Chemicals and antibodies

Radioisotope [³H]-glycine was purchased from Perkin Elmer Life Scientific (Boston, MA). XLA-5407, XLA1393, Flag-M2 agarose beads, N-ethylmaleimide, sodium fluoride, phenylmethanesulfonyl fluoride, aprotinin, and leupeptin were from Sigma-Aldrich Co. (St. Louis, MO). Rabbit polyclonal antibody to GlyT1 was kindly donated by Detlev Boison and Dietmar Benke (University of Zurich). Rabbit antibody to GlyT2 was a customized antibody from Dr. Manuel Miranda’s laboratory (University of Texas at El Paso). HRP-conjugated secondary antibodies were from Promega (Madison, WI).

GlyT1 and GlyT2 Plasmids

GlyT1 was kindly donated by Bruno Giros (University of Italy). GlyT1 and GlyT2 were tagged with Flag and 10XHis epitope at the N-termini (FH-GlyT1 and FH-GlyT2) as previously described (^{Miranda, Wu, Sorkina, Korstjens & Sorkin, 2005}). Resulting constructs were cloned into pcDNA 3.1 and Deoxyribonucleic Acid (DNA) sequence was verified by automatic deoxynucleotide sequencing.

Cell culture and stable expression of GlyT1 and GlyT2

Porcine aortic endothelial (PAE) cells were selected to generate stable cell lines, and they were kindly provided by Dr. A. Sorkin (University of Colorado at Denver). PAE cells were grown at 37 ºC and 5% CO₂ in Ham’s F-12 medium containing 10% fetal bovine serum (FBS) and 100 µg/ml gentamicin. PAE cells were grown to 50% - 80% confluence and transfected with appropriate plasmids using Effectene, according to manufacturer’s recommendations (Qiagen, Hilden, Germany). PAE cells stably expressing FH-GlyT1 or FH-GlyT2 were selected by growing them in presence of G418 (400 µg/ml). C6 glioma cells (ATCC, Manassas, VA) were grown under the same conditions described for PAE cells but in Ham’s F-12 Kaighn’s modification medium containing 1.25% FBS and 7.5% of horse serum.

Determination of [ ³ H] glycine uptake

Glycine uptake by PAE cell lines (with stable expression of GlyT1 or GlyT2) or by C6 glioma cells was measured as previously described with some modifications (^{Sato, Adams, Betz & Schloss, 1995}). Confluent cells were washed with 0.25 ml of wash buffer containing 10 mM Hepes, pH 7.4, 135 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgSO₄ and 10 mM glucose. Uptake was started by adding 0.25 ml the buffer described above but containing 4 µCi [³H]glycine /ml and 200 µM glycine. Reaction ran for 10 min at 37 ºC, and it was stopped by washing cells twice with ice-cold wash buffer without glycine. This was followed by extraction of [³H]glycine from cells using 0.2 N of NaOH for 1 h. Finally, glycine uptake was determined by scintillation spectroscopy. Total protein concentration was determined by the ^{Bradford (1976)} assay using bovine serum albumin (BSA) as a standard.

Double affinity chromatography purification and Western blotting

PAE cells stably expressing FH-GlyT1 or FH-GlyT2 were grown to confluence in 60 mm culture dishes. Cells were washed twice with Ca²⁺- and Mg²⁺- free cold phosphate-buffered saline (CMF-PBS) and lysed in ice-cold-buffer containing 25 mM Hepes, pH 8.0, 100 mM NaCl, 15 mM imidazole, 10% glycerol, 1% triton X-100, 1% sodium deoxycholate, 10 mM Nethymaleimide, 10 µM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Lysates were cleared by centrifugation at 14 000 xg. FH-GlyT1 and FH-GlyT2 were then purified by double affinity chromatography. First, lysates were incubated with Ni-NTA affinity agarose beads for 1 h and eluted with imidazole. Eluted proteins were then incubated with anti-Flag affinity agarose beads for 4 h and eluted with 100 mM glycine, pH 3.5. Purified proteins were then subjected to 8.0% SDS-PAGE, followed by transfer into a nitrocellulose membrane (^{Laemmli, 1970}; ^{Towbin, Staehelin & Gordon, 1979}). Membranes were blocked with 5% non-fat dry milk for 1 h. Presence of GlyT1 and GlyT2 was determined by western blotting using the appropriate primary antibodies followed by incubation with corresponding HRP-conjugated secondary antibodies. For sensitive detection, enhanced chemiluminescence (ECL) technique was used according to manufacturer’s instructions (Pierce Biotechnology, Rockford, IL).

Immunofluorescence staining and confocal microscopy

Cells were grown overnight on glass coverslips until they were sub-confluent and were attached to coverslip. Cells were then washed with CMF-PBS, fixed with freshly prepared 4% paraformaldehyde for 15 min at room temperature, and mildly permeabilized by incubation in CMF-PBS containing 0.1% Triton X-100 and 0.5% BSA at room temperature for 3 min. Then, cells were incubated with primary antibodies in CMF-PBS containing 0.5% BSA at room temperature for 1 h, and subsequently incubated for 30 min with secondary antibody labeled with Alexa 488 (Jackson Laboratories, West-Glove, PA). Both primary and secondary antibody solutions were precleared by centrifugation at 14 000 xg for 20 min. After immunostaining, coverslips were mounted in Mowiol (Calbiochem). Images were captured utilizing a laser-scanning confocal microscope (LSM 700; Zeiss) using the 64X objective and assisted with ZEN 2009 software (Zeiss, New York, NY). High-resolution images were acquired through corresponding filter channel (Alexa 488). Fluorescence intensity was measured in 10 cells by pixel in an area of 5 mm2, using three independent experiments. Final arrangement of all images was accomplished using Adobe Photoshop software.

Statistical Analysis

Statistical and kinetic analyses were performed using Sigma Plot 10 software (Systat Software). Unless otherwise stated, glycine uptake or fluorescence intensity values are represented as the mean ± SE of at least three independent experiments, with statistical significance of p < 0.05 determined by Student's paired t test.

Pharmacological analysis of glycine uptake in C6 glioma cells

Uptake of [³H]glycine by C6 glioma cells has been previously demonstrated. Furthermore, it was also shown to be downregulated by treatment with phorbol 12-myristate 13-acetate (PMA) (^{Gomeza et al., 1995}). Therefore, it has been made [³H]glycine uptake assay using C6 glioma cells and employing PMA in order to analyze kinetics of the uptake (figure 1). Kinetic enzymatic analysis suggested that glycine uptake may be mediated by a high-affinity glycine transporter because of low K_m value (248 ± 113) computed in this analysis. In addition, K_m values were very similar after treatment with PMA (K_m 255 ± 120; black circles) or vehicle (K_m 248 ± 113; white circles). However, V_max values were shown to be different after PMA treatment (3.4 ± 0.8 in PMA-treated cells and 4.3 ± 1 in cells treated with vehicle). This is consistent with previous reports for other members of SLC6 family of transporters, where downregulation in glycine uptake is due to a reduction in the V_max, but not in the transporter affinity (^{Huff, Vaughan, Kuhar & Uhl, 1997}).

Source: Author’s own elaboration.

Figure 1 Effect of PMA in glycine uptake in C6 glioma cells. 

Cells were incubated with vehicle (DMSO, white circles) or 1 µM PMA (black circles) for 1 h, followed by determination of glycine uptake. The assay was performed in presence of increasing concentrations of cold glycine (25 µM - 400 µM) in a buffer with a constant concentration of 4 µCi/ml of [³H]glycine at 37 ºC. Kinetic parameters were calculated using the Michaelis-Menten equation and Sigma Plot 10. Values represent mean of 4 independent determinations ± SE (^{Vargas-Medrano, Castrejón-Téllez, Plenge, Ramírez & Miranda, 2011}).

Given data provided by kinetic analysis, and since GlyT1 has already been found to be expressed in C6 glioma cells, we hypothesized that GlyT2 may also be found and expressed in this cell line. In order to verify this hypothesis, were performed a series of glycine uptake assays using C6 glioma cells in presence of selective inhibitors to GlyT1 or GlyT2 respectively. XLA-5407 (0.25 µM), a selective inhibitor to GlyT1, inhibited ~40% of the total glycine uptake (figure 2a, *p < 0.001). Alternatively, GlyT2 inhibitor XLA-1393 (0.75 µM) also inhibited ~40% of total glycine uptake (figure 2b, **p = 0.003). This data strongly suggested presence of two different high-affinity glycine transporters in C6 glioma cells. Selective inhibitors to GlyT1 or GlyT2 were previously described using other models (^{Atkinson et al., 2001}; ^{Aubrey & Vandenberg, 2001}), and therefore, validation for this model became necessary. In order to determine tested inhibitors selectivity (XLA-5407 and XLA-1393), were performed dose-response curves testing XLA-5407 and XLA1393 inhibitors in PAE cells stably expressing GlyT1 and GlyT2. Not surprisingly, both compounds were selective but at a different concentration (figures 2c and 2d). In PAE cells with stable expression of GlyT1, XLA-5407 inhibited ~90% of total glycine uptake at 0.25 µM. This concentration did not affect glycine uptake mediated by GlyT2 (figure 2c). Moreover, 1 µM of XLA-1393 inhibited ~80% of glycine uptake mediated by GlyT2 but did not affect GlyT1 activity (figure 2d). With this analysis, we were able to determine concentrations for a selective inhibition of GlyT1 or GlyT2, but not both.

Source: Author’s own elaboration.

Figure 2 Inhibition of glycine uptake in C6 glioma cells by selective inhibitors to GlyT1 or GlyT2. 

Glycine uptake from untreated cells was normalized to 100% of glycine uptake and selective inhibitors (a) 0.25 µM ALX-5407 (inhibitor to GlyT1) or (b) 0.75 µM ALX-1393 (inhibitor to GlyT2) significantly inhibited glycine uptake mediated by C6 glioma cells. *p = < 0.001, **p = 0.003 compared to control (untreated cells). Bars represent the mean ± SE, n = 8. GlyT1 or GlyT2 selective inhibitors were further validated by performing dose-response curves with ALX-5407 (c) or ALX-1393 (d) using stable cell lines expressing GlyT1 or GlyT2. Point values represent mean ± SE, n = 4. (^{Vargas-Medrano et al., 2011}).

Immunoprecipitation and immunoblotting analysis of GlyTs in C6 glioma cells

Pharmacological analysis indicated endogenous expression of both GlyT1 and GlyT2 in C6 glioma cells. In order to support our initial hypothesis and pharmacological experiments, were conducted immunoprecipitation and immunoblotting analysis using selective antibodies to GlyT1 or GlyT2. Confluent C6 glioma cells were lysed and endogenous GlyTs were immunoprecipitated using selective antibodies. FH-tagged GlyT1 and FH-GlyT2 (figures 3a and 3b) purified from PAE cells with stable expression of transporters served as positive controls. As hypothesized, bands at about ~100 kDa were detected in lysates from C6 glioma cells, corresponding to molecular weights of GlyT1 and GlyT2, respectively, which was also very similar to molecular weights displayed by purified FH-GlyT1 and FH-GlyT2 (figures 3c and 3d). Immunoreactivity signal detected for transporters in C6 glioma cells was very weak, indicating a low amount of transporter expression in these cells. Use of this experimental approach made it obvious that endogenous GlyT1 and GlyT2 expression in C6 glioma cells is very low when compared to levels of a stable expression system, such as positive control used (PAE cells). To evaluate tested antibodies specificity, we purified FH-GlyT1 and FH-GlyT2 from stable cell lines by double affinity chromatography. Purified GlyT1 and GlyT2 were divided into two tubes and then loaded into two western blots as diagram indicates (figure 3e). Immunoblots were incubated with selective antibodies to GlyT1 or GlyT2 (figure 3f). Not surprisingly, GlyT1 antibody selectively detected only FH-GlyT1, while GlyT2 antibody specifically detected FH-GlyT2, which supports antibodies specificity.

Source: Author’s own elaboration.

Figure 3 Immunoprecipitation of GlyT1 or GlyT2 from C6 glioma cells total lysates. 

Schematic representations of N-terminal Flag, Histagged GlyT1 (a) and GlyT2 (b) are shown. FH-GlyT1 (c) and FH-GlyT2 (d) were purified from PAE cells in a 60 mm tissue culture dish and loaded as positive controls in the first lane of each western blot. Immunoprecipitated GlyT1 and GlyT2 from C6 glioma cells grown on a 100 mm tissue culture dish were loaded in second lane. Antibodies selectivity was validated by purifying FH-GlyT1 and FH-GlyT2 using double affinity chromatography (e) as described in methods. Purified proteins were spread into two western blots (f). As shown, antibodies were highly-selective for detecting FH-GlyT1 and FH-GlyT2. Solid lines follow GlyT1 and dotted lines follow GlyT2 (^{Vargas-Medrano et al., 2011}).

Detection GlyT1 and GlyT2 in C6 glioma cells by confocal microscopy

In order to further support our previous findings about endogenous expression of GlyT1 and GlyT2 in C6 glioma cells, were conducted an immunofluorescence staining using highly selective GlyT1 and GlyT2 antibodies followed by confocal microscopy analysis of stained cells. Endogenous GlyT1 and GlyT2 were both detected in C6 glioma cells (figures 4a and 4b). In addition, a positive control was performed by staining PAE cells stably expressing GlyT1 or GlyT2 (figure 4c and 4d). To further investigate levels of GlyT expression in C6 glioma cells, was performed a quantitative analysis of stained C6 glioma cells using the ZEN software (Zeiss). Results showed that endogenous GlyT2 immunofluorescence signal was ~2 folds higher than the signal detected for endogenous GlyT1 (figure 4e).

Source: Author’s own elaboration.

Figure 4 Visual detection of the endogenous GlyT1 and GlyT2 in C6 glioma cells. 

Immunofluorescence staining of GlyT1 (a) or GlyT2 (b) with selective antibodies in C6 glioma cells is shown. As positive controls, PAE cells stably expressing FHGlyT1 (c) and FH-GlyT2 (d) were immunostained and are shown in corresponding panels. In panel e, a quantitative plot of fluorescence intensity by C6 glioma cells for staining of endogenous GlyT1 and GlyT2 is shown indicating that endogenous GlyT2 (eGlyT2) is expressed in higher amounts when compared to endogenous GlyT1 (eGlyT1). Bars represent the mean ± SE, n = 10, *p = 0.025 when compared to eGlyT2 (^{Vargas-Medrano et al., 2011}).

Acknowledgements

We thank the Cell Culture and High Throughput Screening staff, Biomolecule Analysis, and Genomic Analysis Core Facilities (University of Texas at El Paso) for support provided. Also want to thank our mentor Dr. Manuel Miranda (University of Texas at El Paso) for his support and training.