Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Abstract

Taurine is actively transported at the retinal pigment epithelial (RPE) apical membrane in an Na(+)- and Cl(-)-dependent manner. Diabetes may alter the function of the taurine transporter. Because nitric oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we asked whether NO would alter the activity of the taurine transporter in cultured ARPE-19 cells. The activity of the transporter was stimulated in the presence of the NO donor 3-morpholinosydnonimine. The stimulatory effects of 3-morpholinosydnonimine were not observed during the initial 16-h treatment; however, stimulation of taurine uptake was elevated dramatically above control values with 20- and 24-h treatments. Kinetic analysis revealed that the stimulation was associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study provides the first evidence of an increase in taurine transporter gene expression in human RPE cells cultured under conditions of elevated levels of NO.

Fig. 1

Laser scanning confocal microscopic immunolocalization of taurine transporter in cultured human ARPE-19 cells and mouse retina. ARPE-19 cells were grown to confluency on laminin-coated chamber slides and processed for immunohistochemistry using a primary antibody against taurine transporter followed by an FITC-labeled secondary antibody. A: optical section of cells taken in a vertical plane (x,z). Bright band of fluorescence is indicative of positive detection of the antibody in cultured cells. Top and bottom arrows at left of A point to apical and basal surfaces of cells. B: optical section of cells taken in a horizontal plane (x,y) shows taurine transporter on apical retinal pigment epithelial (RPE) surface. C: horizontal section of cells incubated with peptide that had been preincubated with antibody against taurine transporter (negative control) shows no positive staining. Original magnifications ×600.

Fig. 2

Laser scanning confocal microscopic immunolocalization of taurine transporter in intact mouse retinal tissue. Eyes of ICR albino mice were frozen in OCT embedding medium, and cryosections were prepared and subjected to immunohistochemical detection of taurine transporter using a commercially available antibody. A: hematoxylin-and-eosin-stained cryosection of outer portion of normal mouse retina showing outer layers of the retina. B: immunolocalization of taurine transporter. Arrow points to intense labeling of the RPE; arrow-head points to labeling of inner segments (IS). Magnification ×400. ONL, outer nuclear layer; OS, outer segments; CH, choroid.

Fig. 3

Effects of NO donors on uptake of taurine in cultured ARPE-19 cells. A: uptake of [³H]taurine (80 nM) in the absence (control) or presence of 1 mM 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP) for 24 h. B: dose-response relationship for effect of SIN-1 on uptake of 80 nM [³H]taurine (treatment time 24 h). Values are means ± SE for 4 determinations from 2 independent experiments.

Fig. 4

Time course of stimulation of taurine uptake by SIN-1. ARPE-19 cells were exposed to 1 mM SIN-1 for various lengths of time, and uptake of [³H]taurine (80 nM) was determined. Parallel experiments were carried out with cells cultured similarly, but in the absence of SIN-1. Results are given as percentage of taurine uptake measured in corresponding control cells not treated with SIN-1. Values are means ± SE for 4 determinations from 2 independent experiments.

Fig. 5

Effects of antioxidants and nitric oxide (NO) scavengers on SIN-1-induced stimulation of taurine uptake in ARPE-19 cells. Confluent cells were treated with or without 1 mM SIN-1 for 24 h. Cells were incubated at the same time in the presence or absence of ascorbate, glutathione, or methylene blue. Uptake of [³H]taurine (80 nM) was measured for 15 min. Values are means ± SE for 3 determinations from 2 independent experiments.

Fig. 6

Immunodetection of nitrotyrosine in ARPE-19 cells exposed to SIN-1. ARPE-19 cells were grown to confluency on laminin-coated chamber slides and then exposed to 1 mM SIN-1 for 2 h. Cells were processed for immunohistochemistry using a primary antibody against nitrotyrosine followed by an FITC-labeled secondary antibody. A: SIN-1 treated cells. B: control cells. Magnification ×600.

Fig. 7

Kinetic analysis of taurine uptake in control and SIN-1-treated ARPE-19 cells. Confluent cells were treated with or without 1 mM SIN-1 for 24 h. Uptake of taurine was measured for 15 min over a taurine concentration range of 0.5–20 μM. Values are means ± SE for 3 determinations from 3 independent experiments. Results are presented as plots describing the relationship between taurine concentration and taurine uptake rate and also as Eadie-Hofstee plots: V/S vs. V, where V is taurine uptake (in pmol·mg protein⁻¹·15 min⁻¹) and S is taurine concentration (in μM).

Fig. 8

Effects of cycloheximide and actinomycin D on SIN-1-induced increase in taurine transporter activity. Confluent ARPE-19 cells were pretreated with cycloheximide (100 μg/ml) or actinomycin D (10 μg/ml) for 2 h and then incubated with these compounds in the presence or absence of 1 mM SIN-1. Uptake of [³H]taurine (80 nM) was then measured for 15 min. Values are means ± SE for 3 determinations from 3 independent experiments.

Fig. 9

Analysis of steady-state levels of mRNA for taurine transporter (TAUT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in control (−SIN-1) and SIN-1 treated (+SIN-1) ARPE-19 cells. Confluent cells were treated with or without 1 mM SIN-1 for 24 h. Poly(A)⁺ RNA was then isolated from these cells and used for semiquantitative RT-PCR. Primer pairs specific for human TAUT mRNA and human GAPDH mRNA were used. RT-PCR was done with a wide range of PCR cycles (9–30). Resultant products were run on a gel and then subjected to Southern hybridization with ³²P-labeled cDNA probes specific for TAUT and GAPDH. Hybridization signals were quantified using the STORM PhosphorImaging System, and intensities that were in the linear range with the PCR cycle number were used for analysis. GAPDH-specific band intensity was used as an internal control. Ratios of TAUT-specific band intensity to GAPDH-specific band intensity were then compared between control and SIN-1 treated cells. Ratio in control cells was taken as 1.

Fig. 10

Nuclear run-on transcription assay. ARPE-19 cells were incubated for 24 h with or without 1 mM SIN-1. Nuclei were isolated, and nascent RNA was labeled with [α-³²P]UTP. This radiolabeled RNA was then hybridized to membranes with cross-linked human taurine transporter cDNA. Membranes were hybridized overnight at 68°C and exposed to X-ray film.