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Role of membrane lipids in regulation of vulnerability to oxidative stress in PC12 cells: implication for aging
Free Radical Biology & Medicine, Vol. 30, No. 6, pp. 671– 678, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(00)00513-X
Original Contribution ROLE OF MEMBRANE LIPIDS IN REGULATION OF VULNERABILITY TO OXIDATIVE STRESS IN PC12 CELLS: IMPLICATION FOR AGING NATALIA A. DENISOVA,* IPPOLITA CANTUTI-CASTELVETRI,* WALEED N. HASSAN,† K. ERIC PAULSON,*† JAMES A. JOSEPH*
and
*United States Department of Agriculture, Jean Mayer Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA; and †Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA (Received18 September 2000; Revised 17 November 2000; Accepted 12 December 2000)
Abstract—Previously, we reported that PC12 cells showed increased vulnerability to oxidative stress (OS) induced by H2O2 (as assessed by decrements in calcium recovery, i.e., the ability of cells to buffer Ca2⫹ after a depolarization event) when the membrane levels of cholesterol (CHL) and sphingomyelin (SPH) were modified to approximate those seen in the neuronal membranes of old animals. The present study was designed to examine whether the enrichment of the membranes with SPH-CHL and increased cellular vulnerability to OS are mediated by neutral SPH-specific phospholipase C (N-Sase) and the intracellular antioxidant GSH. The results showed a significant up-regulation of N-Sase activity by both low (5 M) and high (300 M) doses of H2O2. However, under high doses of H2O2 the up-regulation of N-Sase is accompanied by a significant increase in reactive oxygen species and by a decrease in intracellular GSH. The enrichment of membranes with SPH-CHL significantly potentiated the effects of high doses of H2O2, by further reducing the intracellular GSH and further up-regulating the N-Sase activity. Furthermore, repleting intracellular GSH with 20 mM N-acetylcysteine treatment was sufficient to attenuate the effect of a low dose of H2O2 on Ca2⫹ recovery in SPH-CHL-treated cells. Thus, these results suggested that age-related alterations in the membrane SPH-CHL levels could be important determinants of the susceptibility of neuronal cells to OS. © 2001 Elsevier Science Inc. Keywords—Oxidative stress, Membrane cholesterol and sphingomyelin, Neutral sphingomyelinase, Glutathione, PC12 cells, Free radicals
changes in calcium homeostasis, i.e., enhanced Ca2⫹ flux through voltage-gated calcium channels, decreased activity of plasma membrane Ca2⫹-ATPase and Na⫹/Ca2⫹ exchanger, decreased Ca2⫹ transport in mitochondria, and a significant loss of calcium binding proteins has been observed. These all result in an impaired homeostasis of intracellular calcium with increased levels of free intracellular Ca2⫹ that may ultimately lead to cell death [8 –14]. Recent research from our laboratory demonstrated that an SPH-specific phospholipase C (ceramide-phosphocholine phosphodiesterase, EC 3.1.4.12) induced production of reactive oxygen species (ROS) in a cellular model, and may be directly involved in the loss of the ability to regulate intracellular Ca2⫹ homeostasis [15]. In addition, increases in the metabolism of membrane SPH, associated with increased levels of its metabolites, such as sphingosine-1-phosphate (SPP), may be particularly important in increasing the vulnerability of PC12 cells to
INTRODUCTION
Past research indicates that aging is often accompanied by a significant impairment in behavioral performance and neuronal plasticity [1– 4]. While the etiology of these impairments is not known, we hypothesized that agerelated alterations in membrane molecular structure may contribute to alterations in calcium (Ca2⫹) homeostasis, and ultimately lead to the increased neuronal vulnerability to OS seen in aging. Recent research from our laboratory suggests that modifications of membrane lipid composition with CHL and SPH to levels similar to those seen in neuronal membranes in aging [5,6] dramatically increased cells’ vulnerability to OS as reflected by an impairment in Ca2⫹ homeostasis [7]. During aging, Address correspondence to: Natalia A. Denisova, Ph.D., USDAHNRCA at Tufts University, 711 Washington Street, Boston, MA 02111, USA; Tel: (617) 556-3147; Fax: (617) 556-3222; E-Mail: [email protected]. 671
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OS as measured by Ca2⫹ recovery. Although the mechanisms responsible for the effect of SPP on Ca2⫹ recovery under OS remain unknown, our results suggest that GSH may play a critical role. In fact, it has been shown that a decrease in GSH levels may result in Ca2⫹-mediated cell death in PC12 cells [16]. The GSH/GSSG system plays a pivotal role in the control of the cellular redox status, and is the primary defense mechanism for the removal of peroxides in the brain [17–19]. Recently, an inhibitory effect of GSH on N-Sase activity has been suggested [20 –23]. Highly purified bovine N-Sase activity was effectively blocked by GSH [24]. Increase in intracellular GSH levels induced by treatment with N-acetyl-L-cysteine (NAC) significantly inhibited N-Sase under hypoxic conditions in PC12 cells [22]. In fact, up-regulation of membrane N-Sase may significantly affect the membrane SPH levels and consequently the CHL distribution within the membrane. Preliminary results in rats showed that specific areas of the brain presented different markers of age-related vulnerability to OS, such as brain-area-specific changes in levels of SPH-CHL, ROS production, membrane MDA/HNE, GSH levels, and Ca2⫹ homeostasis [25]. Therefore, given that membrane lipid composition can be significantly affected by the aging process [27–34] and this can modulate cellular sensitivity to OS through alterations in GSH, we designed the present experiments to evaluate whether the enrichment of membranes with SPH-CHL, which increases cell vulnerability to OS as reflected through calcium activity (recovery), is mediated by GSH and N-Sase. MATERIALS AND METHODS
Materials PC12 cells were a generous gift of Dr. Tischler (Tufts University, Boston, MA, USA). RPMI-1640, horse serum, fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). All reagents used were of the highest grade available. Organic solvents were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Cholesterol (nonesterified), sphingomyelin (acylated, 25.5% stearic, and 42.2% nervonic acids) and albumin (fatty acid content 0.01%) as well as all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2⬘,7⬘dichlorofluorescin diacetate (DCFH-DA), monochlorobimate (MBCL), and Amplex Red sphingomyelinase assay kit were obtained from Molecular Probes, Inc. (Eugene, OR, USA).
Cell culture and treatment PC12 cells were seeded onto 100 mm plates coated with rat-tail collagen (type VII) and grown at 37°C under an atmosphere of 5% CO2/95% air, in complete growth medium (GM: RPMI-1640 supplemented with 2 mM glutamine, 10% heat-inactivated horse serum, 5% FBS, 120 U/ml penicillin, and 120 g/ml streptomycin). All PC12 cells were of passage 50 –54. PC12 membrane lipid composition was modified as described previously [7]. Briefly, PC12 cells were incubated for 1 h at 37°C under one of the following conditions: (i) 500 M SPH in GM, (ii) 660 M CHL in GM, or (iii) 500 M SPH-660 M CHL. At the conclusion of the incubation period, cells were washed three times with fresh GM to remove any lipids that were not incorporated into the cells. A subset of these cultures was then exposed to different concentrations of H2O2 (0, 5, 300 M) for 30 min. The H2O2 was prepared immediately prior to the experiments from a concentrated (9.8 M) stock solution. At the conclusion of the incubation period, the medium with H2O2 was removed and the plates were washed with fresh GM. Postnuclear fraction was isolated from PC12 cells by centrifugation of the cell homogenate for 10 min at 1000 g at 4°C. GSH levels The GSH level in PC12 cells was analyzed by using the fluorescent method with MBCL as described previously [15]. Briefly, PC12 cells were plated in a 96-well plate (102–104 cells per well), 0.04 U/ml GST and 50 M MBCL were added and cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader (PerSeptive Biosystem, Framingham, MA, USA) with ex 360 nm and em 460 nm. In order to increase GSH levels in PC12 cells, we employed N-Acetylcysteine (NAC). PC12 cells (104 cells per well) were plated in a 96-well plate, and cultured with 20 mM NAC for 24 h. The results were expressed as a relative percent of MBCL fluorescence in control, untreated with H2O2, cells. DCF fluorescence In order to measure the ROS production in PC12 cells under OS, we used the DCFH-DA method as described previously [15]. Briefly, DCFH-DA crosses the cell membrane and is enzymatically hydrolyzed by intracellular esterases to nonfluorescent DCFH. In the presence of ROS, DCFH is rapidly oxidized to the highly fluorescent 2⬘,7⬘-dichlorofluorescein (DCF). PC12 cells (104 cells per well in a 96-well plate) were loaded with DCFH-DA (final concentration 100 M) for 30 min,
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washed, and treated under one of the experimental conditions followed by exposure to H2O2 at 37°C. Fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 485 nm and em 530 nm (slit 20 and 25 for excitation and emission, respectively). The results were expressed as a relative percent of DCF-fluorescence in control, untreated with H2O2, cells.
Data analysis
Sphingomyelinase activity
The interaction of membrane lipids with oxidative stress
Neutral SPH-specific phospholipase C (ceramidephosphocholine phosphodiesterase, EC 3.1.4.12) activity was assessed in postnuclear fraction by using the commercially available Amplex Red fluorescent method for 96-well plates. Postnuclear fraction was obtained by low-speed centrifugation as described previously [31]. Each reaction contained 50 M Amplex Red reagent, 1 U/ml HRP, 0.1 U/ml choline oxidase, 4 U/ml of alkaline phosphatase, 0.25 mM SPH, and the aliquot of analyzed samples. Cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex/em at 530/590 nm. Sphingomyelinase from Staphylococcus aureus (0 – 40 mU/ml) was employed as a standard. Activity was calculated based on a calibration curve, adjusted per milligram of protein and expressed as milliunits of sphingomyelinase activity per milligram of protein. Ca2⫹ imaging Ca2⫹ image analysis was performed as previously described [7,26]. Briefly, PC12 cells were treated under one of the experimental conditions, washed with fresh media, and loaded with Fura-2/acetoxymethyl ester (2 M) in loading medium (99% RPMI-1640, 1% FBS) for 45 min at 37°C with 5% CO, followed by 30 min incubation in Krebs-Ringer buffer (KRB; 1.3 mM CaCl2; 131 mM NaCl; 1.3 mM MgSO4; 5.0 mM KCL; 0.4 mM KH2PO; 6.0 mM glucose; 20 mM HEPES; pH 7.4). Simultaneous images of cells at ex 340/380 nm and em 510 nm were captured by using SIMCA, a software package designed by Compix (Mars, PA, USA). After 30 s the cells were depolarized by KCl (30 mM final concentration). The cells were imaged for a total of 600 s. Pixel-by-pixel comparisons of the captured images were made using SIMCA, and a ratio of Ca2⫹-bound Fura (340 nm excitation wavelength) to unbound Fura (380 nm excitation wavelength) was generated for each pair of images. Our previous study showed that recovery, i.e., the ability of the cells to remove 80% of [Ca2⫹]I influx, was the calcium parameter that was the most sensitive to OS and to membrane lipid modifications [7]. For this reason, in the present study, recovery was selected as the physiological indicator of Ca2⫹ activity in PC12 cells.
Data were analyzed by ANOVA and Fisher’s LSD test. All statistical analyses were done using SYSTAT software (SPSS Inc., Chicago, IL, USA). RESULTS
The level of oxidative stress was evaluated in PC12 cells exposed to OS induced by low (5 M) or high (300 M) doses of H2O2 by measuring the level of the intracellular antioxidant GSH (Fig. 1). OS induced by H2O2 has a significant effect on the level of GSH [F(2,64) ⫽ 66.689; p ⬍ .001]. In fact, exposure of PC12 cells to either low or high doses of OS for 30 min significantly decreased GSH (Fig. 1, Control vs. Control-tr. with 5 M H2O2, p ⬍ .001; Control vs. Control-tr. with 300 M H2O2, p ⬍ .001). In addition, we observed a significant differential effect of lipid treatments on GSH [F(3,64) ⫽ 3.710; p ⫽ .016]. Although treatment of PC12 cells with either SPH or CHL alone did not affect the intracellular level of GSH in PC12 cells (Fig. 1), the results showed that the modification of the membrane with both SPH-CHL, significantly decreased intracellular GSH when compared to control, even in the absence of OS (Fig. 1, Control vs. SPH-CHL, p ⱕ .01), and had a synergistic effect with high doses of OS further decreasing the intracellular levels of GSH (Fig. 1, 300 M H2O2 vs. SPH-CHL/300 M H2O2; p ⱕ .05). Furthermore, the effect of SPH-CHL under high OS was significantly different from the effect of CHL alone (Fig. 1, SPHCHL-tr. with 300 M H2O2 vs. CHL cond.-tr. with 300 M H2O2; p ⱕ .01); however, it was not different from the effect of SPH on GSH under the same conditions. In addition to measuring the intracellular GSH we also evaluated the intracellular response to OS by measuring ROS. The results in the present study showed that low doses of OS did not affect intracellular level of ROS (Fig. 2). Short-term exposure (10 min) of PC12 cells to high doses of OS did not affect ROS either (data not shown). However, longer exposure (30 min) to high doses of OS significantly increased the intracellular levels of ROS (Fig. 2, Control vs. Control-tr. with 300 M H2O2, p ⱕ .001). Our results show that although the modification of the membrane lipid composition with SPH-CHL significantly potentiated the effect of high doses of OS on intracellular GSH this effect was not mediated by increased levels of ROS as measured by DCF fluorescence (Fig. 2).
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Fig. 1. GSH level was assessed by using a fluorescent method with MBCL as described in Material and Methods. Briefly, PC12 cells were plated in a 96-well plate, cultured with 660 M CHL, 500 M SPH, or combinations of 500 M SPH and 660 M CHL for 60 min followed by 30 min exposure to 5 M or 300 M H2O2. 0.04U/ml GST and 50 M MBCL were added and cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 360 nm and em 460 nm. The results were expressed as a relative percent of MBCL fluorescence in control, untreated with H2O2, cells. Values are means ⫾ SEM of eight individual experiments. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
The interaction of membrane lipids with oxidative stress and N-Sase activity Figure 3 represents the effects of OS and of membrane lipid modifications on N-Sase activity in a partially purified postnuclear preparation from PC12 cells. Our results show a profound effect of OS on N-Sase activity
[F(2,24) ⫽ 230.851; p ⱕ .001]. In fact, a significant up-regulation of N-Sase activity in PC12 cells by low and high doses of OS was observed (Fig. 3, Control vs. Control-tr. with 5 M H2O2, p ⱕ .01; Control vs. Control-tr. with 300 M H2O2, p ⱕ .001). Moreover, the modification of the membrane lipid structure in PC12 cells significantly affected N-Sase activity [F(3,24) ⫽
Fig. 2. Oxidative stress in PC12 cells was evaluated by using DCFH-DA as described in Materials and Methods. Briefly, PC12 cells were plated in a 96-well plate, loaded with DCFH-DA, treated under one of the experimental conditions, exposed either to 5 M or 300 M H2O2 for 30 min, and cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 485 nm and em 530 nm. The results were expressed as a relative percent of DCF fluorescence in control, untreated with H2O2, cells. Values are means ⫾ SEM of eight individual experiments. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
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Fig. 3. Neutral SPH-specific phospholipase C activity was assessed by using the commercially available Amplex Red fluorescent method for 96-well plates. Briefly, PC12 cells (106 cells per each condition) were cultured with 660 M CHL, 500 M SPH, or combinations of 500 M SPH and 660 M CHL for 60 min followed by 30 min exposure to 5 M or 300 M H2O2. The postnuclear fraction was tested for N-Sase activity. Each reaction contained 50 M Amplex Red reagent, 1 U/ml HRP, 0.1 U/ml choline oxidase, 4 U/ml of alkaline phosphatase, 0.25 mM SPH, and the aliquot of analyzed samples. PC12 cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 530 nm and em 590 nm. Sphingomyelinase from Staphylococcus aureus (0 – 40 mU/ml) was employed as a standard. Values are means ⫾ SEM of three individual experiments performed in duplicate. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
141.412; p ⱕ .001]. The results showed a differential effect of CHL and SPH on N-Sase activity. On one hand, PC12 cells treatment with CHL significantly decreased N-Sase activity even in the absence of OS, and attenuated the effects of low and high doses of OS (Fig. 3, Control vs. CHL; p ⱕ .01; 5 M H2O2 vs. CHL/5 M H2O2 p ⱕ 0.001; 300 M H2O2 vs. CHL/300 M H2O2 p ⱕ .001). On the other hand, the modification of the membrane with SPH alone did not affect N-Sase activity and it significantly potentiated the effects of high doses of OS on the N-Sase enzymatic activity when in the concomitant presence of CHL (Fig. 3, 300 M H2O2 vs. SPH-CHL/300 M H2O2; p ⱕ .001). The effect of GSH on N-Sase and calcium activity in PC12 cells Since the modification of the membrane SPH-CHL levels show the most profound effect on intracellular GSH levels, and on N-Sase activity, we explored this membrane lipid modification to test the hypothesis that increased GSH levels in PC12 cells may be sufficient to alter the effect of age-related membrane lipid modification (e.g., SPH/CHL co-treatment) on the “dysregulation” of calcium activity as reflected by Ca2⫹ recovery, and consequently decrease the cellular vulnerability to OS. Figure 4 represents GSH, N-Sase, and calcium recovery in PC12 cells co-cultured with 20 mM NAC.
NAC is a compound known as an antioxidant and efficient thiol source for GSH. Treatments of PC12 cells with 20 mM NAC significantly increased intracellular GSH levels compared to control (Fig. 4A, Control vs. 20 mM NAC cond.; p ⱕ .0001) and attenuated the effect of low and high doses of OS on GSH [F(2,78) ⫽ 41.986, p ⱕ .001]. Moreover, pretreatment with NAC significantly attenuated the synergistic effect of SPH-CHL cotreatment and OS on GSH levels (Fig. 4A, see SPH-CHL cond., p ⱕ .001). Figure 4B represents the N-Sase activity in PC12 cells treated with NAC. Although our results show that repletion of GSH with NAC was sufficient to down-regulate the N-Sase activity in PC12 cells (untreated with lipids) when exposed to 5 M H2O2 (Fig. 4B, see Control vs. Control-tr. with NAC, p ⱕ .001), it did not alter the effect of high (300 M H2O2) doses of OS on this enzyme activity. Figure 4C represents the effects of 5 and 300 M H2O2 on calcium recovery in the presence or absence of NAC and SPH-CHL co-treatments. Exposure to 5 or 300 M H2O2 reduced in a dose-dependent manner the cellular ability to buffer excess calcium after depolarization induced by 30 mM KCl. Surprisingly, repletion of GSH with NAC did not decrease the vulnerability of PC12 cells, untreated with lipids, to OS as reflected by recovery. PC12 simultaneous treatment with SPH-CHL in the absence of OS challenge greatly reduced the ability of the cells to recover from a depolarization event. However, when the cells were exposed to both 20 mM NAC
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Fig. 4. PC12 cells were cultured with 20 mM NAC for 24 h, followed by SPH-CHL treatment, exposed to 5 M or 300 M H2O2 (see Figs. 1 and 3 for details). (A) GSH levels; (B) N-Sase activity; (C) Ca2⫹ recovery data. Values are means ⫾ SEM of eight individual experiments. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
and 5 M H2O2, the recovery was restored to control levels; however, the NAC-induced GSH repletion was ineffective in restoring calcium buffering ability after exposure to SPH-CHL/300 M H2O2 conditions. DISCUSSION
Membrane molecular structure, compartmentalization and physical properties may be crucial in the regulation of
cellular vulnerability to OS and intracellular signaling. Recently it has been shown that aging significantly affects properties of brain membranes. The aging process is accompanied by a decrease in membrane width [27], a change in membrane a symmetry [28], a change in membrane phospholipid and fatty acid composition [29 –33], and a significant modification of the dynamic of cholesterol within the membrane and sphingolipid-cholesterol microdomain [34]. The consequences of alterations in membrane cholesterol and sphingolipid-cholesterol microdomains have become extremely important. There is strong evidence supporting the hypothesis that sphingolipid-cholesterol enriched domains exist in vivo in most cells [35,36]. Moreover, evidence is accumulating that the interaction between sphingolipids and CHL and the consequent formation of sphingolipid-cholesterol enriched microdomains may be essential for the cellular functional activity [35,36]. In the presence of CHL the SPH-CHL membrane domains become more ordered and less fluid than bulk cell membranes because of the lipid-lipid interactions [37]. It has been shown that the association of molecules with SPH-CHL microdomains may significantly alter physical properties of protein molecules [37,38]. For instance, the association of caveolin with SPH-CHL microdomains makes this protein detergent-insoluble. The important role of caveolin in the mediation of cellular response to injury has been recently discussed [39]. Although there are several attractive hypotheses about compartmentalization of sphingolipid signaling in CHLSPH microdomains [36,40], the mechanism remains unknown. We hypothesized that differential brain-regionspecific alterations in the membrane SPH-CHL levels may contribute to the neuronal vulnerability to OS in aging. Our research suggests that the alterations in the membrane CHL may significantly affect intracellular signal transduction through alterations in G-protein activity [31], dopamine release [41], and membrane physical properties [27]. Moreover, high-CHL diet intervention potentiated the effect of aging on intracellular signaling in Fisher 344 rats [42]. A significant brainregion-specific alteration in N-Sase activity is seen in aging [43]. Membrane fractions isolated from the striatum and hippocampus of Fisher 344 rats were significantly enriched in N-Sase. In addition, the activity of this enzyme was up-regulated in aging. Our results obtained in a model system (PC12 cells) support our hypothesis. The enrichment of the cellular membranes with SPH-CHL to approximate those seen in neuronal membranes of old animals significantly increased their vulnerability to OS. Although in the present study we did not observe an interaction of lipid treatments with intracellular ROS as measured by DCF fluorescence, our previous results showed that modification
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of PC12 membrane lipid composition with SPH-CHL significantly increased membrane lipid peroxidation, as measured by membrane levels of conjugated dienes and the phospholipid membrane asymmetry [7]. Thus, these two observations taken together suggest that the modification in membrane lipid composition does not produce OS per se, but it makes the cells more vulnerable to an internal/external oxidative stimulus. In addition, previously we showed that the alterations in SPH-CHL membrane levels also significantly affected phospholipid membrane asymmetry, potentiated the effects of high doses of OS on membrane lipid peroxidation, and decreased the cellular ability to regulate calcium activity [7]. These effects were mediated by SPH metabolites and intracellular GSH [15]. Ceramide and sphingosine significantly increased ROS production, whereas SPP decreased GSH. However, it is possible that other mechanisms may be involved. Recently, the important role of SPP phosphohydrolase in the regulation of cells’ fate has been discussed [44]. It has been suggested that phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate. The present study shows that CHL-SPH modifications can significantly potentiate the effects of high doses of OS by depleting intracellular GSH. The inhibitory effect of increased GSH levels on N-Sase reported in this study are in agreement with other model systems [20 –22,45]. Our results show a significant correlation between decreased GSH levels and increased N-Sase activity in PC12 cells with modified SPH-CHL membrane domains under high doses of OS. Moreover, increases in GSH induced by NAC significantly attenuate the effect of low (physiological) doses of OS on cellular vulnerability. However, increases in GSH were not sufficient to compensate the effect of high doses of OS on cell viability. Although in our study high doses of OS up-regulated N-Sase, it is possible that this effect is Sase-isoform- and OS-dose-dependent. For example, recently an inhibitory effect of OS on N-Sase associated with intracellular membranes has been reported [46]. Multiple forms of signaling sphingomyelinases have been described in a recent review by Levade and Jaffrezou [47]. More and more evidence is suggesting that various sphingomyelinases may participate in defined yet different cellular functions. It is possible that membrane SPH-CHL microdomains and Sase associated with these domains play an important role in age-related neuronal diseases and in mediation of amyloid toxicity. Thus, a differential effect of aging on membrane lipid composition in brain regions may create abnormalities in the membrane molecular structure and consequently determine the fate of the interaction between ROS and intracellular signal transduction molecules. GSH may
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play an important role in this interaction under physiological levels of OS and consequently alter cells’ functional activity as reflected by calcium recovery. Acknowledgements — This work was supported by United States Department of Agriculture Intramural Grant. The authors would like to acknowledge Dr. Donna Bielinski, Derek Fisher, and Ryan Casler for their valuable help in preparation of this manuscript.
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ABBREVIATIONS
OS— oxidative stress CHL— cholesterol SPH—sphingomyelin N-Sase—neutral SPH-specific phospholipase C (ceramidephosphocholine phosphodiesterase, EC 3.1.4.12) ROS—reactive oxygen species NAC—N-acetyl-L-cysteine SPP—sphingosine-1-phosphate DCF—2⬘,7⬘-dichlorofluorescein DCFH-DA—2⬘,7⬘-dichlorofluorescin diacetate MBCL—monochlorobimate GM— growth medium ex/em— excitation/emission wavelengths FBS—fetal bovine serum KRB—Krebs-Ringer buffer
PII S0891-5849(00)00513-X
Original Contribution ROLE OF MEMBRANE LIPIDS IN REGULATION OF VULNERABILITY TO OXIDATIVE STRESS IN PC12 CELLS: IMPLICATION FOR AGING NATALIA A. DENISOVA,* IPPOLITA CANTUTI-CASTELVETRI,* WALEED N. HASSAN,† K. ERIC PAULSON,*† JAMES A. JOSEPH*
and
*United States Department of Agriculture, Jean Mayer Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA; and †Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA (Received18 September 2000; Revised 17 November 2000; Accepted 12 December 2000)
Abstract—Previously, we reported that PC12 cells showed increased vulnerability to oxidative stress (OS) induced by H2O2 (as assessed by decrements in calcium recovery, i.e., the ability of cells to buffer Ca2⫹ after a depolarization event) when the membrane levels of cholesterol (CHL) and sphingomyelin (SPH) were modified to approximate those seen in the neuronal membranes of old animals. The present study was designed to examine whether the enrichment of the membranes with SPH-CHL and increased cellular vulnerability to OS are mediated by neutral SPH-specific phospholipase C (N-Sase) and the intracellular antioxidant GSH. The results showed a significant up-regulation of N-Sase activity by both low (5 M) and high (300 M) doses of H2O2. However, under high doses of H2O2 the up-regulation of N-Sase is accompanied by a significant increase in reactive oxygen species and by a decrease in intracellular GSH. The enrichment of membranes with SPH-CHL significantly potentiated the effects of high doses of H2O2, by further reducing the intracellular GSH and further up-regulating the N-Sase activity. Furthermore, repleting intracellular GSH with 20 mM N-acetylcysteine treatment was sufficient to attenuate the effect of a low dose of H2O2 on Ca2⫹ recovery in SPH-CHL-treated cells. Thus, these results suggested that age-related alterations in the membrane SPH-CHL levels could be important determinants of the susceptibility of neuronal cells to OS. © 2001 Elsevier Science Inc. Keywords—Oxidative stress, Membrane cholesterol and sphingomyelin, Neutral sphingomyelinase, Glutathione, PC12 cells, Free radicals
changes in calcium homeostasis, i.e., enhanced Ca2⫹ flux through voltage-gated calcium channels, decreased activity of plasma membrane Ca2⫹-ATPase and Na⫹/Ca2⫹ exchanger, decreased Ca2⫹ transport in mitochondria, and a significant loss of calcium binding proteins has been observed. These all result in an impaired homeostasis of intracellular calcium with increased levels of free intracellular Ca2⫹ that may ultimately lead to cell death [8 –14]. Recent research from our laboratory demonstrated that an SPH-specific phospholipase C (ceramide-phosphocholine phosphodiesterase, EC 3.1.4.12) induced production of reactive oxygen species (ROS) in a cellular model, and may be directly involved in the loss of the ability to regulate intracellular Ca2⫹ homeostasis [15]. In addition, increases in the metabolism of membrane SPH, associated with increased levels of its metabolites, such as sphingosine-1-phosphate (SPP), may be particularly important in increasing the vulnerability of PC12 cells to
INTRODUCTION
Past research indicates that aging is often accompanied by a significant impairment in behavioral performance and neuronal plasticity [1– 4]. While the etiology of these impairments is not known, we hypothesized that agerelated alterations in membrane molecular structure may contribute to alterations in calcium (Ca2⫹) homeostasis, and ultimately lead to the increased neuronal vulnerability to OS seen in aging. Recent research from our laboratory suggests that modifications of membrane lipid composition with CHL and SPH to levels similar to those seen in neuronal membranes in aging [5,6] dramatically increased cells’ vulnerability to OS as reflected by an impairment in Ca2⫹ homeostasis [7]. During aging, Address correspondence to: Natalia A. Denisova, Ph.D., USDAHNRCA at Tufts University, 711 Washington Street, Boston, MA 02111, USA; Tel: (617) 556-3147; Fax: (617) 556-3222; E-Mail: [email protected]. 671
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OS as measured by Ca2⫹ recovery. Although the mechanisms responsible for the effect of SPP on Ca2⫹ recovery under OS remain unknown, our results suggest that GSH may play a critical role. In fact, it has been shown that a decrease in GSH levels may result in Ca2⫹-mediated cell death in PC12 cells [16]. The GSH/GSSG system plays a pivotal role in the control of the cellular redox status, and is the primary defense mechanism for the removal of peroxides in the brain [17–19]. Recently, an inhibitory effect of GSH on N-Sase activity has been suggested [20 –23]. Highly purified bovine N-Sase activity was effectively blocked by GSH [24]. Increase in intracellular GSH levels induced by treatment with N-acetyl-L-cysteine (NAC) significantly inhibited N-Sase under hypoxic conditions in PC12 cells [22]. In fact, up-regulation of membrane N-Sase may significantly affect the membrane SPH levels and consequently the CHL distribution within the membrane. Preliminary results in rats showed that specific areas of the brain presented different markers of age-related vulnerability to OS, such as brain-area-specific changes in levels of SPH-CHL, ROS production, membrane MDA/HNE, GSH levels, and Ca2⫹ homeostasis [25]. Therefore, given that membrane lipid composition can be significantly affected by the aging process [27–34] and this can modulate cellular sensitivity to OS through alterations in GSH, we designed the present experiments to evaluate whether the enrichment of membranes with SPH-CHL, which increases cell vulnerability to OS as reflected through calcium activity (recovery), is mediated by GSH and N-Sase. MATERIALS AND METHODS
Materials PC12 cells were a generous gift of Dr. Tischler (Tufts University, Boston, MA, USA). RPMI-1640, horse serum, fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). All reagents used were of the highest grade available. Organic solvents were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Cholesterol (nonesterified), sphingomyelin (acylated, 25.5% stearic, and 42.2% nervonic acids) and albumin (fatty acid content 0.01%) as well as all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2⬘,7⬘dichlorofluorescin diacetate (DCFH-DA), monochlorobimate (MBCL), and Amplex Red sphingomyelinase assay kit were obtained from Molecular Probes, Inc. (Eugene, OR, USA).
Cell culture and treatment PC12 cells were seeded onto 100 mm plates coated with rat-tail collagen (type VII) and grown at 37°C under an atmosphere of 5% CO2/95% air, in complete growth medium (GM: RPMI-1640 supplemented with 2 mM glutamine, 10% heat-inactivated horse serum, 5% FBS, 120 U/ml penicillin, and 120 g/ml streptomycin). All PC12 cells were of passage 50 –54. PC12 membrane lipid composition was modified as described previously [7]. Briefly, PC12 cells were incubated for 1 h at 37°C under one of the following conditions: (i) 500 M SPH in GM, (ii) 660 M CHL in GM, or (iii) 500 M SPH-660 M CHL. At the conclusion of the incubation period, cells were washed three times with fresh GM to remove any lipids that were not incorporated into the cells. A subset of these cultures was then exposed to different concentrations of H2O2 (0, 5, 300 M) for 30 min. The H2O2 was prepared immediately prior to the experiments from a concentrated (9.8 M) stock solution. At the conclusion of the incubation period, the medium with H2O2 was removed and the plates were washed with fresh GM. Postnuclear fraction was isolated from PC12 cells by centrifugation of the cell homogenate for 10 min at 1000 g at 4°C. GSH levels The GSH level in PC12 cells was analyzed by using the fluorescent method with MBCL as described previously [15]. Briefly, PC12 cells were plated in a 96-well plate (102–104 cells per well), 0.04 U/ml GST and 50 M MBCL were added and cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader (PerSeptive Biosystem, Framingham, MA, USA) with ex 360 nm and em 460 nm. In order to increase GSH levels in PC12 cells, we employed N-Acetylcysteine (NAC). PC12 cells (104 cells per well) were plated in a 96-well plate, and cultured with 20 mM NAC for 24 h. The results were expressed as a relative percent of MBCL fluorescence in control, untreated with H2O2, cells. DCF fluorescence In order to measure the ROS production in PC12 cells under OS, we used the DCFH-DA method as described previously [15]. Briefly, DCFH-DA crosses the cell membrane and is enzymatically hydrolyzed by intracellular esterases to nonfluorescent DCFH. In the presence of ROS, DCFH is rapidly oxidized to the highly fluorescent 2⬘,7⬘-dichlorofluorescein (DCF). PC12 cells (104 cells per well in a 96-well plate) were loaded with DCFH-DA (final concentration 100 M) for 30 min,
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washed, and treated under one of the experimental conditions followed by exposure to H2O2 at 37°C. Fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 485 nm and em 530 nm (slit 20 and 25 for excitation and emission, respectively). The results were expressed as a relative percent of DCF-fluorescence in control, untreated with H2O2, cells.
Data analysis
Sphingomyelinase activity
The interaction of membrane lipids with oxidative stress
Neutral SPH-specific phospholipase C (ceramidephosphocholine phosphodiesterase, EC 3.1.4.12) activity was assessed in postnuclear fraction by using the commercially available Amplex Red fluorescent method for 96-well plates. Postnuclear fraction was obtained by low-speed centrifugation as described previously [31]. Each reaction contained 50 M Amplex Red reagent, 1 U/ml HRP, 0.1 U/ml choline oxidase, 4 U/ml of alkaline phosphatase, 0.25 mM SPH, and the aliquot of analyzed samples. Cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex/em at 530/590 nm. Sphingomyelinase from Staphylococcus aureus (0 – 40 mU/ml) was employed as a standard. Activity was calculated based on a calibration curve, adjusted per milligram of protein and expressed as milliunits of sphingomyelinase activity per milligram of protein. Ca2⫹ imaging Ca2⫹ image analysis was performed as previously described [7,26]. Briefly, PC12 cells were treated under one of the experimental conditions, washed with fresh media, and loaded with Fura-2/acetoxymethyl ester (2 M) in loading medium (99% RPMI-1640, 1% FBS) for 45 min at 37°C with 5% CO, followed by 30 min incubation in Krebs-Ringer buffer (KRB; 1.3 mM CaCl2; 131 mM NaCl; 1.3 mM MgSO4; 5.0 mM KCL; 0.4 mM KH2PO; 6.0 mM glucose; 20 mM HEPES; pH 7.4). Simultaneous images of cells at ex 340/380 nm and em 510 nm were captured by using SIMCA, a software package designed by Compix (Mars, PA, USA). After 30 s the cells were depolarized by KCl (30 mM final concentration). The cells were imaged for a total of 600 s. Pixel-by-pixel comparisons of the captured images were made using SIMCA, and a ratio of Ca2⫹-bound Fura (340 nm excitation wavelength) to unbound Fura (380 nm excitation wavelength) was generated for each pair of images. Our previous study showed that recovery, i.e., the ability of the cells to remove 80% of [Ca2⫹]I influx, was the calcium parameter that was the most sensitive to OS and to membrane lipid modifications [7]. For this reason, in the present study, recovery was selected as the physiological indicator of Ca2⫹ activity in PC12 cells.
Data were analyzed by ANOVA and Fisher’s LSD test. All statistical analyses were done using SYSTAT software (SPSS Inc., Chicago, IL, USA). RESULTS
The level of oxidative stress was evaluated in PC12 cells exposed to OS induced by low (5 M) or high (300 M) doses of H2O2 by measuring the level of the intracellular antioxidant GSH (Fig. 1). OS induced by H2O2 has a significant effect on the level of GSH [F(2,64) ⫽ 66.689; p ⬍ .001]. In fact, exposure of PC12 cells to either low or high doses of OS for 30 min significantly decreased GSH (Fig. 1, Control vs. Control-tr. with 5 M H2O2, p ⬍ .001; Control vs. Control-tr. with 300 M H2O2, p ⬍ .001). In addition, we observed a significant differential effect of lipid treatments on GSH [F(3,64) ⫽ 3.710; p ⫽ .016]. Although treatment of PC12 cells with either SPH or CHL alone did not affect the intracellular level of GSH in PC12 cells (Fig. 1), the results showed that the modification of the membrane with both SPH-CHL, significantly decreased intracellular GSH when compared to control, even in the absence of OS (Fig. 1, Control vs. SPH-CHL, p ⱕ .01), and had a synergistic effect with high doses of OS further decreasing the intracellular levels of GSH (Fig. 1, 300 M H2O2 vs. SPH-CHL/300 M H2O2; p ⱕ .05). Furthermore, the effect of SPH-CHL under high OS was significantly different from the effect of CHL alone (Fig. 1, SPHCHL-tr. with 300 M H2O2 vs. CHL cond.-tr. with 300 M H2O2; p ⱕ .01); however, it was not different from the effect of SPH on GSH under the same conditions. In addition to measuring the intracellular GSH we also evaluated the intracellular response to OS by measuring ROS. The results in the present study showed that low doses of OS did not affect intracellular level of ROS (Fig. 2). Short-term exposure (10 min) of PC12 cells to high doses of OS did not affect ROS either (data not shown). However, longer exposure (30 min) to high doses of OS significantly increased the intracellular levels of ROS (Fig. 2, Control vs. Control-tr. with 300 M H2O2, p ⱕ .001). Our results show that although the modification of the membrane lipid composition with SPH-CHL significantly potentiated the effect of high doses of OS on intracellular GSH this effect was not mediated by increased levels of ROS as measured by DCF fluorescence (Fig. 2).
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Fig. 1. GSH level was assessed by using a fluorescent method with MBCL as described in Material and Methods. Briefly, PC12 cells were plated in a 96-well plate, cultured with 660 M CHL, 500 M SPH, or combinations of 500 M SPH and 660 M CHL for 60 min followed by 30 min exposure to 5 M or 300 M H2O2. 0.04U/ml GST and 50 M MBCL were added and cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 360 nm and em 460 nm. The results were expressed as a relative percent of MBCL fluorescence in control, untreated with H2O2, cells. Values are means ⫾ SEM of eight individual experiments. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
The interaction of membrane lipids with oxidative stress and N-Sase activity Figure 3 represents the effects of OS and of membrane lipid modifications on N-Sase activity in a partially purified postnuclear preparation from PC12 cells. Our results show a profound effect of OS on N-Sase activity
[F(2,24) ⫽ 230.851; p ⱕ .001]. In fact, a significant up-regulation of N-Sase activity in PC12 cells by low and high doses of OS was observed (Fig. 3, Control vs. Control-tr. with 5 M H2O2, p ⱕ .01; Control vs. Control-tr. with 300 M H2O2, p ⱕ .001). Moreover, the modification of the membrane lipid structure in PC12 cells significantly affected N-Sase activity [F(3,24) ⫽
Fig. 2. Oxidative stress in PC12 cells was evaluated by using DCFH-DA as described in Materials and Methods. Briefly, PC12 cells were plated in a 96-well plate, loaded with DCFH-DA, treated under one of the experimental conditions, exposed either to 5 M or 300 M H2O2 for 30 min, and cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 485 nm and em 530 nm. The results were expressed as a relative percent of DCF fluorescence in control, untreated with H2O2, cells. Values are means ⫾ SEM of eight individual experiments. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
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Fig. 3. Neutral SPH-specific phospholipase C activity was assessed by using the commercially available Amplex Red fluorescent method for 96-well plates. Briefly, PC12 cells (106 cells per each condition) were cultured with 660 M CHL, 500 M SPH, or combinations of 500 M SPH and 660 M CHL for 60 min followed by 30 min exposure to 5 M or 300 M H2O2. The postnuclear fraction was tested for N-Sase activity. Each reaction contained 50 M Amplex Red reagent, 1 U/ml HRP, 0.1 U/ml choline oxidase, 4 U/ml of alkaline phosphatase, 0.25 mM SPH, and the aliquot of analyzed samples. PC12 cell-associated fluorescence was monitored on a Cyto Fluor Multi-Well Plate Reader with ex 530 nm and em 590 nm. Sphingomyelinase from Staphylococcus aureus (0 – 40 mU/ml) was employed as a standard. Values are means ⫾ SEM of three individual experiments performed in duplicate. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
141.412; p ⱕ .001]. The results showed a differential effect of CHL and SPH on N-Sase activity. On one hand, PC12 cells treatment with CHL significantly decreased N-Sase activity even in the absence of OS, and attenuated the effects of low and high doses of OS (Fig. 3, Control vs. CHL; p ⱕ .01; 5 M H2O2 vs. CHL/5 M H2O2 p ⱕ 0.001; 300 M H2O2 vs. CHL/300 M H2O2 p ⱕ .001). On the other hand, the modification of the membrane with SPH alone did not affect N-Sase activity and it significantly potentiated the effects of high doses of OS on the N-Sase enzymatic activity when in the concomitant presence of CHL (Fig. 3, 300 M H2O2 vs. SPH-CHL/300 M H2O2; p ⱕ .001). The effect of GSH on N-Sase and calcium activity in PC12 cells Since the modification of the membrane SPH-CHL levels show the most profound effect on intracellular GSH levels, and on N-Sase activity, we explored this membrane lipid modification to test the hypothesis that increased GSH levels in PC12 cells may be sufficient to alter the effect of age-related membrane lipid modification (e.g., SPH/CHL co-treatment) on the “dysregulation” of calcium activity as reflected by Ca2⫹ recovery, and consequently decrease the cellular vulnerability to OS. Figure 4 represents GSH, N-Sase, and calcium recovery in PC12 cells co-cultured with 20 mM NAC.
NAC is a compound known as an antioxidant and efficient thiol source for GSH. Treatments of PC12 cells with 20 mM NAC significantly increased intracellular GSH levels compared to control (Fig. 4A, Control vs. 20 mM NAC cond.; p ⱕ .0001) and attenuated the effect of low and high doses of OS on GSH [F(2,78) ⫽ 41.986, p ⱕ .001]. Moreover, pretreatment with NAC significantly attenuated the synergistic effect of SPH-CHL cotreatment and OS on GSH levels (Fig. 4A, see SPH-CHL cond., p ⱕ .001). Figure 4B represents the N-Sase activity in PC12 cells treated with NAC. Although our results show that repletion of GSH with NAC was sufficient to down-regulate the N-Sase activity in PC12 cells (untreated with lipids) when exposed to 5 M H2O2 (Fig. 4B, see Control vs. Control-tr. with NAC, p ⱕ .001), it did not alter the effect of high (300 M H2O2) doses of OS on this enzyme activity. Figure 4C represents the effects of 5 and 300 M H2O2 on calcium recovery in the presence or absence of NAC and SPH-CHL co-treatments. Exposure to 5 or 300 M H2O2 reduced in a dose-dependent manner the cellular ability to buffer excess calcium after depolarization induced by 30 mM KCl. Surprisingly, repletion of GSH with NAC did not decrease the vulnerability of PC12 cells, untreated with lipids, to OS as reflected by recovery. PC12 simultaneous treatment with SPH-CHL in the absence of OS challenge greatly reduced the ability of the cells to recover from a depolarization event. However, when the cells were exposed to both 20 mM NAC
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Fig. 4. PC12 cells were cultured with 20 mM NAC for 24 h, followed by SPH-CHL treatment, exposed to 5 M or 300 M H2O2 (see Figs. 1 and 3 for details). (A) GSH levels; (B) N-Sase activity; (C) Ca2⫹ recovery data. Values are means ⫾ SEM of eight individual experiments. (a) different from control, untreated with H2O2; (b) different from control treated with 5 M H2O2; (c) different from control treated with 300 M H2O2.
and 5 M H2O2, the recovery was restored to control levels; however, the NAC-induced GSH repletion was ineffective in restoring calcium buffering ability after exposure to SPH-CHL/300 M H2O2 conditions. DISCUSSION
Membrane molecular structure, compartmentalization and physical properties may be crucial in the regulation of
cellular vulnerability to OS and intracellular signaling. Recently it has been shown that aging significantly affects properties of brain membranes. The aging process is accompanied by a decrease in membrane width [27], a change in membrane a symmetry [28], a change in membrane phospholipid and fatty acid composition [29 –33], and a significant modification of the dynamic of cholesterol within the membrane and sphingolipid-cholesterol microdomain [34]. The consequences of alterations in membrane cholesterol and sphingolipid-cholesterol microdomains have become extremely important. There is strong evidence supporting the hypothesis that sphingolipid-cholesterol enriched domains exist in vivo in most cells [35,36]. Moreover, evidence is accumulating that the interaction between sphingolipids and CHL and the consequent formation of sphingolipid-cholesterol enriched microdomains may be essential for the cellular functional activity [35,36]. In the presence of CHL the SPH-CHL membrane domains become more ordered and less fluid than bulk cell membranes because of the lipid-lipid interactions [37]. It has been shown that the association of molecules with SPH-CHL microdomains may significantly alter physical properties of protein molecules [37,38]. For instance, the association of caveolin with SPH-CHL microdomains makes this protein detergent-insoluble. The important role of caveolin in the mediation of cellular response to injury has been recently discussed [39]. Although there are several attractive hypotheses about compartmentalization of sphingolipid signaling in CHLSPH microdomains [36,40], the mechanism remains unknown. We hypothesized that differential brain-regionspecific alterations in the membrane SPH-CHL levels may contribute to the neuronal vulnerability to OS in aging. Our research suggests that the alterations in the membrane CHL may significantly affect intracellular signal transduction through alterations in G-protein activity [31], dopamine release [41], and membrane physical properties [27]. Moreover, high-CHL diet intervention potentiated the effect of aging on intracellular signaling in Fisher 344 rats [42]. A significant brainregion-specific alteration in N-Sase activity is seen in aging [43]. Membrane fractions isolated from the striatum and hippocampus of Fisher 344 rats were significantly enriched in N-Sase. In addition, the activity of this enzyme was up-regulated in aging. Our results obtained in a model system (PC12 cells) support our hypothesis. The enrichment of the cellular membranes with SPH-CHL to approximate those seen in neuronal membranes of old animals significantly increased their vulnerability to OS. Although in the present study we did not observe an interaction of lipid treatments with intracellular ROS as measured by DCF fluorescence, our previous results showed that modification
Membrane lipids and oxidative stress
of PC12 membrane lipid composition with SPH-CHL significantly increased membrane lipid peroxidation, as measured by membrane levels of conjugated dienes and the phospholipid membrane asymmetry [7]. Thus, these two observations taken together suggest that the modification in membrane lipid composition does not produce OS per se, but it makes the cells more vulnerable to an internal/external oxidative stimulus. In addition, previously we showed that the alterations in SPH-CHL membrane levels also significantly affected phospholipid membrane asymmetry, potentiated the effects of high doses of OS on membrane lipid peroxidation, and decreased the cellular ability to regulate calcium activity [7]. These effects were mediated by SPH metabolites and intracellular GSH [15]. Ceramide and sphingosine significantly increased ROS production, whereas SPP decreased GSH. However, it is possible that other mechanisms may be involved. Recently, the important role of SPP phosphohydrolase in the regulation of cells’ fate has been discussed [44]. It has been suggested that phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate. The present study shows that CHL-SPH modifications can significantly potentiate the effects of high doses of OS by depleting intracellular GSH. The inhibitory effect of increased GSH levels on N-Sase reported in this study are in agreement with other model systems [20 –22,45]. Our results show a significant correlation between decreased GSH levels and increased N-Sase activity in PC12 cells with modified SPH-CHL membrane domains under high doses of OS. Moreover, increases in GSH induced by NAC significantly attenuate the effect of low (physiological) doses of OS on cellular vulnerability. However, increases in GSH were not sufficient to compensate the effect of high doses of OS on cell viability. Although in our study high doses of OS up-regulated N-Sase, it is possible that this effect is Sase-isoform- and OS-dose-dependent. For example, recently an inhibitory effect of OS on N-Sase associated with intracellular membranes has been reported [46]. Multiple forms of signaling sphingomyelinases have been described in a recent review by Levade and Jaffrezou [47]. More and more evidence is suggesting that various sphingomyelinases may participate in defined yet different cellular functions. It is possible that membrane SPH-CHL microdomains and Sase associated with these domains play an important role in age-related neuronal diseases and in mediation of amyloid toxicity. Thus, a differential effect of aging on membrane lipid composition in brain regions may create abnormalities in the membrane molecular structure and consequently determine the fate of the interaction between ROS and intracellular signal transduction molecules. GSH may
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play an important role in this interaction under physiological levels of OS and consequently alter cells’ functional activity as reflected by calcium recovery. Acknowledgements — This work was supported by United States Department of Agriculture Intramural Grant. The authors would like to acknowledge Dr. Donna Bielinski, Derek Fisher, and Ryan Casler for their valuable help in preparation of this manuscript.
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ABBREVIATIONS
OS— oxidative stress CHL— cholesterol SPH—sphingomyelin N-Sase—neutral SPH-specific phospholipase C (ceramidephosphocholine phosphodiesterase, EC 3.1.4.12) ROS—reactive oxygen species NAC—N-acetyl-L-cysteine SPP—sphingosine-1-phosphate DCF—2⬘,7⬘-dichlorofluorescein DCFH-DA—2⬘,7⬘-dichlorofluorescin diacetate MBCL—monochlorobimate GM— growth medium ex/em— excitation/emission wavelengths FBS—fetal bovine serum KRB—Krebs-Ringer buffer