HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 18, 19315-19326, April 30, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/18/19315    most recent M311172200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jayanthi, L. D. Articles by Ramamoorthy, S. Search for Related Content PubMed PubMed Citation Articles by Jayanthi, L. D. Articles by Ramamoorthy, S.

Regulated Internalization and Phosphorylation of the Native Norepinephrine Transporter in Response to Phorbol Esters

EVIDENCE FOR LOCALIZATION IN LIPID RAFTS AND LIPID RAFT-MEDIATED INTERNALIZATION^*

Lankupalle D. Jayanthi, Devadoss J. Samuvel, and Sammanda Ramamoorthy

From the Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, October 10, 2003 , and in revised form, January 29, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The effects of norepinephrine in the brain and periphery are terminated primarily by active reuptake of the catecholamine via cocaine- and amphetamine-sensitive norepinephrine transporters (NETs). Activation of protein kinase C (PKC) down-regulates NET by sequestering it from the plasma membrane, although the underlying mechanism is not yet known. Previously, we showed robust expression of endogenous NETs in rat placental trophoblasts (Jayanthi, L. D., Vargas, G., and DeFelice, L. J. (2002) Br. J. Pharmacol. 135, 1927-1934). Here we report a significant reduction in native NET function and surface expression in these cells following phorbol ester (-PMA) treatment. The -PMA-mediated down-regulation of NET occurs by a rapid sequestration of NETs from the plasma membrane and is calcium-independent. Reversible biotinylation experiments revealed a significant enhancement of NET endocytosis following -PMA treatment. Chemical treatments and expression of dominant negative mutants of dynamin 1 and 2 failed to prevent the -PMA effect, suggesting a clathrin-independent pathway. In contrast, treatment with the cholesterol-disrupting agent filipin, which blocks caveolae/lipid raft-mediated internalization, completely blocked the -PMA-mediated NET sequestration. Discontinuous sucrose density gradient centrifugation revealed NET in the lipid raft fractions. Following -PMA treatment, there was reduced NET levels in the lipid raft fractions suggesting that cholesterol-rich lipid rafts mediate PKC-triggered NET internalization. Metabolic labeling and immunoprecipitation studies revealed that NET phosphorylation is stimulated severalfold by PKC activation and protein phosphatase 1/2A inhibition. Together, these findings demonstrate for the first time that in trophoblasts (i) PKC activation regulates NET function and surface expression by an enhanced internalization process that is lipid raft-mediated and (ii) PKC and protein phosphatase(s) modulation regulates NET phosphorylation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
NET,¹ which belongs to GAT1/NET family of Na⁺/Cl^--dependent transporters, is selectively expressed on noradrenergic nerve terminals and exerts spatial and temporal control over the actions of NE (1, 2). NET is also an important target for various antidepressants and psychostimulants (3). Functional silencing of NET through genetic manipulations results in altered behavior when animals are stressed or challenged pharmacologically (hypersensitivity to psychostimulants and opiates) (4, 5). NE uptake sites and NET activity are also compromised in cardiovascular diseases and brain disorders (6-8). A point mutation in NET (A457P in exon 9 of the NET gene) has been shown to be associated with orthostatic intolerance (9), whereas another study shows the absence of this specific mutation in all the orthostatic intolerance patients examined (10). The A457P mutation disrupts surface expression of mutant and wild type NETs when coexpressed in Chinese hamster ovary cells (11).

Diverse stimuli, including neuronal activity, peptide hormones, and second messengers elevated after receptor activation, regulate NET. For example, activation of PKC down-regulates the activity of NET and results in redistribution of NET from the plasma membrane (12, 13). Activation of PKC leads to a reduction in amine transport capacity, revealed by a change in maximal transport with little or no change in substrate affinity (12-14). Similarly, inhibition of protein phosphatase 1/2A (PP1/PP2A) reduces amine transport activity, and stimulates transporter phosphorylation (15). PP2Ac exists in physical complexes containing biogenic amine transporters including NET (16). NET bears multiple consensus sites for PKC phosphorylation on putative cytoplasmic domains (3). Determinants within the C-terminal region of NET dictate NET trafficking, stability, and activity (17). Although membrane trafficking is implicated in PKC-mediated regulation of NET proteins, it is not yet known whether NET cell surface losses are due to increased internalization (endocytosis) or decreased recycling. It is also not known whether NET undergoes regulated phosphorylation.

Although dynamin-dependent clathrin-mediated internalization is involved in the regulation of DAT and GAT1 trafficking (18-20), such a mechanism is not known for NET. Certain endocytic pathways utilize glycosylphosphatidylinositol-anchored membrane proteins (lipid rafts) and caveolae to concentrate and internalize molecules such as folate (21). The association of multiple signaling molecules with caveolae and lipid rafts suggests their role in coordinating signal transductions with internalization of molecules (22).

Human placenta expresses both SERT and NET (23-25), and we have recently developed trophoblast cultures from the rat placenta that robustly express endogenous NET (26). In this study, we tested the hypothesis that the -PMA-induced down-regulation of NET in trophoblasts occurs by an enhanced internalization process and that this internalization is dynamin-dependent. Our results demonstrate that in trophoblasts, PKC activation enhances NET internalization and that the internalization occurs in a calcium- and clathrin (dynamin)-independent manner. Surprisingly, cholesterol-disrupting agents prevented the -PMA-induced NET internalization. In addition, we show that NET protein is present in lipid raft fractions and demonstrate that NETs internalize via lipid rafts following -PMA treatment. Finally we provide evidence that PKC and PP1/PP2A regulate the phosphorylation of native NETs. These findings suggest that NET function in the trophoblast is regulated by endogenous kinases and phosphatases, and the regulation of NET may play a role in placental control of amine entry or exit in order to cope with the fetal demands, such as fetal growth, and the maternal insults, such as drug abuse by the pregnant mother.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials
- and -PMA were from Alexis Biochemicals (San Diego, CA). Rat NET polyclonal antibody was raised against the peptide WERVAYGITPENEHHLVAQRDVR, amino acids 535-557 of rNET supplied by the commercial vendor, Invitrogen. Mouse monoclonal antibodies to dynamin 1, dynamin 2, caveolin 1, and PKC were from BD Biosciences. Mouse monoclonal antibody to PKC was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-HA antibody was from Roche Applied Science. Polyclonal anti-GFP and anti-Myc antibodies were from Clontech (Palo Alto, CA). Mouse anti-TfR antibody was from Zymed Laboratories Inc. (South San Francisco, CA). All other chemicals were from Sigma unless otherwise noted.

Rat Placental Trophoblast Cultures
Placentas were isolated from pregnant rats at gestation day 17, and trophoblasts were isolated as described earlier (26). All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Placental tissue was minced and digested with 0.1% w/v collagenase plus 0.002% w/v DNase in 10 ml of M199 medium containing Hanks' salts. Tissue was dispersed in 1x Ca^2+- and Mg^2+- free Hanks' balanced salt solution containing 0.1% BSA followed by filtration through 4 layers of 150-µm nylon cloth. Trophoblasts were isolated by discontinuous Percoll gradient (Pharmacia Fine Chemicals, Piscataway, NJ) (10-70%) centrifugation and cultured in a mixture of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells seeded in 24-well cell culture plates (100,000 cells/well) or 35-mm dishes or 6-well plates (500,000 cells/dish or well) were allowed to grow in an atmosphere of 95% air, 5% CO₂ and used for the experiments.

Immunostaining
Trophoblasts grown on glass coverslips were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in PBS/Ca-Mg (phosphate-buffered saline with 0.5 mM CaCl₂ and 1 mM MgCl₂) for 15 min at room temperature (RT), rinsed with PBS/Ca-Mg, and quenched with 50 mM NH₄Cl in PBS/Ca-Mg. The cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at RT and blocked with 2% BSA in 0.1% Triton X-100 in PBS/Ca-Mg. Trophoblasts were incubated with the primary antibody (polyclonal anti-rNET, raised against rNET sequence (WERVAYGITPENEHHLVAQRDVR) in PBS containing 0.1% Triton X-100 and 2% BSA for 1 h at RT. After washing the cells three times with PBS/Ca-Mg, 1:1000 dilution of the secondary antibody (Cy3-conjugated donkey anti-rabbit IgG, Jackson ImmunoResearch Laboratories Inc, West Grove, PA) in PBS containing 0.1% Triton X-100 and 2% BSA was added and incubated for 1 h at RT. The cells were washed as before with PBS, mounted on glass slides in Aqua-Poly/Mount (Polysciences, Inc., Warrington, PA), and examined using a Zeiss Axiovert 135 confocal microscope.

Treatments and NE Uptake Assays
Trophoblasts were treated with the vehicle or -PMA (0.5 µM) for 30 min at 37 °C. Other treatments with various reagents were performed as described in appropriate places. Uptake measurements were performed by incubating the trophoblasts for 10 min at 37 °C, with [³H]NE (35.0 Ci/mmol L-[7,8-³H]norepinephrine, Amersham Biosciences) in 0.5 ml of Krebs-Ringer/HEPES (KRH) buffer, pH 7.4 (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 10 mM HEPES, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 5 mM Tris, and 10 mM D-glucose), containing 100 µM ascorbic acid and 100 µM pargyline. Assays were terminated by removing the radiolabel and by rapid washings of cells three times with 1 ml of ice-cold KRH buffer. Cells were solubilized in 0.5 ml of 1% SDS, and the accumulated [³H]NE was quantified by liquid scintillation counting (Beckman Coulter Inc., CA). 50 nM of [³H]NE was used in all experiments except for kinetic studies, where uptake of NE was measured over a concentration of 0.1 to 10 µM NE with 50 nM of [³H]NE and the rest substituting with nonradioactive NE. Specific NE uptake was measured by subtracting the NE uptake measured in the presence of 1 µM desipramine (DS) from the total NE uptake measured in the absence of DS. For kinetic analysis of NE uptake in trophoblasts, the values were plotted as picomoles of NE uptake versus concentration of NE, and the data represent the mean ± S.E. from three experiments performed in triplicate on different batches of trophoblast cultures. Substrate K_m and V_max values for NE uptake were determined by nonlinear least square fits (Kaleidagraph, Synergy Software, Reading, PA) with the generalized Michaelis-Menten equation: , where V = transport velocity, [S] = substrate (NE) concentration, and n represents the Hill coefficient.

Cell Surface NET Measurements
Nisoxetine Binding—To estimate the change in cell surface density of NET in placental trophoblasts, binding of [³H]nisoxetine (86.0 Ci/mmol [N-methyl-³H]nisoxetine, Amersham Biosciences), a specific radioligand for NET, was measured using both intact trophoblasts and isolated membrane fractions. Trophoblasts were treated with the vehicle or -PMA as described under "Treatments and NE Uptake Assays," washed with ice-cold binding buffer (10 mM Na₂HPO₄, 120 mM NaCl and 5 mM KCl, pH 7.4 containing 0.32 M sucrose), and incubated with increasing concentrations (0.1-5 nM) of [³H]nisoxetine in binding buffer on ice for 2 h (saturates by 2 h). At the end of the incubations, the cells were washed, lysed, and measured for radioactivity.

For [³H]nisoxetine binding in membranes, trophoblast cultures in 35-mm dishes were treated with the vehicle or -PMA as described under uptake assays, and the cells were collected in ice-cold phosphate-buffered saline (PBS). Pelleted cells were resuspended in ice-cold binding buffer (10 mM Na₂HPO₄, 120 mM NaCl, and 5 mM KCl, pH 7.4) containing a mixture of protease inhibitors (1 µg/ml each of aprotinin, leupeptin, and soybean trypsin inhibitor, 1 µM each of pepstatin and iodoacetamide, 250 µM phenylmethylsulfonyl fluoride) and centrifuged at 20,000 x g. The resulting pellet was homogenized in 4 ml of binding buffer with a Polytron (Brinkmann Instruments) at 25,000 rpm for 5 s, and the homogenate was centrifuged at 20,000 x g for 30 min. The resulting pellet (membrane fraction) was resuspended in ice-cold binding buffer, and membrane protein was estimated by the Bradford method (Bio-Rad). Membranes (50 µg of protein) were incubated with increasing concentrations (0.1-5nM)of[³H]nisoxetine for 2 h (saturates by 2 h)at 4 °C and collected (Brandel, Gaithersburg, MD) on GF/B glass filters (Whatman, Clifton, NJ) presoaked in 0.3% polyethyleneimine. Filters were washed, and bound radioactivity was measured by liquid scintillation counting. Nonspecific binding, defined as the binding in the presence of 10 µM DS, was subtracted from total binding to obtain specific binding to whole cells as well as to membranes. Scatchard analysis was used to estimate ligand K_d and binding site density (B_max).

Surface Biotinylation—Although nisoxetine has been used as a NET ligand for binding studies to quantify cell surface NET density (12, 27), nisoxetine may enter the cell even at 4 °C. Cell surface biotinylation and immunoblot analyses were employed (28) to quantify more accurately the amount of NET protein distributed between cell surface and intracellular pools. Trophoblasts subjected to various treatments were washed and incubated with the cell membrane-impermeable reagent, sulfo-NHS-biotin (1.5 mg/ml, Pierce) for 1 h at 4 °C 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). The biotinylating agent was removed by incubating twice with 100 mM glycine for 30 min. Cells were washed with PBS/Ca-Mg and lysed at 4 °C with 400 µl of RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate) containing protease inhibitors. Lysates were centrifuged at 20,000 x g for 30 min at 4 °C, and the supernatants were incubated with monomeric avidin beads for 1 h at room temperature. The beads were washed three times with RIPA buffer, and adsorbed proteins were eluted in 50 µl of Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.01% bromphenol blue). An aliquot (40 µl) of total cell lysate from each sample and all (50 µl) of the avidin-bound samples were analyzed by immunoblotting with NET-specific antibody. To visualize intracellular NET, proteins from unbound fractions were precipitated with trichloroacetic acid, and equal amounts of protein were analyzed by immunoblotting with NET antibody. To validate the surface localization of biotinylated NET protein, blots were stripped and reprobed with anticalnexin antibody (1:1000, Stressgen Biotechnologies, Victoria, British Columbia, Canada). Band intensities were quantified using NIH Image (version 1.62). Exposures were precalibrated to ensure quantitation within the linear range of the film, and multiple exposures were taken to validate linearity of quantitation. Values of total, nonbiotinylated, and surface NET proteins were normalized using levels of calnexin immunoreactivity in total cell extract, and values were averaged across three experiments.

Calcium Depletion
In one set, trophoblasts were treated with the membrane-permeant Ca²⁺ chelator BAPTA-AM (10 µM) in Ca²⁺-free KRH buffer for 2 h at 37 °C to deplete both external and internal Ca²⁺ (29). After BAPTA-AM treatment, -PMA (0.5 µM final concentration) or the vehicle was added, and cells were incubated at 37 °C for 30 min in the continued presence of BAPTA-AM. In another set, trophoblasts were incubated at 37 °C with 0.001% dimethyl sulfoxide (Me₂SO) (vehicle for BAPTA-AM) in Ca²⁺ (2.2 mM) containing KRH buffer for 2 h. After the treatment, -PMA (0.5 µM final concentration) or the vehicle was added, and cells were incubated at 37 °C for another 30 min. Following treatments, uptake assays and biotinylation experiments were performed as described above.

Reversible Biotinylation (Internalization Assay)
Reversible biotinylation was performed as described (30). Trophoblasts were cooled rapidly to 4 °C to inhibit endocytosis by washing with cold PBS and surface-biotinylated with a disulfide-cleavable biotin (sulfo-NHS-SS-biotin; Pierce), and free biotinylating reagent was removed by quenching with glycine. NET endocytosis was initiated by incubating the cells with prewarmed media containing -PMA or the vehicle for indicated times at 37 °C. At the end of each time point, the reagents were removed, and fresh pre-chilled media were added to stop the endocytosis. The cells were then washed and incubated twice with 250 µM sodium 2-mercaptoethanesulfonate (MesNa), a reducing agent in PBS/Ca-Mg for 20 min to dissociate the biotin from cell surface-resident proteins via disulfide exchange. To define total biotinylated NETs, one dish of biotinylated trophoblasts was not subjected to reduction with MesNa and directly processed for extraction followed by isolation by avidin beads. To define MesNa-accessible NETs, another dish of cells was treated with MesNa immediately (at 0 time) following biotinylation at 4 °C to reveal the quantity of surface NET biotinylation that MesNa can reverse efficiently.

At the end of the treatments, trophoblasts were solubilized in RIPA, and biotinylated NETs were separated from nonbiotinylated proteins by using monomeric avidin beads. Biotinylated proteins were eluted from beads and resolved by SDS-PAGE. NET proteins in the fractions were visualized with NET-specific antibody as described under "Surface Biotinylation." NET bands were scanned, and the band densities were quantified by NIH Image 1.62 software (National Institutes of Health).

Manipulations (Chemical Treatments) to Block NET Internalization
Trophoblasts were subjected to various conditions that inhibit endocytosis mediated by either dynamin (clathrin-coated pits) (30) or caveolae/lipid rafts (31, 32). Trophoblasts were incubated with cycloheximide (30 µg/ml) for 30 min prior to treatment with -PMA to prevent the confounding effects of protein synthesis (18). Procedures to inhibit endocytosis were also performed for 30 min prior to experimentation coincident with cycloheximide treatment. The reagents and conditions to block clathrin-mediated endocytosis are as follows: (i) Control buffer, KRH buffer; (ii) K⁺ depletion, trophoblasts were incubated at 37 °C for 5 min in hypotonic shock solution (serum-free DMEM and distilled water in 1:1 ratio) and then rinsed and incubated with K⁺ depletion buffer (50 mM HEPES; 100 mM NaCl, pH 7.4; 1 mM CaCl₂;1mM MgCl₂); (iii) Hypertonic medium, 0.5 M sucrose in KRH buffer; (iv) chemical inhibition by (a) concanavalin A (ConA) (250 µg/ml) and (b) monodansyl cadaverine (MDC) (50 µM). The reagents and conditions to block caveolae/lipid raft-mediated endocytosis are as follows: (i) filipin (5 µg/ml) and (ii) nystatin (5 µg/ml) in KRH assay buffer. Treatments with appropriate vehicles were carried out for controls. Following the treatments, trophoblasts were treated with vehicle or -PMA as described before and used for NE uptake measurement and biotinylation studies as described above. Because trophoblasts also express Na⁺/Cl^--dependent taurine transporters, and PKC activation down-regulates the taurine transporter,² the effect of these manipulations on -PMA-mediated inhibition of taurine transport was examined. In addition, following filipin treatment, -PMA induced translocation of PKC isoforms ( and ) was measured as described by Karl and Divald (33) to determine whether PKC signaling pathways remain intact in response to cholesterol-depleting agents.

K44A Dominant Negative Dynamin and Caveolin Mutant Expression
Trophoblasts were tested for the endogenous expression of dynamin 1, dynamin 2, and caveolin 1 by immunoblotting with monoclonal antibodies to dynamin 1, dynamin 2, and caveolin 1, respectively. Trophoblasts were transiently transfected with K44A mutant cDNAs of dynamin 1 (HA-tagged) (34) or dynamin 2 (GFP-tagged) (35), or Myc-tagged S80E mutant cDNA of caveolin 1 (36), or HA-tagged DGV mutant cDNA of caveolin 3 (32), or empty vectors (control) using FuGENE 6 Transfection Reagent (Roche Applied Bioscience), and cultures were maintained for 48 h prior to experimentation (0.25 µg/well in 24-well plates or 1 µg/well in 6-well plates). Cells grown in 24-well plates were used for NE uptake measurements, and cells grown in 6-well plates were used for biotinylation experiments to assess the ability of these mutants to block -PMA-mediated NET endocytosis. To determine whether dominant negative dynamin mutants impaired clathrin-mediated endocytosis, TfR internalization was assessed by surface biotinylation experiments using trophoblasts transfected with vector alone or K44A dynamin 1 mutant. To determine whether dominant negative mutants of caveolin impaired caveolin-dependent endocytosis, cholera toxin B (CTXB) internalization was assessed. Trophoblasts transfected with vector alone, S80E caveolin 1, or DGV caveolin 3 were incubated at 4 °C with horse-radish peroxidase-conjugated CTXB (4 µg/ml PBS/Ca-Mg) for 45 min. Unbound CTXB was removed by washing with cold PBS/Ca-Mg. Internalization of bound CTXB was carried out by incubating the cells in PBS/Ca-Mg at 37 °C for 2.5 h. Following the incubation, biotinylation experiments were performed on the trophoblasts as described under "Surface Biotinylation." Equal amounts (150 µg) of protein from total lysates, all (50 µl) of the avidin-bound samples, and equal amounts (300 µl concentrated to 150 µl) of unbound fractions were dotted on a nitrocellulose membrane and developed with ECL reagents (37). Dot blots were scanned, and the dot densities were quantified by NIH Image 1.62 software (National Institutes of Health).

Lipid Raft Isolation
Lipid rafts were isolated from vehicle or -PMA-treated trophoblasts using the protocol described earlier (38) with few modifications. Trophoblasts were lysed in 1.5 ml of MBS (25 mM MES and 150 mM NaCl, pH 6.5) containing 1% Triton X-100 and a mixture of protease inhibitors (1 µM pepstatin A, 250 µM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) by using Dounce homogenizer with 10 up and down strokes at 4 °C. Equal volumes of 80% (w/v) sucrose in MBS were added to the homogenates. Cell lysates in 40% sucrose were placed at the bottom of SW41 centrifuge tubes and overlaid successively with 4 ml of 30% sucrose and 3 ml of 5% sucrose. The tubes were centrifuged at 188,000 x g for 18 h at 4 °C, and 1-ml fractions were collected from the top. The pellets were resuspended in 1 ml of MBS by passing through 27-gauge needle. Proteins from collected fractions were trichloroacetic acid-precipitated, washed in ether, air-dried, and subjected to 4-15% linear gradient SDS-PAGE. After transfer to polyvinylidene difluoride membrane, the presence of NET and other proteins was visualized by immunoblotting with specific antibodies.

Metabolic Labeling and Immunoprecipitation (Phosphorylation Assay)
Trophoblasts were incubated at 37 °C in phosphate-free DMEM for 1 h and then with 1 mCi/ml of carrier-free [³²P] orthophosphate (Amersham Biosciences) for 2 h (39). -PMA (0.5 µM) or okadaic acid (10 nM) or vehicle was added to the medium, and the incubation was continued at 37 °C for 30 min. Cells were washed with cold PBS and lysed in 400 µl of RIPA buffer containing protease inhibitors and phosphatase inhibitors (10 mM NaF, 50 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 µM of okadaic acid). Extracts were centrifuged at 20,000 x g for 30 min at 4 °C, and the supernatants were precleared using 100 µl of Protein A-Sepharose beads (39). NET protein was immunoprecipitated overnight at 4 °C by the addition of NET-specific antibody on end-over-end continuous mixing, followed by a 1-h incubation with Protein A-Sepharose beads (3 mg in 100 µl in RIPA buffer) at 22 °C (room temperature). The immunoadsorbents were washed with ice-cold RIPA buffer, extracted with 50 µl of Laemmli, and resolved by SDS-PAGE (10%). The radiolabeled proteins were detected by autoradiography, and digitized autoradiograms were evaluated on multiple film exposures to ensure quantitation within the linear range of film exposure.

Statistical Analyses
Statistical significance for NE uptake values and band densities were calculated using Student's t test, and comparisons were made between two groups (each treatment compared with respective vehicle control).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Endogenous NET Expression in Rat Placental Trophoblasts—Recently, we have successfully developed a primary cell culture model system of rat placental trophoblasts (Fig. 1A) to study NET function in a native cell environment. By using this native system, we demonstrated that rat trophoblasts exhibit Na⁺ and Cl^--dependent NE transport that is DS-sensitive (26). We have also shown the presence of an 85-kDa immunoreactive NET protein band in trophoblast cultures as a major protein (26) by Western blot analyses. We now directly identified NET protein expression by immunostaining with a primary antibody raised against the rNET (amino acids 535-557) (Fig. 1B). NET immunostaining was blocked by preincubation with the peptide against which the antibody was raised (Fig. 1C), indicating the presence of NET in native cells.

View larger version (56K): [in this window] [in a new window]   FIG. 1. NET expression in rat placental trophoblast cultures. Photographs of NET immunoreactivity in rat placental trophoblasts. Polyclonal NET antibody raised against a rat NET sequence was used to immunostain the trophoblasts as described under "Experimental Procedures." A, live cell image of trophoblasts in culture shows single nucleated cytotrophoblasts and differentiated multinucleated trophoblasts. B, immunostaining of trophoblasts with rNET antibody (amino acids 535-557) reveals specific NET expression. C, peptide antigen that was used to raise the NET antibody completely blocks NET immunostaining.

View larger version (26K): [in this window] [in a new window]   FIG. 2. PKC activation decreases NE uptake capacity. A, PMA specificity. Trophoblasts treated with the PKC activator, -PMA (0.5 µM), or -PMA (0.5 µM), the inactive isomer of -PMA, or staurosporine (1 µM) alone or pretreated with 1 µM staurosporine for 20 min before -PMA treatment for 30 min and NE uptake assays were performed as described under "Experimental Procedures." B, kinetic analysis. Kinetic experiments for NE uptake were performed as described under "Experimental Procedures." To define specific NE transport via NETs, parallel uptake assays were carried out in the presence of 1 µM DS. -PMA treatment decreased the maximal velocity (Vmax) by 30% with little or no change in affinity (Km) of the transporter for NE. Data derived from three separate experiments, each in triplicate, are given as mean ± S.E. (*, p < 0.05). Inset, Eadie-Hofstee plot.

View larger version (26K): [in this window] [in a new window]   FIG. 3. -PMA reduces NET-binding sites and cell surface NET protein. A, nisoxetine binding using whole trophoblasts. Trophoblasts were treated with vehicle or 0.5 µM -PMA for 30 min, and whole cell binding experiments using [3H] nisoxetine were performed as described under "Experimental Procedures." Kinetic analysis of nisoxetine binding to whole cells shows a change in Bmax values without any changes in Kd values. B, nisoxetine binding using trophoblast membranes. Total membranes were prepared from trophoblasts treated with vehicle or 0.5 µM -PMA for 30 min, and membrane binding experiments using [3H] nisoxetine were performed as described under "Experimental Procedures." The kinetic analysis of membrane binding experiments shows no changes in Kd or Bmax values. Nonspecific binding of [3H] nisoxetine was defined by using 10 µM DS. Data from three separate experiments performed in triplicate are given as mean ± S.E. C, Biotinylation. Trophoblasts were treated with -PMA or vehicle before biotinylation with sulfo-NHS-biotin as described under "Experimental Procedures." Aliquots (40 µl) of total and entire eluate (50 µl) from avidin beads were loaded onto gels, and the blot was probed with an affinity-purified NET-specific antibody. A representative blot indicates two species of NET-specific bands at 85 kDa, and one species at 48 kDa are identified in the trophoblasts. Stau, staurosporine. D, proteins from avidin-unbound fractions were trichloroacetic acid-precipitated, and equal amount proteins were subjected to SDS-PAGE followed by immunoblotting with NET-specific antibody. A representative blot shows NET protein levels in intracellular pools. E, biotinylation of NETs was quantified using NIH image, and the densities of 85-kDa band from three separate experiments are presented as mean ± S.E. Asterisks indicate significant loss of cell surface NET (C) and a concomitant increase in intracellular NET (D) by -PMA treatment compared with vehicle treatment (Student's t test; p < 0.05).

Calcium-independent Down-regulation of NET—-PMA (0.5 µM) produced 30% inhibition of NE uptake in the presence of Ca²⁺ (regular Ca²⁺ containing KRH buffer) (Fig. 4A). In trophoblasts, calcium depletion by BAPTA-AM or EGTA has been used to block the regulation of 15-hydroxyprostaglandin dehydrogenase by corticotrophin-releasing hormone (40). In contrast, the -PMA-mediated inhibition of NE uptake was observed even after 2 h of treatment of cells with 10 µM BAPTA-AM in Ca²⁺-free KRH buffer to deplete intracellular calcium stores (Fig. 4A). Surface biotinylation experiments confirmed that removal of Ca²⁺ did not prevent -PMA-induced NET sequestration (Fig. 4B).

View larger version (43K): [in this window] [in a new window]   FIG. 4. -PMA-mediated NET down-regulation is calcium-independent. Trophoblasts were treated with 10 µM membrane-permeable Ca2+-chelator BAPTA-AM in calcium-free KRH buffer for 2 h followed by incubation in the presence or absence of 0.5 µM -PMA for 30 min, and NE uptake assays and biotinylation experiments were performed as described under "Experimental Procedures." A, uptake data are presented as mean ± S.E. of three experiments carried out in triplicate. B, a representative blot from biotinylation experiments shows no significant change in -PMA-induced loss of NET surface density by calcium removal. Biotinylated NETs (85 kDa) were quantified using NIH image, and band densities from three separate experiments are shown in the lower panel as the mean ± S.E. Values from -PMA treatments were compared with vehicle treatments using Student's t test (*, p < 0.05).

View larger version (21K): [in this window] [in a new window]   FIG. 5. NET internalization is enhanced following -PMA treatment. A, reversible biotinylation demonstrates profiles of NET internalization in the trophoblasts. Lane 1 represents total NET biotinylated on the cell surface at the end of the incubation with sulfo-NHS-SS-biotin (no MesNa reversal, see "Experimental Procedures"). Lane 2 shows the effectiveness of MesNa in reversing the surface biotinylation immediately after it occurs. Biotinylated cells were incubated at 37 °C with vehicle alone or 0.5 µM -PMA for 5 min (lanes 3 and 4) and 30 min (lanes 5 and 6). B, biotinylated NET (85 kDa) bands were quantified using NIH image, and the percentage internalized (compared with total surface NET) in three separate experiments are shown as the mean ± S.E. Values from -PMA treatments were compared with vehicle treatments using Student's t test (*, p < 0.05).

View larger version (37K): [in this window] [in a new window]   FIG. 6. Chemical agents that block caveolae/lipid raft-mediated endocytosis but not the clathrin-mediated pathway block the -PMA-induced loss of cell surface NET. Trophoblasts were treated with vehicle or -PMA following treatments with chemical agents, and NE uptake assays and biotinylation experiments were carried out as described under "Experimental Procedures." A, uptake data are presented as mean ± S.E. of three experiments carried out in triplicate. B, representative blot of three separate biotinylation experiments shows no significant change in -PMA-induced loss of NET surface density by treatments with ConA or MDC that prevent clathrin-mediated endocytosis but a significant inhibition by filipin that blocks caveolae/lipid raft-mediated endocytosis. Lower panel represents quantitative values of mean band (85 kDa) densities obtained from three measurements. Asterisks indicate significant block of -PMA-mediated inhibition of NE uptake and loss of NET protein from the cell surface by filipin compared with the control (Student's t test; *, p < 0.05).

View larger version (36K): [in this window] [in a new window]   FIG. 7. Dominant negative mutants of dynamin and caveolin effectively block clathrin- and caveolae-mediated endocytosis, respectively. A, TfR internalization. Trophoblasts were transfected with K44A dynamin 1 or the vector, and the transfected cells were treated with vehicle or -PMA as described under "Experimental Procedures." Biotinylation followed by immunoblotting with anti-TfR antibody was carried out as described under "Experimental Procedures." A representative blot from three separate biotinylation experiments is shown. B, bar graph. Biotinylation was quantified using NIH image, and the densities of TfR band from three separate experiments are presented as mean ± S.E. Asterisks indicate a significant reduction in surface TfR level following -PMA treatment in vector-transfected cells and a significant block of -PMA-induced TfR sequestration in K44A dynamin (K44A-dyn) 1 transfected trophoblasts compared with vector control (Student's t test; *, p < 0.05). C, CTXB internalization. Trophoblasts were transfected with S80E caveolin 1 or DGV caveolin 3 or the vector as described under "Experimental Procedures." Transfected cells were incubated with peroxidase-conjugated CTXB, and the internalization of CTXB was measured as described under "Experimental Procedures." A representative blot from three separate dot blot experiments is shown. D, bar graph. Dot densities of internalized (intracellular) CTXB were quantified by NIH image, and mean values from three separate experiments are presented as mean ± S.E. Asterisks indicate significant block of CTXB internalization by S80E caveolin 1 (cav 1) and DGV caveolin 3 (cav 3) mutants compared with vector control (Student's t test; *, p < 0.05).

View larger version (60K): [in this window] [in a new window]   FIG. 8. Filipin does not alter -PMA-mediated PKC signaling. Trophoblasts were first subjected to treatments with filipin or ConA or K+ depletion and then treated with -PMA or the vehicle as described under "Experimental Procedures." A, taurine uptake. Taurine uptake was measured using [3H]taurine by similar uptake assays described under "Experimental Procedures." Specific taurine transport was calculated by subtracting [3H]taurine uptake measured in the presence of 100 µM guanidinoethylsulfinate, a specific inhibitor of taurine transporter, from the uptake measured in the absence of guanidinoethylsulfinate. Uptake data are presented as mean ± S.E. of three experiments performed in triplicate. Asterisks indicate significant block of -PMA-mediated inhibition of taurine uptake by ConA treatment and K+ depletion compared with control treatment (Student's t test; *, p < 0.05). B, PKC translocation. PKC translocation in response to -PMA was measured following the treatment with -PMA or the vehicle in the presence or absence of filipin as described under "Experimental Procedures." Cyt, cytosol; PM, plasma membrane. One representative blot resulting from immunoblotting with anti-PKC antibody shows no changes in -PMA-induced PKC translocation by filipin treatment. Another representative blot from immunoblotting with anti-PKC antibody shows no changes in the -PMA-induced translocation of PKC isoform by filipin.

To examine the role of lipid rafts in the -PMA-mediated effects, discontinuous sucrose gradient centrifugation was used to measure the amount of NET and other marker proteins in the isolated fractions (lipid rafts and nonlipid rafts). Immunoblot analyses of the proteins in the isolated fractions revealed that NET proteins are present in lipid raft fractions (fractions 4 and 5) (Fig. 9A). Markers for lipid rafts such as GM1 and caveolin were also detected in these fractions (Fig. 9). Treatment of trophoblasts with -PMA decreased the levels of NET in the lipid raft fractions, with concomitant increases of NET protein in the nonlipid raft fractions (fractions 7-9) (Fig. 9). These results provided further evidence for the involvement of lipid rafts in the PKC-mediated NET internalization.

View larger version (33K): [in this window] [in a new window]   FIG. 9. Lipid rafts contain NET proteins and -PMA redistributes lipid raft-associated NET. Trophoblasts were treated with vehicle (A), and 0.5 µM -PMA (B) for 30 min at 37 °C. Treated cells were solubilized in MBS containing 1% Triton X-100 and subjected sucrose gradient centrifugation as described under "Experimental Procedures." 10 fractions of 1 ml were collected along with the pelletted material (P). Fractions 1-10 are from top to bottom. Trichloroacetic acid-precipitated proteins from these fractions were analyzed by 4-15% linear gradient SDS-PAGE and Western blotting for the distribution of different proteins using indicated antibodies. Aliquots from total homogenate (T) and resuspended pellet (P) were also loaded on the gels. Sucrose concentrations are shown at the bottom. Representative Western blots of three separate experiments corresponding to each protein are shown.

View larger version (22K): [in this window] [in a new window]   FIG. 10. -PMA-induced NET phosphorylation. A, trophoblasts were treated with -PMA (0.5 µM), okadaic acid (10 nM), or the vehicle for 30 min, and NE uptake assays were performed as described under "Experimental Procedures." Values of percent inhibition of NE uptake are presented as mean ± S.E. of three separate experiments carried out in triplicate. Values from -PMA- or okadaic acid-treated cells were compared with respective vehicle-treated cells using Student's t test (*, p < 0.05). B, metabolically labeled trophoblasts were treated with vehicle, -PMA (0.5 µM), or okadaic acid (10 nM) for 30 min and subjected to immunoprecipitations with NET antiserum, preimmune serum (Preimm), or NET antiserum preabsorbed (Pre-absorb) by the antigen column or protein A-Sepharose (Prot. A Seph.) or an irrelevant (Irrel) antibody (anti-calnexin) followed by autoradiography as described under "Experimental Procedures." Phosphorylated protein bands corresponding to NET protein are shown. C, the fold increase in NET phosphorylation is shown in arbitrary units. The intensity of phosphorylated band is increased 3-fold by -PMA or okadaic acid treatments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PKC plays a pivotal role in a transmembrane signal transduction chain found ubiquitously in mammalian cells. At least 12 isoforms of PKC have been identified to date. These are classified as the classical or conventional PKCs (isoforms , 1, 2, and ) that require phosphatidylserine, diacylglycerol, and Ca²⁺ for activation; the novel PKCs (isoforms , , , , and µ) that require phosphatidylserine and diacylglycerol only; and the atypical PKCs (isoforms , , and ) that require neither diacylglycerol nor Ca²⁺ (46). Placental tissue expresses several isoforms of PKC that include , , , , , and , and PKC-mediated phosphorylation has been implicated in the regulation of cytoskeletal elements (47) and amino acid transporters (48) in placenta. Phorbol esters and other tumor promoters mimic natural activators of both conventional PKCs and novel PKCs but do not activate atypical PKCs (49). Fresh or cultured placental trophoblasts in particular express , , and isoforms of PKC, and following treatment with -PMA, only and isoforms but not isoform translocate from the cytosol to the plasma membrane (33). This suggests that the -PMA-mediated inhibition of NE uptake in trophoblasts occurs following activation of either conventional PKCs or novel PKCs. However, complete removal of calcium (both external and internal) did not block the effect of -PMA suggesting that Ca²⁺-independent novel PKC isoforms might be involved in the regulation of placental NET. Under similar conditions of calcium removal, the -PMA-induced inhibition of paroxetine-sensitive [³H]5HT uptake by trophoblasts was completely blocked.² These results indicate the specific involvement of Ca²⁺-independent PKC isoforms in the regulation of NET in the trophoblast. In SK-N-SH cells, PKC-linked muscarinic acetylcholine receptors regulate NET activity in a calcium-independent manner (12). In contrast, the phosphatidylinositol 3-kinase-linked and insulin-mediated regulation of NET is calcium-dependent (29).

The decrease in cell surface NET density observed following -PMA treatment in the present study could be due to either enhanced internalization or reduced recycling. The data from reversible biotinylation experiments support the hypothesis that decreased cell surface NET protein after -PMA treatment occurs via enhanced internalization of the transporter. -PMA-induced NET down-regulation did not involve dynamin, and clathrin-mediated internalization as K⁺ depletion or hypertonic sucrose or treatment with ConA and MDC were ineffective. In addition, expression of dominant negative mutants of dynamin 1 and dynamin 2 in trophoblasts also had no effect on -PMA-mediated NET down-regulation. However, the effective block of -PMA-mediated inhibition of taurine transport by ConA treatment or K⁺ depletion suggests that these manipulations to inhibit clathrin-mediated endocytosis were effective on another transporter protein of the same gene family. In addition, expression of dominant negative dynamin mutant effectively blocked TfR internalization suggesting that K44A dynamin was also effective in inhibiting clathrin-mediated endocytosis. It is well known that TfR internalizes via clathrin-mediated endocytosis, and dominant negative dynamin mutants effectively block TfR internalization (50). Earlier, it has also been shown that PKC activation accelerates TfR internalization (51). Coexpression of human NET and K44A mutant of dynamin 1 in HEK-293 cells completely blocked the effect of -PMA on NE transport as well as NET sequestration (Table I), suggesting that the mechanisms underlying PKC-induced internalization are cell type-specific. The expression of K44A dynamin 1 and dynamin 2 mutants was confirmed by immunoblotting with anti-HA and anti-GFP antibodies. Trophoblasts as well as HEK-293 cells expressed similar levels of endogenous dynamin proteins (based on immunoreactivity to anti-dynamin antibodies) and also transfected dominant negative mutant dynamin proteins (based on immunoreactivity to anti-HA and anti-GFP antibodies) ruling out the possibility of discrepancy of expression efficiencies between cell types (data not shown). These results collectively demonstrate that the PKC-mediated NET sequestration in trophoblasts occurs by dynamin-independent endocytic mechanisms. Although many GPCRs are internalized via clathrin-dependent mechanisms, M2 muscarinic acetylcholine receptors are internalized by clathrin-independent mechanisms (50).

Although treatments that disrupt dynamin and clathrin-dependent pathways were without effect, exposure of cells to filipin blocked the PKC-mediated NET down-regulation in trophoblasts. Under similar conditions of filipin treatment, the -PMA-induced inhibition of taurine uptake was unaltered suggesting that the filipin effect is specific to NET down-regulation. In addition, phorbol ester-induced translocation of PKC or PKC from the cytosol to the plasma membrane remained intact following filipin treatment. Together, these results ruled out the possibility that filipin, being a cholesterol-depleting agent, can block -PMA-mediated NET internalization by altering the membrane integrity and hence altering the PKC signaling. Surprisingly, neither the dominant negative S80E mutant of caveolin 1 nor the DGV mutant of caveolin 3 blocked the -PMA effects on NET when transfected into trophoblasts. However, the inhibition of CTXB internalization by caveolin mutants indicates that these mutants were effective in inhibiting the caveolae-mediated endocytosis. CTXB by binding to the cell surface ganglioside GM1 internalizes via caveolae, and S80E caveolin 1 mutant has been shown to block CTXB uptake by adipocytes (36). Expression of mutant proteins of caveolin 1 and caveolin 3 was confirmed by immunoblotting with anti-Myc and anti-HA antibodies, respectively.

The presence of NET in lipid rafts and the reduction of NET levels following PKC activation (Fig. 9) strongly suggest that NET internalization in the trophoblast occurs via lipid rafts. The signals and the spatial regulation of signaling in lipid rafts are distinctly different from that of caveolae (22, 52, 53), and therefore, signaling machinery specific to lipid rafts may be linked to PKC-mediated NET down-regulation. Recently, lipid raft-mediated internalization has been demonstrated for glucose transporter (GLUT4) (54, 55). GLUT4 is similar to NET and DAT with respect to regulation by phosphatidylinositol 3-kinase- and insulin-mediated regulation (29, 56). Membrane cholesterol modulates SERT activity in HEK-293 cells (57). However, cholesterol appears to be involved in stabilizing transporter proteins but not in trafficking regulation. Thus, this is the first report in the Na⁺/Cl^--dependent transporter family that is to identify lipid raft-mediated internalization as a mechanism underlying native NET regulation by PKC in the trophoblast.

Although the present findings show that native NET undergoes phosphorylation in response to PKC activation or PP1/2A inhibition, it is not known whether phosphorylation of NET leads to its internalization. DAT and SERT undergo phosphorylation (42, 39), and SERT substrates modulate the degree of phosphorylation (58). DAT is phosphorylated on N-terminal serine residues in rat striatum (59), and N-terminal truncation of DAT abolishes PKC-mediated phosphorylation without impairing its internalization in HEK-293 cells (60). However, in Chinese hamster ovary cells, N-terminal phospho-sites are required for the regulation of DAT function and phosphorylation by several kinases (61). NET contains multiple consensus sites for several kinases, including PKC, that are distinct from those present in DAT or SERT. It is therefore possible that NET is regulated by mechanisms that are different for those of DAT and SERT. Whereas native systems that endogenously express transporters are great assets to identify the endogenous stimuli controlling the transporter function, heterologous expression systems are useful for asking mechanistic questions with regard to understanding the relationships among transporter trafficking, phosphorylation, and regulation. Current studies are aimed at understanding these complex relationships using phospho-site mutants of NET and should provide insights into our understanding of NET regulation.

Protein phosphatases also regulate the function and phosphorylation state of the NET in trophoblasts (Fig. 10). In addition, in recent studies we have isolated native NET as a complex with PP2Ac, and we observed that the associated PP2A dephosphorylates phospho-NET.³ The fact that monoamine transporters interact with PP2A (16) suggests a role for protein phosphatases in monoamine transporter phosphoregulation. Recently, Uhl et al. (62) reported that KEPI and GBPI, members of a family of PKC-dependent inhibitors of PP1 and PP2A, alter DAT activity. It is possible that phorbol esters could alter PKC-mediated effects on proteins such as KEPI, and may provide alternative pathways for PKC-mediated transporter regulation.

Lipid rafts are residents of a number of signaling molecules (22) that may have appropriate regulatory machinery for PKC-mediated NET regulation. It is tempting to speculate that a part of NET protein exists as a readily available pool in cholesterol-rich lipid rafts that are available for regulation by PKC. In addition, lipid rafts may operate as an obligatory transition station before transporter internalization. NETs may be regulated by different mechanisms in neuronal versus non-neuronal tissues depending upon the needs and functional significance of that particular organ. As placenta is an example of a non-innervated tissue, placental control of amine transport via lipid raft-mediated NET regulation may be one of the underlying mechanisms by which placenta regulates physiologic catecholamine levels. Future studies examining the signals and molecules linked to this regulatory machinery may provide insight into the participation of NET in the placental control of NE levels.

	FOOTNOTES

 
^* This work was supported by National Institute of Mental Health Grant MH62612 (to S. R.), a National Alliance for Research on Schizophrenia and Depression (NARSAD) Young Investigator award, and a University Research Committee award from Medical University of South Carolina (to L. D. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physiology and Neuroscience, Medical University of South Carolina, Charleston, SC 29425. Tel.: 843-792-8542; Fax: 843-792-1066; E-mail: jayanthi{at}musc.edu.

¹ The abbreviations used are: NET, norepinephrine transporter; NE, norepinephrine; DAT, dopamine transporter; GAT1, -aminobutyric acid transporter; SERT, serotonin transporter; DS, desipramine; ABDU, arbitrary band density units; -PMA, -phorbol 12-myristate 13-acetate; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; GFP, green fluorescent protein; HA, hemagglutinin; RT, room temperature; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; MesNa, sodium 2-mercaptoethanesulfonate; ConA, concanavalin A; MDC, monodansyl cadaverine; CTXB, cholera toxin B; MES, 4-morpholineethanesulfonic acid; TfR, transferrin receptor; rNET, rat NET.

² L. D. Jayanthi, D. J. Samuvel, and S. Ramamoorthy, unpublished observations.