(Received for publication, September 18, 1995; and in revised form, February 7, 1996)
From the
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.
Ceramide is emerging as an important bioeffector lipid molecule (1, 2, 3, 4, 5) . The
action of a number of extracellular agents as well as stress stimuli
such as 1,25-dihydroxyvitamin D
, tumor 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, 7, 8, 9, 10, 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-ceramide (
)that this molecule demonstrates structural and
stereospecific cellular activities(17, 18) . The most
significant specificity was demonstrated with the lack of activity of
C
-dihydroceramide (17, 19) which differs
from C
-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
-ceramide but not
C
-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, 25, 26, 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 in the formation of sphingosine and free fatty acids(1, 28, 29) .
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.
Figure 1: Structures of D-e-ceramide, D-e-MAPP, and L-e-MAPP. The structure of D-erythro-ceramide is shown vertically-inverted to allow ease of comparison with the configuration of the polar head groups of D- and L-e-MAPP. Also shown are the Fisher projections for D-e-ceramide and L-e-MAPP. In absolute configuration, D-e-ceramide corresponds to L-e-MAPP whereas D-e-MAPP has the enantiomeric configuration.
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.
Figure 2: Effects of D- and L-e-MAPP on cell growth. HL-60 cells were treated with 5 µM of either D-e-MAPP or L-e-MAPP and growth was monitored over the indicated time range (A). The effects of 5 µMD- and L-e-MAPP on cell cycle progression was determined at 48 h (B).
In order to evaluate if D-erythro-MAPP modulated cell cycle progression,
HL-60 cells were treated with 5 µMD-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/G
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
/M (from 10
to 3%) phases of the cell cycle (Fig. 2B). On the other
hand, 5 µML-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
/G
arrest in cell cycle progression resulting
in growth suppression.
Figure 3:
Effects of D- and L-e-MAPP on CAPP. The serine/threonine phosphatase
activity of CAPP was determined using as substrate myelin basic protein
phosphorylated by protein kinase A. , C
-ceramide;
, D-MAPP;
, L-MAPP.
Figure 4:
Effects of D- and L-e-MAPP on endogenous levels of ceramide. HL-60 cells were
treated with 5 µMD- or L-e-MAPP, and lipids were extracted for ceramide
measurements at the indicated time points. A, the effects of D- and L-e-MAPP on total ceramide levels
standardized to total levels of lipid phosphate as described under
``Experimental Procedures.'' , control;
, D-MAPP;
, L-MAPP. B, TLC separation
of diacylglycerol kinase products of synthetic D-erythro-C
-ceramide, D-erythro-C
-dihydroceramide, and
ceramide from bovine brain sphingomyelin. C, relative changes
in the levels of ceramide and dihydroceramide following the addition of D-e-MAPP to cells. Ceramide and dihydroceramide were
quantitated as the respective phosphates.
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 dihydroceramide phosphate whereas the lower spot co-migrated with D-erythro-C
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.
Figure 5:
Uptake and metabolism of D- and L-MAPP. HL-60 cells were treated with tritium-labeled D- and L-e-MAPP as described under
``Experimental Procedures.'' A, back-extraction of D-e-MAPP taken up by cells. HL-60 cells were
incubated with 5 µM tritium-labeled D-e-MAPP, and radioactivity associated with cells was
determined before (no wash) or following the indicated number of
washes. B, effect of duration of exposure of HL-60 cells to D-e-MAPP on growth suppression. HL-60 cells were
treated with 5 µMD-e-MAPP which was
back-extracted with 5 washes at the indicated time points. C,
total uptake of D-e-MAPP and L-e-MAPP. HL-60 cells were treated with the indicated
concentrations of tritium-labeled D- and L-e-MAPP, and total radioactivity associated with the
cell pellet was determined. , 5 µMD-MAPP;
&cjs2100;, 30 µMD-MAPP; &cjs2108;, 5 µML-MAPP; &cjs2113;, 30 µML-MAPP. D, TLC separation of intact tritium-labeled D- and L-e-MAPP and their metabolites. Cells were treated
with 5 µMD- or L-e-MAPP, and
lipids were extracted at the 24-h time point and resolved on TLC as
described under ``Experimental Procedures.'' E, time
dependence of metabolism of D- and L-e-MAPP.
HL-60 cells were treated with 5 µMD- or L-e-MAPP. Intact compounds and their metabolites were
resolved by TLC, and radioactivity associated with each spot was
determined by scintillation counting of the excised spots.
, D-MAPP;
, L-MAPP;
, L-MAPP (upper
spot);
, myristoyl-CoA.
Next, the uptake of tritium-labeled D- and L-e-MAPP was examined. To this end, D- and L-e-MAPP labeled in the N-myristoyl group were synthesized and used for uptake and metabolism studies. The addition of 5 or 30 µM 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 µM) and 26% of L-e-MAPP (5 µM) 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 µM (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 µMD- 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 N-linked myristate. On the other hand, D-e-MAPP does not appear to be a substrate for this enzyme.
Figure 6:
Effects of D- and L-e-MAPP on alkaline and acid ceramidase activity in vitro. Ceramidase activity was determined from total
homogenates of HL-60 cells as described under ``Experimental
Procedures.'' A, pH dependence of ceramidase activity
from HL-60 cells. Ceramidase activity using tritium-labeled
C-ceramide was measured in vitro using sodium
acetate, HEPES, and CHES buffers over a pH range of 4.5 to 9.0. B, substrate preference of ceramidase in vitro.
Tritium-labeled C
-ceramide, D-e-MAPP, or L-e-MAPP were used as substrates in an in vitro ceramidase assay at 50 µM concentrations as described
under ``Experimental Procedures.'' Activity was determined by
measuring release of the tritium-labeled acyl chains. C,
inhibition of acid ceramidase by D-e-MAPP and N-oleoylethanolamine. Ceramidase activity was evaluated in
vitro at pH 4.5 using 50 µM C
ceramide
as a substrate in the presence of increasing concentrations of D-e-MAPP or N-oleoylethanolamine. Data are
presented as mean activity normalized to control activity in samples
treated with vehicle alone. D, inhibition of alkaline
ceramidase by D-e-MAPP and N-oleoylethanolamine. Ceramidase activity was evaluated in
vitro at pH 9.0 using 50 µM C
ceramide
as a substrate in the presence of increasing concentrations of D-e-MAPP (D-MAPP) or N-oleoylethanolamine (NOE). Data are presented as
mean activity normalized to control activity in samples treated with
vehicle alone.
Next,
the ability of D- and L-e-MAPP to interfere
with hydrolysis of C-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
approximately
500 µM) than D-e-MAPP (Fig. 6C). The effects of N-oleoylethanolamine
are consistent with the previously reported K
of
700 µM(42) . D-e-MAPP
demonstrated much greater inhibition of alkaline ceramidase with an
IC
of 1-5 µM (Fig. 6C). D-e-MAPP displayed an apparent in vitro
K
for alkaline ceramidase of 2-13
10
µM. At a final concentration of 5
µM, 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
µM (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
µM (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
mMN-oleoylethanolamine 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 1). As a control, PMMP, a previously established
inhibitor of cerebroside synthase, induced significant inhibition of
this enzymatic activity (Table 1). 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).
Figure 7: Effects of fumonisin B1 on growth suppression by D-e-MAPP. HL-60 cells were treated with either vehicle or 3 µMD-MAPP in the presence of the indicated concentrations of fumonisin B1. Cell growth was determined at 48 h.
Figure 8: Interaction of D- and L-e-MAPP in cells. A, the effects of D-e-MAPP on cellular metabolism of L-e-MAPP. HL-60 cells were treated with 3 µM tritium-labeled L-e-MAPP with or without 3 µMD-e-MAPP. Metabolism of L-MAPP was determined as described under ``Experimental Procedures.'' B, concentration dependence of inhibition of L-e-MAPP metabolism in cells. The upper spot on TLC reflects a major metabolite of L-MAPP that probably represents triacylglycerol.
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 ceramidase. 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 consequences 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.
Figure 9: Scheme of ceramide metabolism and known inhibitors. Ceramide can be interconverted to sphingomyelin, cerebroside, or sphingosine through the action of at least 6 different enzymatic activities: 1) sphingomyelin synthase; 2) sphingomyelinase; 3) cerebroside synthase; 4) cerebrosidase; 5) ceramidase; 6) ceramide synthase.
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.