered in the aqueous phase (Table2) which most likely represents formation of [^3H]ethanolamine. Consistent with this interpretation, about half of the label in the organic phase was in PE, with the remainder distributed mostly among free sphingosine, ceramide, and sphingomyelin (Table2).



When the cells were also treated with 50 µM FB(1), there was a reduction in the amount of label in sphingomyelin (6.7-fold) and ceramide (1.4-fold) due to inhibition of [1-^3H]sphingosine acylation (Table2). The disintegrations/minute in the aqueous phase increased 30-40%, as expected for increased phosphorylation (as shown above) and cleavage of the [1-^3H]sphingosine; however, the disintegrations/minute in PE were somewhat lower than in the cells that were not treated with FB(1). This seeming contradiction was explained by analysis of the mass of the long-chain bases in the cells. When the mass of unlabeled sphinganine and sphingosine is used to estimate the specific activity of the [^3H]long-chain bases (Table2), the specific activity is 45% lower for the FB(1)-treated cells than for the controls. Therefore, the mass of PE that would correspond to the disintegrations/minute (Table2) would be 580 pmol/mg protein in FB(1)-treated cells and 400 pmol/mg protein in control cells. These calculations are approximations because the specific activities of all of the relevant intermediates are not known; nonetheless, this increase in PE synthesis from ^3H-long-chain bases in the FB(1)-treated cells is almost identical to the findings from [^3H]serine labeling and the inhibition of [^14C]ethanolamine incorporation (i.e. 30%, as described above).

The catabolism of exogenous [1-^3H]sphingosine and reutilization for PE synthesis even in cells that were not treated with FB(1) was examined further (Fig.13). At the earliest time point examined (15 min), over half of the cell-associated radiolabel was found in the aqueous phase of the lipid extract. Sphingosine, ceramide, sphingomyelin, and PE accounted for most of the radiolabel in the organic phase. The time course for the disintegrations/minute in sphingosine and ceramide versus PE and sphingomyelin suggested a precursor-product relationship.


Figure 13: [1-^3H]Sphingosine uptake and metabolism. [1-^3H] Sphingosine was added as a 1:1 (mol/mol) sphingosine-BSA complex to a final concentration of 1 µM at time 0 in fresh culture medium (DMEM). Radioactivity associated with the total cell extract, the aqueous phase recovered from the two-phase cellular lipid extraction, and the individual lipids were determined as described under ``Experimental Procedures.'' Results are expressed as the mean ± S.D. of triplicate samples.




DISCUSSION

The findings of this study are summarized by the scheme shown in Fig.14. Replacement of conditioned with fresh culture medium on J774A.1 cells induces a rapid burst of long-chain bases from at least two sources: sphinganine and its derivatives (sphinganine 1-phosphate, N-acyl-sphinganines, etc.) that are newly synthesized, and sphingosine and its derivatives (sphingosine 1-phosphate and N-acyl-sphingosines) that are produced by the turnover of existing complex sphingolipids. Since we first described this change with J774A.1 cells(27, 73, 74, 75) , we have noted similar bursts in Swiss 3T3 cells(28) , primary cultures of rat hepatocytes, and mouse peritoneal macrophages (data not shown), and Lavie et al.(29) have made similar observations with NIH-3T3 cells, A431 cells, and NG108-15 cells. While the biological significance (if any) of this apparently common phenomenon is unknown, it has provided a useful model system for dissecting the factors that influence the appearance and fate of free long-chain bases in cells.


Figure 14: An overview of endogenous long-chain base metabolism in J774A.1 macrophages. Shown are the interrelationships found by this study. The abbreviations represent 3-ketosphinganine (KA), sphinganine (Sa), N-acyl-sphinganine or dihydroceramide (N-Acyl-Sa), ceramide (N-Acyl-So), sphingosine- and sphinganine 1-phosphates (Sa-P and So-P), PE, beta-fluoro-alanine (betaF-ala), and FB(1). This figure illustrates the elements of this pathway that are most relevant to this article, but does not include all of the intracellular reactions of sphingolipid metabolism (such as the turnover of sphingolipids in the plasma membrane), nor does it show all elements of their topologic distribution (for example, the first step of glycolipid synthesis occurs on the cytosolic leaflet of the Golgi).



The 4-trans double bond of sphingosine is inserted subsequent to the formation of N-acyl-sphinganine (dihydroceramide) during sphingolipid biosynthesis de novo(35, 48, 60) ; thus, free sphinganine appears as an intermediate of the de novo pathway, with sphingosine arising from hydrolysis of complex sphingolipids. The findings of this study confirm this pathway because the amounts of sphinganine (but not sphingosine) were affected by beta-F-alanine (as an inhibitor of serine palmitoyltransferase) (49) as well as by varying the amounts of serine. The strong dependence of long-chain base biosynthesis on the availability of this precursor has been seen in a variety of cell types (mouse LM cells, hepatocytes, and J774 macrophages) (50, 51, this study), which suggests that this may be one of the factors that regulates this pathway. If so, sphingolipid metabolism may be affected by hormonal and nutritional factors that govern amino acid metabolism. There have been no reports of hormones affecting de novo long-chain base synthesis; however, a recent study noted that insulin augmented the accumulation of sphinganine in FB(1)-treated Swiss 3T3 cells(28) .

N-Acylation appears to account for the majority of sphinganine metabolism, particularly after the initial burst has dissipated. Substantial amounts of sphinganine appeared first in dihydroceramides and more complex sphingolipids, and sphingosine later became the major long-chain base of the complex sphingolipids. A similar observation has been made with LM cells(35) . Apparently, the 4,5-double bond is not required for addition of the headgroups, yet ultimately appears in the backbones of complex sphingolipids. Evidence for an enzyme activity that adds a double bond to dihydroceramides has been reported(60) , but our findings raise the possibility that as-yet-uncharacterized enzyme(s) may act also on more complex substrates. Alternatively, newly made (sphinganine-containing) sphingolipids may be turned over rapidly (61) and the double bond added before the (dihydro)ceramide backbone is reutilized. Hannun and co-workers (12, 14) have speculated that formation of ceramides as intermediates of the de novo biosynthetic pathway could be deleterious because they can affect cell growth and induce apoptosis. Therefore, introduction of the 4,5-double bond after addition of headgroups to dihydroceramides would avoid this potential danger.

In J774A1 cells, free sphingosine is derived primarily from hydrolysis of complex sphingolipids in an acidic compartment(s), perhaps reflecting the endosomal and/or lysosomal turnover of sphingolipids. A source of the sphingosine appears to be sphingomyelin, based on the labeling studies. However, the amounts of free sphingosine are only a small percentage of the cellular sphingolipid mass; therefore, turnover of other sphingolipids could also contribute.

We estimate that approximately 30% of endogenous sphingosine generated during the burst period is reincorporated into sphingolipids; this relatively low reutilization agrees well with the rapid (<15 min) generation of water-soluble metabolites and the substantial utilization of [1-^3H]sphingosine for phosphatidylethanolamine biosynthesis. The difference in sphingosine and sphinganine metabolism may be related to the location of the enzymes involved in acylation and phosphorylation. Along with serine palmitoyltransferase and the NADPH-dependent 3-ketosphinganine reductase(62, 63) , N-acyl-sphingosine (sphinganine) transferase is localized to the external leaflet of the ER; sphingosine kinase, however, has been found in cytosol and localized to microsomal membranes(56, 57, 64, 65) , and sphingosine 1-phosphate lyase is associated mainly with the endoplasmic reticulum(66) . Therefore, sphingosine that is liberated in lysosomes (or other acidic compartments), and perhaps also in the plasma membrane(67) , must move to other intracellular locations to be metabolized further, providing opportunities for it to be phosphorylated and cleaved.

Our findings demonstrate that the burst, alone or in combination with inhibition of N-acylation of sphingoid bases by FB(1), results in an augmented flux of long-chain bases toward the 1-phosphates and cleavage, which in turn apparently affects the cellular ethanolamine phosphate pool used for PE biosynthesis via the CDP-ethanolamine pathway. Based on the changes that FB(1) treatment induced in PE labeling (and on the assumption that the other PE biosynthetic pathways were not affected), sphingolipid degradation contributed approximately 0.1-1 nmol of ethanolamine/hour to total PE synthesis. This rate is feasible considering the amounts of long-chain bases that are formed by de novo synthesis and shuttled toward phosphorylation and cleavage. Therefore, although the quantitative significance of this pathway is still unclear, this study indicates that sphinganine could be a significant source of ethanolamine in some cell types.

The formation of such large amounts of the long-chain base 1-phosphates is also intriguing because sphingosine 1-phosphate has potent effects on cells (reviewed in (16, 17, 18) ). Spiegel and co-workers have shown that sphingosine 1-phosphate stimulates proliferation of quiescent Swiss 3T3 fibroblasts(68) , and have suggested that this may be mediated through several pathways, including release of intracellular calcium(25) , increases in phosphatidic acid levels(58) , and modulation of AP1(26) . They have also found that sphingosine 1-phosphate (as well as sphingosine) increases in Swiss 3T3 cells treated with platelet-derived growth factor(16) . Studies in permeabilized cells have implicated sphingosine 1-phosphate in the release of calcium from intracellular stores(15) , and Ghosh et al.(17) have reported that the conversion of sphingosine to sphingosine 1-phosphate by the sphingosine kinase associated with the endoplasmic reticulum activates release of stored calcium.

It is tempting to speculate that these changes in long-chain bases and their derivatives (both the 1-phosphates and N-acyl-derivatives) affect the behavior of J774 cells. The increases in sphingosine and sphingosine 1-phosphate after changing the medium are comparable to the amounts seen in platelet-derived growth factor-treated Swiss 3T3 cells (16) and stimulation of Swiss 3T3 cell growth by FB(1)(28) . In other cell systems, sphingosine and sphinganine are growth inhibitory and cytotoxic at these levels (69) , and ceramides have been found to have diverse effects, from growth stimulation (70) to induction of apoptosis(71) . In recent studies(72, 76) , we have found that J774 cells undergo changes in phorbol dibutyrate binding and a number of other cellular responses that would be predicted to occur when the amounts of sphingosine increase after changing the medium. Thus, the burst may play a role in the generally known (but rarely mentioned) anomalies in cell behavior after changing the medium that necessitate that many types of cells be given several hours to ``calm down'' before beginning an experiment.


CONCLUDING REMARKS

Two findings of this study should be borne in mind by investigators who are analyzing the role of free long-chain bases and their derivatives (N-acyl-compounds, 1-phosphates, and probably others) as mediators of the action of hormones, growth and differentiation factors, and cytokines. First, it appears that many cells undergo large increases in these compounds after changing the culture medium; therefore, care must be taken to distinguish between this perturbation and the response that is of interest. Second, many factors differentially alter the metabolism of sphinganine versus sphingosine, ceramide versus dihydroceramide, etc. Therefore, analyses of these compounds by methods that do not distinguish between the presence of the 4,5-trans double bond will not determine whether an agonist is producing the presumed bioactive product: for example, the use of diglyceride kinase to analyze ceramides as ceramide-1-[P]phosphate does not discriminate between ceramide released from existing sphingolipids and dihydroceramide produced via stimulated de novo biosynthesis. While these questions are complex, the approaches described in this article should be helpful in distinguishing among the possibilities.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM33369 and GM46368 (to A. H. M.) and a National Science Foundation Graduate Research Fellowship and National Institutes of Health Training Grant GM08367 (to E. R. S.). Portions of this work were presented in abstract form at the 1992 and 1993 Experimental Biology meetings in Anaheim, CA, and New Orleans, LA, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, 4113 Rollins Research Center, Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-5978; Fax: 404-727-3954; amerril{at}unix.cc.emory.edu.

^1
The abbreviations used are: FBS, fetal bovine serum; FB(1), fumonisin B(1); DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; OPA, o-phthaldialdehyde; DTT, dithiothreitol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; HPLC, high performance liquid chromatography; dpm, disintegrations/minute; TLC, thin layer chromatography; PBS, phosphate-buffered saline.

^2
L. Warden, unpublished observations.


ACKNOWLEDGEMENTS

We thank Lisa Warden for useful discussions about the sphingosine burst and the roles of ammonia and conditioned medium in these phenomena.


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