(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol as an inhibitor of ceramidase.

In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-Derythro-2-(N-myristoylamino)-1-phenyl-1-propanol (Derythro-MAPP). Addition of 5 μMD-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G/G phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceramidase to an extent similar to that seen with C-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC of 1-5 μM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC > 500 μM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.

mor necrosis factor ␣, interleukin-1␤, neurotrophins, the Fas ligand, dexamethasone, serum withdrawal, chemotherapeutic agents, and ␥-irradiation causes an elevation in the endogenous levels of ceramide (1, 6 -11). A role for endogenous ceramide in mediating, at least in part, the actions of these stimuli on cell differentiation, apoptosis, and growth suppression is supported by the ability of exogenous analogs of ceramide to induce these biologic responses in the respective cell types (1,(12)(13)(14)(15)(16).
Additional evidence for a physiologic function for endogenous ceramide has come from studies examining the specificity of action of ceramide analogs. Thus, it has been shown with C 2 -ceramide 1 that this molecule demonstrates structural and stereospecific cellular activities (17,18). The most significant specificity was demonstrated with the lack of activity of C 2dihydroceramide (17,19) which differs from C 2 -ceramide by lacking the trans 4 -5 double bond in the sphingoid base; otherwise, these two molecules display identical stereochemistry at the two chiral carbons (C-2 and C-3). Moreover, the cellular uptake and metabolism of these two compounds is nearly identical (17), suggesting that the lack of activity of dihydroceramide is due to inability to interact with relevant intracellular targets. Indeed, C 2 -ceramide but not C 2 -dihydroceramide is able to activate ceramide-activated protein phosphatase (CAPP) in vitro (18,20).
In addition, some of the cellular activities of ceramide have been mimicked by metabolic manipulation of endogenous ceramide levels. Thus, the addition of bacterial sphingomyelinase, which cleaves outer leaflet sphingomyelin and causes accumulation of membrane ceramide, has been shown to mimic at least some of the effects of exogenous cell permeable ceramides (21,22). Moreover, PDMP and related compounds, which inhibit cerebroside synthase (23) and also cause accumulation of ceramide, result in cellular activities shared with ceramide. These experimental approaches have lent further credence to a role for endogenous ceramide in cell regulation. They also point to the potential versatility in regulation of ceramide levels. Indeed, ceramide occupies a central position in sphingolipid metabolism. Complex sphingolipids derive from ceramide through various enzymatic reactions that add various head groups to the 1-hydroxyl position (1, 24 -27). The breakdown of these sphingolipids through sequential metabolic reactions also results in the formation of ceramide. In turn, ceramide can be degraded further through the action of ceramidases resulting * This work was supported in part by National Institutes of Health Grants GM43825 and DK45067. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In other studies, we have examined a number of synthetic ceramide analogs based on N-acylated phenylaminopropanols (30). Two of these analogs, D-erythro-MAPP and L-erythro-MAPP, were of particular interest because they demonstrated enantiomeric selectivity of action. However, examination of the absolute stereochemistry of these molecules ( Fig. 1) discloses that D-erythro-MAPP has an absolute configuration corresponding to L-erythro-ceramide, whereas L-erythro-MAPP has an absolute configuration corresponding to D-erythro-ceramide. The discrepancy in nomenclature arises from the various methods employed in assigning configuration of stereoisomers and the chiral carbon atom used as a reference (31)(32)(33)(34)(35)(36). Because of this confusion, it is preferable to base configuration primarily on the R/S system for each chiral atom to eliminate any ambiguities. Therefore, we wondered whether D-and L-e-MAPP were direct mimics of ceramide or whether their mechanism of action could be through interference with ceramide-metabolizing enzymes such that the enantiomer resembling the natural ceramide would be a substrate for one of these enzymes whereas its mirror image (D-e-MAPP) may interfere with ceramide metabolism.
In this study we provide evidence demonstrating that L-e-MAPP is a substrate for an alkaline ceramidase whereas D-e-MAPP inhibits this enzyme resulting in substantial elevation in intracellular levels of ceramide. These studies provide further support for physiologic functions of endogenous ceramide; especially in growth regulation. They also provide important and novel tools for studying non-acid ceramidases.

Methods
Proliferation and Cell Cycle Studies-HL-60 human myelocytic leukemia cells were grown in RPMI 1640 medium containing 10% fetal calf serum at 37°C in a 5% CO 2 incubator. For proliferation and cell cycle studies, cells were resuspended at a density of 2 ϫ 10 5 cells/ml in serum-free media containing insulin (5 mg/liter) and transferrin (5 mg/liter) and sodium selenite (5 g/liter) for 2-4 h. Cells were then treated with the indicated compounds. Ethanol concentration was always maintained at less than 0.1%. Cell proliferation was determined by counting cells using a hemacytometer, cell viability was evaluated by trypan blue dye exclusion, and cell cycle analysis was determined by propidium iodide flow cytometry as described (37).
Synthesis and Labeling of D-e-MAPP and L-e-MAPP-D-and L-MAPP were prepared by acylation of D-and L-erythro-2-amino-1-phenyl-1-propanol with myristoyl chloride as described (30 (38). A crude product was purified via the flash chromatography method (EM-Science silica gel; 40 -63 m) using a methylene chloride/methanol system with increasing polarity from 100:0 to 96:4 and were crystallized from ethanol. The purity of the obtained compound was assessed by TLC analysis using Merck precoated Silica Gel 60 F-254 plates and methylene chloride/methanol, 93:7, or chloroform/methanol, 4:1, solvent systems to develop plates. TLC spot detection was by iodine vapor and 5% potassium permanganate in 1 N potassium hydroxide. Structure of nonradioactive amides was verified by proton-NMR ( 1 H NMR), mass spectroscopy (MS), and CD spectra. Specific activity of the obtained N-[ 3 H]acyl derivatives was ϳ3-4 ϫ 10 4 dpm/nmol, and the purity was close to 100%.
Uptake and Metabolism Studies--HL-60 cells were grown as described and treated with 3 H-labeled compounds at concentrations ranging from 0 to 10 M. At the indicated time points, cell pellets were separated from media and 3 H radioactivity was counted.
Metabolism Studies-Lipids from cells treated with 3 H-labeled compounds were extracted (Bligh & Dyer method), dried, resuspended in chloroform/methanol (10:1, v/v), and subjected to separation by TLC in chloroform/methanol (4:1, v/v). Spots containing radioactive compounds were scraped and radioactivity was measured using a scintillation counter.
Measurement of Ceramidase Activity in HL-60 Cells-Ceramidase activity in HL-60 cells was measured by a modification of the method of Gatt and Yavin (39). Cells were disrupted by sonication in 0.25 M sucrose, 1 mM EDTA, and centrifuged at 10,000 ϫ g for 10 min after which the supernatant was centrifuged at 100,000 ϫ g for 60 min. Ten l of 1 mM ceramide substrate ([ 3 H]C 16 -ceramide) was mixed with 100 l of Triton X-100 (0.1%) in chloroform/methanol, 2:1, and 100 l of 0.2% sodium cholate in chloroform/methanol, 2:1, after which the solvent was evaporated under N 2 at 70°C. To this mixture, 30 l of water was added and the tubes were heated for 5 s in an 80°C water bath and then put on ice. Fifty l of the appropriate buffer depending on desired pH was added followed by 20 l of 50 mM MgCl 2 and 100 l of cell extract. In experiments using inhibitors, the desired inhibitor was added in ethanol with either the substrate or directly to the final solution. The reaction mixture was incubated for 1 h at 37°C. The 3 H-fatty acid products of ceramidase were separated by the addition of 2 ml of Dole's solution (isopropyl alcohol, heptane, NaOH), 1.2 ml of heptane, and 1 ml of water. After vortexing and centrifugation, the upper phase was discarded and the lower phase was washed twice with heptane and the upper phase discarded each time. Finally, 1 ml of 1 N H 2 SO 4 , and 2 ml of heptane were added, and after vortexing and centrifugation the upper phase was transferred for counting by liquid scintillation.
Glucocerebrosidase and Glucocerebroside Synthase Assays-In vitro assays for glucocerebrosidase and for cerebroside synthase were performed as described (23).
CAPP-The activity of CAPP was determined as described previously (40) using myelin basic protein (phosphorylated by protein kinase A) as a substrate.

Cellular Activity of D-and L-Erythro-MAPP-
In structurefunction analysis of ceramide-mediated growth suppression using amides of phenylaminoalcohols, we employed D-and Lerythro-MAPP. In these studies, HL-60 cells were treated with 5 M of either D-e-MAPP or L-e-MAPP, and growth was determined at the indicated time points ( Fig. 2A). D-erythro-MAPP produced a time dependent suppression of growth that was predominantly characterized by a cytostatic effect on proliferation. On the other hand, L-erythro-MAPP was largely inactive ( Fig. 2A).
In order to evaluate if D-erythro-MAPP modulated cell cycle progression, HL-60 cells were treated with 5 M D-e-MAPP, and cell cycle analysis was performed at 48 h using propidium iodide flow cytometry. These studies showed that D-erythro-MAPP produced a significant increase in the population of cells in the G 0 /G 1 phase of the cell cycle (from 60 to 73%) with a corresponding drop in the population of cells in S (from 30 to 24%) and G 2 /M (from 10 to 3%) phases of the cell cycle (Fig. 2B). On the other hand, 5 M L-e-MAPP was largely without effect (Fig. 2B). These studies show that a predominant effect of D-e-MAPP is the induction of a G 0 /G 1 arrest in cell cycle progression resulting in growth suppression.
Effects on CAPP-Since D-e-MAPP mimicked the cellular activities of ceramide, we investigated if D-e-MAPP mimicked the in vitro activity of ceramides. We have shown that in vitro ceramides of varying N-linked chain lengths activate a serine/ threonine protein phosphatase (CAPP), with a specificity matching the specificity of the cellular activities of ceramides (41). CAPP appears to belong to the heterotrimeric subfamily of the PP2A family of protein phosphatases (20). Addition of C 6ceramide to CAPP in vitro resulted in a concentration-dependent stimulation of activity (Fig. 3). On the other hand, neither D-e-MAPP nor L-e-MAPP activated CAPP in vitro (Fig. 3), raising the possibility that D-e-MAPP may not function as a direct analog of ceramide.
Effects of D-and L-MAPP on Endogenous Levels of Ceramide-Since D-erythro-MAPP, the bioactive enantiomer, corresponds in absolute configuration to L-erythro-ceramide (the unnatural enantiomer of ceramide (Fig. 1), and since it lacks in vitro activity with CAPP, we wondered whether D-e-MAPP mimicked ceramide biology by modulating endogenous ceram-ide levels. Therefore, HL-60 cells were treated with 5 M D-e-MAPP, ethanol vehicle, or L-e-MAPP for 0 -24 h. At the indicated time points (Fig. 4A), total lipids were extracted and endogenous ceramide levels were measured by the Escherichia coli diacylglycerol kinase assay. In control cells, there were no changes in endogenous ceramide levels, and in cells treated with L-e-MAPP there were very modest changes (ϳ20%) in ceramide levels; especially at the early time points (Fig. 4A). However, the addition of D-e-MAPP resulted in a time dependent accumulation of endogenous levels of ceramide reaching approximately 3-fold of baseline by the 24-h time point. These results demonstrate that, as compared to L-e-MAPP, D-e-MAPP selectively regulates endogenous ceramide levels.
The results from the diacylglycerol kinase assay disclosed that D-e-MAPP selectively modulated the levels mostly of one of two major species of ceramide phosphate (the product of ceramide transformation by the diacylglycerol kinase assay) as detected by TLC (Fig. 4B). The upper spot of ceramide phosphate co-migrated with synthetic D-erythro-C 18 dihydroceramide phosphate whereas the lower spot co-migrated with Derythro-C 18 ceramide phosphate. Fig. 4C shows that the addition of D-e-MAPP resulted in selective enhancement in the levels of the lower spot of ceramide phosphate. Based on this co-migration, the results suggest that D-e-MAPP causes a selective increase in ceramide but not dihydroceramide levels. It should be noted, however, that the structure of these species of ceramide phosphate has not been determined conclusively.
Uptake and Metabolism of D-and L-MAPP-In order to explore further the reasons for the discrepancies in the activities of D-and L-MAPP and to gain insight into the possible enzymes involved in modulating ceramide levels in response to D-e-MAPP, studies were conducted examining the uptake and me-tabolism of D-and L-MAPP. In the first set of studies, the effect of duration of exposure of HL-60 cells to D-e-MAPP on the bioactivity of the molecule was determined. D-e-MAPP added to cells could be washed off by repeated sedimentation and washing of cells. Thus, after five cycles of washing, less than 10% of D-MAPP remained associated with the cell pellet compared to unwashed cells (Fig. 5A). The continuous exposure of HL-60 cells to D-MAPP resulted in dramatic growth inhibition (Fig.  5B) as compared to vehicle-treated cells. Exposure of HL-60 cells to D-MAPP for only 15 min did not affect cell growth, whereas exposure for progressively longer intervals resulted in increased growth suppression (Fig. 5B). These results show that the ability of D-e-MAPP to inhibit growth requires prolonged exposure of cells to this compound and suggest that the effects of D-e-MAPP require continuous interactions with intracellular targets (such as ceramide-metabolizing enzymes).
Next, the uptake of tritium-labeled D-and L-e-MAPP was examined. To this end, D-and L-e-MAPP labeled in the Nmyristoyl group were synthesized and used for uptake and metabolism studies. The addition of 5 or 30 M of either D-or L-e-MAPP resulted in a dose-and time-dependent increase in uptake of these molecules (Fig. 5C). At 24 h, approximately 20% of D-e-MAPP (5 M) and 26% of L-e-MAPP (5 M) were taken up by cells.
The partitioning of the label taken up by cells into the lipid organic phase was evaluated next. It was found that the majority of the label in D-e-MAPP or L-e-MAPP remained associated with the organic phase over a 0 -72-h duration at concentrations ranging from 5 to 30 M (data not shown).
Next, the metabolism of D-and L-e-MAPP was evaluated. To this end, organic lipids were extracted from cells treated with 5 M D-or L-e-MAPP at the indicated time points (Fig. 5D), and lipids were resolved on TLC. D-e-MAPP showed little metabolism and remained intact throughout the duration of the experiment (Fig. 5, D and E). On the other hand, L-e-MAPP underwent significant metabolism in a time-dependent fashion such that by 24 h, more than 70% of the L-e-MAPP taken up by cells was metabolized (Fig. 5, D and E). Two major breakdown products of L-e-MAPP were resolved by TLC. One metabolite co-migrated with myristoyl-CoA and not with other neutral or polar lipids. The other metabolite (upper spot in Fig. 5D) comigrated with triacylglycerol. These results are of 2-fold significance. First, they suggest that the major reason for the lack of activity of L-e-MAPP may be due to its relatively rapid metabolism. Second, they show that L-e-MAPP is a substrate for endogenous ceramidases that are capable of releasing the Nlinked myristate. On the other hand, D-e-MAPP does not appear to be a substrate for this enzyme.
Effects of D-and L-e-MAPP on Ceramide-metabolizing Enzymes-Since the above results show that L-e-MAPP is a potential substrate for ceramidase, whereas D-e-MAPP is not and since D-e-MAPP caused a time-dependent accumulation of endogenous ceramide levels, we suspected that D-e-MAPP may serve as an inhibitor of this enzyme. Therefore, in order to evaluate the interactions of D-and L-e-MAPP with ceramidase, we conducted in vitro studies using cytosolic and membrane fractions from HL-60 cells. Initially, ceramidase activity was examined for pH dependence using tritium-labeled C 16 -ceramide as substrate. Ceramidase activity was found to exist predominantly in the membrane fraction at two separate pH optima of 4.5 and 9.0 (Fig. 6A). The alkaline ceramidase activity was very similar to that described in fibroblasts and cerebellar tissue (42,43). In initial studies, the ability of this ceramidase to utilize D-e-and L-e-MAPP as substrates was investigated. D-e-MAPP was not a substrate for the alkaline ceramidase (Fig.  6B). In contrast, L-e-MAPP was readily hydrolyzed by cerami- dase to a level equivalent to that seen with C 16 -ceramide as a substrate (Fig. 6B). These studies corroborate the cellular metabolism studies showing that L-e-MAPP is a substrate for ceramidases in vitro and in cells.
Next, the ability of D-and L-e-MAPP to interfere with hydrolysis of C 16 -ceramide by ceramidase at pH 4.5 and 9.0 was evaluated. Inhibition of ceramidase activity by D-e-MAPP was compared to that by N-oleoylethanolamine, a putative inhibitor of acid ceramidase (42). N-Oleoylethanolamine was found to be a better inhibitor of acid ceramidase (IC 50 approximately 500 M) than D-e-MAPP (Fig. 6C). The effects of N-oleoylethanolamine are consistent with the previously reported K i of 700 M (42). D-e-MAPP demonstrated much greater inhibition of alkaline ceramidase with an IC 50 of 1-5 M (Fig. 6C). D-e-MAPP displayed an apparent in vitro K i for alkaline ceramidase of 2-13 ϫ 10 6 M. At a final concentration of 5 M, D-e-MAPP inhibited alkaline ceramidase in vitro by approximately 60% whereas N-oleoylethanolamine displayed less than 10% inhibition of alkaline ceramidase in vitro at final concentrations up to 50 M (Fig. 6D). In contrast, D-e-MAPP was a relatively poor inhibitor of acid ceramidase, with less than 10% inhibition of acid ceramidase at 50 M (Fig. 6C). These results demonstrate that D-e-MAPP is a much superior inhibitor of alkaline ceramides than N-oleoylethanolamine and displays specificity for in vitro inhibition of alkaline ceramidase over acid ceramidase. These results are consistent with the use of 0.5 mM N-oleoyleth-anolamine to inhibit mitogenesis (44) which is 100-fold higher than the concentration of D-e-MAPP used to inhibit cell growth in Fig. 2.
In addition to ceramidase, inhibition of cerebroside synthase and sphingomyelin synthase or stimulation of sphingomyelinase or cerebrosidase could result in elevations in endogenous ceramide levels. Therefore, the effects of D-and L-e-MAPP were examined on these enzyme activities in vitro and in cells. Neither D-nor L-e-MAPP caused inhibition of cerebroside synthase activity (Table I). As a control, PMMP, a previously established inhibitor of cerebroside synthase, induced significant inhibition of this enzymatic activity (Table I). Also, D-and L-e-MAPP did not activate or modulate the activity of ␤-glucosidase (data not shown). In addition, neither D-nor L-e-MAPP modulated the endogenous levels of sphingomyelin (data not shown) arguing against an effect of either of these molecules on sphingomyelinase or sphingomyelin synthase. In vitro, neither D-nor L-e-MAPP modulated the activity of neutral or acidic sphingomyelinases (data not shown). Finally, fumonisin B1, an inhibitor of ceramide synthase (45), did not inhibit the effects of D-e-MAPP on growth (Fig. 7).  (Fig. 8A). The effects of D-e-MAPP on metabolism of L-e-MAPP were dose dependent with most of the inhibition occurring at levels of D-e-MAPP of 1-3 M (Fig. 8B). Therefore, these studies demonstrate that D-e-MAPP inhibits the metabolism of L-e-MAPP in cells; through inhibition of the involved ceramidase.

DISCUSSION
The results from this study demonstrate that D-e-MAPP is an inhibitor of alkaline ceramidase both in vitro and in cells. D-e-MAPP underwent time-and concentration-dependent uptake by cells, but the compound was very poorly metabolized. This was accompanied by a time-and concentration-dependent elevation in endogenous levels of ceramide. In vitro, D-e-MAPP had little effect on other ceramide metabolizing enzymes including sphingomyelinase and glucocerebroside synthase, and it was a very poor inhibitor of acid ceramidase. The activity of D-e-MAPP against alkaline ceramidase was at least 100-fold more potent than oleoylethanolamine, a previously determined inhibitor of the acid ceramidase. In cells, D-e-MAPP did not modulate the levels of sphingomyelin. Furthermore, the effects of D-e-MAPP on growth were not modulated by fumonisin B1, an inhibitor of ceramide synthase; suggesting that D-e-MAPP does not activate this enzyme in cells. In contradistinction, L-e-MAPP, the enantiomer of D-e-MAPP, which demonstrated similar uptake, underwent time-dependent degradation in cells resulting in the release of the N-linked myristate as detected by the formation of myristoyl-CoA. The enzyme responsible for this activity is most probably ceramidase as suggested by the significant metabolism of L-e-MAPP in vitro by alkaline cerami- dase. These results demonstrate that D-e-MAPP acts as an indirect analog of ceramide through modulation of endogenous ceramide levels. This is further supported by the lack of activity of D-e-MAPP on ceramide-activated protein phosphatase.
These studies provide the first clues as to the possible cellular and biochemical function of alkaline ceramidase. Two ceramidase have been described in the literature. The better known is an acid ceramidase that has a predominantly lysosomal localization (46). The deficiency in the activity of this acid ceramidase results in Farber's disease, a lysosomal sphingolipidoses characterized by accumulation of ceramide and possibly other sphingolipids in lysosomes (47). In addition, alkaline ceramidases with broad pH optima have been described in several tissues including kidney, brain, liver, and epidermis (28,29,42,43). The acid ceramidase appears to function in the biodegradative pathways of sphingolipid metabolism; probably in the process of recycling membranes and membrane lipids through the endolysosomal pathway. On the other hand, the role and significance of alkaline ceramidase has been poorly studied. The current study demonstrates the capability of regulating endogenous ceramide levels through modulation of alkaline ceramidase activity; with significant biologic conse-quences that mimic those obtained with exogenous ceramides.
These results, coupled to the emerging role of ceramide as an important bioeffector molecule, are beginning to provide a perspective on possible functions of non-acid ceramide-metabolizing enzymes. Neutral sphingomyelinases have been implicated in the hydrolysis of the specific pool of sphingomyelin (48) resulting in the formation of ceramide in response to a number of extracellular agents and agonists. The fate of this generated ceramide has not been investigated thoroughly. Since continuous and progressive accumulation of ceramide has been correlated with the onset of apoptosis and other growth suppresser activities (8,19,38), the need must arise for further metabolism of ceramide in order to regulate ceramide responses. In this context, the operation of alkaline ceramidases may play important roles in attenuating ceramide signals. The alkaline ceramidase has in addition the capability of regulating the non-lysosomal levels of sphingosine. Thus, activation of this ceramidase will result in attenuation of ceramide levels and possibly an increase in sphingosine levels. Reciprocally, inhibition of this enzyme will result in accumulation of ceramide levels as shown in this study with the possible concomitant decrease in sphingosine levels.
The selective modulation of D-e-ceramide levels by D-e-MAPP when compared to D-e-dihydroceramide (Fig. 4C) may be of particular significance. Since analogs of D-e-ceramide but not of D-e-dihydroceramide are biologically active, this selectivity may imply a special role of alkaline ceramidase in cell regulation. The mechanism of this selectivity has not been defined. It may result from either substrate selectivity or from co-localization of the enzyme with ceramide (and not dihydroceramide). These possibilities are currently under investigation.
The current results also suggest that D-e-MAPP may emerge as a useful inhibitor of alkaline ceramidase. This inhibitor demonstrates stereospecificity of action such that its enantiomer (L-e-MAPP) does not inhibit the enzyme in vitro or in cells. This specificity should allow better evaluation of what functions of D-e-MAPP are attributable to inhibition of ceramidase. The major usefulness of ceramidase inhibitors will derive from studies that examine different approaches aimed at modulating endogenous levels of ceramide. As shown in Fig. 9, ceramide plays a central role in sphingolipid biosynthesis and degradation (1,49). Currently, a number of pharmacologic approaches are available to modulate endogenous ceramide levels. For example, D-erythro-sphingosine serves as a specific precursor to ceramide. Thus, the addition of sphingosine results in substantial elevations in ceramide levels (49,50). This is also accompanied in some cell systems by the formation of sphingosine phosphate (51)(52)(53)(54). However, the usefulness of sphingosine as a pharmacologic agent is hampered somewhat by its multiple cellular targets of action including protein kinase C (55,56). However, only D-erythro-sphingosine appears to function as a substrate for ceramide synthase (49) whereas inhibition of protein kinase C, and most other cellular targets identified so far, does not seem to disclose stereospecific action (36). Therefore, activities that are specific to D-erythro-sphingosine raise the possibility that they may be mediated through incorporation of D-erythro-sphingosine into ceramide. In addition, fumonisin B1 is an inhibitor of ceramide synthase (45). This agent is useful in evaluating pathways resulting in de novo ceramide formation. The inhibitor can also be used to distinguish those effects of sphingosine that are mediated through the direct formation of ceramide from sphingosine since those effects would be anticipated to be inhibitable by fumonisin B1. A point of caution with fumonisin B1 derives from the fact that this compound will eventually inhibit not only ceramide formation but all complex sphingolipids. Therefore, prolonged use of this inhibitor could result in attenuating ceramide levels secondarily to attenuation of other sphingolipids. Finally, PDMP and PMMP have emerged as important and biologically active specific inhibitors of glucocerebroside synthase. These compounds inhibit glucocerebroside synthase activity in vitro and in cells resulting in attenuation of glycosphingolipid levels and significant increases in the levels of ceramide (57). In cells, these molecules have demonstrated a wide range of cellular activities including growth suppression (57), cell cycle arrest (58), inhibition of neuronal differentiation (59), modulation of adhesion receptors (60), and inhibition of experimental metastasis (61). Although it has been difficult to determine whether these effects are a consequence of decreased glycosphingolipids or increased ceramide, many of these effects appear to be shared by ceramide itself as well as by extracellular agents that activate sphingomyelinases and cause the accumulation of ceramide. Therefore, multiple approaches already exist that allow modulation of intracellular levels of ceramide. While none of these approaches is foolproof, used judiciously and in the aggregate, they can provide significant insight into the consequences of changes in endogenous ceramide and sphingolipid levels.
The availability of D-e-MAPP as inhibitors of ceramidase should provide an important tool in distinguishing the effects of endogenous ceramide from those of sphingosine. In many cell systems, sphingosine and ceramide induce similar activities such as growth suppression, and induction of Rb dephosphorylation (37,62), whereas in other situations, sphingosine may have effects opposite to those of ceramide such as activation of phospholipase D (63,64) which is inhibited by ceramide (65)(66)(67)(68)(69). Therefore, a major question with many of the extracellular agents that induce changes in ceramide levels relates to whether these are possibly due to further degradation of ceramide into sphingosine (and possibly to subsequent metabolites such as sphingosine 1-phosphate). Availability of ceramidase inhibitors should allow further examination of these questions. In addition, determination if the activities of exogenously added ceramide analogs is due to possible further metabolism to sphingosine can now be assessed with D-e-MAPP. Effects due to ceramides themselves or other metabolites not a result of further degradation to sphingosine should not be inhibited by D-e-MAPP but actually should be expected to be enhanced by D-e-MAPP. On the other hand, effects occurring as a result of further metabolism to sphingosine should be prevented by the action of D-e-MAPP.
Finally, the availability of ceramidase inhibitors should provide important tools for the study of alkaline ceramidases in vitro and in cells. The availability now of stereospecific inhibitors (D-e-MAPP) and stereospecific substrates (L-e-MAPP) for ceramidase should provide a significant tool for evaluating the in vitro biochemical mechanism of action of ceramidase. Also, D-e-MAPP, which is active in cells, should be a valuable tool in studying regulation of alkaline ceramidase as discussed above.
In conclusion, this study defines a specific inhibitor of ceramidase in vitro and in cells, and provides important insight into possible physiologic significance of regulation of ceramidases in cells.