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Research ArticleArticle

Acute Regulation of Norepinephrine Transport: II. PKC-Modulated Surface Expression of Human Norepinephrine Transporter Proteins

Subramaniam Apparsundaram, Sally Schroeter, Elena Giovanetti and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1998, 287 (2) 744-751;

Abstract

Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, β-PMA or β-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km . Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, β-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetineKd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.

Presynaptic reuptake of l-NE by Na+- and Cl−-dependent NETs is largely responsible for the efficient termination of neurotransmission at noradrenergic synapses (Axelrod and Kopin, 1969;Iversen, 1971; Graefe and Bönisch, 1988; Trendelenburg, 1991). In addition to its physiological role, NET is also a target for tricyclic antidepressants, cocaine and amphetamine (Pacholczyk et al., 1991; Barker and Blakely, 1995). The critical role played by NETs in noradrenergic transmission raises the question as to whether the function of NETs may be tightly regulated in vivo and compromised in disease states (Barker and Blakely, 1995). At present little is known about the molecular mechanisms underlying NET regulation, though neuronal activity and hormonal signals have been reported to influence NET function (Barker and Blakely, 1995). The molecular cloning of a NET cDNAs (Pacholczyk et al., 1991;Lingen et al., 1994; Fritz et al., 1998), their stable and transient expression in nonneuronal cell lines (Melikianet al., 1994; Melikian et al., 1996; Ngugen and Amara, 1996), and the development of NET-specific antibodies (Melikianet al., 1994; Bruss et al., 1995), provide new tools to explore mechanisms of NET regulation. hNET is a protein of 617 amino acids modeled with cytoplasmic NH2 and COOH termini and 12 membrane spanning hydrophobic domains (Pacholczyk et al., 1991; Bruss et al., 1995). Multiple Ser/Thr phosphorylation sites exist in the cytoplasmic NH2 and COOH as well as within internal domains (Pacholczyk et al., 1991). Increasing evidence suggests a role for PKC in acute modulation of multiple members of the Na+- and Cl−-coupled neurotransmitter transporter gene family including GABA, DA and 5HT transporters (Corey et al., 1994;Osawa et al., 1994; Zhang et al., 1997;Ramamoorthy et al., 1998a). In a previous study (see accompanying paper) we used cell-surface radioligand binding studies to demonstrate that activation of mAChR in the human noradrenergic neuroblastoma SK-N-SH acutely regulates cell-surface density of hNETs. In these cells, mAChR regulation of hNET involves PKC and direct activation of PKC with phorbol esters also affects surface hNET density. Unfortunately, SK-N-SH cells lack sufficient quantities of hNET protein to validate our conclusions using biochemical and immunological approaches. In our study, we adopted heterologous expression systems where the level of hNET protein expression would permit assessment of hNET protein trafficking. Our findings, arising from radioligand binding, surface biotinylation and confocal imaging of immunolabeled transporters, argue for a major role for change in surface distribution as underlying the reductions in NE transport capacity observed after acute PKC activation.

Methods

Materials

Reagents used to manipulate second messengers and protein kinases were obtained from the following sources: actinomycin D, cycloheximide, desipramine, dopamine, l-NE, staurosporine (Sigma Chemical Co., St. Louis, MO); bisindolylmaleimide I, β-PMA (Alexis Biochemicals, San Diego, CA); U-0521 (Upjohn, Kalamazoo, MI). l-[7,8-3H]noradrenaline (37 Ci/mmol), [N-methyl-3H]nisoxetine (86 Ci/mmol) and [32P]orthophosphate were obtained from Amersham (Arlington Heights, IL). Trans [35S]-label (1200 Ci/mmol) was obtained from ICN Biomedicals (Kalamazoo, MI). Protein-A Sepharose was obtained from Pharmacia Biotech, Costa Mesa, CA. Other reagents were of analytical purity and were obtained from standard sources.

Cell Culture and l-NE Uptake Assays

hNET stably-transfected HEK-293 (HEK-hNET) and LLC-PK1 (LLC-hNET) cells were generated and maintained as described previously (Melikian et al., 1994; Galli et al., 1995). For uptake studies, cells were plated at 100,000 cells/well on poly-d-lysine coated, 24-well tissue culture plates (Becton Dickinson, Lincoln Park, NJ) 2 days before experiments. In some studies, cells were plated at 500,000 cells/well on poly-d-lysine coated 6-well plates. Transient transfection of hNET cDNA (2 μg/well) in COS-7 cells and l-NE uptake assays were carried out as previously described (Melikian et al., 1994). For uptake assays, culture medium was removed by aspiration and cells were washed with 2 ml KRH buffer (130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM HEPES, pH 7.4). Cells were then preincubated at 37°C in KRH containing 10 mM d-glucose, 100 μM pargyline, 10 μM U-0521 and 100 μM ascorbic acid for 10 min. After the equilibration period, cells were incubated at 37°C with assay buffer containing either modulating agents or appropriate vehicle. l-NE transport assays were initiated by the addition of 1 μM [3H]-l-NE for 10 min at 37°C and terminated by three rapid washes with ice-cold KRH buffer. Cells were lysed in Optiphase Supermix scintillation cocktail (Wallac, Gaithersburg, MD) and accumulated radioactivity directly quantified in a microplate liquid scintillation counter (Microbeta, Wallac). Nonspecific [3H]NE uptake, defined as the accumulation in the presence of 1 μM desipramine, was subtracted from total uptake to define hNET-specific accumulation. Nonlinear curve fits of data (Kaleidagraph, Synergy Software, Reading, PA) for uptake used the generalized Michaelis-Menten model V = Vmax[S]n/[S]n + [K]n. Results presented on the effects of modulators on NE uptake arise from experiments using vehicle-treated cells, assayed in parallel. Statistical analyses were performed comparing mean transport values or kinetic constants using INSTAT software (Software Inc., GraphPad, San Diego, CA).

Quantitative Estimation of hNET Surface Density

Antagonist binding assays in intact cells and membrane fractions.

To assess surface density of hNET proteins, an intact cell paradigm was implemented using [3H]nisoxetine (see accompanying paper). HEK-hNET cells were treated with modulating agents or appropriate vehicle as described in transport assays and then transferred to 4°C, washed with ice-cold binding buffer [100 mM NaCl, 50 mM Tris (pH 8.0), ascorbic acid 100 μM]. Cells were incubated with 0.5 ml of ice-cold binding buffer for 20 min. After 20 min, cells were incubated in 0.5 ml of ice-cold binding buffer containing [3H]nisoxetine (0.01–10 nM) at 4°C for 1 hr. Binding assays were terminated by washing the cells three times with ice-cold binding buffer. Cell extracts were prepared with 0.5 ml of 1% SDS or 1 ml of Optiphase Supermix scintillation cocktail and bound radioactivity quantified using scintillation spectrometry. A portion of the cell extracts were analyzed for protein content (Bradford assay, BioRad, Hercules, CA). Nonspecific binding was defined using 10 μM DA, which gave the same values as 1 μM desipramine. Radioligand binding to total cell membrane was carried out as described previously (Galli et al., 1995). After treatments, cells were washed and scrapped into ice-cold PBS and pelleted at 1600 ×g for 4 min. The medium was discarded and the pellet resuspended in ice-cold binding buffer (100 mM NaCl, 50 mM Tris, ascorbic acid 100 μM, pH 8.0) by trituration and the cells were homogenized with a Polytron (Brinkman, Westbury, NY) at 25,000 revs/min for 5 sec.

Centrifugation, resuspension and homogenization were repeated and a sample of suspension was removed for protein determination by the Bradford method (BioRad). Initial studies with total cell membranes isolated from hNET stably expressing HEK-293 cells, demonstrated linearity of specific binding up to 100 μg membrane protein per tube and subsequent assays used 50 μg/tube. Nisoxetine saturation analyses were generated as described for intact cells. Assays were terminated after 2 hr incubation at 4°C by rapid filtration (Brandel, Gaithersburg, MD) over GF/B glass-fiber filters (Whatman, Clifton, NJ), presoaked in 0.5% polyethylenimine (Sigma). Filters were washed in ice-cold binding buffer and bound radioactivity measured using liquid scintillation counting (Beckman, Fullerton, CA). Nonspecific binding, defined as the binding in the presence of 1 μM desipramine or 10 μM DA which yielded equivalent data, was subtracted from total binding to define specific binding. Nonlinear curve fits of binding data (Kaleidagraph, Synergy Software, Reading, PA) using the equation, B = Bmax[S]n/[S]n + [K]nand the Scatchard linear transformation were used to estimate ligandKd and site density (Bmax). Statistical analysis was performed using Student’s t test (INSTAT Software, Inc., Graphpad, San Diego, CA).

hNET immunoblots and biotinylation.

Cell-surface biotinylation (Melikian et al., 1996; Ramamoorthy et al., 1998b) was performed to assess potential redistribution of transporter protein in regulation experiments. Parental HEK-293 or HEK-hNET cells were seeded on poly-d-lysine coated 6-well plates at 500,000 cells/well 48 hr before treatments, washed with 37°C KRH and incubated with vehicle or β-PMA for 30 min in KRH at 37°C for the times indicated and cell surface protein biotinylation was carried out as previously described (Qian et al., 1997). Cells were washed quickly with warm KRH and then treated with sulfo-succinimidobiotin (1.5 mg/ml, Pierce Chemical Co., Rockford, IL) at 4°C for 1 hr in PBS/Ca-Mg (138 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 9.6 mM Na2HPO4, 1 mM MgCl2, 0.1 mM CaCl2, pH 7.3). Biotinylating reagents were removed by washing at 4°C with 100 mM glycine in PBS/Ca-Mg twice, the reaction further quenched by incubation with 100 mM glycine for 30 min and then cells were washed with PBS/Ca-Mg rapidly three times before lysis with 250 μl/well Radio Immuno Precipitation Assay buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% sodium dioctyl sulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, supplemented with protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μM pepstatin, 1 mg/ml soybean trypsin inhibitors, 1 mM iodoacetamide and 250 μM PMSF) for 1 hr at 4°C with constant shaking. Lysates were centrifuged at 20,000 ×g for 30 min at 4°C and supernatant incubated with monomeric avidin beads (Pierce; 175 μl beads/1250 μl supernatant) for 1 hr at room temperature. Beads were washed three times with Radio Immuno Precipitation Assay and adsorbed proteins eluted with 50 μl Laemmli loading buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercapto ethanol and 5% bromophenol blue) for 30 min at room temperature. A total of 40 μl of total cell lysate, lysates after incubation with avidin beads, the last wash and the bead eluate (50 μl) were separated by SDS-polyacrylamide gel electrophoresis (10%) and immunoblotted with N430 NET antibody (0.25 μg/ml) using 1:3000 goat-anti-rabbit horseradish peroxidase-conjugated secondary antibody (Melikian et al., 1994). To validate the surface localization of biotinylated NET protein, blots were stripped [62.5 mM Tris-HCl, pH 6.8, 2% SDS and 100 mM β-mercaptoethanol (BME)] for 30 min at 50°C, washed with PBS twice for 10 min, reblocked in 5% dry milk for 1 hr and probed with anti-calnexin (1:1000, Stressgen, Victoria, BC, Canada) followed by goat-anti-rabbit horseradish peroxidase conjugated secondary antibody (1:3000). Immunoreactive bands were visualized by ECL on Hypersensitive ECL film (Amersham Corp., Arlington Heights, IL) and scanned bands were quantitated using ImageQuant (Molecular Dynamics, Sunnyvale, CA). Exposures were precalibrated to insure quantitation within the linear range of the film and multiple exposures were taken to validate linearity of quantitation. Values of hNET total, nonbiotinylated and surface protein were normalized using levels of calnexin immunoreactivity in total cell extracts and values averaged across three experiments.

Visualization of hNET Protein Redistribution via Immunofluorescence/Confocal Microscopy

Changes in the subcellular localization of hNET proteins after exposure to modulators of PKC activity were examined via confocal microscopy after immunofluorescent labeling of NETs with N430 antibody (Melikian et al., 1994). LLC-hNET or HEK-hNET cells were plated onto sterile, 3-aminopropyltriethoxysilane-treated coverslips (10000 cells/12-mm coverslip) in 24-well tissue culture plates. After 5 days, cells were treated with β-PMA and staurosporine or vehicle in the same manner as for transport assay experiments. After drug treatment, the cells were fixed in methanol for 10 min and washed three times in 0.05M (pH 7.2) TBS. Nonspecific binding was blocked for 30 min at room temperature with 5% NDS containing 0.4% Nonidet P-40 to permeabilize the cells. Cells were incubated with either affinity-purified N430 polyclonal antibody at 5 μg/ml or ZO-1 monoclonal antibody (1:20) (Peralta et al., 1996) in 5% NDS/TBS overnight at 4°C. After washing six times in TBS, Cy-3 conjugated donkey anti-rabbit secondary antibodies (1:600 in 5% NDS/TBS) were applied for 1 hr at room temperature. Finally, the cells were washed six times with TBS, dipped in phosphate buffer and mounted on slides with Polyaquamount (Polysciences, Warrington, PA). Confocal images of the labeled cells were captured with one, 8-sec scan through the vertical center of the cell layer using a Zeiss LSM 410 equipped with internal He/Ne and external Ar/Kr lasers (VUMC Cell Imaging Resource supported by CA 68485 and DK 20593).

Results

PKC activators reduce l-NE transport capacity in hNET transfected cell lines.

HEK-293 and LLC-PK1 cells stably expressing hNET cDNA (HEK-hNET and LLC-hNET, respectively) have been characterized previously for their l-NE transport kinetics, antagonist sensitivities, associated ionic currents and protein expression (Asano et al., 1993; Melikian et al., 1994; Galli et al., 1995). We explored the effect of PKC-activating phorbol esters on l-NE transport in both stable cell lines as well as in transiently transfected COS-7 cells. Treatment of HEK-hNET cells with β-PMA produces both a time- and concentration-dependent reduction in l-NE uptake (fig. 1, A and B). Significant reductions in l-NE uptake are observed within 10 min of β-PMA treatment (1 μM) and maximal reductions (39 ± 2%) achieved with 30 min exposure. β-PMA concentrations as low as 1 nM (30-min incubation) significantly reduce NE transport and maximal inhibition of NE uptake is obtained with 100 nM β-PMA. The effects of PMA are stereospecific as the PKC-inactive isomer α-PMA failed to alter l-NE uptake (fig. 1C). Another PKC-active phorbol ester, PDBu, also exhibited stereospecific inhibition of l-NE uptake with β-PDBu (1 μM; 30 min) inducing a 37 ± 5% reduction in l-NE uptake, whereas, α-PDBu does not alter l-NE uptake under the same conditions. The PKC inhibitor, staurosporine, which at 1 μM does not affect basal l-NE uptake in HEK-hNET cells, abolishes the β-PMA-induced inhibition of l-NE transport (fig. 1D). Similar results were obtained with another PKC inhibitor, bisindolylmaleimide I (1 μM) (data not shown). Although less efficacious when compared to NE transport regulation, β-PMA (1 μM, 30 min) also reduced activity of three other endogenous Na+-dependent transport systems (l-alanine, 76 ± 3%; l-glutamate uptake, 76 ± 2%; glycine, 78 ± 2%, relative to control); however, Na+-independent l-leucine transport was unaffected by phorbol ester treatments (110 ± 2%, relative to control), suggesting that the effect of PKC activators cannot be generalized to all transport systems expressed by these cells.

Figure 1
Figure 1

Reduction of l-NE uptake by β-PMA in hNET stably transfected HEK 293 (HEK-hNET) cells. A, Time-dependence; B, concentration-dependence; C, stereospecificity and D, staurosporine sensitivity of the effect of phorbol esters on l-NE uptake in HEK-hNET cells. Cells were treated with β-PMA for indicated times and assayed for l-NE (1 μM, 5 min) uptake as described in “Methods.” Maximal inhibition of l-NE uptake was observed after a total incubation time of 30 min with 1 μM of β-PMA. The effect of phorbol esters on l-NE uptake in transporter transfected HEK-293 cells is stereospecific. Cells were preincubated separately with α and β isomers of PMA and PDBu for 20 min before the addition of l-NE (1 μM; 10 min). Data are presented as means ± S.E.M. of three experiments performed in triplicate. Staurosporine (1 μM) produces concentration-dependent attenuation of β-PMA- (1 μM) induced reduction in l-NE uptake. Staurosporine was added 20 min before the addition of β-PMA. Parallel assays were carried out in the presence of 1 μM desipramine to define specific uptake. Data presented are means ± S.E.M. of three separate experiments performed in triplicate. *P < .05; two-tailed Student’s t test.

Kinetic analysis of the effect of β-PMA (1 μM; 30 min) on l-NE transport in HEK-hNET cells indicates that β-PMA significantly reduces the Vmax of l-NE transport with little or no change in NEKm (table 1). Similar findings to those described above for HEK-hNET cells were obtained with β-PMA treatment of LLC-hNET and hNET transiently transfected COS-7 cells (table 1). β-PMA (1 μM, 30 min) produces a 42, 26 and 38% decrease in Vmax of l-NE transport in HEK-hNET, LLC-hNET and hNET transiently transfected COS-7 cells, respectively. Both basal and β-PMA-induced reductions in NET activity are unaffected by pretreatment of cells with the mRNA synthesis inhibitor, actinomycin D (10 μM; 20 min) or the translation inhibitor cycloheximide (10 μM; 2 hr) (table 2) suggesting that β-PMA induced reduction of NET activity is primarily a posttranslational process.

View this table:
Table 1

Effect of β-PMA on kinetics of l-NE uptake in hNET stably and transiently transfected cell lines

View this table:
Table 2

Reduction in NE transport induced by β-PMA in HEK-hNET cells is protein and mRNA synthesis independent

PKC activators reduce plasma membrane expression of hNETs in stably transfected HEK-293 and LLC-PK1 cells.

Reduction in NE transport capacity after β-PMA treatment could arise from a reduction in transport efficiency, a reduction in the number of functional carriers in the plasma membrane, or both. To further address this issue, we determined the effect of β-PMA (1 μM; 30 min) on the surface density of binding sites for the NET-selective antagonist, [3H]nisoxetine (Tejani-Butt, 1992; Cheetham et al., 1996). Previously, we used this paradigm to demonstrate that activation of PKC and muscarinic M3 acetylcholine receptors in human noradrenergic neuroblastoma SK-N-SH reduces the surface expression of hNET. In our study, we determined [3H]nisoxetine binding to intact HEK-hNET cells and total membrane fractions isolated from lysed cells after treatment with either vehicle or β-PMA (fig. 2, A and B). As found with SK-N-SH cells, specific [3H]nisoxetine binding to intact cells is abolished by the hydrophilic NET substrate DA suggesting that [3H]nisoxetine binding in this paradigm reports cell-surface transporters. Scatchard analysis of [3H]nisoxetine binding to intact hNET cells reveals aKd of 9.2 ± 1.3 nM and Bmax of 7.2 ± 0.51 pmol/mg protein. In intact HEK-hNET cells, β-PMA treatment (1 μM; 30 min) reduces the Bmax of [3H]nisoxetine binding (∼30% reduction) with no change in nisoxetineKd . Scatchard analysis of [3H]nisoxetine binding to lysed membrane fractions reveals single-site kinetics with a Kd of 8.7 ± 1.1 nM, equivalent to affinity estimates from intact cell studies. The density of [3H]nisoxetine binding sites is greater than detected with intact cells (Bmax of 16.1 ± 2.1 pmol/mg protein) suggesting at least ∼55% of NETs are intracellular or otherwise inaccessible to displacement by DA. More importantly, β-PMA treatment fails to alter the density (Bmax) of [3H]nisoxetine binding sites in membrane fractions (fig.2B).

Figure 2
Figure 2

β-PMA selectively reduced density of [3H]nisoxetine binding sites in intact HEK-hNET cells. A, Cells were incubated with vehicle or β-PMA (1 μM; 30 min), followed by incubation with [3H]nisoxetine (0.01–30 nM) as described in “Methods.” Nonspecific binding was defined by using 100 μM dopamine and was subtracted from total binding. B, Cells were incubated with vehicle or β-PMA (1 μM; 30 min), followed by preparation of membrane fractions as described in “Methods.” A total of 80 μg of membrane suspension was incubated with 0.5 ml of [3H]nisoxetine (0.01–30 nM) at 4°C for 4 hr as described in “Methods.” Nonspecific binding was defined by using 100 μM dopamine and was subtracted from total binding. Plots show Scatchard analysis of the [3H]nisoxetine binding of a representative experiment. Bmax and Kd of [3H]nisoxetine binding in the presence and absence of drug treatment obtained in three separate experiments are presented as mean ± S.E.M. Asterisks indicates statistically significant changes as compared to vehicle controls (P < .05, Student’st test).

To confirm that β-PMA-induced reductions in NE uptake capacity and [3H]nisoxetine binding density occur as a result of changes in surface expression of hNETs, we determined the effect of β-PMA on the population of hNET proteins accessible to the membrane impermeant biotinylation reagent, sulfo-NHS-biotin (Melikian et al., 1994, 1996). In HEK-hNET cells, both 80- and 54-kDa forms of hNETs are detected by immunoblot of total extracts (fig.3A). These reflect fully and partially N-glycosylated forms of hNET protein, respectively (Melikian et al., 1994). Biotinylation per se has no effect on immunoreactivity of hNET protein to the N430 antibody (Melikianet al., 1996) and immunoreactivity for the endoplasmic reticulum marker calnexin is not appreciably recovered in biotinylated (cell-surface) fractions. The 80-kDa species constitutes ∼70% of total hNET protein with ∼23% of the total 80-kDa species recovered in plasma membrane fractions, as determined from blots normalized for loading with the intracellular marker calnexin. Approximately 45% of the total 54-kDa species is found on the cell surface (fig. 3A), consistent with previous evidence that immature NETs are expressed in the plasmalemma of transfected cells (Melikian et al., 1994). Whereas β-PMA has no effect on the level of total hNET protein observed in the unfractionated extracts, β-PMA produces a decrease in biotinylated fractions of comparable magnitude (∼20%) for both 80-kDa and 54-kDa hNET species (Figure 3C and 3D). We also observe a small, but significant, increase in the nonbiotinylated pool of hNET protein after β-PMA treatments, evident for both isoforms.

Figure 3
Figure 3

Effect of β-PMA on cell-surface abundance of hNET protein. A, hNET immunoblot of total, nonbiotinylated and biotinylated (cell-surface) protein in hNET transfected HEK-293 cells. Cell were treated with 1 μM β-PMA or vehicle for 30 min before biotinylation with sulfo-NHS-biotin. Aliquots of total, nonbiotinylated and wash fractions were loaded, whereas the entire eluate (50 μl) from streptavidin beads was loaded as the biotinylated sample and blots were probed with hNET specific antibody N430 as described previously. Two species of hNET specific bands, 80 and 54 kDa, are identified in hNET transfected HEK-293 cells. B, Immunoblot with anti-calnexin antibody. After detection of hNET, the blot was stripped and probed to identify the endoplasmic reticular membrane protein calnexin. Intensity of calnexin specific band was used to normalize hNET specific immunoreactivity in vehicle- and β-PMA-treated cells. C, Density of hNET specific band at 80 kDa in total, nonbiotinylated and cell-surface fractions from PMA-treated cells. Data are presented as means ± S.E.M. of three experiments as percent of vehicle-treated controls. D, Density of hNET specific band at 80 kDa in total, nonbiotinylated and cell-surface fractions from PMA-treated cells. Data are presented as means ± S.E.M. of three experiments as percent of vehicle-treated controls. The asterisk indicates statistically significant changes as compared to vehicle controls (P < .05, Student’s ttest).

Next we sought to visualize a possible steady-state redistribution of hNET protein triggered by activation of PKC using confocal microscopy of immunolabeled cells. We performed these studies in LLC-hNET cells since the polarity of hNET expression had been established in previous studies (Gu et al., 1996). In addition, their more columnar organization, as compared with HEK-293 cells, allows for greater certainty in obtaining optical sections through both the plasma membrane and cytoplasm of individual cells. Nonetheless, qualitatively similar results were found using HEK-hNET cells (data not shown). When LLC-hNET cells are treated with vehicle before immunolabeling with N430 antibody, hNET immunoreactivity is most prominent in the plasma membrane but is also present within cytoplasmic compartments (fig.4A). Images captured after β-PMA (1 μM; 30 min) treatment reveal substantially diminished plasma membrane labeling and a concomitant increase in cytoplasmic labeling (fig. 4B). Incubation of LLC-hNET cells with staurosporine alone (1 μM, 30 min) produced no changes in the labeling pattern seen in vehicle-treated cells (fig. 4C). However, incubation with staurosporine completely prevented the redistribution of hNET immunoreactivity associated with β-PMA treatment (fig. 4D). In Z-series reconstructions, plasma membrane hNET immunoreactivity was associated largely with basolateral domains as reported by Gu and coworkers (Gu et al., 1996), and manipulations of hNET membrane expression noted above did not redistribute protein to the apical membrane (data not shown). Unlike hNET, the plasma membrane protein ZO-1 (Peralta et al., 1996) is unchanged by β-PMA application, indicating that the effects of β-PMA on hNET redistribution is not generalizable to all other membrane proteins (fig. 4, E–F).

Figure 4
Figure 4

Effect of β-PMA on the cell-surface hNET density in LLC-hNET cells. Cells were labeled with N430 antibody, and the cellular localization of hNETs was examined by confocal microscopy in control and treated cells as described in “Methods.” The predominant plasma membrane localization of hNET immunoreactivity seen in vehicle-treated LLC-hNET cells (A) is diminished after exposure to β-PMA, with a concomitant increase in cytoplasmic labeling (B). Staurosporine produced no changes in the labeling pattern seen in vehicle-treated cells (C) but prevented the effects of β-PMA (D). Plasma membrane localization of membrane protein ZO-1 (E) was unaffected by PMA treatment (F). Cells were treated with vehicle or β-PMA (1 μM) for 30 min in the presence and absence of 1 μM staurosporine. Staurosporine was added 20 min before the addition of β-PMA.

Discussion

After the demonstration of carrier-mediated accumulation of catecholamines by noradrenergic neurons, now known to be largely mediated by high-affinity, desipramine-sensitive NETs (Graefe and Bönisch, 1988; Trendelenburg, 1991), evidence was presented for an activity-dependent regulation of these transporters (Gillis, 1963;Rorie et al., 1989; Eisenhofer et al., 1990;Stjärne et al., 1994). Evidence also exists that NETs can be regulated by circulating and paracrine stimuli (Peach et al., 1969; Boyd et al., 1986; Vatta et al., 1993; Yang et al., 1997). To date, molecular mechanisms underlying activity and hormonally regulated NE uptake have not been established, although several second messenger-linked pathways such as those downstream of cAMP, cGMP and nitric oxide have been reported to influence NE transport (Bunn et al., 1992; Kaye et al., 1997). Previously we have demonstrated that activation of mAChRs in human noradrenergic neuroblastoma, SK-N-SH cells, results in reduced NE transport activity via PKC-dependent and -independent pathways (accompanying paper). Based on studies evaluating cell-surface hNET density using [3H]nisoxetine binding, we proposed that mAChR-mediated reduction in NE transport arises from a loss of NET proteins from the cell-surface. The loss of cell-surface hNETs was independent of transporter mRNA transcription or protein translation and could not be explained by degradation of transporter protein since [3H]nisoxetine binding in membrane fractions remained unaffected by mAChR activation. However, our inability to detect NET proteins in SK-N-SH cells using NET-specific antibody, perhaps as a result of low levels of NET expression, limits our investigation of mechanisms underlying acute regulation of NET activity. In our study using hNET-transfected mammalian cells (lacking muscarinic receptors), we reevaluated the effects of acute phorbol esters on hNET expression and gathered evidence in support of the regulated trafficking of hNET proteins.

As in SK-N-SH cells, β-PMA produces rapid reduction in the maximal capacity (Vmax) of l-NE transport in stably transfected cells without significantly altering l-NE Km , findings independent of changes in hNET transcription or translation. The lack of effect of β-PMA on endogenous leucine transport in HEK-293 cells indicates a degree of specificity for hNET though we observe some modulation of endogenous Na+-coupled transporters in these cells. The ability of β-PMA to regulate endogenous alanine, glutamate and glycine transport in HEK cells in a manner similar to that of transfected hNET suggests that these carriers may share a common mode of regulation. However, the inability to modulate leucine transport argues against a general perturbation on membrane protein trafficking. Next, we used a [3H]nisoxetine binding paradigm to evaluate regulated surface expression of NET proteins. This paradigm involves determination of [3H]nisoxetine binding in intact cells with incubations conducted at low temperature to limit endocytosis of the ligand. In addition, we determined nonspecific binding using the hydrophilic transporter substrate, DA, that does not cross the plasma membrane in appreciable quantities at reduced temperatures. Our estimate of surface density of hNETs in HEK-hNET (3.8 × 105 sites per cell) coupled with [3H]nisoxetine binding in membrane fractions indicate that ∼45% of NETs in hNETs reside on the cell-surface. If all the cell-surface hNETs contribute to transport of [3H]NE, the ratio of Vmax/Bmax gives an estimate of cycle rate equal to 1.8 cycles/sec, consistent with data established for native rat NET in PC12 cells (Harder and Bönisch, 1984) and our own estimates from SK-N-SH cells (Apparsundaram et al., accompanying paper). We cannot rule out that the isolation of total membranes from cells may also have increased site availability by the removal of ionic or proteinaceous inhibition of binding by more thorough washing. We also may have incurred losses of NET protein during membrane preparation such that the intracellular pool of protein would actually be greater. More importantly, our observation that β-PMA produces a decrease in Bmax of [3H]nisoxetine binding with little or no change in the Kd in intact cells, but not in membrane fractions, suggests that the loss of binding in intact cells is not due to degradation of transporters but more likely attributable to a loss of cell-surface transporters subsequent to transporter internalization. The reduction in Bmax we observe (30%) matches well the loss in transport capacity (39%) after PKC activation. Similarly, Zahniser and coworkers (Zhu et al., 1997) have noted that even the lipophilic ligand mazindol can report changes in transporter surface density after β-PMA treatments, perhaps because of a requirement for extracellular levels of Na+-to support high-affinity mazindol binding.

Previously we have shown that hNET stably-transfected cells express NETs varying in their N-glycosylation state (Melikian et al., 1994). We presume that under conditions of native expression with lower levels of NET protein synthesized, that all NET protein would be fully processed. Though the 80-kDa isoform is the predominant hNET in these cells at steady state, both 80- and 54-kDa forms are expressed at the cell surface as described in COS cells (Melikianet al., 1996). We observe that β-PMA reduces the abundance of biotinylated hNETs. In contrast to previous studies with transfected hSERT, the biotinylation paradigm fails to reveal as extensive a loss of surface NETs as expected from transport and binding studies, perhaps due to quantitation inaccuracies arising from multiple isoforms at the cell-surface. Alternatively, a fraction of NETs may be functionally inactivated and inaccessible to [3H]nisoxetine but accessible to sulfosuccinimidobiotin. However, the phenomenon of NET redistribution is corroborated by the dramatic internalization of hNETs detected by confocal microscopy in which application of β-PMA induces a decrease in cell-surface labeling with a concomitant increase in cytoplasmic labeling of hNETs in either LLC-hNET or HEK-hNET cells. This is not a nonspecific effect of β-PMA on membrane protein trafficking per se as phorbol ester treatment fails to alter surface expression of other cell-surface proteins including ZO-1. We have also visualized increased cytoplasmic staining in β-PMA-treated LLC-PK1 cells transfected with the rat serotonin transporter (Schroeter S and Blakely RD, unpublished observations).

Our data derived from cell-surface biotinylation, radioligand binding analysis and confocal microscopy are most consistent with reduced surface expression of transporter protein as an important mechanism contributing to diminution of NET activity after PKC activation. Similar conclusions have been reached in studies with PKC activators and inhibitors for a number of members of the Na+- and Cl−-coupled transporter gene family. Thus, treatment ofXenopus laevis oocytes with PKC activators induces movement of GABA transporter proteins between cytoplasmic membrane vesicles to surface membranes (Corey et al., 1994; Quick et al., 1997). Activation of PKC also induces translocation of DATs from cell-surface to cytoplasm in X. laevis oocytes (Zhuet al., 1997). Similarly we have shown that PKC treatment results in a down-regulation of hSERT-associated currents and transport activity in HEK-hSERT cells, paralleled by a translocation of cell-surface transporters to the cytoplasmic pool (Qian et al., 1997). Activation of PKC by phorbol esters causes phosphorylation and redistribution of dopamine and serotonin transporters in transfected LLC-PK1 and HEK-293 cells, respectively (Huff et al., 1997; Vaughan et al., 1997;Ramamoorthy et al., 1998a). Canonical phosphorylation sites for PKC (T30, T58, S259, S502, S579), PKA (S502, S579) and PKG (T30, T58, T258, T259, S502, S579) are located on the putative cytoplasmic domains of hNETs. Using in vitro phosphorylation approaches we were able to demonstrate phosphorylation of purified NH2and COOH termini of hNETs by PKC (Apparsundaram and Blakely, 1996). In preliminary studies, we have obtained evidence that phosphorylated NETs can be immunoprecipitated from metabolically labeled, transfected cells after phorbol ester treatment (Apparsundaram S, preliminary studies), although it remains to be determined whether this labeling is required for changes in transporter surface expression. In summary, our findings of altered surface expression of NET proteins in transfected cells support models of regulated hNET trafficking developed in noradrenergic SK-N-SH cells and suggest that, in vivo, hNET proteins may redistribute to influence NE clearance capacity as a consequence of presynaptic receptor and kinase activation.

Acknowledgments

The authors thank Dr. Heinz Bönisch for suggestions regarding nisoxetine binding, Dr. Gary Rudnick for donating LLC-hNET cells, Dr. S. Ramamoorthy for comments on the manuscript, Dr. Peter Dempsey for the kind gift of ZO-1 antibody and Ms. Qiao Han and Ms. Denise Malone for technical assistance.

Footnotes

  • Send reprint requests to: Dr. Randy D. Blakely, Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-6600.

  • ↵1 This work was supported by NINDS Award NS33373 and MH58921 to R.D.B.

  • Abbreviations:
    DA
    dopamine
    NE
    norepinephrine
    NET
    norepinephrine transporters
    PKC
    protein kinase C
    hNET
    human NET
    mAChR
    muscarinic M3 acetylcholine receptor
    KRH
    Krebs-Ringers-HEPES
    PBS
    phosphate-buffer saline
    TBS
    Tris-buffered saline
    NDS
    normal donkey serum
    PDBu
    phorbol-12,13-dibutyrate
    PMA
    phorbol-12-myristate-13-acetate
    SDS
    sodium dioctyl sulfate
    U-0521
    3,4-dihydroxymethyl propiophenone
    PMSF
    phenylmethylsulfonyl fluoride
    • Received January 13, 1998.
    • Accepted June 8, 1998.

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Acute Regulation of Norepinephrine Transport: II. PKC-Modulated Surface Expression of Human Norepinephrine Transporter Proteins

Subramaniam Apparsundaram, Sally Schroeter, Elena Giovanetti and Randy D. Blakely
Journal of Pharmacology and Experimental Therapeutics November 1, 1998, 287 (2) 744-751;
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