Inhibition of the Neutral Magnesium-dependent Sphingomyelinase by Glutathione

Abstract

Sphingomyelin hydrolysis through the activation of sphingomyelinases has become a potentially important signaling pathway with the product ceramide implicated in the regulation of cell growth, differentiation, apoptosis, and inflammatory responses. However, little is known about the regulation of sphingomyelinases. In this study, we show that the magnesium-dependent, neutral pH-optimum and membrane-associated sphingomyelinase (N-SMase) is inhibited, in a dose-dependent manner, by glutathione (GSH) at physiological concentrations with a greater than 95% inhibition observed at 5 mm GSH. The inhibitory effect of GSH was reproduced by γ-glutamyl-cysteine, but not the cysteinyl-glycine fragment of GSH. The S-modified GSH analogs were as effective as GSH in inhibiting the N-SMase. On the other hand, neither dithiothreitol nor β-mercaptoethanol had any effect on the N-SMase, suggesting that the sulfhydryl in GSH is not required for inhibition of N-SMase. GSH had no effect on the acid pH-optimum SMase, whereas dithiothreitol inhibited the acid SMase. These results suggest that in cells the N-SMase is inactive in the presence of physiological concentrations of GSH (1–20 mm). Finally, treatment of cultured Molt-4 cells with the GSH synthesis inhibitor,l-buthionine-(SR)-sulfoximine, resulted in a time-dependent depletion of GSH, accompanied by an increased hydrolysis of sphingomyelin and production of ceramide. Since GSH depletion is observed in a variety of cells in the process of cellular injury and apoptosis, these studies suggest that depletion of GSH may be an important mechanism in activation of N-SMase. This mechanism may therefore bring together the fields of oxidative stress and signaling through products of sphingomyelin hydrolysis. Sphingomyelin hydrolysis through the activation of sphingomyelinases has become a potentially important signaling pathway with the product ceramide implicated in the regulation of cell growth, differentiation, apoptosis, and inflammatory responses. However, little is known about the regulation of sphingomyelinases. In this study, we show that the magnesium-dependent, neutral pH-optimum and membrane-associated sphingomyelinase (N-SMase) is inhibited, in a dose-dependent manner, by glutathione (GSH) at physiological concentrations with a greater than 95% inhibition observed at 5 mm GSH. The inhibitory effect of GSH was reproduced by γ-glutamyl-cysteine, but not the cysteinyl-glycine fragment of GSH. The S-modified GSH analogs were as effective as GSH in inhibiting the N-SMase. On the other hand, neither dithiothreitol nor β-mercaptoethanol had any effect on the N-SMase, suggesting that the sulfhydryl in GSH is not required for inhibition of N-SMase. GSH had no effect on the acid pH-optimum SMase, whereas dithiothreitol inhibited the acid SMase. These results suggest that in cells the N-SMase is inactive in the presence of physiological concentrations of GSH (1–20 mm). Finally, treatment of cultured Molt-4 cells with the GSH synthesis inhibitor,l-buthionine-(SR)-sulfoximine, resulted in a time-dependent depletion of GSH, accompanied by an increased hydrolysis of sphingomyelin and production of ceramide. Since GSH depletion is observed in a variety of cells in the process of cellular injury and apoptosis, these studies suggest that depletion of GSH may be an important mechanism in activation of N-SMase. This mechanism may therefore bring together the fields of oxidative stress and signaling through products of sphingomyelin hydrolysis. Sphingolipids have emerged as important components of signal transduction pathways involved in a variety of cellular processes. This is best illustrated in the case of the sphingomyelin (SM) 1The abbreviations used are: SM, sphingomyelin; SMase, sphingomyelinase; N-SMase, neutral magnesium-dependent SMase; A-SMase, acid SMase; DTT, dithiothreitol; BSO,l-buthionine-(SR)-sulfoximine. cycle (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar), which has become a cornerstone in studying the role of sphingomyelin, ceramide, and other related products in cellular regulation. In this pathway, a number of extracellular agents such as tumor necrosis factor α (2Kim M.-Y. Linardic C. Obeid L. Hannun Y. J. Biol. Chem. 1991; 266: 484-489Abstract Full Text PDF PubMed Google Scholar), 1,25-dihydrovitamin D3 (1Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar), interleukin 1β (3Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Abstract Full Text PDF PubMed Google Scholar, 4Mathias S. Younes A. Kan C.-C. Orlow I. Joseph C. Kolesnick R.N. Science. 1993; 259: 519-522Crossref PubMed Scopus (391) Google Scholar), nerve growth factor (5Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (549) Google Scholar), chemotherapeutic agents (6Strum J.C. Small G.W. Pauig S.B. Daniel L.W. J. Biol. Chem. 1994; 269: 15493-15497Abstract Full Text PDF PubMed Google Scholar, 7Zhang J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (294) Google Scholar), and serum deprivation (8Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar) results in activation of sphingomyelinases (SMases), which causes the hydrolysis of sphingomyelin, leading to the generation of ceramide. In turn, ceramide has been suggested to play important roles in differentiation, cell cycle arrest, apoptosis, inflammation, and the regulation of eukaryotic stress responses (9Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar). Although the ceramide generated in response to the action of extracellular agents appears to derive from the hydrolysis of sphingomyelin, not much is known about the mechanisms regulating the activity of the involved SMases. To date, at least five types of SMases have been identified. An acidic sphingomyelinase (A-SMase) was first discovered and found to have an optimum pH at 5.0 (10Spence M.W. Adv. Lipid Res. 1993; 26: 3-23PubMed Google Scholar). Although the acidic sphingomyelinase has been primarily found to reside in the lysosomes, it has also been detected as a soluble form in cytosol and extracellular media (11Quintern L.E. Weitz G. Nehrkorn H. Tager J.M. Schram A.W. Sandhoff K. Biochim. Biophys. Acta. 1987; 922: 323-336Crossref PubMed Scopus (114) Google Scholar). This enzyme has been cloned, and it is deficient in Neimann-Pick cells (12Kitagawa T. Jpn. J. Hum. Genet. 1987; 32: 55-69Crossref Scopus (4) Google Scholar). It is also suggested to play a role in radiation-induced apoptosis (13Haimovitz-Friedman A. Kan C.-C. Ehleiter D. Persaud R.S. McLoughlin M. Fuks Z. Kolesnick R.N. J. Exp. Med. 1994; 180: 525-535Crossref PubMed Scopus (855) Google Scholar) and has been described to be activated by Fas (14Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (598) Google Scholar) and tumor necrosis factor-α (15Schutze S. Potthof K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar). A neutral pH-optimum and magnesium-dependent SMase (N-SMase) has also been described (16Chatterjee S. Adv. Lipid Res. 1993; 26: 25-47PubMed Google Scholar). This enzyme is associated with the plasma membrane. It is found to be activated in response to tumor necrosis factor-α, Fas, Ara-C, and serum deprivation, and its activation appears to be closely related to growth suppression and apoptosis (6Strum J.C. Small G.W. Pauig S.B. Daniel L.W. J. Biol. Chem. 1994; 269: 15493-15497Abstract Full Text PDF PubMed Google Scholar,8Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 17Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R.A. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar, 18Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1992; 78: 1005-1015Abstract Full Text PDF Scopus (678) Google Scholar). Recently, a cytosolic magnesium-independent neutral SMase was partially purified from HL-60 cells following treatment with vitamin D3 (19Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar). In addition, a zinc-dependent acidic SMase was detected in sera from a variety of species (20Spence M.W. Byers D.M. Palmer F.B. St C. Cook H.W. J. Biol. Chem. 1989; 264: 5358-5363Abstract Full Text PDF PubMed Google Scholar), and recent studies suggest that it is derived from the lysosomal A-SMase (21Schiessel S.L. Schuchman E.H. Williams K., J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Finally, an alkaline pH-optimum SMase has been described in intestinal cells (22Nyberg L. Duan R.D. Axelson J. Nilsson A. Biochim. Biophys. Acta. 1996; 1300: 42-48Crossref PubMed Scopus (77) Google Scholar). In ongoing studies on characterizing N-SMase, we discovered that this enzyme was inhibited in vitro by physiologically relevant concentrations of glutathione (GSH) and it was activated in cells depleted of GSH. Glutathione, a tripeptide (l-γ-glutamyl-l-cysteinyl-glycine), is the most abundant nonprotein thiol-containing small molecule in mammalian cells. It plays a critical role in cellular defense against oxidative stress by inactivating free radials, reactive oxygen species, and a variety of cytotoxic electrophiles including alkylating agents. GSH functions as an antioxidant by directly reacting with free radicals, conjugating with various agents to form S-substituted adducts nonenzymatically or through the action of GSHS-transferase, and serves as a substrate for GSH peroxidase (23Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (400) Google Scholar). Under normal conditions, cells maintain a high level of intracellular GSH (1–20 mm). In this study, we report on the effects of GSH on N-SMase. The implications of inhibition of N-SMase by GSH will be discussed. Molt-4 human leukemia cells were obtained from ATCC (Rockville, MD). RPMI 1640 and fetal calf serum were purchased from Life Technologies, Inc. Glutathione and related compounds were from Sigma. Leukotrienes were from Cayman Chemicals (Ann Arbor, MI). Stripped rat brains were from Pel-Freez Biologicals (Rogers, AR). DEAE-Sepharose was from Pharmacia Biotech Inc. [N-methyl-14C]Sphingomyelin was synthesized as described (19Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar). Molt-4 cells were in RPMI 1640 with fetal calf serum at in a with were at a of and to Molt-4 cells were with and with a in mm pH mm 5 mm of and The was for at and the was in in The was a DEAE-Sepharose in A mm pH mm mm and the the were by to in by a of from in A. were and for of N-SMase and The of the N-SMase the DEAE-Sepharose was greater than SMases from rat rat brains were used and the was the as that for Molt-4 cells as described were at The activity of N-SMase and A-SMase were a (19Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar). N-SMase enzyme of to with mm pH was with of in in of pH 5 mm The for at and was by the of of by of a of the was and the by A-SMase activity was in of mm pH The of the product in the was by as described (19Okazaki T. Bielawska A. Domae N. Bell R.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 4070-4077Abstract Full Text PDF PubMed Google Scholar). Molt-4 cells were at in for and with or for various cells were and cellular by the of and J. PubMed Scopus Google Scholar). was a as described J. Alter N. Reed J.C. Borner C. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5325-5328Crossref PubMed Scopus (294) Google Scholar, S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 17Tepper C.G. Jayadev S. Liu B. Bielawska A. Wolff R.A. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar). level was as described (8Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). cellular were by a of The sphingomyelin were by and for Sphingomyelin was an and was to GSH was by the PubMed Scopus Google Scholar) of F. PubMed Scopus Google Scholar). cells were in of The were by at for at To the GSH of the were with mm pH mm mm and in a of of glutathione the in at was and used to the of GSH in the by to a generated with known of GSH was by the PubMed Scopus Google Scholar) serum as The of GSH and was by N-SMase with GSH for a and an of the was on a with a of and as by and A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The compounds were by with and with of the of N-SMase, we the effect of agents on the in of the To this we partially purified the N-SMase from the of Molt-4 cells which have little cellular acidic SMase On DEAE-Sepharose of SMase activity were A-SMase and had a small of had the activity of N-SMase that of and no A-SMase The N-SMase in was purified and the N-SMase in was the N-SMase in was used for this The partially purified N-SMase was with the agents of and this was by with of sphingomyelin substrate in a in the presence of 5 mm The product a with substrate was by and its by The was the of the of enzyme of and used in this in of N-SMase for at with concentrations of GSH from to mm resulted in an inhibition of the N-SMase a GSH inhibition was detected at concentrations of GSH whereas GSH mm to a inhibition of N-SMase or β-mercaptoethanol at concentrations from to mm were in inhibiting the N-SMase these results that physiological concentrations of GSH inhibitory to the neutral magnesium-dependent sphingomyelinase and that this inhibitory of GSH not to be a of thiol-containing of N-SMase by GSH. effect of agents on N-SMase N-SMase of from Molt-4 cells was for at with the concentrations of GSH, DTT, or β-mercaptoethanol in of pH by for at with in a mm pH and 5 mm The product was and as described from in for GSH inhibition of N-SMase. Molt-4 cell N-SMase was at with mm GSH for to the of activity of and of The inhibition by GSH of the N-SMase was by of GSH with the no was GSH at 5 mm inhibited the enzyme activity by the enzyme was with GSH for from 5 to the of inhibition increased such that with 5 for and resulted in a and of the enzyme with GSH for than not in inhibition not for than 5 of inhibition was observed for GSH concentrations and 5 mm To effects of on of the enzyme or GSH, the following were of N-SMase, in the of GSH, for to at to the of activity not the enzyme activity not of GSH following with N-SMase for to at the to SMase not any of GSH to or cysteinyl-glycine not To the of GSH inhibition of N-SMase, enzyme was with mm GSH for at and for SMase activity or 5 to a of GSH of mm activity N-SMase activity on of was GSH. These results show that inhibition is and that the of inhibition by is not a of of GSH or an in the the for GSH inhibition of the N-SMase by the effects on the enzyme activity of GSH related in and at concentrations to mm were in inhibiting the enzyme a of action for its effect on the N-SMase. An inhibitory was observed with concentrations of from to and this was by a for concentrations and concentrations at mm and to was with that of GSH the enzyme was with the GSH fragment a of action was concentrations from to cysteinyl-glycine was partially inhibitory of the which was by of its inhibition at concentrations from to closely the for In to at concentrations cysteinyl-glycine not show inhibition N-SMase. The effect of fragment of GSH, γ-glutamyl-cysteine, on the enzyme activity was to that of GSH that was such that the for inhibition was from mm for this fragment mm for These results that the of GSH required for inhibition in the of the which reproduced the effects of GSH on N-SMase. Since and β-mercaptoethanol not the we the for the free sulfhydryl on GSH. these we used GSH, and the GSH GSH and GSH. GSH and GSH were as GSH in inhibiting the enzyme glutathione was effective than GSH in inhibiting the with an of and mm with mm for GSH The greater of not to from of to of GSH. was than as as GSH. the inhibition of N-SMase activity by was not in an by with In this the at important in cellular we also effects on the activity of the N-SMase in in of and physiologically relevant concentrations had no effect on the activity of the these results suggested that the for inhibiting the N-SMase was and that the free sulfhydryl on the GSH molecule was not for the inhibitory we the of GSH with the substrate sphingomyelin and the To the of GSH with sphingomyelin, enzyme was with mm GSH by with of in concentrations of GSH from to mm to of inhibition a of substrate of the of the inhibitory effect of GSH on the N-SMase, we not for these However, concentrations of substrate not inhibition by GSH, the of inhibition not to be of the the enzyme was with mm GSH by with of sphingomyelin in the presence of mm a inhibition was observed and mm GSH a of the inhibition not to be of a the inhibitory effect of GSH was to the N-SMase. To this we on a DEAE-Sepharose the A-SMase and N-SMase from rat which of in the of the A-SMase was found in with a small in and and of the N-SMase The N-SMase in was than that in and was free of A-SMase activity we the effect of GSH on the activity of enzyme from (A-SMase) and with mm GSH, N-SMase from rat was inhibited by GSH with a inhibition at mm GSH, was In GSH to mm had little effect on the activity of A-SMase from rat the activity of the acid SMase as T. K. J. PubMed Scopus Google Scholar). results for GSH and were obtained for purified human A-SMase of not Finally, we the effect of the of GSH in cells on the hydrolysis of and the generation of ceramide. Molt-4 cells were with for and the intracellular GSH was in treatment of cells with resulted in a time-dependent in the intracellular level of GSH. The GSH level of the Molt-4 cells to and of that of cells at and ceramide in the cells was an in the cellular ceramide level was observed as as of cells with The ceramide level at and treatment was than that of cells and that of A in was observed in cells with for 5 that the ceramide generated was to the activation of a In this we that glutathione at physiologically relevant concentrations the neutral magnesium-dependent sphingomyelinase but not the acidic sphingomyelinase and that depletion of cellular GSH results in the hydrolysis of and generation of ceramide. The inhibition of the neutral magnesium-dependent sphingomyelinase not to be a of thiol-containing agents dithiothreitol and β-mercaptoethanol were the free sulfhydryl of GSH is not required inhibition was observed with the GSH, as as with with analogs and of GSH suggest that the for inhibition reside in the of GSH. This in inhibiting N-SMase that GSH may as a of N-SMase. However, partially purified sphingomyelinases were used in this study, we the of an mechanism of action for GSH inhibition of the N-SMase. The mechanism for GSH inhibition of the N-SMase to be results a number of of intracellular concentrations of free GSH in most cell types a and mm B. in and Scholar, A. Science. PubMed Scopus Google Scholar). In the of intracellular GSH level is to be mm and the This to that and conditions, the N-SMase in the cells as an inactive enzyme in the presence of GSH. of GSH from the cell may the inhibition by GSH, in activation of N-SMase. of GSH has been described for agents such as oxidative and alkylating tumor necrosis factor-α, and Fas in various cell types C. I. S. 1995; PubMed Scopus Google Scholar). These cells to high of Biophys. 1994; PubMed Scopus Google Scholar), the cell and to the alkylating R.S. 1994; PubMed Scopus Google Scholar), and cells with and synthesis L. S. G. M.R. Biophys. Res. 1995; PubMed Scopus Google Scholar). In to GSH may be by from the cell as has been observed with Fas, which causes a of GSH from cells. with GSH apoptosis or become to various agents R.S. 1994; PubMed Scopus Google Scholar, P. J. Biol. 1995; Google Scholar, M. Res. 1993; PubMed Scopus Google Scholar). and that depletion of GSH in a cell it to apoptosis R. H. T. Science. 1993; PubMed Scopus Google Scholar). the intracellular GSH through increased synthesis or of GSH or GSH appears to cells from by the cytotoxic agents. In that a human cell to Fas inhibition of its GSH synthesis T. S. N. S. K. J. 1996; 26: PubMed Scopus Google Scholar). Since activation of N-SMase to the generation of ceramide, in GSH may ceramide through N-SMase. In turn, this may the various and of the of the cell of GSH with cellular responses. These cell (5Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (549) Google Scholar), apoptosis L.M. Linardic Hannun Y.A. Science. 1993; 259: PubMed Scopus Google Scholar), cell cycle (8Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar), G. Pushkareva M.Y. Jayadev S. J.M. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar), activation of a R.T. Hannun Y.A. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and a S. Kolesnick R.N. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar), inhibition of cellular J.Y. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), Bielawska A. Obeid L.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and cellular Lee J.Y. Bielawska A. Obeid L.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of ceramide has been to the activation of N-SMase in studies with serum (8Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar), apoptosis C.G. Jayadev S. Liu B. Bielawska A. Wolff R.A. Yonehara S. Hannun Y.A. Seldin M.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8443-8447Crossref PubMed Scopus (325) Google Scholar), and cellular studies Lee J.Y. Bielawska A. Obeid L.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The that GSH depletion is observed in most of these that the of cellular GSH at least in ceramide show in Molt-4 the GSH synthesis not as the GSH of these but also causes the hydrolysis of and generation of ceramide. These that GSH may play a role in regulating hydrolysis and ceramide generation in cells. a to this N-SMase in the cells may inactive GSH is This against the that physiological N-SMase be activated the depletion of GSH. a mechanism may for N-SMase by the inhibition by GSH. the intracellular GSH and the level of free intracellular is to be in the high In studying the of by human and S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that the intracellular of was In by S.C. J. N. J. 1992; PubMed Scopus Google Scholar), the of GSH to was found to be to with the that the intracellular GSH in is mm and that intracellular from of the cell is A. Science. PubMed Scopus Google Scholar), the level of intracellular is and in the the inhibition by of N-SMase observed in vitro may not be relevant but be at this depletion of GSH and has been in studies H. S. H. A. T. J. 1991; Google Scholar, D. S. J. 1992; PubMed Scopus Google Scholar, J.M. M. Res. Google Scholar). the in vitro inhibition of N-SMase by the observed in this may not have much physiological is as a of the of GSH with reactive oxygen with GSH, in cells at a much to the activity of glutathione which the of GSH (23Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (400) Google Scholar, Adv. Enzymol. Google Scholar). In this study, was found to be effective than GSH in inhibiting N-SMase, whereas in cells the of is to be in the of in cells normal N-SMase is to be inhibited by GSH, but not In inhibition of N-SMase by GSH as in this serves as a important involved in the regulation of mammalian stress the oxidative and the A of agents and the hydrolysis of sphingomyelin and the generation of ceramide leading to cell cycle and In addition, of these agents and the depletion of GSH in the of is required to the GSH and sphingomyelinases at the cellular M. Obeid for critical of this