Glucosylceramide and Glucosylsphingosine Modulate Calcium Mobilization from Brain Microsomes via Different Mechanisms

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673–21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were ∼10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease. We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673–21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were ∼10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease. Sphingolipids (SLs) 1The abbreviations used are: SL, sphingolipid; DTT, dithiothreitol; GalCer, galactosylceramide; GalSph, galactosylsphingosine; GlcCer, glucosylceramide; GlcSph, glucosylsphingosine; GPCR, G-protein-coupled receptor; GSL, glycosphingolipid; InsP3, inositol 1,4,5-trisphosphate; InsP3R, inositol 1,4,5-trisphosphate receptor; LacCer, lactosylceramide; LC, long chain; RyaR, ryanodine receptor; ER, endoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; MOPS, 4-morpholinepropanesulfonic acid. act as structural components of cell membranes and as bioactive molecules, functioning as both first and second messengers (1Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Google Scholar, 2Kolesnick R. J. Clin. Invest. 2002; 110: 3-8Google Scholar). Of the signaling pathways regulated by SLs, Ca2+ homeostasis has received wide attention due largely to observations that sphingosine, sphingosine 1-phosphate, and sphingosylphosphorylcholine modulate Ca2+-homeostasis via the edg receptors, a class of plasma membrane G-protein-coupled receptors (GPCRs) (3Meyer zu Heringdorf D. Himmel H.M. Jakobs K.H. Biochim. Biophys. Acta. 2002; 1582: 178-189Google Scholar, 4Young K.W. Nahorski S.R. Semin. Cell Dev. Biol. 2001; 12: 19-25Google Scholar, 5Sharma C. Smith T. Li S. Schroepfer Jr., G.J. Needleman D.H. Chem. Phys. Lipids. 2000; 104: 1-11Google Scholar, 6Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Google Scholar). However, complex glyco-SLs (GSLs) and their lyso-derivatives (Fig. 1) have also been implicated in regulating Ca2+ homeostasis (7Ledeen R.W. Wu G. Lu Z.H. Kozireski-Chuback D. Fang Y. Ann. N. Y. Acad. Sci. 1998; 845: 161-175Google Scholar, 8Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Google Scholar). Of the lyso-GSLs, galactosylsphingosine (GalSph, psychosine) (9Catalan R.E. Miguel B.G. Calcerrada M.C. Ruiz S. Martinez A.M. Biochem. Biophys. Res. Commun. 1997; 238: 347-350Google Scholar, 10Liu R. Farach-Carson M.C. Karin N.J. Biochem. Biophys. Res. Commun. 1995; 214: 676-684Google Scholar, 11Okajima F. Kondo Y. J. Biol. Chem. 1995; 270: 26332-26340Google Scholar, 12Himmel H.M. Meyer zu Heringdorf D. Windorfer B. van Koppen C.J. Ravens U. Jakobs K.H. Mol. Pharmacol. 1998; 53: 862-869Google Scholar, 13Sakano S. Takemura H. Yamada K. Imoto K. Kaneko M. Ohshika H. J. Biol. Chem. 1996; 271: 11148-11155Google Scholar) has been shown to mobilize Ca2+ from intracellular stores, possibly via activation of the ryanodine receptor (RyaR), the major Ca2+-release channel of the endoplasmic reticulum (ER), although this has never been unambiguously proven. Recent studies (reviewed in Ref. 14Ledeen R.W. Wu G. Neurochem. Res. 2002; 27: 637-647Google Scholar) have shown that complex GSLs, such as ganglioside GM1, can potentiate the activity of a nuclear envelope Na+-Ca2+ exchanger (15Xie X. Wu G. Lu Z.H. Ledeen R.W. J. Neurochem. 2002; 81: 1185-1195Google Scholar), and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) can be modulated by gangliosides GM1 and GM3 (16Wang Y. Tsui Z. Yang F. Glycoconj. J. 1999; 16: 781-786Google Scholar, 17Wang Y. Tsui Z. Yang F. FEBS Lett. 1999; 457: 144-148Google Scholar). Since GSLs and lyso-GSLs accumulate in the sphingolipidoses, in which neuronal function is often severely impaired (18Gravel R.A. Kaback M.M. Proia R. Sandhoff K. Suzuki K. Suzuki K. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York2001: 3827-3876Google Scholar, 19Beutler E. Grabowski G.A. Scriver C.R. Sly W.S. Childs B. Beaudet A.L. Valle D. Kinzler K.W. Vogelstein B. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. II. McGraw-Hill Inc., New York2001: 3635-3668Google Scholar), and since altered Ca2+ homeostasis has been implicated in a number of neurodegenerative diseases (20Rizzuto R. Curr. Opin. Neurobiol. 2001; 11: 306-311Google Scholar, 21Paschen W. Frandsen A. J. Neurochem. 2001; 79: 719-725Google Scholar), determination of the molecular mechanisms by which GSLs or lyso-GSLs modulate intracellular Ca2+ signaling may be a prerequisite for determining the mechanism leading from GSL accumulation to neuronal cell dysfunction and/or death. We recently demonstrated (22Korkotian E. Schwarz A. Pelled D. Schwarzmann G. Segal M. Futerman A.H. J. Biol. Chem. 1999; 274: 21673-21678Google Scholar) that the simplest GSL, glucosylceramide (GlcCer), upon its accumulation in cultured neurons in a chemically induced model of type 2/3 Gaucher disease (the neuronopathic forms of Gaucher disease, Refs. 23Erikson A. Bembi B. Schiffmann R. Zimran A. Gaucher's Disease. 10. Bailliere Tindall, London1997: 711-723Google Scholar and 24Vellodi A. Bembi B. de Villemeur T.B. Collin-Histed T. Erikson A. Mengel E. Rolfs A. Tylki-Szymanska A. J. Inherit. Metab. Dis. 2001; 24: 319-327Google Scholar), increases Ca2+ mobilization from intracellular stores, presumably via the RyaR. As a result, neurons with elevated GlcCer levels showed enhanced sensitivity to agents that induce cell death via Ca2+ mobilization (22Korkotian E. Schwarz A. Pelled D. Schwarzmann G. Segal M. Futerman A.H. J. Biol. Chem. 1999; 274: 21673-21678Google Scholar, 25Pelled D. Shogomori H. Futerman A.H. J. Inherit. Metab. Dis. 2000; 23: 175-184Google Scholar). In the current study we systematically examine the effects of GlcCer, other GSLs and their lyso-derivatives on Ca2+ release from isolated rat brain microsomes, and demonstrate that GlcCer and its lyso-derivative, glucosylsphingosine (GlcSph) (Fig. 1), both modulate Ca2+ release but via different molecular mechanisms, and by a different mechanism to that of galactosylsphingosine (GalSph), none of which involve GPCRs. Moreover, Ca2+ release was also enhanced in human brain microsomes obtained from a type 2 Gaucher disease patient, in which GlcCer levels are elevated ∼10-fold. Materials—C8-GlcCer (N-octanoyl-d-glucosylsphingosine), C8-galactosylceramide (C8-GalCer; N-octanoyl-d-galactosylsphingosine) and C8-lactosylceramide (C8-LacCer; N-octanoyl-d-lactosylsphingosine) werefrom Avanti Polar Lipids, Alabaster, AL. Natural long acyl-chain (LC)-LacCer (porcine), sphingosine 1-phosphate, sphinganine 1-phosphate and sphingosylphosphorylcholine were from Matreya, Pleasant Gap, PA. C8-Ceramide (C8-Cer; N-octanoyl-d-sphingosine), LC-GlcCer (from human Gaucher spleen), LC-GalCer (from bovine brain), GalSph, GlcSph, antipyrylazo III, A23187, heparin, inositol 1,4,5-trisphosphate (InsP3), palmitoyl CoA, creatine phosphokinase, phosphocreatine, ATP, and NAD were from Sigma. GDPβS and pertussis toxin A protomer were from Calbiochem, Darmstadt, Germany. Aminopropyl (LC-NH2, 100 mg) and weak cation exchanger (LC-WCX, 100 mg) columns were from Supelco (Bellefonte, PA). Ryanodine was from either Alomone Labs, Jerusalem, Israel, or from Sigma. 3Hryanodine (109 Ci/mmol) and 3Hacetic anhydride (9.7 Ci/mmol) were from Amersham Biosciences. Brain Microsomes—Wistar rats, obtained from the Weizmann Institute Breeding Center, were sacrificed, their brains removed, separated into cerebral cortex and cerebellum, rapidly frozen in liquid N2, and stored at –80 °C. Microsomes (from 10–12 gm of tissue) were prepared essentially as described (26Betto R. Teresi A. Turcato F. Salviati G. Sabbadini R.A. Krown K. Glembotski C.C. Kindman L.A. Dettbarn C. Pereon Y. Yasui K. Palade P.T. Biochem. J. 1997; 322: 327-333Google Scholar) with some modifications. Tissue was suspended at a ratio of 1:4 (w/v) in ice cold 0.32 m sucrose, 20 mm HEPES-KOH, pH 7.0, containing 0.4 mm phenylmethylsulfonyl fluoride, leupeptin (0.8 μg/ml), and aprotinin (1.4 TIU) (buffer A), and homogenized at 4 °C using 8 up and down strokes of a rotating Potter-Elvehjem homogenizer. After centrifugation (700 × gav, 10 min), the resulting pellet (P1) was gently resuspended in one-fourth of the original volume of buffer A, centrifuged (700 × gav, 10 min), and the two supernatants pooled (S1). Mitochondria were removed by centrifugation (8,000 × gav, 45 min) of S1 and the resulting supernatant (S2) centrifuged (115,000 × gav, 90 min) to obtain a microsomal pellet (P3), which was resuspended in 0.4–0.8 ml of buffer A. Protein was determined (27Bradford M. Anal. Biochem. 1976; 72: 248-254Google Scholar), and the microsomes subsequently flash-frozen in liquid N2. Microsomes were stored at –80 °C and used for up to six months after their preparation, during which time there was no change in their activity with respect to Ca2+ release and uptake. Human brain microsomes were prepared exactly as described for rat brain microsomes. Microsomes were prepared from a control human brain of a young adult and from the brain of a type 2 Gaucher patient who died at 1 year of age. Spectrophotometric Assay of Ca2+Uptake and Release—Ca2+ uptake and release was measured by a spectrophotometric assay using the Ca2+-sensitive dye, antipyrylazo (26Betto R. Teresi A. Turcato F. Salviati G. Sabbadini R.A. Krown K. Glembotski C.C. Kindman L.A. Dettbarn C. Pereon Y. Yasui K. Palade P.T. Biochem. J. 1997; 322: 327-333Google Scholar, J. Biol. Chem. Scholar, C. R. Salviati G. Sabbadini R. Palade Brain Res. 1995; Scholar), with some modifications. Brain microsomes in of buffer were added to ml of 8 mm pH 7.0, mm mm and antipyrylazo III, in a containing a to which 1 mm creatine phosphokinase, and mm phosphocreatine, pH 7.0, were Ca2+ uptake and release were measured in a at °C by the at from at The effect of GSLs and lyso-GSLs was by their either to or after Ca2+ for and lyso-GSLs were in and were in G. S. Y. Y. FEBS 1995; Scholar, K. Futerman A.H. Biochim. Biophys. Acta. 2001; Scholar). The or in the and were added as a complex with bovine was in toxin A protomer was added in a containing NAD and In the effect of and was The of Ca2+ from microsomes was as a of Ca2+ in the microsomes, which was obtained by Ca2+ up during the with Ca2+ from the microsomal after of a Ca2+ Ca2+ The of Ca2+ uptake into microsomes was by the of the after of or Ca2+ release was Ca2+ release was to be a increased by the with the as the measured the brain microsomes were resuspended in buffer mm pH 1 m ATP, 100 to a of 1 was as described R.A. Brain Res. Scholar) for 1 at in a volume of of buffer containing of microsomes and was determined by with The was by of ml of buffer mm pH 1 m 100 through in a by two The and the number of receptor were by GlcCer and were J. Pelled D. Futerman A.H. J. Res. Scholar) from the human temporal lobe microsomes used for Ca2+ GlcCer and were in by using a as described J. Pelled D. Futerman A.H. J. Res. Scholar). GSLs and lyso-GSLs were separated by weak cation using a and GSLs subsequently by m in 100 The resulting lyso-GSLs were using mm anhydride containing 3Hacetic anhydride and in as were the lyso-GSLs obtained in the A of this S. and A. H. Futerman, in Ca2+ mobilization from rat brain microsomes was using the Ca2+-sensitive dye, antipyrylazo has been used to Ca2+ release from J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. Scholar), which levels of R. S. Pharmacol. 1997; Scholar), and from brain C. R. Salviati G. Sabbadini R. Palade Brain Res. 1995; Scholar). using a and by the of microsomal membranes with respect to Ca2+ we were to this to Ca2+ release in rat brain microsomes, from which significantly levels of can be its to the Ca2+ in microsomes and be by palmitoyl (Fig. a RyaR Dettbarn C. D. G. J. Scholar, J. 1996; 270: Scholar), to a to that in brain C. R. Salviati G. Sabbadini R. Palade Brain Res. 1995; Scholar). Ca2+ release was enhanced upon with by (Fig. and be by with ryanodine (Fig. a to or that used to Ca2+ release measured using antipyrylazo (26Betto R. Teresi A. Turcato F. Salviati G. Sabbadini R.A. Krown K. Glembotski C.C. Kindman L.A. Dettbarn C. Pereon Y. Yasui K. Palade P.T. Biochem. J. 1997; 322: 327-333Google Scholar, J. Biol. Chem. Scholar, Mol. Pharmacol. 1997; Scholar). In induce Ca2+ release by (Fig. that GlcCer is of the RyaR, but rather its LC-GlcCer enhanced Ca2+ release to a to that of using either palmitoyl or as RyaR and a of (Fig. and were in Ca2+ release, as were a of other a of of the RyaR by of on microsomal release of 4 in a GlcCer induce Ca2+ release by (Fig. a in Ca2+ release S. J. Biol. Chem. Scholar, Palade B. S. J. Biol. Chem. Scholar) was in the of In microsomes, were the of a which increased to in the of both of which be by ryanodine The of Ca2+ was higher in the of and LC-GlcCer in although was LC-GlcCer a in but no was with other the that GlcCer the of GSLs on of Ca2+ release was was was in a We the of GlcCer to mobilize Ca2+ via other Ca2+ release from microsomes, a of the K. Curr. Opin. Neurobiol. 1997; Scholar), which be by the J. Biol. Chem. Scholar) (Fig. (Fig. 10 LC-GlcCer of or 10 LC-GalCer of had effect on the of Ca2+ into microsomes via the we that GlcCer Ca2+ mobilization via the RyaR and via the or Since with or LC-GalCer had effect on the or of 3Hryanodine to the RyaR of 3Hryanodine of for control or microsomes for for LC-GlcCer and for LC-GalCer for control for for LC-GlcCer for LC-GalCer we that GlcCer the and the of ryanodine to the RyaR. Recent studies have demonstrated that RyaR activity can be enhanced by its redox W. G. J. Biol. Chem. 2000; Scholar, J. G. J. Biol. Chem. 2001; Scholar, T. T. Y. J. 2002; Scholar). with the DTT, the of (Fig. to Ca2+ release, and that GlcCer may modulate the redox of the RyaR via its redox sensor S. Wu Y. W. W. Acad. Sci. S. 2002; Scholar, J. Biol. Chem. 1997; Scholar). We the of Ca2+ release from brain obtained from a Gaucher disease type 2 patient to the of studies have suggested that GlcCer in Gaucher brain J. Neurochem. Scholar, Grabowski G.A. Clin. 27: Scholar), but the of accumulation was from compared with a for of GSLs and lyso-GSLs J. Pelled D. Futerman A.H. J. Res. Scholar), and a to in which the of lyso-GSLs is with 3Hacetic 2 higher of GlcCer was in microsomes prepared from the temporal lobe of a type 2 Gaucher patient a neuronopathic compared with a control and of GlcCer and levels in human brain both from control and type 2 and Gaucher is on J. Res. Scholar, J. Neurochem. Scholar, Grabowski G.A. Clin. 27: Scholar, Erikson A. Scholar), we a in GlcCer levels in control human and was also in the Gaucher although at levels GlcCer, with no in control brain microsomes palmitoyl Ca2+ release from Gaucher brain microsomes was significantly higher from human control brain microsomes, and be by to control levels (Fig. demonstrate a and GlcCer accumulation in Gaucher brains and enhanced levels of Ca2+ release via the and levels in human brain type 2 of of of in a In to GlcCer, and stimulated Ca2+ release from rat microsomes, at of higher and in to the of ryanodine to Ca2+ release (Fig. ryanodine was to Ca2+ release (Fig. and Ca2+ release was enhanced by GlcCer that Ca2+ release via a mechanism of the RyaR. However, ryanodine Ca2+ release (Fig. that is a RyaR Ca2+ release be by with GDPβS of or by pertussis toxin μg/ml), of the and that are in the of and on Ca2+ this is by the of a and of and sphingosylphosphorylcholine at 100 to induce Ca2+ release from rat brain microsomes The major of the current study is that GlcCer Ca2+ from microsomes via a mechanism modulation of the activity of a major Ca2+ channel of the ER, the RyaR. be of relevance for the pathophysiology of neuronal forms of Gaucher disease, in which GlcCer is by that GlcCer in microsomes prepared from a type 2 Gaucher disease in which Ca2+ release is also the of GlcCer in the human brain microsomes was to that added exogenously to rat brain microsomes in to Ca2+ release was at levels of of in Gaucher type 2 microsomes of were used Ca2+ release in a volume of 1 a of GlcCer of in the to the used to Ca2+ release added exogenously (Fig. via the RyaR. The for the current study was that upon its accumulation in cultured neurons, GlcCer in neuronal as a in Ca2+ release from intracellular stores was in to or Moreover, neurons were to neuronal and to induced via other which be by with of ryanodine (22Korkotian E. Schwarz A. Pelled D. Schwarzmann G. Segal M. Futerman A.H. J. Biol. Chem. 1999; 274: 21673-21678Google Scholar, 25Pelled D. Shogomori H. Futerman A.H. J. Inherit. Metab. Dis. 2000; 23: 175-184Google Scholar). current that GlcCer Ca2+ release from brain microsomes via the RyaR is with and the of effect of and other GSLs and that GlcCer a in of the RyaR. is by the in the of Ca2+ release upon of microsomes with Ca2+ increases in intracellular Ca2+ J. 1997; Scholar), have been in and in brain S. J. Biol. Chem. Scholar, C. Palade Mol. Pharmacol. 1997; Scholar, C. S. Palade Mol. Pharmacol. Scholar, A. Cell 1998; 23: Scholar), and have been suggested to be of in Ca2+ signaling in the N. R. Acad. Sci. U. S. A. 1999; Scholar). The of and LC-GlcCer to a for GlcCer in the of Ca2+ homeostasis via its of the RyaR. However, is to the in brain microsomes to we as the molecular mechanism by which GlcCer the RyaR, the of to GlcCer modulation of both Ca2+ and of agonist-stimulated Ca2+ release that GlcCer may modulate the redox of the RyaR via its redox A number of have been to have S. Wu Y. W. W. Acad. Sci. S. 2002; Scholar). In the of the RyaR, the channel have been to and J. G. J. Biol. Chem. 2001; Scholar, J. C. G. Acad. Sci. U. S. A. 2001; Scholar), and GlcCer may with the redox sensor W. G. J. Biol. Chem. 2000; Scholar). GlcCer as a or of the RyaR, which is in the ER, GlcCer presumably be in this is to the ER, in the A.H. R.E. Biochem. J. Scholar). However, of the intracellular of GSLs may to be in of that GlcCer a number of with the and other intracellular G. J. Biol. Chem. 2002; 277: Scholar, J. Pelled D. C. S. Futerman A.H. J. 2002; 16: Scholar). a study demonstrated that are in a of rat D. J. D. J. Biochem. J. Scholar) which be to the showed for as that different of GlcCer may be at different In of this are current that microsomes obtained from human Gaucher brain levels of GlcCer, although no of the of microsomes was due to of the human brain GlcCer is the only GSL used in this study that the RyaR, and both as to Ca2+ release from microsomes. was shown to induce Ca2+ release from cultured T. J. Cell Biol. 2001; Scholar) via a cell GPCR, 8 However, is at levels in brain and has a Y. 1996; Scholar). The of effect of GDPβS and pertussis toxin on and Ca2+ release from brain microsomes that act via in microsomes, although the that also to a cell in neurons, as has been suggested in F. Kondo Y. J. Biol. Chem. 1995; 270: 26332-26340Google Scholar), be The of effect in microsomes of and which to cell S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Google Scholar), with the effect of ryanodine on Ca2+ release, the that (9Catalan R.E. Miguel B.G. Calcerrada M.C. Ruiz S. Martinez A.M. Biochem. Biophys. Res. Commun. 1997; 238: 347-350Google Scholar, 10Liu R. Farach-Carson M.C. Karin N.J. Biochem. Biophys. Res. Commun. 1995; 214: 676-684Google Scholar, 11Okajima F. Kondo Y. J. Biol. Chem. 1995; 270: 26332-26340Google Scholar, 12Himmel H.M. Meyer zu Heringdorf D. Windorfer B. van Koppen C.J. Ravens U. Jakobs K.H. Mol. Pharmacol. 1998; 53: 862-869Google Scholar, 13Sakano S. Takemura H. Yamada K. Imoto K. Kaneko M. Ohshika H. J. Biol. Chem. 1996; 271: 11148-11155Google Scholar) as of the RyaR, Ca2+ release via a mechanism of the RyaR. or of the effects of and on Ca2+ mobilization are of relevance in is a of since in are in the Ref. J. Pelled D. Futerman A.H. J. Res. Scholar). was at levels of of in Gaucher type 2 microsomes but was the in a control the of in the human microsomes used for Ca2+ release studies was as in a significantly that to induce Ca2+ release from microsomes added However, lyso-GSLs accumulate at higher levels in GSL such as Gaucher and disease, in the and the lyso-GSLs, rather the GSLs, have been implicated in the mechanisms disease J. Pelled D. Futerman A.H. J. Res. Scholar, K. Neurochem. Res. 1998; 23: Scholar). of the relevance of lyso-GSLs in Ca2+ from microsomes, the of GlcCer compared with both other GSLs and lyso-GSLs on Ca2+ mobilization both in this study in microsomes and in study in cultured neurons (22Korkotian E. Schwarz A. Pelled D. Schwarzmann G. Segal M. Futerman A.H. J. Biol. Chem. 1999; 274: 21673-21678Google Scholar), and study on activation of by GlcCer J. Pelled D. C. S. Futerman A.H. J. 2002; 16: Scholar), that GlcCer is intracellular that in both the of and in intracellular Ca2+ The human control brain used in this study was by the of Brain and Tissue for through