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Am J Physiol Heart Circ Physiol 288: H2381-H2389, 2005. First published December 30, 2004; doi:10.1152/ajpheart.00904.2004
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Norepinephrine induces endoplasmic reticulum stress and downregulation of norepinephrine transporter density in PC12 cells via oxidative stress

Weike Mao, Chikao Iwai, Fuzhong Qin, and Chang-seng Liang

Cardiology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, New York

Submitted 31 August 2004 ; accepted in final form 29 December 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac norepinephrine (NE) uptake is reduced in cardiomyopathy. This change is associated with a decrease of NE transporter (NET) receptor and can be reproduced in PC12 cells by extracellular NE. To study whether this effect of NE is mediated via impaired glycosylation and trafficking of NET in the endoplasmic reticulum (ER), we measured the distribution of glycosylated 80-kDa NET and unglycosylated 46-kDa NET in the membrane and cytosolic fractions of PC12 cells. We found that NE decreased glycosylated NET in both membrane and cytosolic fractions and increased cytosolic unglycosylated NET protein. Similar results were produced by tunicamycin and thapsigargin, two agents that induce ER stress by inhibiting N-glycosylation of membrane proteins and disrupting calcium homeostasis, respectively. Also, like the ER stressors, NE not only increased phosphorylation of both the {alpha}-subunit of eukaryotic initiation factor-2 and its upstream RNA-dependent protein kinase-like ER kinase over 12 h of treatment but also increased ER chaperone molecule glucose-regulated protein 78 and the nuclear transcription factor C/EBP homologous protein. Antioxidants superoxide dismutase and catalase prevented the downregulation of NET proteins and induction of ER stress signals produced by NE but not by tunicamycin or thapsigargin. The results indicate that the downregulation of membrane NET by NE is mediated by decreased N-glycosylation of NET proteins secondary to induction of ER stress pathways by NE-derived oxidative metabolites. Interventions involving the ER stress pathways may provide novel therapeutic strategies for the treatment of sympathetic dysfunction in heart failure.

norepinephrine uptake; superoxide dismutase; glucose-regulated protein 78; eukaryotic initiation factor-2 {alpha}-subunit; protein-like endoplasmic reticulum kinase

NOREPINEPHRINE (NE) transporter (NET), a member of the gene family of Na+/Cl–-dependent plasma membrane transporters, possesses 12 putative transmembrane domains, multiple N-linked glycosylation sites, cytoplasmic NH2 and COOH termini, and cytoplasmic phosphorylation sites (1, 5). It is present in the noradrenergic cell type of the brain, peripheral sympathetic nerve terminals, and some cell types outside of the nervous system such as PC12 cells (26, 32), with a primary function to remove NE from the synaptic space and quickly terminate the actions of extracellular NE on postsynaptic receptors (8). Dysfunction of NET in cardiac sympathetic nerve terminals has been demonstrated in hypertension and congestive heart failure and plays a role in the pathophysiology of heart failure (15, 24, 28). More recently, we have shown (29, 33) that decreases of NET density and NE uptake activity in the cardiac sympathetic nerve terminals and PC12 cells also occur after NE administration and that these effects of NE are mediated via a posttranscription event caused by NE-derived oxidative stress.

The purpose of the present study was to elucidate the posttranscriptional mechanisms by which NE reduces NET density and function. Earlier studies showed that N-glycosylation is important for physical maturation and surface targeting of NET, using site-directed mutagenesis in which mutant NET lacking glycosylation sites exhibited reduced NET expression at the cell surface (35). Inhibition of protein N-glycosylation and synthesis by tunicamycin (11) also has been shown to reduce NET activity and membrane ligand binding (34, 35). Protein glycosylation is known to occur in endoplasmic reticulum (ER). N-glycosylation increases the properly folded transporter proteins and/or protects them from degradation so that they can be efficiently trafficked to plasma membrane. However, when the load of proteins facing the ER is increased beyond its capacity to process the load efficiently, under conditions such as ischemia, hypoxia, viral infection, or oxidative stress (7, 12, 21, 23, 46), ER stress occurs. This leads to two sequences of events known as unfolded protein response and ER overload response, respectively (7, 12, 44). ER stress is characterized by early phosphorylation of the RNA-dependent protein kinase-like ER kinase (PERK) and the {alpha}-subunit of eukaryotic initiation factor-2 (eIF2{alpha}), transcriptional upregulation of genes encoding ER chaperones such as glucose-regulated protein 78 (GRP78) and the transcription factor C/EBP homologous protein (CHOP), and rapid reduction in protein biosynthesis aimed at lowering the load of client proteins (18, 19, 27). We speculate that excessive NE causes ER stress via oxidative stress and prevents full N-glycosylation and trafficking of NET to cell membrane. In the present study, we sought 1) to determine whether the reduction of membrane NET by NE was associated with reduced N-glycosylation, maturation, and trafficking of NET protein to the cell surface by measuring the distribution of the glycosylated (80 kDa) and unglycosylated (46 kDa) NET proteins in the membrane and cytosolic fractions; 2) to study whether NE activated ER stress signals (eIF2{alpha}, PERK, GRP78, and CHOP); 3) to compare the effects of NE to those of tunicamycin and thapsigargin, two classic agents that cause ER stress by inhibiting protein glycosylation (11) and Ca2+-ATPase activity in ER (13), respectively; and 4) to determine whether the effects of NE on ER stress signals could be prevented by catalase and superoxide dismutase, which have been shown to attenuate the NE-induced NET downregulation (33).


   METHODS AND MATERIALS
TOP
 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. RPMI 1640 medium, fetal bovine serum, heat-inactivated horse serum, and HEPES buffer were obtained from GIBCO-BRL (Gaithersburg, MD). NE, cupric sulfate, 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tunicamycin, thapsigargin, catalase, and superoxide dismutase were obtained from Sigma-Aldrich (St. Louis, MO). Protein A/G agarose was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were used: rabbit anti-phospho-eIF2{alpha}, anti-eIF2{alpha}, and anti-phospho-PERK (Cell Signaling, Beverly, MA); rabbit anti-GRP78 and mouse anti-CHOP (Santa Cruz Biotechnology); mouse anti-NET (Mab Technologies, Stone Mountain, GA); and rabbit anti-NET (Chemicon International, Temecula, CA). [3H]NE and [3H]nisoxetine were obtained from NEN Life Science Products (Boston, MA).

Cell cultures and viability assay. PC12 cells were obtained from American Type Culture Collection (Rockville, MD) and cultured as described previously (33). Cells were plated at a density of 1 x 105 cells/well in 12-well plates and grown for 24 h at 37°C. The medium was switched to 2% serum RPMI 1640 overnight for all studies described below.

To determine the optimal sublethal doses for use in this study, we measured cell viability by MTT after the PC12 cells had been incubated with a wide range of concentrations of NE (1, 10, 50, 100, 200, and 500 µM), tunicamycin (0.01, 0.1, 0.2, 0.5, and 5 µg/ml), and thapsigargin (0.01, 0.1, 0.2, 0.5, and 5 µM) for 24 h. Cupric sulfate (1 µM) was added to the NE medium to increase free radical formation and accentuate the reduction of NET downregulation in PC12 cells (33). At the concentration used, cupric sulfate alone had no effect on either free radical production or NET density (33). After 24 h of incubation, MTT (0.5 mg/ml) was added to the culture medium to measure cell viability (33). As we demonstrated previously (33), NE did not reduce cell viability until its concentration exceeded 100 µM. Tunicamycin and thapsigargin did not affect cell viability significantly until their concentrations exceeded 0.2 µg/ml and 0.1 µM, respectively. Thus in this project we chose 100 µM NE, 0.2 µg/ml tunicamycin, and 0.1 µM thapsigargin for further studies. The effects of NE reported here were obtained in the presence of 0.1 µM cupric sulfate unless otherwise indicated.

[3H]NE uptake activity. Uptake of [3H]NE into PC12 cells was determined as described previously (2, 33). Briefly, drug-containing medium was removed after 24 h of incubation. Cells were washed with PBS and preincubated at 37°C for 10 min in a bicarbonate-buffered Krebs-Ringer buffer. The uptake assay was initiated by adding 35.7 nM L-[3H]NE into medium for 15 min at 37°C. After wash, cells were solubilized and counted for radioactivity in a Tri-Carb 2400 TR liquid scintillation counter (Packard Instrument, Meriden, CT). Nonspecific uptake of L-[3H]NE was assessed with 10 µM nisoxetine present during both preincubation and incubation periods. Protein concentration in lysed cells was measured with the bicinchoninic acid protein kit (Pierce, Rockford, IL). We also performed kinetic analysis of NE uptake in PC cells, using Eadie-Hofstee plot analysis to determine the maximal velocity (Vmax) and dissociation constant of NE uptake (33).

Measurement of NET surface density by [3H]nisoxetine binding. To study the effects on NET surface density of PC12 cells (33, 50), PC12 cells were washed twice with ice-cold PBS and homogenized in 4 ml of ice-cold homogenizing buffer (in mM: 50 Tris, pH 7.4, 120 NaCl, 5 KCl) with a Polytron homogenizer (setting 6, 20 s). Homogenates were centrifuged at 600 g and 4°C to remove nuclei and debris. After two washes with centrifugation at 40,000 g for 30 min, the final pellets were suspended in ice-cold binding buffer (in mM: 50 Tris, pH 7.4, 300 NaCl, and 5 KCl). The reaction mixture (500 µl) included the membrane preparation (~50 µg of protein) and [3H]nisoxetine at 3 nM for single-point assay and at a concentration ranging from 0.1 to 10 nM in saturation assays. Nonspecific binding was defined with nisoxetine (10 µM). The mixture was incubated at 4°C for 2 h, and the membrane was rapidly washed three times before being filtered through Whatman GF/B filters on a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersburg, MD). Radioactivity was determined by liquid scintillation counting. Specific binding was calculated as the difference between the binding in the presence and in the absence of 10 µM nisoxetine. The number of binding sites and dissociation constant were calculated with standard radioligand binding site analysis.

Western blot analysis of NET and ER stress signal proteins. We measured the stable glycosylated 80-kDa NET protein expression in both membrane and cytosolic fractions by immunoprecipitation. The PC12 cell membrane pellets were dissolved in ice-cold RIPA buffer (in mM: 10 Tris, 150 NaCl, and 1 EDTA with 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate, pH 7.4) supplemented with PMSF (1 mM), leupeptin (1 µg/ml), and aprotinin (1 µg/ml). The membrane and cytosolic proteins (100–200 µg) were then immunoprecipitated by 2 µl of mouse anti-NET antibody and bound to 15 µl of protein A/G agarose at 4°C for 14 h. The beads were then washed three times with RIPA buffer. Equal amounts of proteins from both membrane and cytosolic fractions were eluted into 2x Laemmli sample buffer at room temperature for 30 min and subjected to Western blot. A single NET band (~80 kDa) was detected by immunoprecipitation. The 46-kDa NET was not detected in the membrane fraction. A whole cell lysate (see below) was used for measuring 46-kDa NET by Western blot analysis.

PC12 cells were lysed in a lysate buffer containing (in mM) 20 Tris·HCl (pH 7.5), 150 NaCl, 1 Na2EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 {beta}-glycerophosphate, 1 Na3VO4, and 1 PMSF with 1% Triton X-100 and 1 µg/ml leupeptin for 30 min on ice. Lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. Supernatants (50–60 µg) were boiled in 1x loading buffer (125 mM Tris·HCl, pH 6.8, 10% SDS, 20% glycerol, 0.05% bromophenol blue, and 1% 2-mercaptoethanol) for 5 min. The samples for Western blot were subjected to 6–12% SDS-polyacrylamide gel electrophoresis and transferred electrically to polyvinylidene difluoride membrane (PerkinElmer, Boston, MA). After blocking, the membranes were incubated with one of the following specific antibodies overnight at 4°C: affinity-purified rabbit anti-NET (1:250), rabbit anti-phospho-eIF2{alpha} (1:1,000), rabbit anti-phospho-PERK (1:500), rabbit anti-GRP78 (1:250), and mouse anti-CHOP (1:100). Membrane was then incubated with a horseradish peroxidase-conjugated secondary antibody, and the signal was detected with a chemiluminescence kit (Cell Signaling). The autoradiograms were scanned on a Microtek model 6800 scanner (Microtek Lab, Carson, CA), and the optical density of bands was determined with a NIH 1.6 gel image program. The optical density readings of samples were normalized to a control sample in arbitrary units.

To study the direct effect of Cu2+ on the ER stress signal proteins, we also added cupric sulfate (1 µM) to PC12 cells and measured the phospho-eIF2{alpha} and GRP78 proteins as described above. The results were compared with control, NE (100 µM) alone, and NE (100 µM) plus cupric sulfate (1 µM).

RT-PCR analysis. Total RNA was isolated with a GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich), following the manufacturer's instruction. cDNA was synthesized by reverse transcriptase, and the first-stranded cDNAs were then used for RT-PCR with an enhanced avian DuraScript RT-PCR kit. The primers used are as follows: sense primer 5'-GTTCTGCTTGATGTGTGTCC-3' and antisense primer 5'-TTTGGTCATTGGTGATGGTG-3' for GRP78; sense primer 5'-TCAGATGAAATTGGGGGCAC-3' and antisense primer 5'-TTTCCTCGTTGAGCCGCTCG-3' for CHOP; and sense primer 5'-GCCAAAAGGGTCATCATCTC-3' and antisense primer 5'-GGCCATCCACAGTCTTCT-3' for the housekeeper gene GAPDH. The PCR reaction for GRP78 and CHOP consisted of an initial denaturation cycle at 94°C for 2 min, followed by 30 cycles for CHOP and GAPDH and 25 cycles for GRP78 containing a denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and elongation at 68°C for 1 min. An additional cycle at 72°C for 7 min ended the PCR process. The PCR products were separated by 5% polyacrylamide gel, dried by vacuum, and exposed to Kodak Biomax MR film at –70°C overnight. The autoradiographs were quantified in arbitrary units as described above.

Effects of superoxide dismutase and catalase. To study the influence of antioxidants on the effects of NE, tunicamycin, and thapsigargin on NET protein and ER signal proteins, we pretreated PC12 cells with superoxide dismutase (20 µg/ml) and catalase (20 µg/ml) for 30 min before adding NE, tunicamycin, and thapsigargin. The methods used to measure NET protein and ER stress-signaling proteins were the same as described above.

Statistics. Experiment data were managed on the RS/I Research System (Bolt, Beraneck, and Newman Software Products, Cambridge, MA), and the results are presented as means ± SE. The statistical significance of differences among the different experimental groups was analyzed by either one-way or two-way analysis of variance. Bonferroni simultaneous intervals for all comparisons were used to determine the statistical significance of difference between two groups. A difference with a P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NE uptake activity and membrane ligand binding sites. Figure 1 shows the effects of NE (100 µM) plus 1 µM cupric sulfate, tunicamycin (0.2 µg/ml), and thapsigargin (0.1 µM) on NE uptake activity of PC12 cells over 24 h of incubation. NE uptake activity decreased in a time-dependent manner, reaching 40–60% of the control at 24 h. Kinetic analysis of [3H]NE uptake revealed a significant decrease of Vmax by NE, tunicamycin, and thapsigargin (Table 1), with no changes of dissociation constant. Table 1 also shows that PC12 membrane NET ligand binding sites were reduced by the NE, tunicamycin, and thapsigargin treatments. There were no significant changes of dissociation coefficient factor.



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  		Fig. 1. Norepinephrine (NE) uptake activity was reduced in PC12 cells by NE, tunicamycin, and thapsigargin at 6 and 12 h of incubation. Bars denote SE; n = 6 in each group. *P < 0.05 compared with control, {dagger}P < 0.05 compared with baseline values before drug treatment, as measured by 2-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 

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  		Table 1. Effects of NE, tunicamycin, and thapsigargin on Vmax and Bmax of PC12 cells

 
Distribution of N-glycosylated NET proteins. To study the cellular distribution and translocation of NET from the cytoplasm to the plasma membrane of PC12 cells, we measured the 80-kDa glycosylated NET protein in both cell membrane and cytosolic fractions. Figure 2 shows that the 80-kDa NET protein was reduced by NE, tunicamycin, and thapsigargin in both the membrane and cytosolic fractions. In contrast, the unglycosylated 46-kDa NET protein was increased in whole cell lysates by NE, tunicamycin, and thapsigargin at the end of 24-h treatments (Fig. 2).



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  		Fig. 2. Effects of NE, tunicamycin, and thapsigargin on 2 populations of NE transporter (NET) proteins in PC12 cells. Top: NE treatment reduced 80-kDa NET proteins in the membrane and cytosolic fractions of the PC12 cells. Bottom: 46-kDa NET protein was increased in the whole cell lysate after NE, tunicamycin, and thapsigargin treatment. Optical density readings were normalized against a control sample in arbitrary units. Bars denote SE; n = 6–8 in each group. *P < 0.05 compared with the control group without drug treatment, as measured by 1-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 
Upregulation of ER unfolded protein response protein signals. ER stress is associated with increased phosphorylation of eIF2{alpha} and PERK and increased gene expressions of GRP78 and CHOP. Figure 3 shows that addition of cupric sulfate, which had no effect on phospho-eIF2{alpha} or GRP78 in PC12 cells when given alone, potentiated the effects of NE on ER stress signal proteins. Figure 4 shows the time course of the effects of NE plus cupric sulfate on phospho-eIF2{alpha} and GRP78 in PC12 cells compared with tunicamycin and thapsigargin. NE produced a steady increase of phospho-eIF2{alpha} over 12 h, but its effect on GRP78 peaked at 3 h of incubation. Total eIF2{alpha} did not change significantly with time.



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  		Fig. 3. Effects of 1 µM cupric sulfate, 100 µM NE, and 100 µM NE + 1 µM cupric sulfate on phospho-eukaryotic initiation factor-2 {alpha}-subunit (eIF2{alpha}) (top) and glucose-regulated protein 78 (GRP78) (bottom) protein expression in PC12 cells. Optical density readings were normalized against a control sample in arbitrary units. The effects of NE were potentiated by addition of cupric sulfate, which had no direct effects on either protein. Bars denote SE; n = 5 in each group. *P < 0.05 compared with the control group without NE or cupric sulfate treatment, {dagger}P < 0.05 compared with NE alone, as determined by 1-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 


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  		Fig. 4. Effects of NE (top), tunicamycin (middle), and thapsigargin (bottom) on phospho-eIF2{alpha} (left) and GRP78 (right) proteins of PC12 cells. Optical density readings were normalized against a control sample in arbitrary units. NE and tunicamycin produced a gradual increase of phospho-eIF2{alpha} over 12 h. The effect of thapsigargin on phospho-eIF2{alpha} was short-lived, reaching a peak at 3 h. Bars denote SE; n = 6 in each group. *P < 0.05 compared with control before treatments (0 h), as measured by 1-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 
NE, tunicamycin, and thapsigargin also increased phosphorylation of the eIF2{alpha} kinase PERK (Fig. 5). The figure also shows that the activation of PERK was gradual, reaching a peak by 6–12 h of incubation. Tunicamycin and thapsigargin also increased CHOP protein at 6 and 24 h of incubation (Fig. 6). However, the effect of 100 µM NE on CHOP protein was modest (20–25%; Fig. 7). A much larger increase of CHOP protein occurred when the cells were exposed to a cytotoxic dose of NE (500 µM; Fig. 7).



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  		Fig. 5. Effects of NE (top), tunicamycin (middle), and thapsigargin (bottom) on phospho-protein kinase-like endoplasmic reticulum kinase (PERK) protein of PC12 cells. Left: representative Western blots. Optical density readings were normalized against a control sample in arbitrary units. Right: statistical summaries of the data. NE, tunicamycin, and thapsigargin produced a gradual increase of phospho-PERK over 12 h. Bars denote SE; n = 6 in each group. *P < 0.05 compared with control before treatments (0 h), as measured by 1-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 


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  		Fig. 6. Effects of NE, tunicamycin, and thapsigargin on C/EBP homologous protein (CHOP) protein of PC12 cells at 6 (top) and 24 (bottom) h of incubation. Left: representative Western blots. Optical density readings were normalized against a control sample in arbitrary units. Right: statistical summaries of the data. Bars denote SE; n = 6 in each group. *P < 0.05 compared with control before treatments (0 h), as measured by 1-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 


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  		Fig. 7. Effects of NE on CHOP protein in PC12 cells. Optical density readings were normalized against a control sample in arbitrary units. NE produced little or no increase of CHOP proteins at low concentrations (≤100 µM) but increased CHOP protein almost 6-fold at high concentration (500 µM). Bars denote SE; n = 6 in each group. *P < 0.05 compared with control without NE treatment, as measured by 1-way analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 
Upregulation of GRP78 and CHOP mRNA. To study whether the increases of GRP78 and CHOP proteins occurred at the transcriptional level, we also measured GRP78 and CHOP mRNA expression by RT-PCR. mRNA levels were normalized for loading condition by the housekeeping gene GAPDH mRNA. Figure 8 shows that GRP78 mRNA expression was increased two- to threefold by NE, tunicamycin, and thapsigargin over the first 24 h of incubation. Tunicamycin and thapsigargin also increased CHOP mRNA. However, at the dose used, NE produced only a slight increase of CHOP mRNA (31 ± 7%). When a large dose of NE (500 µM) was used, CHOP mRNA increased fivefold (498 ± 85%).



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  		Fig. 8. Effects of NE (top), tunicamycin (middle), and thapsigargin (bottom) on GRP78 (left) and CHOP (right) mRNAs of PC12 cells. GRP78 and CHOP mRNAs were normalized by the housekeeper gene GAPDH mRNA. Bars denote SE; n = 6–7 in each group. *P < 0.05 compared with control before treatments (0 h), as measured by analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 
Effects of superoxide dismutase and catalase on NE-induced changes of NET and ER stress-signaling proteins. Table 2 shows that superoxide dismutase and catalase prevented the reductions of membrane and cytosolic 80-kDa NET and increase of 46-kDa NET produced by NE. The antioxidants also prevented the increases of phospho-eIF2{alpha} and GRP78 proteins (Fig. 9). In contrast, the antioxidants had no effects on the actions of tunicamycin or thapsigargin on NET (Table 2), phospho-eIF2{alpha}, and GRP78 (Fig. 9) protein expressions in PC12 cells.


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  		Table 2. Effects of superoxide dismutase and catalase on NET proteins of PC12 cells

 


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  		Fig. 9. Effects of superoxide dismutase (SOD) and catalase treatment on the increases of phospho-eIF2{alpha} (top) and GRP78 (bottom) protein produced by NE, tunicamycin, and thapsigargin. Optical density readings were normalized against a control sample in arbitrary units. The antioxidants abolished the increase of phospho-eIF2{alpha} and GRP78 produced by NE, but not those by tunicamycin and thapsigargin. Bars denote SE; n = 6–8. *P < 0.05 compared with control without treatment, {dagger}P < 0.05 compared with values without SOD and catalase treatment, as measured by analysis of variance and Bonferroni simultaneous confidence intervals for all comparisons.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ER is an important cellular organelle responsible for posttranslational processing of newly synthesized secretory and membrane proteins and maintenance of important cellular homeostasis (7, 18, 19). When the ER environment is disturbed by increased protein synthesis, altered glycosylation, perturbed Ca2+ homeostasis, and/or deprivation of glucose or other sugars, unfolded or misfolded proteins accumulate in the ER lumen and initiate a classic self-protective unfolded protein response, or the ER stress response (9, 12). This process induces the transcription of a large number of genes encoded for protein secretion and processing such as GRP78 (23, 27, 30), which serve to enhance the folding of nascent proteins and promote cell survival. However, if the damage is too severe for recovery, ER overload response occurs, probably as a result of overaccumulation of proteins in the ER membrane. This causes increased release of Ca2+ and reactive oxygen species signaling from the ER lumen (12–14, 22), followed by activation of transcriptional factors and a number of proapoptotic pathways (9, 38, 43, 44).

The unfolded protein response is characterized by a series of coordinated activations of multiple proteins, including double-stranded RNA-activated protein kinase, PERK, and eIF2{alpha}. The eIF2-GTP participates in the ribosomal recognition of the start codon. When eIF2 is phosphorylated by PERK, it binds to eIF2B with high affinity and acts to prevent the recycling of eIF2-GDP to eIF-GTP. The lower eIF-GTP level reduces assembly of 43S translation initiation complex and attenuates protein synthesis (9, 23), thus allowing the cell sufficient time to correct the misfolding and unfolding of the proteins resulting from ER stress before synthesizing additional proteins. In this study, we measured the unique increases of phosphorylation of PERK and eIF2{alpha} and GRP78 as markers for unfolded protein response activation.

Our present study demonstrates that the reductions of neuronal NE uptake activity and NE ligand binding site density by extracellular NE were associated with a decrease of N-glycosylated 80-kDa NET protein in both cell membrane and cytosolic fractions of PC12 cells. The 80-kDa isoform is the predominant NET in the cells at steady state (1, 34). Cell surface biotinylation in PC12 cells indicates that only a minor fraction of NET is present at the cell surface; most NET is associated with secretory granules inside the cells (26). The 46-kDa NET is the unglycosylated core NET protein that was increased inside the cells after NE treatment. The findings are consistent with reduced glycosylation and increased intracellular retention of NET in PC12 cells. These changes produced by NE were also accompanied by induction of GRP78 and CHOP proteins and components of the PERK-dependent protein translation attenuation pathway. Furthermore, because these changes could be attenuated by superoxide dismutase and catalase, our present study provides evidence linking NE-induced oxidative stress to ER stress in mediating the reduction of membrane NET density and NE uptake activity in PC12 cells.

GRP78 is an ER lumen-resident chaperone protein that facilitates protein folding, stabilizes calcium homeostasis, and protects cells against oxidative stress and apoptotic death (23, 30, 31, 49). We found that, at a dose that produced no significant cell death, NE increased GRP78 at both mRNA and protein levels. In contrast, at this dose NE produced only a slight (20–25%) increase of CHOP expression. CHOP, also known as growth arrest- and DNA damage-inducible gene 153 (GADD153), is a nuclear transcription factor that is expressed at low levels under physiological conditions. It is upregulated during severe and prolonged ER stress and plays an important role in cell arrest and apoptosis (4, 16, 39, 45–47). In our study, we found that CHOP protein was increased markedly only when a dose of NE large enough to cause cell death (500 µM) was used. The findings suggest that GRP78 is differentially activated in the unfolded protein response induced by lower doses of NE. However, when the doses of NE are increased, ER stress is too severe to repair, and the transcription factor CHOP is activated. GRP78 is an antiapoptotic protein (30, 31), whereas CHOP is proapoptotic (4, 39).

The mechanisms by which NE induces ER stress have not been fully elucidated. However, as shown by the effects of antioxidants on NET and ER stress signals in the present study, NE probably acts by a prooxidant effect on the cells. NE is capable of producing oxygen free radicals by both enzymatic and nonenzymatic pathways. Cu2+ was added to NE in our present experiments to enhance hydroxyl radical production in the presence of H2O2 through the Fenton reaction. Other transition metal ions present in the tissue, such as Fe2+, also have been shown to exaggerate reactive oxygen species formation by catecholamines. NE and its oxidative metabolites have been shown to inhibit mitochondrial metabolism, deplete cellular ATP, and impair ER Ca2+-ATPase function (10, 17). Increased cytosolic Ca2+ presumably stimulates neuronal NO synthesis, which may in turn cause further depletion of ER Ca2+ and exaggerate the ER stress (30, 40). NO itself can induce ER stress and inhibit NE uptake (25, 40). Its intermediate metabolite, peroxynitrite, is also highly toxic and has been shown to impair the function of various proteins through nitration of tyrosine residues (36). Furthermore, ER-resident proteins such as GRP78, protein disulfide isomerase, and calreticulin are susceptible to oxidative damage (14, 20, 21, 42). Oxidation of the ER proteins may dissociate GRP78 from PERK and IRE1 (6) and cause further activation of the ER stress signals (19, 21, 42). Our results are consistent with a recent study showing that overexpression of copper/zinc superoxide dismutase protects ischemic rat brain from neuronal degeneration, ER protein oxidative damage, and ER stress response (20, 21). Edaravone, a free radical scavenger that inhibits hydroxyl- and iron-dependent lipid peroxidation, also has been shown to attenuate ER stress by inhibiting eIF2{alpha} phosphorylation and induction of GPR78 and CHOP in mouse hypoxic/ischemic brain (41). The close interaction between reactive oxygen species and ER stress deserves further study. Furthermore, our present experiments showed that the effects of NE in the presence of cupric sulfate cannot be explained by a direct action of Cu2+, because qualitatively similar changes were produced by NE without cupric sulfate. Cu2+ alone at 1 µM had no effect on NET density, phospho-eIF2{alpha}, or GRP78 in PC12 cells.

N-glycosylation plays an important role in the synthesis, maturation, trafficking, and reorientation of the binding site to initiate substrate cycles of a variety of neurotransmitter transporters. Blockade of cellular N-glycosylation by tunicamycin has been shown to reduce protein stability and trafficking in NET-expressing cell lines as well as human NET-transfected cells (34). Failure of glycosylation has also been found to reduce the protein half-life of GLUT1 glucose transporter (3) and GLYT1 glycine transporters (37) and loss of the membrane binding site for serotonin transporter (48). Site-directed mutagenesis to produce mutant NET lacking glycosylation sites resulted in reduced expression of NET at the cell surface, secondary to reduced protein stability, and increased intracellular retention. (34, 35). Our present experiments extend the observation with tunicamycin to NE and thapsigargin, confirming the importance of N-glycosylation of NET in the regulation of NET synthesis and function.

In summary, our study demonstrates that ER stress and its associated interruption of N-glycosylation are implicated in the reduction of membrane-bound NET in PC12 cells produced by extracellular NE. Furthermore, because the effects of NE on unfolded protein response and NET downregulation are reduced by superoxide dismutase and catalase, our present study supports a role of oxidative stress in ER stress. Excessive NE differentially activates a ER stress-related signal pathway with priority on phosphorylation of eIF2{alpha}, PERK, and GRP78. Manipulation of the ER stress pathway signals may provide novel therapeutic interventions for the treatment of sympathetic NET dysfunction in cardiovascular disease.


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The study was supported in part by an American Heart Association postdoctoral fellowship grant and National Heart, Lung, and Blood Institute Grant HL-68151.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C.-s. Liang, Univ. of Rochester Medical Center, Cardiology Unit, Box 679, 601 Elmwood Ave., Rochester, New York 14642 (E-mail: chang-seng_liang{at}urmc.rochester.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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TOP
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 RESULTS
 DISCUSSION
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  1. Amara SG, Sonders MS, Zahniser NR, Povlock SL, and Daniels GM. Molecular physiology and regulation of catecholamine transporters. Adv Pharmacol 42: 164–168, 1998.
  2. Apparsundaram S, Sung U, Price RD, and Blakely RD. Trafficking-dependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transporter in SK-N-SH cells. J Pharmacol Exp Ther 299: 666–677, 2001.[Abstract/Free Full Text]
  3. Asano T, Takata K, Katagiri H, Ishihara H, Inukai K, Anai M, Hirano H, Yazaki Y, and Oka Y. The role of N-glycosylation in the targeting and stability of GLUT1 glucose transporter. FEBS Lett 324: 258–261, 1993.[CrossRef][ISI][Medline]
  4. Barone MV, Crozat A, Tabaee A, Philipson L, and Ron D. CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest. Genes Dev 8: 453–464, 1994.[Abstract/Free Full Text]
  5. Bauman PA and Blakely RD. Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking, stability, and activity. Arch Biochem Biophys 404: 80–91, 2002.[CrossRef][ISI][Medline]
  6. Berlett BS and Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316, 1997.[Free Full Text]
  7. Berridge MJ. The endoplasmic reticulum: a multifunctional signal organelle. Cell Calcium 32: 235–249, 2002.[CrossRef][ISI][Medline]
  8. Blakely RD, De Felice LJ, and Hartzell HC. Molecule physiology of norepinephrine and serotonin transporters. J Exp Biol 196: 263–281, 1994.[Abstract/Free Full Text]
  9. Breckenridge DG, Germain M, Mathai JP, Nguyen M, and Shore GC. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 22: 8608–8618, 2003.[CrossRef][ISI][Medline]
  10. Burke WJ, Kristal BS, Yu BP, Li SW, and Lin TS. Norepinephrine transmitter metabolite generates free radicals and activates mitochondrial permeability transition: a mechanism for DOPEGAL-induced apoptosis. Brain Res 787: 328–332, 1998.[CrossRef][ISI][Medline]
  11. Chang JY and Korolev VV. Specific toxicity of tunicamycin in induction of programmed cell death of sympathetic neurons. Exp Neurol 137: 201–211, 1996.[CrossRef][ISI][Medline]
  12. Cudna RE and Dickson AJ. Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol Bioeng 81: 56–65, 2003.[CrossRef][ISI][Medline]
  13. Doutheil J, Treiman M, Oschlies U, and Paschen W. Recovery of neuronal protein synthesis after irreversible inhibition of the endoplasmic reticulum calcium pump. Cell Calcium 6: 419–428, 1999.
  14. Dreher D, Vargas JR, Hochstrasser DF, and Junod AF. Effects of oxidative stress and Ca2+ agonists on molecular chaperones in human umbilical vein endothelial cells. Electrophoresis 16: 1205–1214, 1995.[CrossRef][ISI][Medline]
  15. Esler M, Rumantir M, Kaye D, Jennings G, Hastings J, Socratous F, and Lambert G. Sympathetic nerve biology in essential hypertension. Clin Exp Pharmacol Physiol 28: 986–989, 2001.[CrossRef][ISI][Medline]
  16. Ferri KF and Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol 3: E255–E263, 2001.[CrossRef][ISI][Medline]
  17. Fu W, Luo H, Parthasarathy S, and Mattson MP. Catecholamines potentiate amyloid {beta}-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis. Neurobiol Dis 5: 229–243, 1998.[CrossRef][ISI][Medline]
  18. Harding HP, Calfon M, Urano F, Novoa I, and Ron D. Transcriptional and translational control in the mammalian unfolded protein response. Annu Rev Cell Dev Biol 18: 575–599, 2002.[CrossRef][ISI][Medline]
  19. Harding HP, Zhang Y, and Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397: 208–211, 1999.[CrossRef][Medline]
  20. Hayashi T, Saito A, Okuno S, Ferrand-Drake M, Dodd RL, Nishi T, Maier CM, Kinouchi H, and Chan PH. Oxidative damage to the endoplasmic reticulum is implicated in ischemic neuronal cell death. J Cereb Blood Flow Metab 23: 1117–1128, 2003.[ISI][Medline]
  21. Hayashi T, Saito A, Okuno S, Ferrand-Drake M, Dodd RL, and Chan PH. Oxidative injury to the endoplasmic reticulum in mouse brains after transient focal ischemia. Neurobiol Dis 15: 229–239, 2004.[CrossRef][ISI][Medline]
  22. He H, Lam M, McCormick TS, and Distelhorst CW. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol 138: 1219–1228, 1997.[Abstract/Free Full Text]
  23. Kaufman RJ. Stress signal from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13: 1211–1233, 1999.[Free Full Text]
  24. Kawai H, Mohan A, Hagen J, Dong E, Armstrong J, Stevens SY, and Liang CS. Alterations in cardiac adrenergic terminal function and {beta}-adrenoceptor density in pacing-induced heart failure. Am J Physiol Heart Circ Physiol 278: H1708–H1716, 2000.[Abstract/Free Full Text]
  25. Kaye DM, Wiviott SD, Kobzik L, Kelly RA, and Smith TW. S-nitrosothiols inhibit neuronal norepinephrine transport. Am J Physiol Heart Circ Physiol 272: H875–H883, 1997.[Abstract/Free Full Text]
  26. Kippenberger AG, Palmer DJ, Comer AM, Lipski J, Burton LD, and Christie DL. Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: evidence for its association with secretory granules in PC12 cells. J Neurochem 73: 1024–1032, 1999.[CrossRef][ISI][Medline]
  27. Lee AS. Mammalian stress response: induction of the glucose-regulated protein family. Curr Opin Cell Biol 4:267–273, 1992.[CrossRef][Medline]
  28. Liang CS. Sympatholysis and cardiac sympathetic nerve function in the treatment of congestive heart failure. J Am Coll Cardiol 42:549–551, 2003.[Free Full Text]
  29. Liang CS, Rounds NK, Dong E, Stevens SY, Shite J, and Qin F. Alterations by norepinephrine of cardiac sympathetic nerve terminal function and myocardial {beta}-adrenoceptor sensitivity in the ferret: normalization by antioxidant vitamins. Circulation 102: 96–103, 2000.[Abstract/Free Full Text]
  30. Liu H, Bowes RC III, van de Water B, Sillence C, Nagelkerke JF, and Stevens JL. Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J Biol Chem 272: 21751–21759, 1997.[Abstract/Free Full Text]
  31. Liu H, Miller E, van de Water B, and Stevens JL. Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death. J Biol Chem 273: 12858–12862, 1998.[Abstract/Free Full Text]
  32. Lorang D, Amara SG, and Simerly RB. Cell-type specific expression of catecholamine transporters in the rat brain. J Neurosci 14: 4903–4914, 1994.[Abstract]
  33. Mao W, Qin F, Iwai C, Vulapalli R, Kang PC, and Liang Cs. Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells. Am J Physiol Heart Circ Physiol 287: H29–H39, 2004.[Abstract/Free Full Text]
  34. Melikian HE, Ramamoorthy S, Tate CG, and Blakely RD. Inability to N-glycosylate human norepinephrine transporter reduces protein stability, surface trafficking, and transporter activity but not ligand recognition. Mol Pharmacol 50: 266–276, 1996.[Abstract]
  35. Melikian HE, McDonald JK, Gu H, Rudnick G, Moore KR, and Blakely RD. Human norepinephrine transporter. Biosynthetic studies using a site-directed polyclonal antibody. J Biol Chem 269: 12290–12297, 1994.[Abstract/Free Full Text]
  36. Nakazawa H, Fukuyama N, Takizawa S, Tsuji C, Yoshitake M, and Ishida H. Nitrotyrosine formation and its role in various pathological conditions. Free Radic Res 33: 771–784, 2000.[CrossRef][ISI][Medline]
  37. Olivares L, Aragon C, Gimenez C, and Zafra F. The role of N-glycosylation in the targeting and activity of the GLYT1 glycine transporter. J Biol Chem 270: 9437–9442, 1995.[Abstract/Free Full Text]
  38. Oyadomari S, Koizumi A, Takeda K, Gotoh T, Akira S, Akira E, and Mori M. Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes. J Clin Invest 109: 525–532, 2002.[CrossRef][ISI][Medline]
  39. Oyadomari S and Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11: 381–389, 2004.[CrossRef][ISI][Medline]
  40. Oyadomari S, Takeda K, Takiguchi M, Gotoh T, Matsumoto M, Wada I, Akira S, Araki E, and Mori M. Nitric oxide-induced apoptosis in pancreatic {beta} cells is mediated by the endoplasmic reticulum stress pathway. Proc Natl Acad Sci USA 98: 10845–10850, 2001.[Abstract/Free Full Text]
  41. Qi X, Okuma Y, Hosoi T, and Nomura Y. Edaravone protects against hypoxia/ischemia-induced endoplasmic reticulum dysfunction. J Pharmacol Exp Ther 311: 388–393, 2004.[Abstract/Free Full Text]
  42. Rabek JP, Boylston WH III, and Papaconstantinou J. Carbonylation of ER chaperone protein in aged mouse liver. Biochem Biophys Res Commun 305: 566–572, 2003.[CrossRef][ISI][Medline]
  43. Rao RV, Peel A, Logvinova A, del Rio G, Hermel E, Yokota T, Goldsmith PC, Ellerby LM, Ellerby M, and Bredesen DE. Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78. FEBS Lett 514: 122–128, 2002.[ISI][Medline]
  44. Rao RV, Ellerby HM, and Bredesen DE. Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ 11:372–380, 2004.[CrossRef][ISI][Medline]
  45. Ron D and Habener JF. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant negative inhibitor of gene transcription. Genes Dev 6: 439–453, 1992.[Abstract/Free Full Text]
  46. Ryu EJ, Harding HP, Angelastro JM, Vitolo OV, Ron D, and Greene LA. Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. J Neurosci 22: 10690–10698, 2002.[Abstract/Free Full Text]
  47. Tajiri S, Oyadomari S, Yano S, Morioka M, Gotoh T, Hamada JI, Ushio Y, and Mori M. Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ 11: 403–415, 2004.[CrossRef][ISI][Medline]
  48. Tate CG and Blakely RD. The effect of N-linked glycosylation on activity of the Na+-and Cl–-dependent serotonin transporter expressed using recombinant baculovirus in insect cells. J Biol Chem 269: 26303–26310, 1994.[Abstract/Free Full Text]
  49. Yu Z, Luo H, Fu W, and Mattson MP. The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis. Exp Neurol 155: 302–314, 1999.[CrossRef][ISI][Medline]
  50. Zhu MY, Shamburger S, Li J, and Ordway GA. Regulation of the human norepinephrine transporter by cocaine and amphetamine. J Pharmacol Exp Ther 295: 951–959, 2000.[Abstract/Free Full Text]



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