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Am J Physiol Heart Circ Physiol 287: H29-H39, 2004. First published February 12, 2004; doi:10.1152/ajpheart.01168.2003
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Extracellular norepinephrine reduces neuronal uptake of norepinephrine by oxidative stress in PC12 cells

Weike Mao, Fuzhong Qin, Chikao Iwai, Raju Vulapalli, Peter C. Keng, and Chang-seng Liang

Cardiology Unit, Department of Medicine, and Department of Radiation Oncology, University of Rochester Medical Center, Rochester, New York 14642

Submitted 8 December 2003 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac norepinephrine (NE) uptake activity is reduced in congestive heart failure. Our studies in intact animals suggest that this effect on the cardiac sympathetic nerve endings is caused by oxidative stress and/or NE toxic metabolites derived from NE. In this study, we investigated the direct effects of NE on neuronal NE uptake activity and NE transporter (NET), using undifferentiated PC12 cells. Cells were incubated with NE (1–500 µM) either alone or in combination of Cu2+ sulfate (1 µM), which promotes free radical formation by Fenton reaction for 24 h. NE uptake activity was measured using [3H]NE. Cell viability was determined with the use of Trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, and cellular oxidative stress by dichlorodihydrofluorescein fluorescence and the GSH/GSSG ratio. Cell viability was reduced by NE >100 µM. At lower doses, NE produced oxidative stress and a dose-dependent reduction of NE uptake activity without affecting cell viability significantly. Cu2+, which has no direct effect on NE uptake activity, potentiated oxidative stress and reduction of NE uptake activity produced by NE. This decrease of NE uptake activity was associated with reductions of NE uptake binding sites and NET protein expression by using the radioligand assay and Western blot analysis, but no changes in NET gene expression. In addition, the free-radical scavenger mannitol, and antioxidant enzymes superoxide dismutase and catalase, reduced oxidative stress and attenuated the reductions of NE uptake activity and NET protein produced by NE/Cu. Thus our results support a functional role of oxidative stress in mediating the neuronal NE uptake reducing effect of NE and that this effect of NE on NET is a posttranscriptional event.

Cu2+; reactive oxygen species; antioxidants

INCREASED CARDIAC sympathetic stimulation and depletion of tissue norepinephrine (NE) are two important clinical features in patients with congestive heart failure. The decrease of tissue NE is caused not only by increased neuronal release of NE but also because of reduced biosynthesis of NE and impaired reuptake of NE at the sympathetic nerve endings. These changes are associated with reductions of sympathetic neuronal markers tyrosine hydroxylase and neuropeptide (24, 39, 41). However, the sympathetic nerves are probably structurally intact because there is no change in protein gene product 9.5 (41), which is a panneuronal marker unrelated to sympathetic transmitters (21). In addition, we found that the reduced NE uptake in heart failure is associated with reduction of NE uptake-1 carrier binding site (24), known as NE transporter (NET). NET, a member of the superfamily of Na+/Cl–-dependent transporters (4), acts to remove NE from the synaptic cleft and is responsible for rapid termination of actions of NE after neuronal release. A decrease in NET function is expected to increase interstitial NE concentration. This function of NET is physiologically important in heart failure because NE uptake correlates inversely with myocardial interstitial NE and is associated with downregulation of myocardial {beta}-receptors (13, 14, 27). Furthermore, because the changes in cardiac sympathetic nerve endings were produced by chronic NE infusion in animals without heart failure (14, 40, 41), and because these changes were reduced by administration of antioxidant vitamins or superoxide dismutase (SOD) (40), we speculate that the decrease in NET is produced by NE-derived oxidative stress.

The purpose of the present study was to provide direct evidence that NE reduces neuronal NE uptake activity in the sympathetic neurons, with the use of the undifferentiated rat pheochromocytoma PC12 cells that are devoid of synaptic contacts (20). The PC12 cell expresses abundant NET (9) and is a useful model system to study the direct actions of interventions on NET and the factors that regulate NET expression (43, 74, 75). In this study, we studied whether the decrease of NE uptake activity was associated with reduction of NET binding site or protein. We also investigated whether this action of NE on NE uptake activity was associated with increased intracellular oxidative stress as measured by intracellular dichlorodihydrofluorescein (DCF) fluorescence (23) and the GSH/GSSG ratio (49). To enhance the prooxidant effect of NE, we added Cu2+, which reacts with O2– and H2O2 to form more toxic hydroxyl radicals by Fenton reaction (25, 31, 46). Finally, to investigate the importance of oxidative stress in mediating this effect of NE, we carried out experiments to determine whether the PC12 cells could be protected from the effect of NE by mannitol, a hydroxyl scavenger (6, 73), and antioxidant enzymes SOD and catalase (35).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals

RPMI 1640 medium, fetal bovine serum, heat-inactivated horse serum, HEPES buffer, TRIzol reagent, and Taq DNA polymerase were obtained from GIBCO-BRL (Gaithersburg, MD). NE, Cu2+ sulfate, Trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), PMSF, nisoxetine, desipramine, DuraScript RT-PCR kit, mannitol, catalase, and SOD were obtained from Sigma (St. Louis, MO). 5-(and 6-)-chloromethyl-2',7'-DCF diacetate acetyl ester (CM-H2DCFDA) was obtained from Molecular Probes (Eugene, OR). [3H]NE, [3H]nisoxetine, and [32P]dCTP were obtained from New England Nuclear Life Science Products (Boston, MA).

PC12 Cell Culture

PC12 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in a 75-cm2 flask in HEPES-modified RPMI 1640 medium containing 10% heat-inactivated horse serum and 2.5% fetal bovine serum in a humidified incubator with 95% air-5% CO2 at 37°C. The culture medium was changed three times a week. Cells were subcultured after 4 to 5 days. All experiments were performed using PC12 cells at the passages 6–15.

Experiment Protocol

Effects of NE on PC12 cell viability. For the studies, PC12 cells were first plated in 12-well plates coated with poly-d-lysine at a density of 1 x 105 cells/well at 37°C and then changed to a RPMI medium containing 2% horse serum. To determine the sublethal doses of NE to be used in this study, we exposed the PC12 cells to a wide range of NE concentrations (1, 10, 25, 50, 100, 200, and 500 µM) in the presence and the absence of Cu2+ sulfate (1 µM) for 24 h. The culture medium was then aspirated, and the cells were washed once with phosphate-buffered saline and prepared for the cell viability study with the use of Trypan blue.

For the Trypan blue extrusion method, the PC12 cells were stained with 0.2% Trypan blue for 5 min. Cells unable to extrude Trypan blue and thus stained blue were considered nonviable, whereas those expelling dye and unstained were considered viable. The stained and nonstained cells were counted separately on a hemocytometer under an inverted microscope (model TMS-F, Nikon; Melville, NY). The number of stained cells, expressed as a percentage of total cells, was used as a measure of cell death.

PC12 cell viability was also measured using the MTT assay, which is based on the reduction of MTT to the blue formazan by mitochondria dehydrogenases (1). The viable cells that contain mitochondrial dehydrogenases are stained blue. For this method, MTT (0.5 mg/ml) was added to the PC12 cells in 2% serum RPMI medium and incubated for 2 h at 37°C. Cells were then rinsed with phosphate-buffered saline and the blue formazan crystals dissolved in isopropanol containing 0.04 N HCl. The absorbance of the formazan crystals was then measured at 570 nm using a SmartSpec 3000 spectrophotometer (Bio-Rad; Hercules, CA). The optical density was calculated and expressed as the percentage of control.

Effects of NE on neuronal NE uptake activity. To determine the effects of NE on neuronal NE uptake activity, we exposed the PC12 cells on 12-well plates coated with poly-D-lysine to three sublethal concentrations of NE (1, 10, and 100 µM) with and without Cu2+ sulfate (1 µM) for 24 h. Untreated PC12 cells were run concurrently as controls. The effects of Cu2+ sulfate (0.1, 1, and 10 µM) were also studied without NE. Additional experiments were carried out to determine the reversibility of NE uptake dysfunction after removal of NE. In these experiments, the NE containing medium was replaced by fresh RPMI with 2% horse serum and incubated for 6 h at 37°C. At the end of experiments, neuronal uptake of NE was measured using the method of Toyahira et al. (67) with minor modifications. Briefly, PC12 cells were washed once with phosphate-buffered saline and preincubated in Krebs-Ringer buffer composed of (in mM) 125 NaCl, 2 KCl, 1.4 MgSO4, 1.2 CaCl2, 1.2 KH2PO4, 20 HEPES, 5 glucose, 1 ascorbic acid, 0.01 pargyline (pH 7.4) at 37°C for 15 min. [3H]NE (35.7 nM, 14 Ci/mmol) was then added to the incubation media at 37°C. After 15 min of incubation, the cells were then washed rapidly with ice-cold Krebs-Ringer solution twice and lysed in 0.5 ml of 1 N NaCl and 0.5 ml of 1 N HCl. The radioactivity was measured in a Tri-Carb 2400 TR liquid scintillation counter (Packard Instrument; Meriden, CT). The specific uptake of [3H]NE was expressed as the difference between the uptake in the absence and in the presence of 10 µM nisoxetine, a highly selective NET inhibitor (66). The dose of nisoxetine was chosen in pilot studies to produce the maximal specific inhibition of NE uptake.

We also performed kinetic analysis of NE uptake in PC12 cells. [3H]NE uptake was measured as above in the absence or presence of 0.1–6 µM unlabeled NE. Saturation analysis of [3H]NE was performed along with Eadie-Hofstee plot analysis to determine the maximal velocity (Vmax) of NE uptake and the dissociation constant (Km). Protein concentration was measured using the bucinchoninic acid protein kit (Pierce; Rockford, IL).

Effects of NE on Oxidative Stress

Total cellular oxidative stress. Total cellular oxidative stress was measured by the ratio of reduced GSH to GSSG using 5,5-dithiobis-2-nitrobenzoic acid as a substrate in a GSH reductase-coupled enzymatic system (49, 63). PC12 cells were washed by phosphate-buffered saline, lysed by 2% picric acid on ice for 30 min, and then centrifuged at 12,000 rpm for 20 min. The supernatant was kept at –70°C for assay. The rate of formation of 2-nitro-5-thiobenzoic acid, which is proportional to the amount of GSH, was determined at a wavelength of 412 nm on the SmartSpec 3000 spectrophotometer. GSSG was measured by masking GSH with 2-vinyl pyridine in the conversion reaction from 5,5-dithiobis-2-nitrobenzoic acid to 2-nitro-5-thiobenzoic acid. The ratio of GSH to GSSG was calculated.

Intracellular reactive oxygen species. Intracellular reactive oxygen species was measured using the fluorescent probe H2DCFDA, as described by Rancourt et al. (59), with a minor modification. After drug exposure, the culture medium was changed to fresh RPMI medium containing 10 µM CM-H2DCFDA and the cells were incubated for an additional 30 min at 37°C. The cells were then treated with trypsin, centrifuged, and resuspended in fresh RPMI medium at 1 x 106/ml. Integrated fluorescence intensity of DCF was quantified using a Coulter Elite flow cytometer with excitation at 488 nm and emission at 530 nm.

Effects of NE on whole cell and cell membrane NE uptake binding site density. PC12 cell NE uptake-1 carrier site density was measured using the high-affinity NET-selective ligand, [3H]nisoxetine, in both the membrane fraction homogenates (65) and whole cells (5). For cell membrane binding, the cells were homogenized and centrifuged at 4°C. Approximately 55 µg of the membrane protein, suspended in 50 mM Tris·HCl buffer (pH 7.4) containing 300 mM NaCl and 5 mM KCl, was incubated in triplicate with 3 nM [3H]nisoxetine (85.5 Ci/mmol) and 6 concentrations of cold nisoxetine (3–100 nM) at room temperature for 60 min. Another structurally unrelated NET inhibitor desipramine was used to determine the nonspecific binding of nisoxetine. A dose of 10 µM desipramine was chosen based on results of pilot studies. The reaction was terminated by the addition of ice-cold Tris buffer. The membranes were rapidly washed three times and filtered through Whatman GF/B filters on a Brandel cell harvester (Biomedical Research and Development Laboratories; Gaithersburg, MD). The filters were dried and counted for [3H]radioactivity by liquid scintillation spectrometry. The specific binding to the NE uptake carrier site was determined by the difference of radioactivity in the absence and presence of desipramine. The number of binding sites and Kd were calculated with the use of EBDA computer software program (47).

For whole cell radioligand binding, PC12 cells were washed twice with binding buffer containing (in mM) 150 NaCl, 5 KCl, 1 MgSO4, 10 Tris, and 0.1% BSA at 25°C. The cells were then incubated for 60 min at 25°C with binding buffer containing 3 nM [3H]nisoxetine and different concentrations of unlabeled nisoxetine (3–100 nM). Desipramine (10 µM) was used to determine nonspecific binding of nisoxetine. After incubation, the solution was removed and cells were washed two times with 1 ml of ice-cold binding buffer and lysed by 500 µl of 1% SDS for 60 min at 37°C. The radioligand activity in cell lysate was counted by liquid scintillation spectrometry and protein concentration was measured by using a bicinchoninic acid (BCA) Protein Assay kit (Pierce; Rockford, IL).

Effects of NE on NET protein and mRNA expression. NET protein from PC12 cell membrane fraction was measured using the Western blot method of Kippenberger et al. (32), with minor modification. After treatment, cells were harvested in phosphate-buffered saline and recovered by centrifugation at 100 g for 5 min. The cells were resuspended in ice-cold homogenization buffer (1 ml/10 cm dish) containing 10 mM HEPES (pH 7.4), 0.15 M NaCl, 1 mM EGTA, 1 mM MgCl2, and 1 mM PMSF, and homogenized with the use of a PCU-2 Polytron homogenizer (setting 6) for 20 s. The solution was centrifuged at 1,000 g at 4°C for 5 min to remove nucleic debris. The supernatant was then centrifuged at 100,000 g for 60 min at 4°C. The membrane pellet was dissolved in radioimmunoprecipitation buffer with 1% Triton and kept in –70°C. The proteins were then separated on 8–10% SDS-PAGE and transferred electrically (100 V, 1 h, 4°C) to polyvinylidene fluoride membranes. The membranes were then treated with affinity-purified primary rabbit anti-rat NET antibody (Chemicon; Temecula, CA; 1:200 dilution) in a 5% milk in Tris-buffered saline solution with 0.1% Tween, followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000 dilution in blocking solution for 1 h at 22°C). Immunoreactive bands were visualized by enhanced chemiluminescence reagents (Amersham; Arlington Heights, IL). The optical intensity of bands was determined with the use of ScanMaker (Microtek; Carson, CA) and the NIH version 1.6 gel Image program. The optical density was normalized to a control sample in arbitrary densitometry unit.

To measure NET mRNA, PC12 cells were lysed for extraction of RNA using TRIzol reagent. The RNA was dissolved in 50 µl of a solution containing 0.5 mM dithiothreitol and 1 U/µl RNase inhibitor. The extracted RNA (5 µg) was denatured for cDNA synthesis by RT with the use of random primers. The purified products of first-strand cDNA were then used for RT-PCR using an enhanced avian DuraScript RT-PCR kit. The PCR reaction (total volume 50 µl) included 0.25 mM dNTPs, 1–2 U/µl Taq DNA polymerase, 4 µM NET or GAPDH primers, and [32P]dCTP 0.25 µCi. The reaction was amplified according the method of Bryan-Lluka et al. (10) in a PTC programmable thermal controller (MJ Research; Watertown, MA) using the following thermal cycles: 95°C for 2 min, 25 cycles of 95°C for 40 s, 60°C for 10 s, and 72°C for 30 s, and then 5 min at 72°C. NET mRNA was detected by an upstream primer 5'-CATCAACTGTGTTACCAGTTTTATT-3' and a downstream primer 5'-AAACATGGCCAGAAGAAAGGTACC-3' (9). The results were normalized by the housekeeping gene GAPDH, which was detected by a second pair of oligo primers: 5'-GCCAAAAGGGTCATCATCTC-3' (upstream) and 5'-GGCCATCCACAGTCTTCT-3' (downstream). The PCR products were separated by a 5% polyacrylamide native gel with a running buffer containing 45 mM Tris-borate and 1 mM EDTA. Gels were dried and exposed to Kodak Biomax MR film at –70°C and autoradiographs were quantified in arbitrary units on a Microtek scanner.

Effects of antioxidants on NE-mediated NE uptake activity, NET density, and protein, and oxidative stress. To study the effects of free radical scavengers on the effects of NE, we pretreated the PC12 cells with 2 mM mannitol, 20 µg/ml SOD, 20 µg/ml catalase, or SOD plus catalase (both 20 µg/ml) for 1 h before the addition of NE and Cu2+ sulfate to the Krebs-Ringer buffer. The cells were incubated for 24 h and then prepared for measurement of NE uptake activity, NET density and protein, and oxidative stress level. The experimental protocols for the latter studies were identical to those described above for NE treatment alone.

Statistics

Experimental data were managed on the RS/1 Research System (Bolt, Beraneck and Newman Software Products; Cambridge, MA) and SYSTAT version 8.0 (SPSS; Chicago, IL), and the results are presented as means ± SE. The statistical significance of the effects of NE and their differences among the different experimental groups were analyzed by either one- or two-way ANOVA, whichever is appropriate. The statistical significance of differences among the groups were determined with the use of Bonferroni simultaneous confidence intervals for all comparisons. A difference with a P < 0.05 was considered statistically significant.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Toxic Effects of NE on PC12 Cells

Figure 1 shows that NE did not affect cell viability at concentration ≤100 µM. However, at higher concentrations, NE caused significant cell death, as judged by both Trypan blue exclusion and MTT assay. Cu2+ (1 µM) potentiated the toxic effect of high doses of NE (200 and 500 µM) on cell death (Trypan blue staining) but did not increase cell loss when added to 1 to 100 µM NE. Thus we limited the NE concentrations for the following studies to 100 µM.



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  		Fig. 1. Dose-dependent effects of norepinephrine (NE) and NE plus Cu2+ (NE/Cu) on the viability of PC12 cells. PC12 cells (2 x 10 5/well) were exposed to either NE alone or NE/Cu for 24 h. Cell viability was measured by either Trypan blue staining (top) or 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (bottom). Bars denote means ± SE; n = 10 for each group in top, and n = 7 for each group in bottom. Two-way ANOVA of the Trypan blue data showed significant effects of NE treatment [variance ratio (F) = 2,980, degrees of freedom (df) = 6, 84; P < 0.001] and Cu2+ interventions (F = 52.2, df = 1, 84; P < 0.001), and statistical significance of the interaction of NE and Cu2+ intervention (F = 6.2, df = 6, 84; P < 0.001). However, similar analysis of the MTT assay data revealed only significant differences among the NE groups (F = 178.2, df = 7, 81; P < 0.001). There was no statistical significance of effects of Cu2+ intervention (F = 1.0, df = 1, 81; P = 0.31) or the interaction between NE treatment and Cu2+ intervention (F = 2.0, df = 7, 81; P = 0.07). *P < 0.05, compared with control; {dagger}P < 0.05, compared with the NE group, as determined by nonsimultaneous 95% confidence intervals for the group means.

 
Effects of NE on NE Uptake Activity of PC12 Cells

PC12 cells were exposed to sublethal doses of NE (1, 10, and 100 µM) in the presence and absence of Cu2+ (1 µM) for 24 h. Figure 2 shows that in the absence of Cu2+, NE had no significant effect on NE uptake activity at 1 µM, but reduced NE uptake activity at 10 and 100 µM. The addition of Cu2+ sulfate (1 µM) to NE in the medium potentiated the inhibitory effect of NE at all doses on NE uptake activity (Fig. 2). The effect of NE/Cu on NE uptake activity was dose dependent. Figure 3 shows the effects of Cu2+ sulfate alone on NE uptake activity in PC12 cells. NE uptake activity was not affected by Cu2+ sulfate at concentrations of 1 µM or less. At a higher dose (10 µM), Cu2+ sulfate decreased NE uptake activity by 35%. The findings suggest that the exaggerated effects of NE/Cu on NE uptake activity shown in Fig. 2 could not be explained by the simple additive effects of NE and Cu2+.



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  		Fig. 2. Reduction of NE uptake activity in PC12 cells produced by either NE alone or NE plus Cu2+ (NE/Cu). Bars denote means ± SE; n = 6 in each group. Two-way ANOVA showed significant effects of NE treatment (F = 36.1, df = 3, 48, P < 0.001) and Cu2+ interventions (F = 35.8, df = 1, 48, P < 0.001), and statistical significance of the interaction of NE/Cu2+ intervention (F = 4.1, df = 3, 48, P = 0.012). *P < 0.05, compared with control; {dagger}P < 0.05, compared with the NE group, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 


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  		Fig. 3. Effects of Cu2+ sulfate on NE uptake activity in PC12 cells. Bars denote means ± SE; n = 8 in each group. One-way ANOVA showed that Cu2+ sulfate (CuSO4) reduced NE uptake activity (F = 4.46, df = 3, 28; P = 0.011). *P < 0.05, compared with control without Cu2+ sulfate, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 
Figure 4, top, shows saturation profiles of [3H]NE to NE/Cu-treated PC12 cells either in the presence or in the absence of 0.1–6 µM unlabeled NE. The linear fit in the Eadie-Hofstee plots (Fig. 4, bottom) indicates a single population of binding sites. The kinetic analysis showed that NE/Cu caused a dose-dependent decrease of Vmax of [3H]NE (Fig. 4, bottom) without affecting Km.



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  		Fig. 4. Top: saturation equilibrium of [3H]NE in the presence of various doses of unlabeled NE in PC12 cells after 24 h of incubation with 0 (control), 1, 10, and 100 µM of NE/Cu (1 µM). Bars denote means ± SE. Bottom: maximal velocity (Vmax) of the NE uptake activity decreased in a dose-dependent fashion in PC12 cells treated with NE and Cu2+ (F = 13.4, df = 3, 32, P < 0.001). Bars denote means ± SE; n = 6 in each group. Inset: Eadie-Hofstee plots of NE uptake in cells at control and after incubation with the highest doses of NE/Cu (100/1 µM). *P < 0.05, compared with control without NE, as determined Bonferroni simultaneous 95% confidence intervals for all comparisons.

 
To study whether the effects of NE/Cu on NE uptake activity are reversible after removal of NE, we changed the NE/Cu-containing incubation medium of the PC12 cells to fresh 2% horse serum culture medium after 24 h of exposure. NE uptake activity was measured at 6 h after removal of the NE/Cu-containing medium and compared with that measured after 24 h of NE/Cu incubation. Table 1 shows that NE uptake activity increased significantly after removal of NE/Cu. NE uptake activity returned to baseline within 6 h of recovery in cells treated with 1 and 10 µM of NE. However, the recovery after removal of NE/Cu in cells treated with 100 µM of NE was incomplete.


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  		Table 1. Recovery of NE uptake activity of PC12 cells 6 h after removal of NE/Cu from incubation medium

 
Effects of NE on Oxidative Stress of PC12 Cells

The effects of NE and NE/Cu after 24 h of incubation on cellular GSH and GSH/GSSG ratio are shown in Fig. 5. NE decreased GSH and GSH/GSSG ratio in PC12 cells. The figure also shows that the effect of NE on the GSH system was exaggerated by addition of Cu2+ (NE/Cu). To simplify study protocols, subsequent experiments were carried out in PC12 cells treated with both NE and Cu2+.



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  		Fig. 5. Changes of GSH and GSH/GSSG ratio in PC12 cells after 24 h of incubation with NE in the presence and absence of Cu2+. Bars denote means ± SE; n = 10 in each group. Two-way ANOVA showed significant effects of NE treatment (F = 24.6, df = 3, 72; P < 0.001) and Cu2+ interventions (F = 35.2, df = 1, 72; P < 0.001), and statistical significance of the interaction of NE and Cu2+ intervention (F = 5.9, df = 3, 72, P < 0.001). *P < 0.05, compared with control; {dagger}P < 0.05, compared with the NE group, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 
The generation of reactive oxygen species within the PC12 cells was also studied after incubation with NE/Cu using the DCF fluorescence method. In a pilot study, we measured DCF fluorescence intensity in PC12 cells after 1, 2, 4, 8, or 24 h of incubation with NE (100 µM) and Cu2+ (1 µM). A peak increase of DCF fluorescence occurred at 2 h of NE/Cu incubation. At 24 h, the fluorescence intensity was still elevated ~70% above the control value. Figure 6 shows that the effects of NE/Cu were dose dependent. At the highest dose studied (100 µM), NE increased DCF fluorescence intensity to 0.61 ± 0.06. The effect was comparable to an increase of DCF fluorescence (0.66 ± 0.13) produced by hydrogen peroxide (100 µM). We also studied the effects of three doses of Cu2+ sulfate (0.1, 1, and 10 µM) on reactive oxygen species in the PC12 cells using intracellular DCF fluorescence. Figure 7 shows that Cu2+ produced no appreciable reactive oxygen species production until the dose was increased to 10 µM.



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  		Fig. 6. Dose-dependent effects of NE plus Cu2+ (1 µM) on intracellular reactive oxygen species formation in PC12 cells, as measured by the integrated DCF fluorescence intensity. Bars denote means ± SE; n = 8 in each group. ANOVA indicates a significant difference of the fluorescence intensity among the groups (F = 8.16, df = 4, 39, P < 0.001). *P < 0.05, compared with control, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 


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  		Fig. 7. Effects of Cu2+ sulfate on intracellular reactive oxygen species formation in PC12 cells, as measured by the integrated dichlorodihydrofluorescein (DCF) fluorescence intensity (n = 6 in each group). Bars denote means ± SE. ANOVA indicates a significant difference of the fluorescence intensity among the groups (F = 15.99, df = 3, 20, P < 0.001). *P < 0.05, compared with control, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 
Effects of NE/Cu on NE Uptake Binding Site Density in PC12 Cell Membrane Fraction and Whole Cell Preparations

Table 2 shows the effects of NE/Cu on the number of [3H]nisoxetine binding sites (Bmax) and Kd in the PC12 cell membrane homogenate. The table shows that NE/Cu produced a dose-dependent reduction of NE uptake binding site density but had no effects on Kd. The addition of Cu2+ alone affected neither Bmax nor Kd of [3H]nisoxetine binding in the membrane fraction.


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  		Table 2. Effects of NE and cupric sulfate on PC12 cell membrane [3H]nisoxetine binding characteristics

 
NE/Cu also reduced the number of NE uptake binding sites in the PC12 whole cell preparation. Figure 8 shows the Scatchard plots of [3H]nisoxetine binding in both cell membrane and whole cell preparations of the PC12 cells treated with and without NE/Cu (100/1 µM). Compared with the 42% reduction in the membrane homogenate, NE uptake site density was reduced to a smaller extent (20%) in the whole cell preparation after 24 h of NE/Cu incubation.



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  		Fig. 8. Scatchard plots of [3H]nisoxetine binding in PC12 cell membrane (n = 6) and whole cell (n = 8) preparations at control and after 24 h of incubation with NE (100 µM) plus Cu (1 µM) (NE/Cu). Bidirectional bars denote means ± SE for both "bound" and "bound/free" axes. Bmax, nisoxetine binding site density. *P < 0.05, compared with control, as determined by nonpaired t-test.

 
Effects of NE/Cu on NET Protein and mRNA Expressions in PC12 Cells

The decrease of NE uptake binding sites in the membrane fraction was confirmed by the Western immunoblots showing a reduction of an 80-kDa NET band (Fig. 9). In contrast, NE/Cu had no significant effect on the PC12 cell NET mRNA expression. Figure 10, top, shows the representative autoradiographs for the NET and GAPDH mRNA bands. The lower panel shows NE/Cu had no effects on the PC12 cell NET mRNA as normalized by GAPDH mRNA.



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  		Fig. 9. Effects of various concentrations of 1 µM NE/Cu on NE transporter (NET) protein expression in PC12 cells. Cells were exposed to three concentrations of NE/Cu2+ for 24 h. An example of the enhanced chemiluminescence NET immunoblot is shown at top. Each well was loaded with 50 µg of membrane protein and blots were probed with the anti-rat NET antibody. The 80-kDa bands identify NET proteins. The optical density was measured in arbitrary unit and summarized on the bottom graph. Bars denote means ± SE (n = 10). One-way ANOVA indicates significant difference among the groups (F = 11.8, df = 3, 36; P < 0.001). *P < 0.05, compared with control with NE/Cu, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 


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  		Fig. 10. Effects of various concentrations of 1 µM NE/Cu2+ on NET mRNA expression in PC12 cells. Cells were exposed to three concentrations of NE/Cu2+ for 24 h and then assayed for NET and GAPDH mRNA expression. Top: representative autoradiographs of the PCR product (2 µl/lane) for NET and GAPDH mRNAs. The optical density of bands was measured in arbitrary unit. Bottom: NET mRNA was normalized by its correspondent GAPDH mRNA. Bars denote means ± SE (n = 6 in each way). ANOVA indicates no significant differences among the groups (F = 0.36, df = 3, 20; P = 0.781).

 
Effects of Antioxidants on NE/Cu-Induced Reductions of NE Uptake Binding Site, NET Protein, and Oxidative Stress

Mannitol, SOD, and catalase were added to the incubation medium for the PC12 cells 1 h before introduction of NE/Cu. Table 3 shows that the antioxidants did not affect the NE uptake activity or GSH/GSSG ratio in a control state without NE/Cu. However, they attenuated the reductions of NE uptake activity and GSH/GSSG ratio produced by NE/Cu. The effects were least pronounced with mannitol, and most marked with SOD plus catalase.


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  		Table 3. Effect of mannitol, SOD, and catalase on NE/Cu-induced reduction of NE uptake activity and GSH/GSSG ratio of PC12 cells

 
We also found that the antioxidants, which had no effects on NE uptake site density or intracellular reactive oxygen species as measured by DCF fluorescence on PC12 cells, prevented the reductions of NE uptake site density (Fig. 11), NET protein (Fig. 12), and DCF fluorescence intensity (Fig. 13) produced by NE/Cu.



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  		Fig. 11. Effects of 100 µM NE and 1 µM Cu2+ (NE/Cu) on cell membrane Bmax in PC12 cells with and without antioxidants superoxide dismutase (SOD) plus catalase or mannitol. Bars denote means ± SE (n = 6 in each group). ANOVA indicates that NE/Cu and antioxidants produced statistically significant effects (F = 20.3, df = 5, 30; P < 0.001). *P < 0.05, compared with control without NE and antioxidants; {dagger}P < 0.05, compared with NE/Cu without antioxidants, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 


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  		Fig. 12. Effects of 100 µM NE and 1 µM Cu2+ (NE/Cu) on NET protein expression in control PC12 cells and PC12 cells treated with antioxidants SOD plus catalase or mannitol. Bars denote means ± SE (n = 10 in each group). ANOVA indicates that statistically significant differences exist among the groups (F = 9.43, df = 3, 36; P = 0.001). *P < 0.05, compared with control without NE; {dagger}P < 0.05, compared with NE/Cu without antioxidants, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 


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  		Fig. 13. Effects of 100 µM NE and 1 µM Cu2+ (NE/Cu) on intracellular reactive oxygen species as measured by DCF fluorescence intensity in PC12 cells with and without antioxidants SOD plus catalase or mannitol (n = 8 in each group). ANOVA indicates that NE/Cu and antioxidants produced statistically significant effects (F = 13.5, df = 5, 54; P < 0.001). *P < 0.05, compared with control without NE and antioxidants; {dagger}P < 0.05, compared with NE/Cu without antioxidants, as determined by Bonferroni simultaneous 95% confidence intervals for all comparisons.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that NE produces a partially reversible reduction of neuronal NE uptake activity in a dose-dependent manner in PC12 cells. Kinetic analysis of [3H]NE uptake revealed a reduction in Vmax of NE uptake without changes of dissociation constant. This change in NE uptake activity was associated with reduction of [3H]nisoxetine binding site and NET protein expression. These changes are caused probably by posttranscriptional factors because NE had no effect on NET mRNA of the PC12 cells. In addition, because the effect of NE on NE uptake activity was associated with an increase in oxidative stress, and exaggerated by the transition ion Cu2+, we speculate that the reduction of NE uptake activity is caused, at least in part, by NE-derived oxidative metabolites and stress. This is further supported by our findings that the reductions of NE uptake activity and NET protein expression by NE/Cu were attenuated by the free radical scavenger mannitol, and antioxidant enzymes catalase and SOD.

The PC12 cell line has been used extensively for studies of sympathetic nerve function and monoamine neurotransmitters. The cells contain not only the enzymes that synthesize dopamine and NE, but also the storage granules and NE uptake-1 carrier sites like the sympathetic nerve endings (20). The NET cDNA cloned from the PC12 cells encodes a membrane protein of 617 amino acids (9). It shows high level of homology to the human NET in the brain. However, unlike the sympathetic nerve endings in the intact animal or isolated organ, the undifferentiated PC12 cells have no synaptic contacts with other cell types and are not influenced by inflammatory granulocytes that are invariably present in animal tissues or tissue enzymes that metabolize NE. These effects of NE on the PC12 cells can be attributed directly to an action of the extracellular NE or its autooxidation products.

NET is a Na+/Cl–-dependent 12-transmembrane domain protein located in the brain and the postganglionic sympathetic nerve terminals (4, 43). It is the site of action of psychotropic drugs, such as desipramine and cocaine, which block the NET reuptake of NE, increase interstitial NE, and potentiate the stimulation of postsynaptic receptors (36). These agents have been shown to exaggerate the pressor response to NE (39, 41). Knockout of the NET gene in transgenic mice reduces NE reuptake and clearance and renders the animals supersensitive to psychostimulants (72). The results of our present study suggest that the number of NE uptake sites and NET are affected by extracellular NE. These findings are consistent with our intact animal studies showing a reduction of cardiac NE uptake activity and downregulation of nisoxetine binding sites after NE infusion and production of heart failure when the myocardial interstitial NE is elevated (13, 14). A decrease of NE uptake in PC12 cells by NE has been reported previously (20). However, the dose of NE employed in that study was large (1 mM), which, as demonstrated in our present study, has a significant cytotoxic effect, making it difficult to interpret the physiological significance of the data. In our study, the doses of NE used to reduce NE uptake activity and NET protein in the presence of Cu2+ were much smaller (1–100 µM) and without significant loss of cell viability. Our study is, however, at variance with a prior study (75), in which NE produced a trend but no statistically significant decrease in the specific binding of [3H]nisoxetine to NET in membrane homogenates of PC12 cells. The number of experiments in that study, however, was small (3 or 4); the difference could become statistically significant if more experiments were included. The same investigators (76, 77) later reported that NE (100 µM) produced significant reductions of NE uptake activity and NET binding and protein expression in human embryonic kidney-293 cells transfected with human NET cDNA (293-hNET cells).

Our study showed that NET mRNA was not affected by 24 h of NE/Cu treatment, a finding also observed in 293-hNET cells in connection with reduction of NET protein (77). Thus the reduction of NET protein produced by NE is a posttranscriptional event. It may be related to decreased synthesis, increased degradation, or abnormal trafficking of the protein to the cell membrane. One possible mechanism may relate to the increased NET endocytosis or internalization of NET receptor by NE-induced protein kinase C activation of transporter phosphorylation (8). Comparisons of our results of [3H]nisoxetine binding site density in whole cells and membrane homogenates suggest that internalization may account in part for the decreased membrane NET, but net loss of NET protein also occurred in PC12 cells after NE/Cu treatment.

The doses of NE we employed in the study are large to produce the desired effect in PC12 cells. Similar or even larger doses of NE have been used by other investigators using the PC12 cells in culture (20, 74–76). However, unlike the healthy PC12 cells, which possess a strong antioxidative stress system, the failing myocardium has reduced antioxidant capacity (57) and probably is much more susceptible to the detrimental effects of NE or its oxidation products on the cardiac sympathetic nerve endings. Furthermore, unlike the short-term (24 h) exposure of the PC12 cells to NE as shown in this study, the failing myocardium in humans or animals with congestive heart failure is exposed to the elevated interstitial NE for months or even years. Under the latter condition, the amount of NE needed to produce the observed sympathetic nerve terminal dysfunction probably is much smaller than what we employed in this study. Prior studies in animals have shown that the myocardial interstitial NE is elevated (3–8 nmol/l) in congestive heart failure (13, 33) or myocardial ischemia-reperfusion (62). It could increase up to 15–30 nmol/l when the animals were given tyramine or exogenous NE (34, 50). Studies have also shown that interstitial NE in the myocardium after coronary ischemia-reperfusion (53) and sympathetic nerve stimulation (51) could reach levels high enough to generate hydroxyl free radicals. A positive correlation exists between the release of NE and the formation of hydroxyl free radicals in the heart (51). Thus we believe that results of our present study are clinically relevant and pathophysiologically important.

The exact mechanisms by which NE reduces NET protein in PC12 cells are not known. A recent study (48) showed that inability to N-glycosylate the human NET reduces protein stability, surface trafficking, and NET activity. Whether NE treatment affects N-glycosylation in PC12 cells is not known. Furthermore, because the degree of NE uptake inhibition produced by NE was greater than the reduction of NET protein, one cannot exclude a possible effect of NE/Cu on functional modification of NET protein. Studies (3, 16) have shown that cysteine residues of the NE and dopamine transports are critical determinants of function, and that these residues are sensitive to the actions of reactive oxygen species. Analysis of the amino acid sequence has identified cysteine residue of Cys 351 in NET as important for the inhibitory effect of nitric oxide on NET function (28). Thus it is possible that NE and its oxidation products may reduce NET function by making a conformational change after conjugation with cysteine residues in NET protein.

Although not studied in the present study, the cellular effects of NE probably are not specific to NET in PC12 cells. We have shown in intact animals that exogenous NE administration also reduced other cell membrane-bound receptors such as sarcolemmal Na+-K+-ATPase (30, 33) and myocardial {beta}-adrenergic receptors (14). These effects of NE on other cell surface proteins probably are also mediated via the formation of oxygen free radicals, because like NET, the reductions of Na+-K+-ATPase activity (17, 70) and myocardial {beta}-adrenergic receptors (55) also occur after induction of oxidative stress. However, the molecular mechanisms by which the changes in cell surface proteins are induced by oxidative stress may differ.

Intracellular reactive oxygen species was increased in PC12 cells by NE and NE/Cu in our studies. NE also has been shown to increase intracellular reactive oxygen species production in the cultured neonatal rat cardiomyocytes (45) and exaggerate the prooxidative effect of amyloid {beta}-peptide in cultured hypocampal neurons (18). Our present study did not identify the source of reactive oxygen species produced by NE. However, it is known that free radicals are formed after autooxidation of NE to NE-orthoquinone and hydroquinone (19, 42), which can be enhanced by transition ions such as Cu2+ and Fe2+. Intracellular NE is also subjected to oxidative deamination by mitochondrial monoamine oxidase A to produce hydrogen peroxide (11). Monoamine oxidase metabolites of catecholamines are physiologically important in vivo and have been implicated in the pathogenesis of neurodegenerative disease (12). However, autooxidation probably plays a greater role in the catecholamine toxicity to PC12 cells in culture. Prior studies have shown that the cytotoxic effect of l-DOPA, which was reduced by catalase and SOD, was affected by neither carbidopa, which prevents the conversion of l-DOPA to dopamine, nor monoamine oxidase inhibitors (7). In intact animals, NE-derived autooxidation products also have been shown to be present in the interstitial space of the heart. With the use of the microdialysis technique, Obata and Yamanaka (52) reported that when pargyline, a monoamine oxidase inhibitor, was infused in rat heart, the level of interstitial NE increased gradually. Increases in 2,3- and 2,5-dihydroxylbenzoic acids, products of ·OH reacting with salicylate, also occurred after pargyline administration (52). Sympathetic nerve stimulation increased both NE and dihydroxylbenzoic acids in the cardiac interstitial space (51). The positive correlation between the interstitial NE and dihydroxylbenzoic acids suggests that when more NE is released into the interstitial space, more can be converted to generate reactive oxygen species.

The effects of NE in organ tissues or animals are more complicated than PC12 cells. In addition to the enzymatic and nonenzymatic pathways mentioned above, NE may generate reactive oxygen species by activation of several tissue enzyme pathways, such as xanthine oxidase, mitochondrial cytochrome oxidases, nitric oxide synthase, and NADPH oxidase, via stimulation of the {beta}-adrenergic receptors (58), and formation of tumor necrosis factor-{alpha} and cytokines. cAMP may play a role in the downregulation of NET in the {beta}-adrenergic receptor-mediated response because cAMP has been shown to reduce NE uptake activity and NET protein expression in PC12 cells (10). On the other hand, nitric oxide has been shown to reduce the uptake of [3H]NE in an endothelial cell-PC12 cell co-culture system (28, 29), via S-nitrosylation of NET, a cGMP-independent process.

NE has also been shown to alter the function of peripheral sympathetic nerves (2). It also reduces tyrosine hydroxylase-positive neurons and NE uptake in rat brain stem neuron cultures (44, 61). The neurotoxic effect of catecholamines could be mimicked by hydrogen peroxide and blocked by catalase (61). We suspect that the neurotoxic effect of NE can be reduced or modified by the endogenous antioxidant mechanisms present in the normal tissue. However, when the tissue antioxidant capacity is reduced such as in the failing heart (57), NE is more likely to exert a greater damaging effect on the cells. Depletion of intracellular GSH also promotes formation of free radicals, loss of dopamine storage, and neurodegeneration (54, 64). Inhibition of glutathione synthesis by a {gamma}-glutamylcysteine synthetase inhibitor (15) or antisense oligomers (26) also has been shown to reduce Na+-dependent cellular uptake of dopamine and Na+-K+-ATPase activity in PC12 cells. On the other hand, administration of antioxidants such as SOD and antioxidant vitamins has been shown to attenuate sympathetic nerve ending dysfunction produced by prolonged NE infusion and congestive heart failure (2, 40). These findings are consistent with our present study indicating that the harmful effects of NE on NET protein expression and function were associated with oxidative stress.

Cu2+ is an essential trace element which functions as a cofactor of several important enzymes such as CuZn-SOD, dopamine-{beta}-hydroxylase, and cytochrome c in the body (37, 71). In addition, the reduced form of Cu2+ has been shown to catalyze the formation of hydroxyl radicals in the presence of H2O2 through the Fenton reaction (22). Cu2+ promotes free radical formation by catecholamines (25, 42). This action of Cu2+ is expected to reduce intracellular GSH and SOD and shift the redox balance to prooxidative stress (31, 38). This prooxidative action of Cu2+ results in formation of DNA adducts and oxidative base damage and contributes to neuronal loss (37, 38). In our study, at low doses (≤1 µM) Cu2+ sulfate exerted no significant effects on cell viability, NE uptake activity, or oxidative stress in PC12 cells, but when added to the NE-containing medium, it potentiated oxidative stress produced by NE, and furthered the reduction of NE uptake activity. Systemic administration of CuCl2 also produced no significant effect on NE uptake in brain synaptosomes (68). However, at large doses, Cu2+ is capable of generating reactive oxygen species as shown in our present study (Fig. 3), probably from interaction with NE present in the PC12 cell culture medium, leading to reduction of NE uptake activity. In rat brain synaptosomes, Cu2+ sulfate has been shown to inhibit uptake of monoamines in vitro (69). The reasons for the differences in results among the studies are not known, but probably are related to the different tissues, experimental conditions, and amount of Cu2+ administered.

In the present study, mannitol was used to scavenge hydroxyl radicals produced by NE/Cu, whereas the antioxidant enzymes SOD and catalase acted to convert superoxide anion to H2O2, and from H2O2 to H2O, respectively. Mannitol is a small molecule that is capable of scavenging oxygen free radicals inside the cells, whereas SOD is a large molecule that exerts its actions by inhibiting the oxidant enzymes primarily outside the cells. These agents have been shown to inhibit free radical-induced DNA damage (37, 38, 60). Our results that the antioxidants and free radical scavengers attenuated the PC12 cell NE uptake inhibition produced by NE support the view that the effects of NE are mediated via formation of oxygen free radicals. Likewise, a synthesized SOD and catalase mimetic, EUK-134, completely blocked 6-hydroxydopamine-induced loss of tyrosine hydroxylase-positive neurons and subsequent dopamine uptake decrease (56). However, because antioxidants did not completely prevent the reductions of NE uptake activity and NET protein expression, the effect of extracellular NE on the NET system may not be explained by oxidative stress alone.

In summary, results of our study indicate that extracellular NE reduces NE uptake activity and NET binding site density and protein in PC12 cells. These effects were associated with oxidative stress and could be reduced by the free radical scavenger mannitol and antioxidant enzymes SOD and catalase. Because there was no change in NET mRNA, the inhibitory effect of NE on the NET system probably was a posttranscriptional event, but the exact mechanisms by which NE reduces NE uptake activity remain to be elucidated.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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, NY 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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 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
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