Acid SMase Research Articles

Role of sphingomyelin-MAPKs pathway in heat-induced apoptosis.

The role of sphingomyelinase (SMase) activation and mitogen activated protein kinases (MAPKs) activation in cellular apoptosis was investigated during the hyperthermic treatment of HL-60 human leukemia cells. Treating the cells for 1 h at 43(o)C caused more than 50% of cellular apoptosis within several hours. The neutral-SMase activity in the cells treated for 1 h at 42(o)C was slightly increased but decreased in the cells treated at 43(o)C or 44(o)C for the same period whereas the acid SMase activity was slightly increased after heating the cells at 42(o)C and 43(o)C and markedly increased at 44(o)C for 1 h. Treatment of cells with inhibitors of SMase activation and ceramide formation significantly reduced the heat-induced apoptosis. Three major families of mitogen-activated protein kinases (MAPKs), i.e. ERK1/2, p38 and JNK, were activated by the hyperthermic treatment of cells. Inhibition of ERK1/2 with PD98059 exerted little effect on the heat-induced apoptosis and p38 inhibition with SB203580 slightly lessened apoptosis whereas, inhibition of JNK with SP600125 markedly suppressed the heat-induced apoptosis. These results indicate that heat-shock induced the activation of SMase, particularly acid-SMase, thereby causing apoptosis and that JNK played a pivotal role in heat-induced apoptosis in HL-60 leukemia cells.

Development of intestinal alkaline sphingomyelinase in rat fetus and newborn rat.

Sphingomyelin metabolism is a novel signal transduction pathway related to cell differentiation, proliferation, and apoptosis. Alkaline sphingomyelinase (alk-SMase) is specifically present in the intestinal tract of many species. The enzyme is important in digestion of dietary sphingomyelin. Milk is the only exogenous source of sphingomyelin for an infant, and digestion of milk sphingomyelin may be important for development of intestinal mucosa. It is unknown whether alk-SMase is present before birth and whether it changes after birth and during the suckling period. We studied activities, expression, and distribution of alk-SMase in rat fetus and newborn. The changes of acid and neutral SMase as well as alkaline phosphatase were analyzed for comparison. Little activity of alk-SMase was identified up to gestation day 20, but increased 10 times during the following 2 d. After birth, the activity continued to increase during the following 4 wk. Western blot using IgY antibody against rat alk-SMase failed to identify the enzyme at gestation day 20 but clearly showed the protein at day 22. The distribution pattern of the enzyme along the intestinal tract in fetus was largely the same as in adult animals, but became more pronounced after birth. Short-term weaning had no effect on alk-SMase activity. The activities of acid and neutral SMase were high at gestation day 20 and decreased significantly before birth. The changes of alk-SMase also differed from those of alkaline phosphatase, another brush border enzyme. Thus, we conclude that alk-SMase is rapidly expressed during the last days of gestation and that the newborn rat acquires the ability to digest milk sphingomyelin early in life.

Ceramide, a putative second messenger for nerve growth factor, modulates the TTX-resistant Na(+) current and delayed rectifier K(+) current in rat sensory neurons.

Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin-sensitive adult and embryonic sensory neurons. Using the whole-cell patch-clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml(-1) NGF or 1 microM N-acetyl sphingosine (C2-ceramide) produced a 3- to 4-fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time-dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF-induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX-resistant sodium current (TTX-R I(Na)) by approximately 1.5-fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (I(K)) by approximately 35 %. Ceramide reduced I(K) in a concentration-dependent manner. Isolation of the NGF- and ceramide-sensitive currents indicates that they were delayed rectifier types of I(K). The inflammatory prostaglandin, PGE(2), produced an additional suppression of I(K) after exposure to ceramide (approximately 35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro-inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF-induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.

Inhibition of Tumor Necrosis Factor-induced Cell Death in MCF7 by a Novel Inhibitor of Neutral Sphingomyelinase

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC(50) of 1 microm. It did not inhibit acid SMase at up to at least 150 microm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 microm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 microm of the compound was protected completely from the loss of SM. The addition of 10-20 microm GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 microm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 microm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-kappaB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

Effects of red meat and fiber in high fat diet on activities of sphingomyelinase, ceramidase and caspase-3 in rat colonic mucosa

Red meat and fiber rich foods are the dietary factors most consistently related to colon carcinogenesis. Although several components in these dietary sources may contribute, the biochemical mechanism by which red meat and fiber affect colorectal carcinogenesis has not yet been established. Sphingomyelin metabolism is a novel signal transduction pathway that may have an impact on colonic tumorigenesis. The present study investigated the activity changes of sphingomyelinase (SMase), ceramidase and caspase-3 in colonic mucosa of rats fed on a high fat control diet, the control diet with beef and the control diet with fiber (cellulose). After a three week feeding period the colonic mucosa were scraped and homogenized and enzyme activities were determined. The fiber diet significantly increased the activities of neutral and acid SMases but had no effect on those of alkaline SMase and neutral ceramidase. The beef diet, on the other hand, significantly reduced neutral ceramidase activity, but had no effect on the activities of any SMase. In addition, the beef diet significantly reduced and the fiber diet increased caspase-3 activity in the colonic mucosa when compared with the control diet. The changes of caspase-3 activities were abolished by preincubating the samples with caspase-3 inhibitor. No significant changes of intestinal alkaline phosphatase could be found among the three dietary groups. In conclusion, fiber and red meat in the high fat diet affected in an opposite way the enzymes responsible for sphingomyelin metabolism and apoptosis in the colon. The effects may have implications in colorectal tumorigenesis.

Divergent role of ceramide generated by exogenous sphingomyelinases on NF-κB activation and apoptosis in human colon HT-29 cells

This study examined the role of ceramide generated by exogenous sphingomyelinases (SMases) on transcription nuclear factor-κB (NF-κB) activation and apoptosis in human colon epithelial HT-29 cells. Exogenous neutral (N) and acidic (A) SMase activated NF-κB with different kinetics, accounting for the diverse pattern of DNA binding of NF-κB complexes activated by tumor necrosis factor-α (TNF). NSMase activated predominantly RelA/p52 and RelA/p50 dimers within 30 min, while ASMase activated the p50/p50 homodimer by 20 h. The predominant activation of RelA-containing κB complexes by TNF or NSMase paralleled the induction of interleukin-8. HT-29 cells were sensitive to ASMase and TNF but resistant to NSMase. However, the apoptotic potential of NSMase was masked by NF-κB, as its prior inactivation sensitized HT-29 cells to NSMase. Thus, the generation of ceramide by exogenous SMases participates differentially in inflammation and apoptosis.

Ultraviolet B radiation induces activation of neutral and acidic sphingomyelinases and ceramide generation in cultured normal human keratinocytes

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces an extracellular signal into the cell. During the past few years increasing evidence has shown that the sphingolipid ceramide may play a role as a second messenger in intracellular signal transduction. The ceramide generation via sphingomyelinase (SMase) is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of this study is to investigate whether activation of SMases and generation of ceramide can be induced by UVB radiation in normal human keratinocytes. The present data show that exposure to UVB radiation results in rapid generation of ceramide. The ceramide accumulation starts 15 min after UV exposure and progressively increases up to 24 h. In vitro measurement of SMase activity following exposure to UVB evidences an activation of both neutral and acidic SMases. Moreover, UVB induces apoptosis in normal human keratinocytes as shown by TUNEL technique and FACS analysis. These data indicate that UVB induced ceramide generation and activation of both neutral and acidic SMases, suggesting that sphingolipids metabolism may be involved in the UVB signaling pathway.

Acid Sphingomyelinase Activation Requires Caspase-8 but Not P53 nor Reactive Oxygen Species during Fas-Induced Apoptosis in Human Glioma Cells

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val138 induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS.

Effects of Fat, Beef and Fiber in Diets on Activities of Sphingomyelinase, Ceramidase and Caspase-3 in Rat Colonic Mucosa

Objective: The present study investigates the activity changes of sphingomyelinase (SMase), ceramidase and caspase-3 in colonic mucosa of rats induced by high fat and red meat diets. Method: For a period of 3 weeks, rats were fed protein without fat (control), high fat only, high fat with beef, and high fat with fiber (cellulose) diets. The fat content (22.4%) was constant in the three high fat diets. Then, the colonic mucosae were scraped and homogenized and the activities of SMase, ceramidase and caspase-3 determined. Results: Compared to the control diet, the fat diet and fat with beef diet reduced intestinal alkaline SMase by 80 and 84%, respectively; ceramidase activity by 60 and 92%, respectively, and caspase-3 activity by 40 and 75%, respectively. The activities of acid and neutral SMases were also decreased by fat and fat with beef diets but to a smaller extent than those of alkaline SMase. Supplement of fiber in the fat diet had no effect on the changes of alkaline SMase activity but prevented fat-induced decreases in acid and neutral SMase activities and partially prevented those of ceramidase and caspase-3 activities. The activity of intestinal alkaline phosphatase was not changed by any of the diets. Conclusion: Fat, beef and fiber significantly affect the enzymes responsible for sphingomyelin metabolism and apoptosis in the colon. The effects may have implications in colonic tumorigenesis related to dietary factors.

Localization of neutral ceramidase in caveolin-enriched light membranes of murine endothelial cells

Sphingomyelinase (SMase) and ceramidase (CDase) activities participate in sphingomyelin (SM) metabolism and have a role in the signal transduction of a variety of ligands. In this study evidence is presented that caveolin-enriched light membranes (CELMs) of murine endothelial cells, characterized by high SM, ceramide (Cer) and cholesterol content, bear acid and neutral SMase as well as neutral CDase activities. Localization of neutral CDase in CELMs was confirmed by Western analysis. Notably, cell treatment with cyclodextrin, which depleted cell cholesterol, did not affect acid or neutral SMase activities but significantly enhanced neutral CDase activity in CELMs, indicating a negative role for cholesterol in CDase regulation. These findings suggest that neutral CDase is implicated, together with SMase activities, in the control of caveolar Cer content that may be critical for caveola dynamics.

Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10-40 micrometer), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20-80 micrometer), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.

Phosphoinositide 3-kinase regulates crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways.

The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75(NTR) neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75(NTR)-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75(NTR). In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75(NTR)-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand-induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis

Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress-induced apoptosis. We now show that apoptosis of human macrophages (MΦ) and fibroblasts initiated by oxidized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6-ceramide treatment by inhibiting transcription via Sp1 and AP-2. Moreover, apoptosis was abolished after mmLDL and C6-ceramide treatment of hereditary aSMase-deficient fibroblasts (from Niemann-Pick patients). We suggest that in mmLDL-initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.—Deigner, H.-P., Claus, R., Bonaterra, G. A., Gehr...

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis.

Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress-induced apoptosis. We now show that apoptosis of human macrophages (MPhi) and fibroblasts initiated by oxidized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6-ceramide treatment by inhibiting transcription via Sp1 and AP-2. Moreover, apoptosis was abolished after mmLDL and C6-ceramide treatment of hereditary aSMase-deficient fibroblasts (from Niemann-Pick patients). We suggest that in mmLDL-initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.

Activation of sphingolipid turnover and chronic generation of ceramide and sphingosine in liver during aging

Aging leads to a decreased ability of liver to metabolize drugs and increased expression and secretion of acute phase proteins, such as serum amyloid A (SAA), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). This phenomenon resembles some aspects of the acute phase response of host to inflammation; however, the molecular basis for the similarity is unclear. Ceramide and sphingosine are second messenger mediators of cellular responses to stress and inflammation. In liver, they play important role in mediating acute phase responses to IL-β. In this study, we use HPLC and thin layer chromatography to evaluate the effects of aging on steady-state level of ceramide and sphingosine. We report that both lipids are elevated in liver of old (24 months) as compared to young (5 months) male Fisher 344 rats. To elucidate the mechanism(s) for ceramide elevation, we test the acidic(ASMase) and neutral sphingomyelinase (NSMase) in vitro using NBD-sphingomyelin as an exogenous substrate. SM synthase is also analyzed in vitro using NBD-ceramide and { 3H}-dipalmitoylphosphatidylcholine (DPPC) as exogenous substrates. In accordance with the increases in the mass of ceramide, the activity of acid and neutral SMase is elevated in old animals. Michaelis-Menten analysis of NSMase implies that the apparent activation of this enzyme is caused by an increase in the V max of the enzyme. In contrast, SM synthase activity is lower in old animals as compared to young ones. These results show that aging is accompanied by an elevation in SM turnover and a decrease in its synthesis, resulting in accumulation of pro-inflammatory and growth inhibitory second messenger ceramide. Ceramidase, the only enzyme leading to sphingosine generation, is also measured in vitro using NBD-ceramide as a substrate and liver homogenate as an enzyme source. Its activity is higher in the old rats, as compared to young ones. The acid and neutral forms of the enzyme are affected the most, while the changes in the alkaline enzyme are not significant. The increases in the basal levels of ceramide and sphingosine in old animals may contribute to the onset of inflammatory like state in liver during aging, exemplified by decrease P4502C11mRNA expression and chronic induction of acute phase protein expression.

Activation of neutral sphingomyelinase participates in ethanol-induced apoptosis in Hep G2 cells.

The mechanism underlying ethanol-induced apoptosis in liver cells is not clear. Sphingomyelin (SM) metabolism is a novel signal transduction pathway that has an impact on apoptosis in many cell types. We investigated whether the SM pathway is involved in ethanol-induced apoptosis in the liver. Hep G2 cells were treated with ethanol followed by assaying apoptosis, sphingomyelinase (SMase) activity, caspase-3 activity, and the changes of SM content in the cells. We found that ethanol dose-dependently increased apoptosis and the effect was accompanied by increases of caspase-3 activity and neutral SMase activity. At concentrations of 80 and 160 mM, ethanol significantly increased caspase-3 activity by 120% and neutral SMase activity by 24%. The activity of acid SMase was only slightly increased without statistical significance. C(2)-ceramide, the exogenous SM metabolite, mimicked the effects of ethanol on apoptosis and caspase-3 activation. When the SM content was determined 24 h after treatment with ethanol, its level was 15% lower than that of controls. The results indicate that metabolism of SM triggered by neutral SMase participates in ethanol-induced apoptosis in Hep G2 cells and activation of caspase-3 is involved in the apoptotic pathway.

The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide

We have demonstrated previously that there is an abnormal expression of sphingomyelin (SM) deacylase in the epidermis of patients with atopic dermatitis (ADe). In the present study, we have prepared N-[palmitic acid-1-(14)C]SM and N-[palmitic acid-1-(14)C]glucosylceramide (GCer) to use as substrates and have quantified SM deacylase activity by detecting the release of [(14)C]palmitic acid in extracts of the stratum corneum or the epidermis of ADe patients. In studies using [palmitic acid-1-(14)C]SM as a substrate, a pH dependency of catalytic activity with a peak at pH 5.0 was found. Preparative SDS/PAGE using an extract of ADe epidermis revealed that the molecular mass of SM deacylase is 40000 Da, which is consistent with its apparent molecular mass of 42000 Da estimated by gel-filtration analysis of stratum corneum extracts. Analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, beta-glucocerebrosidase (GlcCDase), sphingomyelinase (SMase) and acid ceramidase were 4.2, 7.4, 7.0 and 5.7, respectively. In enzymic analysis using pI-4.2 SM deacylase partially purified by IEF, which had no detectable contamination with acid ceramidase, GlcCDase or SMase, radio-TLC analysis revealed that radiolabelled sphingosylphosphocholine or [1-(14)C]palmitic acid was enzymically liberated from [choline-methyl-(14)C]SM or N-[palmitoyl-1-(14)C]GCer, respectively, used as substrates. Further the pI-4.2 protein purified from extracts of the stratum corneum of ADe patients was able to hydrolyse N-[palmitoyl-1-(14)C]SM and GCer, but not N-[palmitoyl-1-(14)C]ceramide. These results indicate that a hitherto undiscovered epidermal enzyme, termed here glucosylceramide sphingomyelin deacylase, is expressed in the skin of ADe patients, which plays an important role in ceramide deficiency (including acylceramides) in the stratum corneum.

Seventh Japanese–German workshop on molecular and cellular aspects of carcinogenesis

Seventh Japanese–German workshop on molecular and cellular aspects of carcinogenesis

Human placenta sphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase, induces oxidative stress, glutathione depletion, and apoptosis in rat hepatocytes.

Ceramide has been identified as a putative lipid messenger that mediates diverse cellular processes including cell death. Since glutathione (GSH) depletion is known to sensitize cells to many cytotoxic agents and as a result of the reported regulation of neutral sphyngomyelinase (NSMase) by GSH, the present study compared the role of individual SMases in the induction of oxidative stress, regulation of cellular GSH, and apoptosis of rat hepatocytes. Exposure of cultured rat hepatocytes to exogenous Bacillus cereus sphingomyelinase (bSMase), a neutral SMase, or human placenta sphingomyelinase (hSMase), an acidic SMase (ASMase), generated similar ceramide levels in a dose-dependent manner. However, whereas bSMase increased hepatocellular GSH levels, hSMase depleted GSH stores, an effect that was prevented by monensin and mannose 6-phosphate (M-6-P), suggesting that exogenous hSMase enters hepatocytes by endocytosis and is delivered to an endosomal/lysosomal acidic compartment. Interestingly, despite the differential effect of either SMases on cell GSH levels, both bSMase and hSMase increased gamma-glutamylcysteine synthetase heavy-subunit chain (gamma-GCS-HS) mRNA levels. Consistent with these findings on GSH regulation, hSMase, but not bSMase, generated reactive oxygen species (ROS), being accompanied by mitochondrial depolarization, suggesting that hSMase targeted mitochondria, leading to oxidative stress. Accordingly, hepatocytes displayed a selective sensitivity to hSMase in contrast to bSMase exposure, and depletion of GSH stores enhanced susceptibility to hSMase as a result of potentiation of ROS formation and caspase 3 activation. Thus, these findings reveal the ability of ASMase to induce oxidative stress as a result of the targeting of mitochondria, and that GSH depletion sensitizes hepatocytes to the ASMase-induced apoptosis.

Ceramide mediates radiation-induced death of endothelium.

The sphingomyelin (SM) pathway is an ubiquitous, evolutionarily conserved signaling system, analogous to conventional systems such as the cAMP and phosphoinositide pathways. Ceramide is generated from SM by the action of a neutral or acid SMase, or by de novo synthesis coordinated through the enzyme ceramide synthase. Once generated, ceramide may serve as a second messenger in signaling responses to physiologic or environmental stimuli, or may be converted to a variety of structural or effector molecules. In the radiation response, ceramide serves as a second messenger in initiating apoptosis, while some of its metabolites block apoptosis. In certain cells, such as endothelial, lymphoid and haematopoietic cells, ceramide mediates apoptosis while in others ceramide may serve only as a co-signal for or play no role in the death response. Regulated ceramide metabolism may determine the balance between pro- and anti-apoptotic signals, and hence, the intensity of the apoptotic response, thus constituting a mechanism of radiation sensitivity or resistance. This paradigm may offer new opportunities for modulation of the radiation effects in the treatment of cancer. Chemical modifiers of ceramide metabolism may be useful to enhance the therapeutic effects or reduce the toxicity of radiation treatment.