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Originally published In Press as doi:10.1074/jbc.M510650200 on January 24, 2006 Originally published In Press as doi:10.1074/jbc.M510650200 on January 23, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9460-9470, April 7, 2006
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Intracellular Zinc Release and ERK Phosphorylation Are Required Upstream of 12-Lipoxygenase Activation in Peroxynitrite Toxicity to Mature Rat Oligodendrocytes*

Yumin Zhang{ddagger}, Hong Wang{ddagger}, Jianrong Li{ddagger}, Ling Dong{ddagger}, Ping Xu{ddagger}, Weizhi Chen{ddagger}, Rachael L. Neve§, Joseph J. Volpe{ddagger}, and Paul A. Rosenberg{ddagger}1

From the {ddagger}Department of Neurology and Program in Neuroscience, Children's Hospital and Harvard Medical School, the §Department of Psychiatry, Harvard Medical School, Boston, Massachusetts 02115 and McLean Hospital, Belmont, Massachusetts 02478

Received for publication, September 29, 2005 , and in revised form, December 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxynitrite toxicity has been implicated in the pathogenesis of white matter injury. The mechanisms of peroxynitrite toxicity to oligodendrocytes (OLs), the major cell type of the white matter, are unknown. Using primary cultures of mature OLs that express myelin basic protein, we found that 3-morpholinosydnonimine, a peroxynitrite generator, caused toxicity to OLs. N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a zinc chelator, completely blocked peroxynitrite-induced toxicity. Use of FluoZin-3, a specific fluorescence zinc indicator, demonstrated the liberation of zinc from intracellular stores by peroxynitrite. Peroxynitrite caused the sequential activation of extracellular signal-regulated kinase 42/44 (ERK42/44), 12-lipoxygenase, and generation of reactive oxygen species, which were all dependent upon the intracellular release of zinc. The same cell death pathway was also activated when exogenous zinc was used. These results suggest that in addition to preventing the formation of peroxynitrite, useful strategies in preventing disease progression in pathologies in which peroxynitrite toxicity plays a critical role might include maintaining intracellular zinc homeostasis, blocking phosphorylation of ERK42/44, inhibiting activation of 12-lipoxygenase, and eliminating the accumulation of reactive oxygen species.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxynitrite, the reaction product of nitric oxide and superoxide (1), is a potent oxidant capable of inducing DNA damage (2), lipid peroxidation (3), and protein nitration (4, 5). A traditional view of the action of peroxynitrite is direct attack by oxidation and nitration of molecules vital for cell survival to cause cell death (6). This scenario suggests little opportunity for therapeutic intervention to protect cells exposed to peroxynitrite. An alternative more recent view is that peroxynitrite triggers a multistage signaling cascade that would permit therapeutic intervention.

Stimulation of phospholipase A2 and the subsequent activation of arachidonic acid (AA)2 metabolism by lipoxygenase (LOX) and cycloxygenase (COX) pathways have been implicated in peroxynitrite-induced toxicity in PC12 cells (7, 8). It also has been reported that peroxynitrite toxicity to thymocytes is mediated by zinc release from intracellular stores (9). Very recently, it has been shown that peroxynitrite can induce intracellular zinc release, ROS generation, and toxicity in neurons (10-12). Zinc liberated by methylisothiazolinone, another oxidative agent, has been suggested to cause activation of 12-LOX, extracellular signal-regulated kinase 42/44 (ERK42/44), and ROS generation, leading to neurotoxicity (13). These results suggest that zinc release and activation of arachidonic acid metabolic pathways might be involved in peroxynitrite-induced toxicity.

Myelin forming oligodendrocytes are the major cell type impaired in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (14). Although it is controversial whether blocking nitric oxide production is beneficial in the treatment of MS (15-18), there is consensus that use of scavengers or decomposition catalysts of peroxynitrite can prevent disease progression in experimental allergic encephalomyelitis (17, 19-21). One such compound is uric acid; interestingly, serum uric acid levels were found to be significantly lower in MS patients than in non-MS patients (19). In addition, in a large population survey, the occurrence of MS and gout (hyperuricemic) appeared to be mutually exclusive, suggesting that hyperuricemia may protect against MS (19). These studies raise the possibility that peroxynitrite plays a critical role in the pathogenesis of MS. Nitric oxide and peroxynitrite have previously been reported to cause toxicity to oligodendrocytes (22-24). We have found that mitochondrial dysfunction and the translocation of apoptosis inducing factor from mitochondria to the nucleus are associated with nitric oxide-induced toxicity to oligodendrocytes (OLs) (25). It is currently unknown how peroxynitrite induces injury to oligodendrocytes. Using primary cultures of rat brain OLs that express myelin basic protein, we found that peroxynitrite toxicity to OLs is mediated via a zinc-12-LOX-ROS pathway, and that ERK activation is upstream of 12-LOX activation and ROS generation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—3-Morpholinosydnonimine (SIN-1), peroxynitrite, carboxyl-PTIO, and FeTMPyP were obtained from Cayman Chemical Co. (Ann Arbor, MI). Newport Green, FluoZin-3, dihydrorhodamine 123, and 2',7'-dichlorohydrofluorescein diacetate (DCF) were purchased from Molecular Probes, Inc. (Eugene, OR). AA-861 was purchased from BioMol Research Laboratories (Plymouth Meeting, PA). N-Benzyl-N-hydroxy-5-phenylpentanamide (BHPP) was a gift from Dr. Lawrence J. Marnett (School of Medicine, Vanderbilt University, Nashville, TN). Plasmid DNAs containing dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) 1 and 2, S222A MEK1 and K101A MEK2, were gifts from Drs. Rony Seger (Weizmann Institute of Science, Israel) and Jeffery Holt (Vanderbilt University, Nashville, TN), respectively. All the culture materials were purchased from Invitrogen. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, and ciliary neurotrophic factor were from Peprotech (Princeton, NJ). All other reagents were obtained from Sigma.

Oligodendrocyte Culture—Primary cultures of OLs were prepared by shaking off the progenitor cells from mixed glial cell cultures as previously described (26). Briefly, mixed primary glial cultures were prepared from 1-2-day-old Sprague-Dawley rats by dissociation of the brains after they were dissected from the pups. Cultures were maintained in Dulbecco's modified Eagle's medium containing 20% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (DMEM 20S) in polylysine-coated 75-cm2 flasks incubated in 95% air, 5% CO2, at 37 °C. The medium was changed 3 times per week until the cells were confluent (7-10 days). The flasks were then shaken for 1 h on an orbital shaker (200 rpm) at 37 °C to remove microglia. They were then changed to new medium and shaken overnight. The oligodendrocyte progenitor cells were detached from the astrocyte layer and resuspended and seeded onto polyornithine-coated plates (96-well (2.3 x 105 cells/plate) and 24-well (3 x 105 cells/plate)) in a basal chemically defined medium (BDM) (Dulbecco's modified Eagle's medium with 1 mg/ml bovine serum albumin (BSA), 50 µg/ml apotransferrin, 5 µg/ml insulin, 30 nM sodium selenite, 10 nM biotin, 10 nM hydrocortisone) plus 10 ng/ml of both platelet-derived growth factor and basic fibroblast growth factor. The cells were maintained in the BDM and the medium was half-changed 3 times per week. For culturing mature OLs, OLs at day 7 were changed to BDM plus 3,3',5-triodo-L-thyronine (T3) (15 nM) and ciliary neurotrophic factor (10 ng/ml) and were half-changed 3 times per week for 2 weeks. At this stage, more than 90% of the cells were myelin basic protein positive (25, 26) and were used for the experiments. The contamination of astrocytes and microglia was 1-2% each.

Toxicity Assay—The survival of the cells after various treatments in 96-well plates were evaluated by visual inspection using phase-contrast microscopy and quantified by using Alamar Blue (Trek Diagnostic Systems, Inc., Westlake, OH), a viability assay that was previously described and validated by cell counting using trypan blue exclusion (27, 28). In all experiments, the culture plates were first washed twice with Hank's balanced salt solution containing 0.1% BSA and then placed in Earle's balanced salt solution (EBSS, which is composed of 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 26 mM NaHCO3, 1 mM NaH2PO4, and 5.5 mM D-glucose). OLs were treated with SIN-1 or peroxynitrite for 2 h or zinc chloride (ZnCl2) for 90 min, washed twice with Hank's balanced salt solution containing 0.1% BSA, and then placed in BDM with T3 and ciliary neurotrophic factor. All drugs were applied 15 min before, during, and after the cells were exposed to SIN-1, peroxynitrite, or ZnCl2. After the cells were incubated for 20-24 h, the culture medium was replaced with EBSS plus a 1:100 dilution of Alamar Blue. After 2 h exposure, the fluorescence of the Alamar Blue solution in each well in the plates was read at room temperature in a fluorescent plate reader (FluoroCount, Packard Instrument Co., Meriden, CT) with excitation wavelength at 530 nm and emission wavelength at 590 nm. The data from Alamar Blue assays matched the results from visual inspection.

Fluorescence Imaging of Intracellular Liberation of Zinc—Changes in intracellular free zinc concentration in OLs were monitored with a high affinity, zinc selective indicator, FluoZin-3 (29). OLs were loaded with FluoZin-3 (1 µM) for 30 min, washed with Hank's balanced salt solution containing 0.1% BSA, and then treated with SIN-1 (1 mM) for various times. The fluorescence imaging of intracellular zinc was monitored immediately using digital fluorescence microscopy with a x20 objective (excitation at 485 nm, emission at 530 nm). For all images, the microscope settings, such as brightness, contrast, and exposure time, were held constant to compare the relative intensity of intracellular zinc fluorescence across all treatment conditions.


Figure 1
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  		FIGURE 1.
SIN-1 toxicity to OLs was blocked by nitric oxide, superoxide, and peroxynitrite scavengers. A, concentration dependence of SIN-1 toxicity to OLs. A representative experiment of six that were performed is shown. In this experiment, the EC50 value of SIN-1 in producing OL toxicity was 300 µM. B, SIN-1 toxicity was mediated by peroxynitrite formation. SIN-1 (500 µM) toxicity was fully prevented by carboxyl-PTIO (C-PTIO, 50 µM), superoxide dismutase together with catalase (S+C, 100 units/ml each), FeTMPyP (10 µM), and uric acid (UA, 1 mM), respectively. ***, p < 0.001 was obtained when the drug-treated groups were compared with the group treated with SIN-1 alone. A representative experiment of three performed is shown.

 
Measurement of 12-LOX Activity—12-LOX activity was measured by quantifying the major metabolic product of 12-LOX, 12-hydroxyeicosatetraenoic acid (12-HETE), by using an enzyme-linked immunosorbent assay kit (Assay Designs Inc., Ann Harbor, MI). OLs were treated with SIN-1 or ZnCl2 for various times or at 60 min with TPEN or U0126 present. After the cultures were washed twice with Hank's balanced salt solution containing 0.1% BSA, arachidonic acid (20 µM) in EBSS was added to the culture, which was then incubated for 30 min. The medium was then collected, extracted, and assayed for 12-HETE according to the manufacturer's protocol. The concentration of 12-HETE in each sample was normalized to the protein concentration of the sample.

Measurement of Intracellular ROS Generation—Intracellular free radical generation was evaluated with DCF (30, 31) and dihydrorhodamine 123 (Rho 123) (Molecular Probes, Eugene, OR) (27, 32). Briefly, after the cells in 96-well plates or on coverslips in 24-well plates were treated with SIN-1 or zinc for various times in the absence or presence of drugs, they were washed with EBSS and loaded with DCF (100 µM) for 30 min or Rho 123 (10 µM) for 20 min in EBSS (95% air, 5% CO2, 37 °C). After the loading solution was removed, the cells in the wells were washed and incubated in EBSS. The fluorescence of the cells in each well was measured and recorded in the fluorescence plate reader as described above (with excitation at {lambda} = 480 nm, emission at {lambda} = 530 nm, and temperature set to 37 °C). For fluorescence imaging of oxidized Rho 123, the oxidized form of dihydrorhodamine, cells plated on coverslips after treatment were immediately visualized using a digital fluorescent microscope equipped with a x20 objective. Cells were visualized by excitation at 490 nm and emission at 515 nm.


Figure 2
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  		FIGURE 2.
Peroxynitrite toxicity to OLs was blocked by a zinc chelator. A, concentration dependence of TPEN protection against SIN-1 (500 µM) toxicity. A representative experiment of three that were performed is shown. The EC50 value of TPEN protection in this experiment was 0.4 µM. B, TPEN protected against peroxynitrite-induced toxicity to OLs. TPEN at 10 µM completely blocked OL toxicity induced by various concentrations of peroxynitrite (100, 300, and 500 µM). **, p < 0.01 and ***, p < 0.001 were obtained when the TPEN-treated groups were compared with the corresponding peroxynitrite groups without TPEN treatment. A representative experiment of three performed is shown. C, TPEN protection against SIN-1-induced toxicity was eliminated by co-administration of equimolar concentrations of ZnCl2 (10 µM), but not FeCl2 (10 µM). ***, p < 0.001 was obtained when the TPEN-treated group was compared with the group treated with SIN-1 alone. A representative experiment of three performed is shown. D, TPEN blocked the increase of FluoZin-3 fluorescence induced by SIN-1. OLs were treated with SIN-1 (1 mM) for various times, and then loaded with FluoZin-3 for 30 min. The time-dependent increase of FluoZin-3 fluorescence is shown. In the presence of TPEN (T), the fluorescence observed at 60 min SIN-1 exposure was diminished (T/S60'). A representative experiment of three performed is shown.

 
HSV Constructs Expressing Wild Type and Mutant MEKs—The wild type and mutant (S222A) MEK1 genes were supplied in the vector pcDNA3 (33) and double digested with HindIII and XhoI. The inserts containing genes were ligated to the herpes simplex virus (HSV) vector between the HindIII and SalI sites. For wild type and mutant (K101A) MEK2 genes that were supplied in the vector pcDNA3 (34), BamHI was used for subcloning. The fragments containing the wild type and mutant genes were inserted into the BamHI site of the HSV vector. All final constructs were confirmed by sequencing.

Infection of Recombinant Herpes Simplex Virus—At day 10 following medium change to BDM with T3 and ciliary neurotrophic factor, OLs were infected overnight with HSV expressing the lacZ gene, wild type, or the dominant-negative (dn) S222A MEK1 (33) and K101A MEK2 (34), and then followed by a complete medium change. Cells were cultured for an additional 24 h to allow the expression of wild type and dominant-negative MEK1 and MEK2. At this time, the virus infection rate was observed to be more than 90% by expression of LacZ. The HSV-infected OLs were then treated with SIN-1 (250 µM) for 2 h, and toxicity was observed at 24 h. For Western blot analysis, HSV-infected OLs were solubilized at 1 h after SIN-1 exposure. Antibody against MEK1/2 was obtained from Cell Signaling, Beverly, MA.

Western Blot Analysis of ERK and p38 Phosphorylation—At various times after SIN-1 or zinc treatment, OLs were placed on ice. After medium aspiration, cells were washed once with ice-cold phosphate-buffered saline, and lysed with lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerolphosphate, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride. An aliquot of cell lysate was removed for later protein determination. Cell lysate was mixed with Laemmli buffer, boiled for 5 min, and stored at -20 °C. Equal amounts of protein were separated by 8% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and then incubated overnight at 4 °C with the primary antibody for phosphorylated ERK42/44, phosphorylated p38, total ERK42/44, or total p38 (Cell Signaling, Beverly, MA) diluted at 1:1000 in TBST containing 5% BSA. After washing 4 times with TBST, the membrane was incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:4000. The membrane was washed again as above and visualized by enhanced chemiluminescence (ECL) according to the manufacturer's protocol (PerkinElmer Life Sciences).

Statistics—Statistical significance was assessed using analysis of variance with the Tukey-Kramer post-hoc multiple comparison test. Statistical analysis was performed using the Instat program from GraphPad Software (San Diego, CA). Representative experiments are shown unless noted otherwise. Experiments were performed with triplicate samples, and the data are expressed as mean ± S.D. or S.E., as is appropriate. All experiments were repeated at least 3 times.


Figure 3
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  		FIGURE 3.
Inhibition of 12-LOX activation blocked peroxynitrite toxicity to OLs. A, inhibitors of 12-LOX, but not cyclooxygenase or cytochrome p450 monooxygenase blocked SIN-1 toxicity to OLs. AA-861 (10 µM) and BHPP (10 µM) significantly blocked SIN-1 (500µM) toxicity. Indomethacin (INDO, 100 µM) and proadifen (PRO, 30 µM) had no protective effect. ***, p < 0.001 was obtained when the AA-861- or BHPP-treated groups were compared with the SIN-1 group alone. A representative experiment of three performed is shown. B, inhibition of 5-LOX had no effect on SIN-1 toxicity to OLs. MK886, a specific 5-LOX inhibitor, did not block SIN-1 toxicity. The same concentrations of AA-861 had a significant protective effect. ***, p < 0.001 was obtained when the AA-861-treated groups were compared with the SIN-1 group alone. A representative experiment of three performed is shown. C, inhibitors of 12-LOX blocked OL toxicity induced by authentic peroxynitrite. ***, p < 0.001 was obtained when the AA-861 or BHPP-treated groups were compared with the peroxynitrite group alone. A representative experiment of three performed is shown. D, SIN-1-induced time-dependent increase of 12-LOX activation. The production of 12-HETE was assayed at various times of SIN-1 exposure. *, p < 0.05 and **, p < 0.01 were obtained when the SIN-1-treated groups at 45 and 60 min were compared with the control group (0 min). A representative experiment of three performed is shown. E, TPEN blocked activation of 12-LOX induced by SIN-1. Exposure of SIN-1 (1 mM)to OLs for the 1-h induced significant increase of 12-LOX activity. TPEN (10 µM) completely blocked the activation of 12-LOX induced by SIN-1. **, p < 0.01 was obtained when the SIN-1-treated group was compared with the control group. A representative experiment of four performed is shown.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SIN-1 Toxicity to OLs Is Mediated by the Formation of Peroxynitrite—SIN-1 is a commonly used peroxynitrite generator, because it can simultaneously generate nitric oxide and superoxide in aerobic conditions (1). It has been reported that the composition of the medium (e.g. serum, vitamins, and amino acids in the cell culture medium) might affect the toxic species produced by SIN-1 exposure (35-37). Therefore, the OL cultures were exposed to SIN-1 in EBSS, which is a biocarbonate-buffered saline. We found that SIN-1 induced OL toxicity in a concentration-dependent manner (Fig. 1A), with an EC50 value of 270 ± 60 µM (n = 6). Similar to our observations in neuronal cultures (38), the toxicity of SIN-1 (500 µM) to OLs was blocked by carboxyl-PTIO (50 µM), a nitric oxide scavenger, or superoxide dismutase (100 units/ml) plus catalase (100 units/ml), which block the accumulation of superoxide (Fig. 1B). This result suggested that the toxic species was not superoxide or nitric oxide alone. Nitric oxide reacts with superoxide to form peroxynitrite in a diffusion-limited reaction. Therefore, we concluded that the toxic species might be peroxynitrite. Indeed, SIN-1 (500 µM) toxicity was completely blocked by a peroxynitrite scavenger, uric acid (1 mM), or a decomposition catalyst of peroxynitrite, FeTMPyP (10 µM) (Fig. 1B).

Zinc Release Is Critical in Peroxynitrite Toxicity to OLs—It has been reported that peroxynitrite causes zinc liberation from thymocytes (9) or neurons (10-12). Therefore, we anticipated that the toxicity of peroxynitrite to OLs might also be mediated by intracellular zinc release. N,N,N,'N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a zinc chelator, blocked SIN-1 (500 µM) toxicity to OLs in a concentration-dependent manner (Fig. 2A). In three experiments, the EC50 value of TPEN protection was 0.9 ± 0.5 µM. TPEN at 10 µM also completely blocked the toxicity of OLs induced by authentic peroxynitrite (Fig. 2B). It is known that TPEN is not exclusively a zinc chelator and can chelate other divalent cations, including iron and copper (39). Co-application of equimolar concentrations of zinc, but not iron, completely blocked the protective effect of TPEN (Fig. 2C), suggesting that iron is not involved in the toxicity of peroxynitrite to OLs. In addition, we found that two specific copper chelators, bathocuprione at 100 µM (39) and diethyldithiocarbamate at 100 µM (40), also have no effect on peroxynitrite toxicity to OLs (data not shown), excluding the possibility that copper is released by peroxynitrite and is responsible for the toxicity to OLs. To further examine the release of zinc by peroxynitrite, OLs were loaded with FluoZin-3 for 30 min, and then treated with SIN-1 (1 mM) for various times. The peroxynitrite-induced time dependent increase of FluoZin-3 fluorescence is indicative of zinc release (Fig. 2D). Zinc release could be detected at 15 min, and reached a maximum at 60 min (Fig. 2D). When TPEN was used together with SIN-1, no increase of fluorescence was observed (Fig. 2D). The same results were also observed when another fluorescent zinc indicator, Newport Green, was used (data not shown).

Activation of 12-LOX Is Induced by Peroxynitrite and Blocked by TPEN—Previous studies have shown that 12-LOX-mediated toxicity is observed in neurons and oligodendrocytes when levels of intracellular glutathione, an important endogenous antioxidant, are depleted (26, 41, 42). A general LOX inhibitor, AA-861 at 10 µM (41, 43) and a more specific 12-LOX inhibitor, BHPP at 10 µM (44, 45), significantly blocked the toxicity of OLs induced by SIN-1 (500 µM) (Fig. 3A). OL survival in the presence of AA-861 and BHPP were 66 ± 2 and 97 ± 5% of control, markedly higher (p < 0.001) than OL survival in the presence of SIN-1 alone (3 ± 2% of control) (n = 3). Because AA-861 is also known as a 5-LOX inhibitor (43), to test whether the protective effect of AA-861 on SIN-1 toxicity is because of its inhibition of 5-LOX, a more specific 5-LOX inhibitor, MK886 (46), was used to compare its effect with AA-861. AA-861 at 1 and 3 µM significantly blocked SIN-1 toxicity. On the contrary, MK886 at 1 and 3 µM had no effect (Fig. 3B). MK886 at 10 µM was toxic to OLs (data not shown), suggesting that 5-LOX activation might be important for cell survival. AA-861 and BHPP also significantly blocked OL toxicity induced by authentic peroxynitrite (100 µM) (Fig. 3C). Similar to OL toxicity induced by glutathione depletion (26), inhibition of other enzymes involved in arachidonic acid metabolism, i.e. COX (indomethacin at 100 µM) and cytochrome P450 monooxygenase (proadifen at 30 µM), had no effect on peroxynitrite toxicity to OLs (Fig. 3A). All these results suggested that 12-LOX was specifically involved. Using an enzyme-labeled immunoassay, we examined the activity of 12-LOX by measuring the production of the end product of 12-LOX metabolism, 12-HETE. We found that exposure of OLs to SIN-1 for 45 and 60 min induced a significant increase of 12-LOX activity (Fig. 3D). In four experiments, the level of 12-LOX activity in SIN-1 (1 mM, 1 h)-treated OLs was 163 ± 9% of control. TPEN at 10 µM blocked the activation of 12-LOX induced by SIN-1 (Fig. 3E). There was no difference in the expression levels of 12-LOX at different times following SIN-1 exposure (data not shown).

TPEN or Inhibitors of 12-LOX Blocked ROS Generation Induced by Peroxynitrite—Zinc has been found to induce ROS generation in neurons (47). Although the mitochondrion is thought to be an important source of ROS (10, 48), arachidonic acid metabolism by the LOX pathway also generates lipid peroxides, causing cell injury and death (41). OLs were incubated with SIN-1 for various times, and then exposed to DCF for 30 min. ROS generation, indicated by an increase of DCF fluorescence, occurred at 60 min, and further increased at 75 and 90 min following SIN-1 exposure (Fig. 4A). Uric acid completely blocked ROS generation (Fig. 4A). Co-application of TPEN or AA-861 also significantly blocked the increase of ROS at 1 h following SIN-1 exposure (Fig. 4B). In three experiments, the levels of ROS in the presence of SIN-1 were 600 ± 134% of control. When TPEN and AA-861 were present, the levels of ROS were 235 ± 25 and 131 ± 38%, respectively. Vitamin E and vitamin K2, which have been shown to block glutathione depletion-induced OL death, possibly by preventing the formation of ROS (32), completely blocked OL toxicity induced by SIN-1 (Fig. 4C). 12-HETE (10 µM), the major metabolic product of 12-LOX, did not cause any toxicity to OLs (data not shown). These results suggested that peroxynitrite causes zinc release and subsequent 12-LOX activation and ROS generation, leading to OL death.


Figure 4
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  		FIGURE 4.
SIN-1-induced ROS generation in OLs is downstream of zinc release and 12-LOX activation. A, ROS was generated by SIN-1 and blocked by uric acid. OLs were treated with SIN-1 for various times in the absence or presence of uric acid (UA, 1 mM), and then exposed to DCF for 30 min. ***, p < 0.001 was obtained when the SIN-1 groups at 60, 75, and 90 min were compared with the control group (0 min). A representative experiment of three performed is shown. B, TPEN or AA-861 blocked ROS generation induced by SIN-1. ***, p < 0.001 was obtained when the SIN-1 group was compared with the control or drug-treated groups. A representative experiment of three performed is shown. C, vitamin E (VE,10 µM) and vitamin K2 (VK2,10 µM) blocked SIN-1 toxicity to OLs. *, p < 0.05 and **, p < 0.01 were obtained when the VE- or VK2-treated groups were compared with the SIN-1 group alone. A representative experiment of three performed is shown.

 
Peroxynitrite-induced ERK42/44 Phosphorylation Is Downstream of Zinc Release, but Upstream of 12-LOX Activation—Activation of mitogen-activated protein kinase (MAPK) has been shown to be required for intracellular ROS generation stimulated by oxidative stress (13, 49, 50). However, different MAPKs, such as ERK or p38, might be activated by different oxidative stimuli (13, 50). Previous studies have found that the metabolic product of 12-LOX, 12-HETE, can activate ERK42/44 (51, 52), and lead to the activation of a signaling cascade (53). We first tested inhibitors of ERK and p38 on the toxicity to OLs induced by peroxynitrite. U0126 (10 µM), an inhibitor of mitogen-activated extracellular-regulated protein kinase kinase (MEK), which blocks the phosphorylation of ERK, completely blocked the toxicity induced by SIN-1 (500 µM) (Fig. 5A). The same result was also observed when another MEK inhibitor, PD98059, was used (data not shown). The inhibitor of p38, SB203580 (10 µM), had no effect on SIN-1 toxicity to OLs (Fig. 5A). SIN-1 caused a time-dependent increase of ERK phosphorylation, which appeared at 30 min, and increased over 2 h (Fig. 5B). ERK phosphorylation remained at 4 or 6 h following SIN-1 exposure, although the intensity of ERK phosphorylation gradually decreased (data not shown). Similarly, SIN-1 also induced p38 phosphorylation in a time-dependent manner (Fig. 5C). Overexpression of dnMEK1 or dnMEK2 partially inhibited the activation of ERK by SIN-1 (500 µM, 1 h) (Fig. 5D). Using densitometry, we found that overexpression of dnMEK1 reduced the phosphorylation levels of ERK42 and ERK44 to 57 ± 5(n = 5) and 35 ± 5% (n = 5) of those in HSV-infected cells expressing lacZ genes. The phosphorylation levels of ERK42 and ERK44 were reduced to 78 ± 8 (n = 5) and 52 ± 4% (n = 5) of those in HSV-infected cells expressing lacZ genes by overexpression of dnMEK2. Overexpression of dnMEK1 and dnMEK2 also significantly blocked SIN-1-induced OL toxicity (Fig. 5E). Infection with recombinant viruses expressing wild type genes had no effect on SIN-1-induced toxicity (data not shown).


Figure 5
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  		FIGURE 5.
Inhibition of ERK phosphorylation blocked SIN-1 toxicity to OLs. A, inhibition of ERK42/44, but not p38, blocked SIN-1 toxicity to OLs. U0126 (10 µM) completely blocked SIN-1 toxicity to OLs. SB203580 (10 µM) had no effect. ***, p < 0.001 was obtained when the U0126-treated group was compared with the SIN-1 group alone. A representative experiment of three performed is shown. B, SIN-1 caused a time-dependent ERK phosphorylation. p-ERK42/44 refers to phosphorylated ERK. A representative experiment of four performed is shown. C, SIN-1 caused time-dependent p38 phosphorylation. p-p38 refers to phosphorylated p38. A representative experiment of three performed is shown. D, overexpression of dominant-negative MEK1 and MEK2 attenuated SIN-1-induced ERK phosphorylation. In the experiment shown, expression of dominant-negative MEK1 (dnMEK1) reduced ERK42 and ERK44 to 47 and 34% of levels observed in LacZ-transfected cells. Expression of dnMEK2 reduced the ERK42 and ERK44 to 58 and 39% of levels observed in LacZ-transfected cells. Similar results were obtained in four experiments. E, overexpression of dominant-negative MEK1 and MEK2 attenuated SIN-1-induced toxicity to OLs. *, p < 0.05 and **, p < 0.01 were obtained when dnMEK1- or dnMEK2-transfected cells were compared with LacZ-transfected cells. A representative experiment of three performed is shown.

 
We next examined the sequence of events relating to zinc release, ERK phosphorylation, and 12-LOX activation. Exposure of OLs to SIN-1 for 1 h induced a large increase of ERK phosphorylation (Figs. 5, C and D, and 6A). As expected, the phosphorylation of ERK was blocked by TPEN, but surprisingly not by AA-861 (Fig. 6A). This result suggested that ERK activation is downstream of zinc release, but upstream of 12-LOX activation, in contrast to observations in methylisothiazolione-induced neurotoxicity (13). Indeed, U0126 significantly blocked the activation of 12-LOX induced by SIN-1 (Fig. 6B), suggesting that the phosphorylation of ERK is necessary for the activation of 12-LOX. In addition, U0126, but not SB203580 completely blocked the generation of ROS induced by SIN-1 (500 µM) (Fig. 6C) or authentic peroxynitrite (data not shown). The generation of ROS was also significantly attenuated by overexpression of dnMEK1 and dnMEK2 (Fig. 6D).

Exogenous Zinc Induced OL Toxicity via an ERK 42/44-12-LOX-ROS Pathway—These results suggested that peroxynitrite toxicity to OLs occurs via an ERK42/44-12-LOX-ROS pathway mediated by zinc release from intracellular stores. We hypothesized that the application of exogenous zinc might induce toxicity to OLs by the same mechanism. Zinc induced OL death (Fig. 7A), with an EC50 value for zinc chloride of 160 ± 18 µM (n = 4). It has been reported that zinc enters neurons via three major routes, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, N-methyl-D-asparatate receptors, and voltage-dependent calcium channels (47). Functional N-methyl-D-asparatate receptors are not present in OLs. 6-cyano-7-nitroquinoxaline-2,3-dione (100 µM), an antagonist of AMPA/kainate receptors, had no effect on zinc-induced toxicity to OLs. However, nifedipine (100 µM), a calcium channel blocker, significantly attenuated the toxicity of zinc (Fig. 7B). These results suggest that zinc enters OLs via voltage-dependent calcium channels. Next, we examined whether application of exogenous zinc could increase the activity of 12-LOX. Exposure of OLs to zinc (300 µM) caused a significant increase in 12-LOX activity at 60 min (170 ± 7% of control) and 90 min (180 ± 12% of control) (Fig. 7C). The toxicity of zinc was significantly (p < 0.001) blocked by the inhibitors of 12-LOX, AA-861 (10 µM), and BHPP (1 µM) (Fig. 7D). To test whether zinc toxicity is due simply to activation of PLA2 (54), we exposed cells to 300 µM arachidonic acid. In mature OLs this concentration of arachidonic acid was not toxic (Fig. 7E), in contrast to its effect on developing OLs (26). Therefore it is unlikely that the effects of zinc were simply because of activation of PLA2. Indeed, inhibitors of PLA2, quinacrine, arachidonyl trifluoromethyl ketone (AACOCF3) (55), and palmitoyl trifluoromethyl ketone (PACOCF3) (56) had no effect on zinc- or peroxynitrite-induced toxicity (data not shown). Instead, we hypothesized that zinc must have a site of action other than this enzyme. We tested the effect of co-exposure (90 min) to non-toxic concentrations of zinc (100 µM) and arachidonic acid (300 µM), and found at these concentrations that these two agents were synergistic in producing toxicity (Fig. 7E). These data suggest that although zinc may have an effect on PLA2, its toxicity to mature OLs is because of its effects on one or more other targets.


Figure 6
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  		FIGURE 6.
SIN-1-induced ERK activation is downstream of zinc release, but upstream of 12-LOX activation. A, TPEN, but not AA-861 blocked SIN-1-induced ERK42/44 phosphorylation. A representative experiment of three performed is shown. B, inhibition of ERK blocked 12-LOX activation induced by SIN-1. **, p < 0.01 was obtained when the SIN-1 group was compared with the control or U0126-treated group. A representative experiment of three performed is shown. C, inhibition of ERK, but not p38 blocked ROS generation induced by SIN-1. **, p < 0.01 and ***, p < 0.001 were obtained when the SIN-1 or SB203580 plus SIN-1-treated group was compared with the control. A representative experiment of three performed is shown. D, overexpression of dominant-negative MEK1 and MEK2 attenuated SIN-1-induced ROS generation. ***, p < 0.001 was obtained when dnMEK1- or dnMEK2-transfected cells were compared with LacZ-transfected cells. A representative experiment of three performed is shown.

 
Consistent with the effect of MAPK inhibitors on SIN-1 toxicity, U0126 (10 µM) completely blocked zinc toxicity, whereas SB203580 had no protective effect (Fig. 8A). Zinc-induced ERK42/44 phosphorylation progressively increased over 2 h (Fig. 8B). p38 was also phosphorylated by zinc exposure (data not shown). The phosphorylation of ERK at 60 min was blocked by nifedipine, but not by AA-861 (Fig. 8C), again suggesting that ERK activation is upstream of 12-LOX activation. Using DCF as an ROS indicator, we found that zinc caused a large increase of DCF fluorescence. In the presence of U0126 or AA-861, no increase of DCF fluorescence was observed (Fig. 8, D and E). The increase of DCF fluorescence was also blocked by vitamin E or vitamin K2 (data not shown), and consistent with this finding, vitamin E or vitamin K2 significantly blocked the toxicity induced by zinc (Fig. 8F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we found that peroxynitrite-induced toxicity to OLs occurs via a pathway that includes zinc release from intracellular stores, phosphorylation of ERK42/44, 12-LOX activation, and ROS generation. These data elucidate the mechanism of peroxynitrite toxicity to OLs and may have implications for the understanding of white matter injury.

Zinc is the second most prevalent trace element in the body and is present in particularly high concentrations in the mammalian brain (47). Zinc is an important component of zinc-dependent transcription factors and zinc-containing proteins. Although zinc is an important constituent of the antioxidant proteins Cu,Zn-superoxide dismutase and metallothionein (57), elevation of intracellular zinc does not increase the endogenous antioxidant capacity of cells. In fact, elevation of zinc has been shown to generate ROS and cause cell injury (48, 58, 59). Peroxynitrite has been reported to cause zinc release from thymocytes (9) and neurons (10-12), and cause mitochondrial dysfunction, ROS generation, and cell death. Similarly, we found peroxynitrite is also capable of liberating zinc from stores in OLs, suggesting that zinc might be an important intermediate in OL toxicity induced by peroxynitrite. Consistent with this notion, we observed that the cell death pathway initiated by peroxynitrite was similar to that induced by use of exogenous zinc. Interestingly, treatment with metallothionein has been found to reduce axonal damage and OL toxicity and facilitate tissue repair during experimental allergic encephalomyelitis, an animal model of MS (60).

Peroxynitrite has been viewed as a highly toxic species, and possibly the principal oxidant produced by cells (1). However, recent studies have suggested that the toxicity induced by peroxynitrite may not be because of peroxynitrite itself, but to the formation of ROS (61, 62). In OLs, both the generation of ROS and the toxicity of peroxynitrite are blocked by chelation of zinc, suggesting that the toxicity of peroxynitrite requires the formation of ROS and is secondary to the release of zinc. It is unclear where and how ROS are generated, although the mitochondrion is believed to be an important source of ROS during oxidative stress (10, 24). Recent studies have suggested that peroxynitrite stimulates PLA2, which then causes release of arachidonic acid from phospholipids in lipid membranes (7, 61). The subsequent metabolism of arachidonic acid via COX and LOX forms hydroperoxides and peroxyl radicals, constituting an alternative pathway of ROS generation (63). Zinc has been reported to bind to and activate PLA2 (54), which leads to ROS generation via the metabolism of AA. We found, however, that several inhibitors of PLA2 were not effective in blocking OL toxicity induced by peroxynitrite or exogenous zinc (data not shown). Inhibition of COX also had no effect. In contrast, inhibition of 12-LOX completely blocked peroxynitrite and zinc-induced toxicity. The accumulation of ROS induced by peroxynitrite or zinc was completely attenuated by the inhibitors of 12-LOX, but not by the inhibitors of PLA2 or COX. These results suggest that zinc may activate 12-LOX, and cause toxic accumulation of ROS.


Figure 7
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  		FIGURE 7.
ZnCl2-induced 12-LOX activation. A, concentration dependence of ZnCl2-induced toxicity to OLs. The EC50 value of ZnCl2 in inducing OL toxicity in this experiment was 150 µM. A representative experiment of four performed is shown. B, nifedipine, but not CNQX blocked ZnCl2-induced toxicity to OLs. Nifedipine at 100 µM significantly blocked ZnCl2 (200 µM)-induced toxicity to OLs. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (100 µM) had no protective effect. ***, p < 0.001 was obtained when the nifedipine plus ZnCl2-treated group was compared with the ZnCl2 alone treated group. A representative experiment of three performed is shown. C, ZnCl2-induced 12-LOX activation in OLs. *, p < 0.05 and **, p < 0.01 were obtained when the ZnCl2 groups at 60 and 90 min were compared with the control group (0 min). A representative experiment of three performed is shown. D, inhibition of 12-LOX significantly blocked ZnCl2-induced toxicity to OLs. ***, p < 0.001 was obtained when the AA-861 (10 µM) or BHPP (1 µM)-treated groups were compared with the ZnCl2 (200 µM) alone group. A representative experiment of three performed is shown. E, ZnCl2 and AA had a synergistic effect in producing toxicity to OLs. Co-application of ZnCl2 (100 µM) and AA (300 µM) caused death of virtually all OLs. ***, p < 0.001 was obtained when the zinc plus AA group was compared with the other groups. A representative experiment of three performed is shown.

 
12-LOX is the primary LOX expressed in the brain (64). Activation of 12-LOX has been found to play a critical role in the oxidative neurotoxicity induced by glutathione depletion, methylisothiazolinone, nitric oxide with glutathione depletion, and beta-amyloid peptide (13, 41, 42, 65, 66). Recently, we also found that activation of 12-LOX is involved in the toxicity of glutathione depletion to OLs (26). All these studies suggest that 12-LOX is a central player in cell toxicity induced by oxidative stress. However, it is still unclear how 12-LOX is activated in these conditions.

MAPK has been suggested as a mediator of oxidative injury to cells (67-70). Previous studies have shown that ERK activation is induced by 12-LOX metabolites (51, 52), and that inhibition of 12-LOX blocked phosphorylation of ERK induced by methylisothiazolinone in neurons (13). However, our studies showed that inhibition of 12-LOX did not block peroxynitrite or zinc-induced phosphorylation of ERK in OLs, suggesting that ERK phosphorylation is upstream of 12-LOX activation. Consistent with this putative sequence of events, prevention of ERK phosphorylation blocked the activation of 12-LOX caused by SIN-1, suggesting the possibility that 12-LOX might be phosphorylated by ERK. It has been reported that 5-LOX, the key enzyme in the biosynthesis of proinflammatory leukotrienes, is phosphorylated by ERK at serine 663 and that inhibitors of ERK phosphorylation reduce the production of 5-LOX metabolic products (71). Although ERK can also phosphorylate PLA2 (68, 71), and lead to arachidonic acid release and subsequent 12-LOX activation, this sequence of events appears unlikely in the present experimental paradigm, because inhibitors of ERK phosphorylation fully blocked the toxicity, whereas inhibitors of PLA2 had no effect. We are now examining whether 12-LOX can be phosphorylated in response to peroxynitrite or zinc, and if so, to determine the functional consequences of this modification. It is currently unclear how peroxynitrite or zinc causes ERK activation. ROS, such as hydrogen peroxide, have been reported to induce ERK phosphorylation (69, 72). However, our study suggests that peroxynitrite-induced ERK activation is not via the formation of ROS, because inhibition of ERK phosphorylation blocked ROS generation (Fig. 6, C and D) and vitamin E had no effect on ERK phosphorylation induced by SIN-1 (data not shown). Infection with recombinant viruses expressing dominant-negative mutants of MEK attenuated ERK phosphorylation, suggesting that Ras and Raf might be the upstream kinases causing ERK phosphorylation. It is also possible that protein kinase C is another upstream kinase, because it has been reported to be activated by zinc and lie upstream of ERK phosphorylation (73).


Figure 8
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  		FIGURE 8.
Zinc-induced ERK activation occurs upstream of 12-LOX activation and ROS accumulation. A, inhibition of ERK, but not p38 blocked ZnCl2-induced toxicity to OLs. U0126 (10 µM) completely blocked ZnCl2 (200 µM)-induced toxicity to OLs. SB203580 (10 µM) had no effect. ***, p < 0.001 was obtained when the U0126-treated group was compared with the ZnCl2 alone group. A representative experiment of three performed is shown. B, zinc caused a time-dependent increase of ERK phosphorylation. A representative experiment of three performed is shown. C, nifedipine, but not AA-861 blocked zinc-induced ERK activation. A representative experiment of three performed is shown. D-E, U0126 and AA-861 completely blocked ROS generation induced by ZnCl2. OLs were exposed to ZnCl2 (200 µM) for 90 min, the ROS generation was qualitatively examined by fluorescence microscopy 20 min after dihydrorhodamine exposure (D) or quantitatively measured by microplate reader 30 min after DCF exposure (E). D1, control; D2, ZnCl2; D3, U0126 (10 µM) plus ZnCl2; D4, AA-861 (10 µM) plus ZnCl2. ***, p < 0.001 was obtained when the ZnCl2 alone group was compared with the control and the drug-treated groups. A representative experiment of three performed is shown. F, vitamin E and vitamin K2 blocked OL toxicity induced by ZnCl2. Vitamin E (VE, 10 µM) or vitamin K2 (VK2, 10 µM) significantly blocked ZnCl2-induced toxicity to OLs. *, p < 0.05 and ***, p < 0.001 were obtained when the VE- and VK2-treated groups were compared with the ZnCl2 alone group. A representative experiment of three performed is shown.

 
Our studies using pharmacological inhibitors and determining the time course of biochemical changes suggest that peroxynitrite toxicity to OLs is via a zinc -> ERK -> 12-LOX -> ROS pathway. However, the death pathway may not be completely described by this simple scheme. It has been reported that both peroxynitrite and zinc can induce the mitochondrial membrane permeability transition pore opening, reduce the respiration of mitochondria, impair enzymes in the tricarboxylic acid cycle and electron transport chain, and cause ATP depletion and ROS generation (6, 10, 48). The toxicity of peroxynitrite to OLs has recently been suggested to be because of, at least in part, the impairment of mitochondrial function (24). In addition, peroxynitrite has also been found to reduce levels of glutathione (74), thus impairing the endogenous antioxidant capacity of cells. We found that 12-LOX activation occurs at 1 h following SIN-1 or zinc exposure, and that levels of glutathione are only reduced at 2 h. Therefore, glutathione depletion does not appear to be a primary cause leading to 12-LOX activation, contrary to what has been reported in neurons (41). However, it is possible that glutathione depletion may enhance the OL toxicity produced by the increase of intracellular free zinc, ERK, and 12-LOX activation.

It is interesting to note that inhibition of p38 has no effect on peroxynitrite toxicity to OLs, although it protects against peroxynitrite toxicity to neurons (12). ERK activation in OLs occurs upstream of 12-LOX, but p38 activation in neurons occurs downstream of 12-LOX activation (12). Inhibitors of caspases blocked peroxynitrite toxicity to neurons (12), but not to OLs (data not shown). These results suggest that different pathways might be involved in peroxynitrite toxicity to OLs and neurons, one leading to apoptotic death and involving p38 activation in neurons and one leading to necrotic death and involving ERK activation in mature OLs. Both, however, appear to critically depend upon the activation of 12-LOX.

Up-regulation of inducible nitric-oxide synthase has been shown in acute MS lesions (75) and in chronic active plaques in experimental allergic encephalomyelitis, an animal model of MS (76). Increased levels of inducible nitric-oxide synthase and nitrotyrosine formation, indicative of peroxynitrite formation, were also found in the cerebrospinal fluid of MS patients, but not in controls (77). Although numerous studies strongly suggest the importance of nitric oxide and peroxynitrite in the pathogenesis of MS, current strategies to prevent peroxynitrite-induced injury are based only on preventing peroxynitrite formation or scavenging peroxynitrite immediately generated by the reaction of nitric oxide and superoxide (17, 19-21, 38, 78, 79). One reason for this relatively restricted approach is that there is so little information available about the mechanisms of peroxynitrite toxicity to OLs (24).

The present study suggests that a specific pathway is involved in the toxicity of peroxynitrite to OLs and this pathway is different from the pathway of peroxynitrite injury to neurons that has recently been described (12). Interfering with the specific steps of this signaling cascade in OLs might prove to be effective in the treatment of MS and other demyelinating and neurodegenerative diseases. Therapeutic strategies derived from the elucidation of this pathway might include maintenance of zinc homeostasis, prevention of phosphorylation of ERK, blockade of the activation of 12-LOX, and elimination of ROS.


   FOOTNOTES
 
* This work was supported by grants from the Muscular Dystrophy Association, the United Cerebral Palsy Research Foundation, the Ron Shapiro Charitable Foundation, and National Institutes of Health Grants HD 18655 and NS 38475. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 300 Longwood Ave, Boston, MA 02115. Tel.: 617-355-6962; Fax: 617-730-0243; E-mail: paul.rosenberg{at}childrens.harvard.edu.

2 The abbreviations used are: AA, arachidonic acid; OL, oligodendrocyte; ERK, extracellular signal-regulated protein kinase; 12-LOX, 12-lipoxygenase; ROS, reactive oxygen species; COX, cycloxygenase; MS, multiple sclerosis; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase; dnMEK, dominant-negative MEK; EBSS, Earle's balanced salt solution; 12-HETE, 12-hydroxyeicosatetraenoic acid; HSV, herpes simplex virus; NOS, nitric-oxide synthase; DCF, 2',7'-dichlorohydrofluorescein diacetate; BSA, bovine serum albumin; BDM, basal chemically defined medium; BHPP, N-benzyl-N-hydroxy-5-phenylpentanamide; T3, 3,3',5-triodo-L-thyronine; PLA2, phospholipase A2; SIN-1, 3-morpholinosydnonimine; TPEN, N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine. Back



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 ABSTRACT
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 RESULTS
 DISCUSSION
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