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Activity and Subcellular Trafficking of the Sodium-Coupled Choline Transporter CHT Is Regulated Acutely by Peroxynitrite

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada (M.P., S.A.G.B., S.S.G.F., R.J.R.); Cell Biology Research Group, Robarts Research Institute, London, Ontario, Canada (M.P., S.A.G.B., F.M.R., S.S.G.F., R.J.R.); and Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand (M.P., C.P.)

Received August 15, 2007; accepted October 29, 2007

	Abstract

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Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC₅₀ = 0.9 ± 0.14 mM and t = 4 min. SIN-1 significantly reduced V_max of choline uptake without altering the K_m. This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.

Several studies link oxidative and nitrosative modifications of cellular constituents to the pathogenesis of neurodegenerative disorders such as Alzheimer disease (AD), Parkinson's disease, and sporadic amyotrophic lateral sclerosis. Peroxynitrite (ONOO^-) is a powerful oxidant that can be formed in vivo from NO and superoxide anion (Beckman, 1994). Because it is highly reactive, ONOO^- can alter the structure and function of cellular proteins, lipids, and other macromolecules by oxidation or nitrosylation. Related to this, increased nitrotyrosine levels have been found in neuronal tissues in AD (Smith et al., 1997), suggesting that ONOO^- may be involved in modifying protein function in this disorder.

Authentic ONOO^-, or ONOO^- produced from 3-morpholinosydnonimine (SIN-1), profoundly inhibits activity of some neurotransmitter transporters, including the human 5-hydroxytryptamine transporter expressed heterologously in COS-7 cells (Bryan-Lluka et al., 2004) and the human dopamine transporter expressed in HEK 293 cells (Park et al., 2002). Inducing oxidative stress by treating cells with H₂O₂ also inhibits dopamine transporter, but this likely occurs by a different mechanism than inhibition caused by ONOO^- (Huang et al., 2003). Oxidative stress mediated by the drug SIN-1 and ONOO^-, but not by H₂O₂, can affect high-affinity choline uptake activity in synaptosomes isolated from electric organ of Torpedo marmorata (Guermonprez et al., 2001). However, the mechanism by which ONOO^- impairs choline transport was not investigated.

It was revealed recently that CHT proteins are located predominantly in intracellular compartments, with only a small proportion of total CHT being present at the cell surface (Lips et al., 2002; Okuda et al., 2002; Nakata et al., 2004; Ribeiro et al., 2005). Moreover, altering the relative subcellular localization and trafficking of CHT proteins between the plasma membrane and intracellular compartments seems to be a critical mechanism for regulating its activity (Apparsundaram et al., 2005; Ribeiro et al., 2005). In the present study, we investigated mechanisms by which oxidative stress mediated by the ONOO^- generator SIN-1 modulates activity of rat CHT. We demonstrate for the first time that SIN-1 decreases choline uptake activity by changing the dynamics of CHT proteins at the cell surface by modifying their internalization and cycling between the cell surface and subcellular compartments.

	Materials and Methods

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Materials. SIN-1 was from BIOMOL Research Laboratories (Plymouth Meeting, PA); 5,10,15,20-tetrakissulfonatophenyl porphyrinato iron(III) (FeTPPS) was from Calbiochem (San Diego, CA); and [methyl-³H]choline chloride (86 Ci/mmol) and [methyl-³H]hemicholinium-3 diacetate (125 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Waltham, MA). EZ-Link sulfo-NHS-SS-biotin and immobilized NeutrAvidin beads were from Pierce Biotechnology (Rockford, IL). All others chemical were from Sigma-Aldrich (St. Louis, MO). SH-SY5Y neuroblastoma cells were purchased from American Type Culture Collection (Manassas, VA), HEK 293 Flp-In cells and all cell culture media, fetal bovine serum (FBS), and reagents were from Invitrogen Canada Inc. (Burlington, ON, Canada). Rabbit polyclonal and mouse monoclonal anti-nitrotyrosine antibodies were obtained from Upstate Biotechnology (Charlottesville, VA). Protein A-Sepharose and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were from Sigma Diagnostics Canada (Mississauga, ON, Canada). Enhanced chemiluminescence immunoblotting reagents were from GE Healthcare Life Sciences (Baie d'Urfé, QC, Canada). The polyclonal antibody against CHT was raised in rabbits by Genemed Synthesis (San Francisco, CA) using a peptide encoding 16 residues (DVDSSPEGSGTEDNLQ) that are conserved at the carboxyl terminus of human and rat CHT as immunogen; the peptide was conjugated to keyhole limpet hemocyanin carrier protein by an amino-terminal cysteine. CHT-specific immunoglobulins were affinity-purified in our laboratory from the crude antiserum on NHS-Sepharose (GE Healthcare) to which the antigenic peptide had been coupled as the binding element. The specificity of this antibody for detection of CHT was described previously by Ribeiro et al. (2005).

Stable Transfection and Selection of HEK 293 and SH-SY5Y Cells. A FLAG epitope-tag (DYKDDDDK) was added to the amino terminus of rat CHT by polymerase chain reaction (Ribeiro et al., 2005); the full-length cDNA for CHT ligated to pSPORT was obtained as a gift from Dr. Takashi Okuda (University College London, London, UK) (Okuda et al., 2000). HEK 293 cell lines stably expressing FLAG-tagged CHT (referred to as HEK 293-CHT cells) were prepared by introducing FLAG-CHT ligated to pcDNA5 into the Flp recombinase target site in HEK-Flp-In cells using Lipofectamine 2000 (Invitrogen Canada Inc.) transfection. Cells were grown in Dulbecco's modified Eagle medium with 10% FBS, 0.1% gentamicin, and 50 µg/ml hygromycin B to select stable transformants. SH-SY5Y neuroblastoma cells were transfected with FLAG-tagged rat CHT ligated into pcDNA3.1(-) using Lipofectamine 2000. Stable transformants (referred to as SH-SY5Y-CHT cells) were selected with 500 µg/ml G-418 (Geneticin; Invitrogen Canada Inc.) for 4 weeks, and subsequently they were maintained in Dulbecco's modified Eagle medium, 10% FBS, 0.1% gentamicin, and 100 µg/ml G-418. Differentiation of SH-SY5Y-CHT cells was induced by supplementing culture media with 10 µM all-trans-retinoic acid (RA) for at least 3 days.

[³H]Choline Uptake Assay. Cells were washed with Krebs-Ringer-HEPES (KRH) buffer, pH 7.4 (124 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgCl₂, 10 mM glucose, and 20 mM HEPES), and then they were incubated at 37°C in the absence or presence of SIN-1 for times specified under Results. After the treatment period, warm KRH buffer containing [³H]choline (0.5 µM; 0.4 Ci/mmol) was added, and incubation was continued for 5 min. For kinetic analysis, choline uptake was measured over a range of choline concentrations between 0.5 and 10 µM, with the specific activity of [³H]choline held constant at 0.4 Ci/mmol. [³H]Choline uptake was terminated by rapidly washing the cells twice with ice-cold KRH. Cells were solubilized in 0.1 N NaOH for 30 to 60 min, and then an aliquot was removed for measurement of radioactivity by liquid scintillation spectrometry. Additional aliquots of the solubilized samples were used for quantification of sample protein content using Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA). HC-3-sensitive [³H]choline uptake was determined by subtracting choline uptake in the presence of HC-3 from total choline uptake and normalizing to sample protein content to be expressed as picomoles per milligram of protein per 5 min.

Cell Toxicity Assays. Lactate dehydrogenase (LDH) activity was measured spectrophotometrically in aliquots of cell lysates or culture medium to determine whether drug treatments resulted in changes in plasma membrane integrity of cells. LDH activity was monitored as a change in absorbance at 340 nm due to the loss of NADH caused by the reduction of pyruvate to lactate coupled to the oxidation of NADH to NAD⁺ as described previously (Baskey et al., 1990). Cells on 12-well plates were treated with SIN-1 at 10, 100, or 1000 µM for 45 min in KRH buffer, pH 7.4, at 37°C. Culture medium was collected for measurement of LDH release from cells, and then cells were lysed in 0.5 ml of 1% Triton X-100 to measure total cellular LDH levels. Released LDH activity in culture medium was expressed as a percentage of total cellular LDH activity. Changes in NADH level in assay solutions were monitored at 340 nm at 30-s intervals for 5 min. The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine whether drug treatments compromised cell viability as described by Calderón et al. (1999). Cells in 96-well plates were washed with warm KRH buffer, and then they were incubated in the presence or absence of SIN-1 for 45 min at 37°C. After incubation of control and treated cells with MTT, cells were rinsed and allowed to air dry before dissolving in 100 µl of 0.04% HCl in isopropanol. The amount of formazan reaction product formed in each well was determined by measuring absorbance at 570 nm. Negative control wells contained medium alone with no cells. The viability of treated cells was presented as a percentage of control vehicle-treated cells.

[³H]HC-3 Binding. Cells were preincubated at 37°C for 45 min in KRH buffer with the addition of vehicle or varying doses of SIN-1, and then they were washed twice with ice-cold KRH and maintained on ice for 10 min. Subsequently, cells were incubated in KRH buffer containing 10 nM [³H]HC-3 (23 Ci/mmol) in the absence (total binding) or presence (nonspecific binding) of 1 µM unlabeled HC-3 for 1 h at 4°C. Cells were then washed rapidly twice with ice-cold KRH to remove unbound [³H]HC-3, and finally they were digested with 0.1 N NaOH. Aliquots of each sample were taken for radioactivity measurement by liquid scintillation spectrometry and for protein quantification. Specific binding was determined as the difference between total binding and nonspecific binding, and it was normalized to sample protein content to be expressed as femtomoles per milligram of protein per hour.

Cell Surface Protein Biotinylation Assay. Cells were treated with vehicle or SIN-1 in KRH buffer at 37°C at doses and times indicated under Results, and then plasma membrane proteins were biotinylated as described previously (Ribeiro et al., 2005). In brief, cells were washed three times with ice-cold PBS/CM (PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂), and then they were incubated with 1 mg/ml sulfo-NHS-SS-biotin in PBS/CM for 1 h at 4°C. Unbound sulfo-NHS-SS-biotin was quenched by washing and incubating cells with 100 mM glycine in ice-cold PBS/CM, followed by washing twice with PBS/CM. Subsequently, biotinylated cells were lysed for 30 min at 4°C in buffer comprised of 1% CHAPS, 0.1 M sodium phosphate buffer, pH 7.2, 10% glycerol, and 150 mM NaCl supplemented with 700 U/ml DNase and protease inhibitors [1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µg/ml each of aprotinin and leupeptin, and 25 µg/ml pepstatin A]. Aliquots of total cell lysates were incubated at 4°C with NeutrAvidin beads for 2 h with gentle mixing. The beads were then washed with lysis solution, and proteins were eluted from the beads with SDS sample buffer (2% SDS, 10% glycerol, 0.1 M sodium phosphate buffer, pH 7.2, 0.001% bromphenol blue, and 2.5% β-mercaptoethanol) for 5 min at 60°C. Aliquots of biotinylated (avidin-bound) proteins, nonbiotinylated proteins, and total cell lysates were separated by SDS-PAGE, and then proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 8% nonfat dry milk in wash buffer (PBS and 0.1% Tween 20) for 30 min, and then they were incubated for 2 h with anti-CHT antibody (1:1000) in wash buffer containing 8% milk. After washing, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG secondary antibody diluted 1:10,000 for 1 h in wash buffer containing 8% milk. Excess antibody was removed by washing, and membranes were processed for chemiluminescence detection using the ECL kit (GE Healthcare). Immunoreactive bands were quantified by densitometry using Scion Image software made available by National Institutes of Health (http://rsb.info.nih.gov/nih-image).

Internalization and Recycling of CHT in Cells. After the biotinylation of plasma membrane proteins, cells were treated rapidly with either vehicle or 1 mM SIN-1 in warm KRH buffer, and then they were transferred to 37°C for varying times to monitor internalization of cell surface proteins. Protein internalization was terminated by transferring culture dishes to 4°C and washing twice with ice-cold PBS/CM. Cells were treated with the membrane-impermeable reducing agent sodium 2-mercaptoethanosulfonic acid (MesNa; 50 mM) in TE buffer (150 mM NaCl, 1 mM EDTA, 0.2% bovine serum albumin, and 20 mM Tris-HCl, pH 8.6) to strip biotin from biotinylated proteins that had not undergone internalization and that were retained at the cell surface. For each experiment, the biotin stripping efficiency was assessed on a plate of cells that was treated in parallel but that had been kept on ice to block internalization of cell surface proteins. In experiments designed to assess the movement of previously internalized biotinylated CHT back to the plasma membrane ("recycling"), biotinylated cell surface proteins were allowed to undergo internalization by incubating cells at 37°C for 15 min, and then the cells were transferred to 4°C and residual biotin was stripped from proteins that remained at the cell surface. Subsequently, cells were returned to a second round of incubation at 37°C in KRH buffer to allow return of internalized biotinylated proteins to the plasma membrane. Treatments in the presence or absence of SIN-1 were initiated at this latter step for times specified under Results. Finally, biotin was removed from previously internalized biotinylated CHT proteins that had returned (recycled) to the cell surface using MesNa in TE buffer as described above, thereby allowing measurement of biotinylated proteins retained within the cells. Biotinylated proteins were separated from nonbiotinylated proteins on NeutrAvidin beads, and then they were analyzed by SDS-PAGE and immunoblotting as described above.

Detection of Tyrosine Nitration in CHT. HEK 293-CHT cells were harvested, and aliquots containing 1 x 10⁶ cells were transferred to microcentrifuge tubes. Cells were then washed with PBS and resuspended in KRH buffer, after which SIN-1, authentic ONOO^-, or vehicle was added with immediate mixing. After 30-min incubation at room temperature, cells were washed three times with PBS, and then they were centrifuged and lysed in CHAPS buffer containing protease inhibitors as described above. Immunoprecipitation was used to test for the presence of CHT-containing nitrotyrosine residues in total cell lysates as described by Tran et al. (2003). In brief, 50 µl of protein A-Sepharose beads was incubated overnight with either rabbit polyclonal anti-nitrotyrosine antibody (1:125) or mouse anti-FLAG antibody (1:125). Subsequently, aliquots of antibody-bound protein A-Sepharose were added to cell lysates (400-500 µg of protein) and incubated overnight at 4°C. Antigen-antibody complexes were recovered by centrifugation, and bound proteins were eluted in double-strength SDS sample buffer with heating at 60°C for 5 min. Proteins were separated by SDS-PAGE, and probed with mouse monoclonal anti-nitrotyrosine antibody (1:1000), or rabbit polyclonal anti-CHT antibody (1:2000), or rabbit polyclonal anti-nitrotyrosine antibody (1 µg/ml); information from these two forms of immunoblot were compared with evaluate whether CHT contained nitrotyrosine residues.

2'7'-Dichlorfluorescein Assay. HEK 293-CHT and SH-SY5Y-CHT cells were seeded in 96-well plates at 5 x 10⁴ cells/well and 1 x 10⁵ cells/well, respectively. On the day of the experiments, cells were incubated with KRH containing 50 µM DCFH-DA for 1 h at 37°C, and then media were aspirated and cells were washed once to remove extracellular DCFH-DA. Cell-permeable DCFH-DA taken up by the cells is converted by intracellular esterases to DCFH by removal of the diacetate moiety. SIN-1, H₂O₂, or vehicle was added to duplicate wells, and fluorescence intensity changes related to oxidation of DCFH to DCF were followed at 5-min intervals for 30 min (_Ex = 485 and _Em = 510 nm). This assay was also used to test whether SOD, catalase, and FeTPPS could reduce DCFH oxidation in cells by adding these agents to culture wells 5 min before the addition of H₂O₂, SIN-1, or vehicle. Changes in fluorescence were calculated by the equation F = (F_t30 - F_t0)/F_t0 x 100.

Membrane Potential Measurements. Effects of SIN-1 on membrane potential of SH-SY5Y cells was monitored with the membrane potential-dependent fluorescent dye Oxonal V, as described by Kukkonen et al. (1996). In brief, cells were harvested in PBS containing 0.02% EDTA, and then they were centrifuged and washed once with Ca²⁺-free KRH buffer. Cell pellets were resuspended in KRH containing 0.5 µM bis-oxonal, and then they were incubated for 30 min in the dark at 25°C with gentle mixing to allow loading of dye into cells. Subsequently, aliquots of cell suspension were placed in a stirred cuvette holder heated at 37°C of a fluorescence spectrophotometer (PTI, South Brunswick, NJ), and fluorescence (_Ex = 490 and _Em = 530 nm) was monitored as a function of time. Vehicle or drug (1 mM SIN-1 or 1 mM ouabain) was added at about 5 min when the baseline had stabilized. At the end of each recording, 10 µM gramicidin was added to the cells to induce maximal depolarization for measurement of the maximum fluorescence emission. Changes in fluorescence indicative of changes in membrane potential of the cells were calculated by F = (F_t30 - F_t0)/F_t0 x 100.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Acute exposure of HEK 293-CHT cells to SIN-1 inhibits HC-sensitive choline uptake. A, cells were preincubated with various concentrations of SIN-1 (10 µM-2 mM) () or with 1 mM H2O2 () for 45 min, and for various times (5-120 min) (B) before choline uptake activity was measured at 0.5 µM[3H]choline. Data from SIN-1-treated cells are normalized to that obtained for control (vehicle-treated) cells (percentage of control), and they are expressed as the mean ± S.E.M. of three to five independent experiments with triplicate measurements in each experiment. Statistically significant differences between control and SIN-1-treated cells are denoted as **, p < 0.01.

View larger version (20K): [in this window] [in a new window]   Fig. 2. SH-SY5Y-CHT cells express HC-3-sensitive choline uptake that increases with differentiation. A, two SH-SY5Y-CHT cell lines were tested, revealing CHT activity at approximately 50% of that found in HEK 293-CHT cells. Wild-type SH-SY5Y cells do not express HC-3-sensitive choline uptake activity constitutively. ***, p < 0.001 compared with wild-type cells; ###, p < 0.001 compared with HEK 293-CHT cells. B, differentiation of SH-SY5Y-CHT cells with retinoic acid treatment for 3 days enhances total (black bars), but not HC-insensitive (white bars), choline uptake activity. Net HC-sensitive, CHT-mediated choline uptake was approximately 5-fold greater in differentiated cells [127.28 ± 17.76 pmol/mg/5 min] compared with undifferentiated cells [26.05 ± 4.68 pmol/mg/5 min]. Data are expressed as mean ± S.E.M. (n = 4 independent experiments). Total choline uptake in differentiated cells was statistically different from that measured in undifferentiated cells and from HC-insensitive choline uptake in both differentiated and undifferentiated cells, denoted as **, p < 0.01. Total choline uptake in undifferentiated cells and HC-insensitive uptake in differentiated and undifferentiated cells were not significantly different from each other.

	Results

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SIN-1 Decreased CHT Activity. To begin, the effect of SIN-1 on CHT was assessed by measuring HC-3-sensitive, high-affinity choline uptake activity in HEK 293-CHT cells. These experiments revealed that SIN-1 significantly decreases CHT activity in a dose-dependent manner, with an IC₅₀ of 0.9 ± 0.14 mM (Fig. 1A). The onset of this inhibition is rapid. Cells were incubated in the presence of 1 mM SIN-1 for up to 120 min with maximal inhibition of choline uptake developing with a t of approximately 4 min. It is noteworthy that the inhibitory effect of SIN-1 plateaus at approximately 50% of control levels, and it remains constant for up to 120 min (Fig. 1B). Unlike SIN-1, treatment of cells with 1 mM H₂O₂ does not alter CHT activity (Fig. 1A).

To determine whether the inhibitory effect of SIN-1 on CHT activity is cell type-specific, we developed lines of SH-SY5Y neuroblastoma cells that stably express FLAG-tagged rat CHT (SH-SY5Y-CHT cells) for comparison with the HEK 293-CHT cells. As illustrated in Fig. 2A, compared with wild-type SH-SY5Y cells, two separate SH-SY5Y-CHT cell lines that were tested displayed substantial HC-sensitive choline uptake activity; this activity was approximately 50% less than that measured in parallel cultures of HEK 293-CHT cells because of lower levels of expression of the transgene in the neural cell lines (data not shown). SH-SY5Y cells grown in the presence of RA differentiate morphologically with the extension of neuritic processes and increased expression of neuron-specific markers (Encinas et al., 1999; Truckenmiller et al., 2001). It is noteworthy that HC-sensitive CHT activity was increased significantly by approximately 5-fold in RA-differentiated SH-SY5Y-CHT cells compared with undifferentiated SH-SY5Y-CHT cells (p < 0.01) (Fig. 2B). HC-insensitive choline uptake was not statistically different between undifferentiated and differentiated cells. As illustrated in Fig. 2B, HC-insensitive choline uptake accounts for approximately 75% of total choline uptake in undifferentiated cells compared with approximately 50% of total uptake in differentiated cells. The mechanisms underlying this are not clear, but it does not relate to changes in steady-state levels of CHT mRNA in the cells (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3. SIN-1 inhibits CHT activity in differentiated SH-SY5Y cells. A, [3H]choline uptake was inhibited in a dose-dependent manner in cells treated for 30 min with SIN-1 at concentrations between 10 µM and 2 mM. Data for SIN-1-treated cells are expressed as percentage of vehicle-treated controls, and they are graphed as mean ± S.E.M. (n = 4). Statistically significant differences compared with control are indicated by *, p < 0.05 (one-way analysis of variance with post hoc Dunnett's multiple comparison test). B, kinetic parameters for HC-3-sensitive [3H]choline uptake in control and SIN-1-treated differentiated SH-SY5Y-CHT cells were estimated using [3H]choline concentrations between 0.5 and 10 µM in the absence or presence of 1 µM HC-3. Data are calculated as nanomoles per milligram of protein per 5 min, and they are expressed as mean ± S.E.M. from five independent experiments with duplicate measurements. Choline uptake data are plotted as a function of [3H]choline concentration, with curves fitted by nonlinear regression analysis using GraphPad Prism 4. SIN-1 (30 min; 1 mM) decreased the Vmax of choline uptake by 25% (*, p < 0.05), with no significant change in Km.C, effect of SIN-1 treatment on plasma membrane integrity and cell viability was tested by monitoring LDH release from cells and MTT metabolism, respectively. As shown, treatment of cells for 30 min with SIN-1 up to a concentration of 1 mM did not significantly alter either of these measures compared with vehicle-treated control cells. Data are expressed as mean ± S.E.M. from at least three independent experiments.

Based on the finding that SIN-1 leads to a rapid and substantial loss of choline uptake activity in CHT-expressing cells, we carried out experiments designed to address three potential mechanisms: 1) changes in the density of transport proteins located at the cell surface, 2) nitration of susceptible tyrosine residues in CHT leading to loss-of-function, or 3) loss of the driving force for choline transport by collapse of the plasma membrane sodium electrochemical gradient or membrane potential.

View larger version (16K): [in this window] [in a new window]   Fig. 4. SIN-1 decreases the binding of [3H]HC-3 to CHT in HEK 293-CHT cells. Cells were treated for 45 min with vehicle or SIN-1 (0.01, 0.1, and 1 mM) before measuring total binding of [3H]HC-3 using 10 nM ligand in the absence or presence of 1 µM HC-3 for estimation of nonspecific binding. Wild-type HEK 293 cells do not express CHT constitutively as seen by the lack of specific [3H]HC-3 binding. Specific binding was determined as the difference between total binding and nonspecific binding, with data normalized to sample protein content. Data are expressed as femtomoles of [3H]HC-3 bound per milligram of protein, and they are graphed as mean ± S.E.M. from five independent experiments with duplicate determinations. Statistically significant differences compared with control group are indicated by *, p < 0.05 or **, p < 0.01. Statistically significant differences compared with 1 mM SIN-1 treatment group are indicated by #, p < 0.05.

View larger version (38K): [in this window] [in a new window]   Fig. 5. SIN-1 treatment decreases the size of the total recycling pool of CHT in HEK 293-CHT cells. A, cells were incubated for varying times at 37°C in the absence or presence of 1 mM SIN-1 in buffer containing sulfo-NHS-SS-biotin to biotinylate CHT proteins when they are present at the cell surface. A representative immunoblot (middle) shows total biotinylated CHT proteins located at the cell surface and internalized into subcellular compartments; total cellular CHT content (nonbiotinylated and biotinylated) (left lane, middle). A representative immunoblot showing biotinylated TfR is shown for the same experimental paradigm (top) with total TfR in cell lysate illustrated in the left lane, top. Actin immunoreactivity in the biotinylated protein fractions was used as a negative control. The absence of actin in these fractions (bottom) indicates that CHT or TfR present is recovered as solubilized biotinylated membrane proteins isolated on NeutrAvidin beads, rather than from contamination of the fractions by intact membranes. Actin immunoreactivity in cell lysates is shown in the left lane, bottom. B, a line graph summarizes time course data from densitometric profiles of the biotinylated CHT pool in control and SIN-1-treated cells normalized to total cellular CHT for five independent experiments.

To corroborate this finding, we used a second approach to quantify cell surface levels of CHT protein and to assess changes after SIN-1 treatment. In these studies, plasma membrane proteins were biotinylated and recovered on NeutrAvidin beads, and then CHT was detected and quantified by immunoblotting with anti-CHT antibody (Ribeiro et al., 2005). To begin, we assessed the effect of SIN-1 treatment on the size of the pool of CHT proteins that becomes biotinylated in HEK 293-CHT cells that were incubated at 37°C for varying times with either vehicle or 1 mM SIN-1 during protein biotinylation. In this experimental paradigm, biotinylated CHT may be located at the cell surface, internalized to subcellular organelles after labeling, or internalized and subsequently returned to the plasma membrane. This combined pool of CHT, termed the recycling pool, is a subpopulation of the total cellular CHT proteins, and it is available for trafficking between cell surface and subcellular organelles to regulate choline uptake activity by fine-tuning plasma membrane CHT levels (Ribeiro et al., 2006). Figure 5A shows a representative immunoblot, and it reveals that SIN-1 substantially reduces the total size of the recycling pool of CHT proteins in a time-dependent manner (Fig. 5, A and B). It is also clear from Fig. 5B that the kinetics of biotinylation of CHT proteins in the recycling pool differs in vehicle-treated compared with SIN-1-treated cells; over a 60-min time course, the rate and magnitude of biotinylation of this CHT pool is greater in control cells compared with cells exposed to SIN-1.

It is important to note from the immunoblot of CHT in Fig. 5A that despite having a calculated molecular mass of approximately 63 kDa, CHT become visible on immunoblots as at least one, and often two or three, bands with masses of approximately 40, 50, and 80 kDa. The 50-kDa protein is the predominant species, with the relative abundance of immunoreactive CHT bands varying depending on expression levels of CHT in cells. As shown in Fig. 5A (middle, left), in HEK 293-CHT cell lysates, immunoreactive CHT proteins are observed with masses of approximately 40 and 50 kDa. It is noteworthy that biotinylated CHT protein (Fig. 5A, middle, right) corresponds to the 50-kDa immunoreactive CHT protein. Also included in this experiment for comparison with CHT was assessment of the effects of SIN-1 on another cell surface receptor, the transferrin receptor (TfR), which is known to undergo clathrin-mediated internalization (Hanover et al., 1985). As illustrated in Fig. 5A (top), SIN-1 also substantially reduces the total size of the biotinylated pool of TfR in a time-dependent manner similar to that observed for CHT.

View larger version (37K): [in this window] [in a new window]   Fig. 6. SIN-1 treatment decreases the amount of CHT protein at the cell surface. A, HEK 293-CHT cells were incubated at 37°C for 45 min in the absence or presence of SIN-1 (0.01, 0.1, or 1 mM) (top, left) or with 1 mM SIN-1 for 5, 15, or 45 min (top, right) before biotinylation of cell surface proteins at 4°C for 1 h. Aliquots of lysates (10 µg of protein) from parallel sets of cells not undergoing biotinylation were immunoblotted to assess total CHT levels (bottom). B, immunoblots of biotinylated CHT were quantified by densitometry and relative absorbance units for band densities from SIN-1-treated cells are expressed as a percentage of cell surface CHT obtained for vehicle-treated control cells. Data shown are from three to five independent experiments, and they represent mean ± S.E.M. Asterisks (**) indicate that data are statistically different from controls at the level of p < 0.01.

SIN-1 Enhances the Internalization of CHT from the Cell Surface. Although it was predicted initially that CHT proteins would be localized mostly to the plasma membrane from where they transport choline into the cell, it is clear that unlike some other neurotransmitter transporters CHT is distributed primarily in intracellular compartments with only a small proportion at the cell surface. Moreover, CHT present at the cell surface is internalized rapidly into intracellular membranous organelles through the endocytic endosomal pathway (Ribeiro et al., 2006). Thus, it is expected that the steady-state plasma membrane level of CHT protein will be determined by the rates at which it is internalized into subcellular compartments and returned back to the plasma membrane. In the present study, we used the cell surface protein biotinylation approach to assess whether SIN-1 alters the rates of either internalization or reinsertion of CHT protein at the plasma membrane. After biotinylation of cell surface proteins at 4°C in the absence of SIN-1, the profiles for CHT internalization were compared in cells incubated at 37°C in the absence (control) or presence of 1 mM SIN-1 for 5 and 15 min; after this incubation, residual biotin was stripped from proteins that remained at the cell surface to ensure that only internalized biotinylated proteins were detected.

Importantly, the data illustrated in Fig. 7 reveal a statistically significant increase in the amount of CHT protein internalized from the plasma membrane in SIN-1-treated SH-SY5Y-CHT cells compared with vehicle-treated control cells. At both 5 and 15 min after the addition of 1 mM SIN-1 to cells at 37°C, the levels of biotinylated CHT were greater than that found in control cells at 37°C (Fig. 7A, bottom, lane 3 versus lane 5, and lane 4 versus lane 6; p < 0.001 and p < 0.05, respectively); an immunoblot of the total cell levels of CHT is shown in Fig. 7A (top) to demonstrate equivalent expression of CHT in each group. The amount of internalized biotinylated CHT was normalized to total cell surface levels of biotinylated CHT in cells incubated at 4°C in the absence of SIN-1 (Fig. 7A, bottom, lane 1). As experimental controls to validate the method, 1) the amount of total cell surface CHT that is biotinylated is shown in Fig. 7A (bottom, lane 1), and 2) one dish of cells was incubated on ice to show that internalization of the protein was negligible at 4°C (data not shown) and that MesNa efficiently stripped biotin from cell surface proteins (Fig. 7A, bottom, lane 2). Typically, one CHT-immunoreactive band with molecular mass of approximately 50 kDa was found on immunoblots of SH-SY5Y-CHT cell lysates (Fig. 7A, top), in contrast to results obtained with HEK 293-CHT cell lysates (Fig. 5A, bottom). Biotinylated CHT from SH-SY5Y-CHT cells corresponded to the immunoreactive CHT band at approximately 50 kDa, similar to that seen with HEK 293-CHT cells.

View larger version (32K): [in this window] [in a new window]   Fig. 7. SIN-1 increases internalization of CHT from the plasma membrane to subcellular compartments in differentiated SH-SY5Y-CHT cells. A, this representative immunoblot (bottom) shows increased internalization of biotinylated CHT at 5 and 15 min after addition of 1 mM SIN-1 compared with control cells. B, line graph displays CHT internalization data normalized to total cell surface CHT computed from four independent experiments. *, p < 0.05 and ***, p < 0.001, data are significantly different from controls, and #, p < 0.05 indicates a statistically significant difference for CHT internalization between 5- and 15-min values in control cells only.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Determination of the return of internalized CHT to the cell surface. After allowing internalization (37°C) for 15 min, two parallel groups of cells were chilled and incubated with MesNa at 4°C to strip biotin from proteins at the cell surface. Cells were then transferred to 37°C, and they were incubated in the presence or absence of 1 mM SIN-1. The decrement in intensity of the internalized, biotinylated CHT band with increasing incubation time (0, 2, 5, and 15 min) at 37°C represents the rate of CHT return back to the plasma membrane. A, data from a representative experiment are shown. B, histogram compiles data from three independent experiments, with data expressed as the percentage of loss of internalized CHT at each time point, thus allowing assessment of the return of CHT to the cell surface.

Effect of SIN-1 On Intracellular Oxidative Stress. To assess whether SIN-1 and its product ONOO^- cause an increase in intracellular oxidative stress, the cell-permeable nonfluorescent probe DCFH-DA was used to monitor levels of reactive oxygen species in control, H₂O₂-, and SIN-1-treated cells. After diffusing into cells, the diacetate moiety is cleaved from DCFH by cytoplasmic esterases, and with increasing oxidative stress in cells DCFH can be oxidized to the fluorescent compound DCF. As shown in Fig. 9, both 500 µMH₂O₂ and 100 µM SIN-1 significantly increased the fluorescence intensity of DCF, confirming that they are membrane-permeable oxidizing agents; induction of DCF fluorescence was greater with SIN-1, suggesting that it is a more powerful oxidizing agent than is H₂O₂. Pretreatment with 100 U/ml catalase, which breaks down H₂O₂, attenuated increases in DCF fluorescence in cells treated with H₂O₂, but not in cells treated with SIN-1. These data indicate that the effects of SIN-1 on CHT were not likely to be mediated by or related to the formation of H₂O₂. In contrast, treatment of cells with either 100 U/ml SOD or 25 µM FeTPPS, which catalyze the decomposition of ONOO^-, significantly reduced the SIN-1-induced increase in DCF fluorescence, supporting the role of ONOO^- in SIN-1 effects on CHT.

View larger version (18K): [in this window] [in a new window]   Fig. 9. DCF assay. HEK 293-CHT cells were incubated with 50 µM DCFH-DA for 1 h at 37°C. After washing away excess extracellular DCFH-DA, cells were treated with 100 and 500 µMH2O2 or 100 µM SIN-1, and the changes in fluorescence intensity were monitored at 5-min intervals for 30 min (excitation, 485 nm and emission, 510 nm). SOD, catalase, and FeTPPS were added to some samples at 5 min before the treatment. Changes in fluorescence were calculated according to the equation F = (Ft30 - Ft0)/Ft0 x 100. As expected, SIN-1 is a more powerful oxidizing agent than is H2O2. Catalase (100 units/ml) significantly reduced the oxidation of DCF in H2O2 (500 µM) treated-cells, but it had no effect in cells treated with SIN-1. However, 100 units/ml SOD, which react with then inhibits the formation of ONOO-, and 25 µM FeTPPS, an ONOO-. scavenger, effectively lowered the oxidation of DCF in cells exposed to SIN-1. The bar graphs shown are means ± S.E.M. (n = 4). *, p < 0.05 and **, p < 0.01 compared with control cells.

Effects of SIN-1 on Membrane Potential. As the velocity of choline uptake by CHT is coupled to the sodium electrochemical gradient, we tested the hypothesis that the inhibitory effect of SIN-1 on CHT activity is related to changes in membrane potential of the cell caused by inhibition of Na⁺/K⁺-ATPase and collapse of transmembrane ionic gradients. Previous studies demonstrated that Na⁺/K⁺-ATPase activity is decreased substantially by both SIN-1 and ONOO^- (Guzman et al., 1995). To test this hypothesis, the effect of SIN-1 on membrane potential of SH-SY5Y cells was evaluated using the bis-oxonal dye. As illustrated in Fig. 10, bis-oxonal fluorescence was not different between vehicle-treated cells and cells treated for 30 min with 1 mM SIN-1. By comparison, treatment of cells with the Na⁺/K⁺-ATPase inhibitor ouabain (1 mM) resulted in increased bis-oxonal fluorescence, suggesting that cells are more depolarized.

View larger version (22K): [in this window] [in a new window]   Fig. 10. SIN-1 did not alter cell plasma membrane potential when investigated using bis-oxonal. Cell suspensions were incubated in KRH buffer, pH7.4, containing 0.5 µM bis-oxonal for 30 min at 25°C. Bis-oxonal fluorescence was monitored as a function of time at wavelengths of 490 nm (excitation) and 530 nm (emission) using a PTI fluorescence spectrophotometer. When the baseline had stabilized, vehicle or drug (SIN-1 or ouabain) was added, and the fluorescence signal was observed continuously for 30 min. A, bar graph represents mean ± S.E.M. of data obtained from three independent experiments. B, representative traces of changes in fluorescence observed during a 30-min incubation with vehicle or drug treatment of cells. Drug or vehicle were added at time = 0 min on these traces, with the baseline having stabilized for several minutes before these additions. Gramicidine (10 µM) was added at the end of each incubation (i.e., at the 30-min time point) to induce membrane depolarization and observed as a large increase in fluorescence intensity.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Several novel observations relating to the regulation of CHT function were made in this study: 1) cell surface levels of CHT are reduced by SIN-1 with this correlating to decreased choline uptake activity and HC-3 binding; 2) SIN-1 enhances the rate of internalization of CHT proteins from plasma membrane to subcellular compartments; similar enhancement of internalization of TfR in SIN-1-treated cells was also observed; 3) CHT that has undergone internalization from the plasma membrane to subcellular organelles can return back to the cell surface with the rate of this not altered by SIN-1; 4) SIN-1 effects on CHT function are not related to nitration of tyrosine residues in the protein or to changes in membrane potential that serves as a driving force for solute transport, and 5) RA-mediated differentiation of SH-SY5Y-CHT cells leads to an approximately 5-fold increase in CHT activity and protein content in cells.

Studying biological effects of ONOO^- in cultured cells is technically challenging due to its short half-life (<1 s at physiological pH) (Radi et al., 2001), thus making the effective exposure time brief and making it difficult to attain the desired dose. Effects of a bolus of ONOO^- may differ from what occurs in vivo where ONOO^- is formed slowly and continuously. As an alternative to using ONOO^- directly, SIN-1 (active metabolite of molsidomine) releases equimolar NO and , leading to ONOO^- generation in well oxygenated aqueous solution at physiological pH. This slow continuous production of ONOO^- from SIN-1 resembles processes occurring during inflammation, ischemic injury and excitotoxicity (Darley-Usmar et al., 1992; Li et al., 2002).

It was reported previously that ONOO^- and SIN-1 inhibit high-affinity choline uptake and ACh synthesis in cholinergic synaptosomes (Guermonprez et al., 2001), but the underlying mechanisms were not investigated. The present study shows that short-term exposure of HEK 293 and SH-SY5Y cells expressing CHT to 1 mM SIN-1 rapidly inhibits choline uptake to approximately 50% of control levels; this agrees with a previous report where synaptosomal HC-sensitive choline uptake was decreased to 50% of control by 0.8 mM SIN-1 (Guermonprez et al., 2001). We observed that the inhibitory effect of SIN-1 on CHT activity plateaus within minutes and remains stable up to 2 h. The reason for this is unclear, but as SIN-1 consumes molecular O₂ with NO generation this could potentially deplete O₂ in media, thereby limiting the maximal effect of SIN-1. Studies on the dynamics of O₂ consumption relative to NO production from SIN-1 have been published (Feelisch et al., 1989; Ullrich et al., 1997), but the time frame for substantial reduction of O₂ in media occurs over hours rather than a few minutes.

Inhibition of HC-sensitive choline uptake by SIN-1 in both non-neural (HEK 293) and neural (SH-SY5Y) cells indicates that the effect is not cell type-specific. However, comparison of HEK 293-CHT and RA-differentiated SH-SY5Y-CHT cells revealed a difference in sensitivity of cells to inhibitory effects of SIN-1 on CHT activity. Although the redox state of HEK 293 and differentiated SH-SY5Y cells was not addressed in this study, both cellular redox state and GSH levels are important defenses against ONOO^- and SIN-1 (Barker et al., 1996; Fass et al., 2004). It is noteworthy that RA treatment can protect PC12 cells from oxidative injury generated by H₂O₂ (Jackson et al., 1991), suggesting that the transition of SH-SY5Y cells to a more differentiated state may contribute to differences in sensitivity of CHT to SIN-1 between HEK 293 and SH-SY5Y cells.

Characteristics of choline uptake measured in SH-SY5Y-CHT cells are similar to those reported in other expression systems such as Xenopus laevis oocytes and transiently transfected cells. Strikingly, RA-induced differentiation of SH-SY5Y-CHT cells markedly enhances CHT activity and protein levels in parallel with morphological differentiation of the cells. As stable introduction of transgenes into recipient cells using traditional plasmid transfection methods normally results in random integration of the gene into the host genome, the effects of RA-induced differentiation was tested on two separate clonal cell lines. The data show that both cell lines respond similarly to RA, suggesting that it is unlikely that the integration site of CHT cDNA is responsible for the RA-induced increase in CHT expression. Further studies are required to elucidate mechanisms by which RA enhances CHT activity and protein levels in SH-SY5Y-CHT cells.

To assess whether SIN-1 and its product ONOO^- cause intracellular oxidative stress, the cell-permeable probe DCFH-DA was used (Crow, 1997; Martin-Romero et al., 2004). Coupling DCFH measures with oxidant inhibitors and scavengers makes it possible to assess oxidant potency and to identify the oxidant mediating SIN-1 effects. Oxidation of nonfluorescent DCFH to its fluorescent product DCF is relatively selective for ONOO^- compared with , H₂O₂, or NO, thereby allowing ONOO^- formation to be monitored. Under the experimental conditions used, it seems that the product of SIN-1 decomposition is ONOO^- rather than H₂O₂; catalase (scavenger of H₂O₂) effectively abolished increased DCF oxidation by H₂O₂, but it did not alter increased fluorescence of DCF in the presence of SIN-1. The interpretation that SIN-1-induced ONOO^- causes oxidative stress is supported by data that SOD (scavenges ) and FeTPPS (scavenges peroxynitrite and uncouples the reaction of NO with O₂ to decrease ONOO^- formation) inhibited oxidation of DCF by SIN-1. In addition, because intracellular DCF is highly oxidized when SIN-1 is added to media, this indicates that intracellular oxidative stress might contribute to SIN-1 effects. DCF fluorescence induced by SIN-1 is severalfold greater than the response to H₂O₂, confirming the potency of pro-oxidant ONOO^-.

A primary effect of ONOO^- on proteins is nitration of tyrosine residues (Beckman et al., 1992). In relation to this, nitrotyrosine immunoreactivity is increased in necropsy AD brain (Smith et al., 1997), indicating that ONOO^- may be involved in the disease pathogenesis. Nitrated residues disrupt normal protein function by several mechanisms, such as altering its ability to undergo tyrosine phosphorylation (Berlett et al., 1996; Nomiyama et al., 2004), modification of protein conformation (Hodara et al., 2004; Reynolds et al., 2005) and altering susceptibility to proteolysis (Hodara et al., 2004; Bar-Shai and Reznick, 2006). We hypothesized that tyrosine nitration in CHT may induce loss-of-function. Rat CHT contains 31 tyrosines that are potential targets for ONOO^--mediated nitration; 12 tyrosines are located in transmembrane domains and 19 are arrayed in extracellular and intracellular loops. Tyrosine nitration in target proteins yields a modification that can be detected immunologically; thus, we used anti-nitrotyrosine antibodies to analyze CHT immunoprecipitated from SIN-1-treated cells. We did not find evidence of nitration of tyrosines in CHT. Similar results (i.e., absence of nitrotyrosine-immunoreactive bands after SIN-1 and ONOO^- treatment) were found with cholinergic synaptosomes or cell lines that overexpress the human dopamine transporter that were exposed to high doses of SIN-1 (Guermonprez et al., 2001; Park et al., 2002). An alternative target in CHT for ONOO^- could be oxidation of cysteine thiols.

We show for the first time that SIN-1 decreases choline uptake by increasing CHT internalization without altering its return to plasma membrane, thus lowering cell surface CHT. Cell surface CHT is determined by its internalization rate into endosomal compartments and by the rate of movement of previously internalized or new CHT to plasma membrane. This latter event is critical for increasing cell surface CHT to mediate rapid enhancement of choline uptake after ACh release, thus facilitating transmitter synthesis to maintain neurotransmission (Ferguson and Blakely, 2004; Ribeiro et al., 2006). Dynamic movement of CHT between plasma membrane and cellular organelles in neurons may involve both regulated and constitutive mechanisms with different functional roles (Ribeiro et al., 2006). In this model, constitutive exocytosis-endocytosis maintains a pool of plasma membrane transporters (estimated at 15% of total CHT), whereas regulated exocytosis of synaptic vesicles delivers CHT to the cell surface during prolonged excitation of cholinergic nerve terminals. An intracellular recycling pool of CHT that can be recruited as needed could explain how cholinergic transmission is maintained during long periods of stimulation (Birks and MacIntosh, 1961). Conversely, changes that enhance CHT internalization and/or decrease its return to plasma membrane could decrease its cell surface levels, lower ACh synthesis and comprise cholinergic communication.

The mechanism by which SIN-1 enhances CHT internalization is unknown, but may involve modification of CHT interaction with cellular proteins and/or endocytic machinery such as adaptor, Rab or clathrin-pathway proteins. After internalization in clathrin-coated pits, CHT seems to be directed to early endosomes as it colocalizes with Rab5 shortly after endocytosis (Ribeiro et al., 2005). In addition to its role in endosomal transport, Rab5 may participate in synaptic vesicle recycling/formation and modulate synaptic vesicle-mediated mobilization of CHT, thus regulating choline uptake (Ferguson et al., 2003; Ribeiro et al., 2003). In this study, SIN-1 also increased TfR internalization from plasma membrane similar to the effect on CHT. TfR is endocytosed by a clathrin-mediated process (Hanover et al., 1985), and although it was shown that SIN-1 decreases cell surface TfR levels (Richardson et al., 1995), a relationship between these events has not been reported. Taken together, our findings suggest that this SIN-1-mediated effect is not specific to CHT, but that it may be generalized to other cell surface proteins that undergo regulated endocytosis by a clathrinmediated mechanism.

	Acknowledgements

 
We thank Drs. Jeffrey Dixon and Stephen Sims for assistance with, and use of, the Photon Technology International fluorescence spectrophotometer for monitoring changes in cell membrane potential.

	Footnotes

 
This research was supported by operating grants to R.J.R. from the Alzheimer Society of Canada and Canadian Institutes for Health Research (CIHR). M.P. was funded by a Scholarship from the Higher Commission on Education, Ministry of Education of Thailand, as part of an exchange program between Mahidol University, Bangkok, Thailand, and the University of Western Ontario, London, Canada. S.A.G.B. is the recipient of a Doctoral Award from CIHR, and S.S.G.F. is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Canada Research Chair in Molecular Neurobiology.

ABBREVIATIONS: ACh, acetylcholine; CHT, high-affinity choline transporter protein; AD, Alzheimer disease; SIN-1, 3-morpholinosydnonimine; HEK, human embryonic kidney; FeTPPS, 5,10,15,20-tetrakissulfonatophenyl porphyrinato iron (III); FBS, fetal bovine serum; DCF, 2',7'-dichlorofluorescein; DCFH, reduced 2',7'-dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescein diacetate; SOD, superoxide dismutase; NHS, N-hydroxysuccinimide; RA, all-trans retinoic acid; KRH, Krebs-Ringer-HEPES; LDH, lactate dehydrogenase; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; PBS, phosphate-buffered saline; PBS/CM, phosphate-buffered saline with 0.1 mM CaCl₂ and 1 mM MgCl₂; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; PAGE, polyacrylamide gel electrophoresis; MesNa, sodium 2-mercaptoethanosulfonic acid; TfR, transferrin receptor.

Address correspondence to: Dr. Rebecca Jane Rylett, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1. E-mail: jane.rylett{at}schulich.uwo.ca

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