Function and regulation of taurine transport at the inner blood–retinal barrier

doi:10.1016/j.mvr.2006.10.003

Microvascular Research

Volume 73, Issue 2, March 2007, Pages 100-106

https://doi.org/10.1016/j.mvr.2006.10.003 Get rights and content

Abstract

In the retina, taurine exerts a number of neuroprotective functions as an osmolyte and antioxidant. The purpose of the present study was to elucidate the taurine transport system(s) at the inner blood–retinal barrier (BRB). [³H]Taurine transport at the inner BRB was characterized using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). The expression of the taurine transporter (TauT) was demonstrated by RT-PCR and immunoblot analyses. The apparent influx permeability clearance of [³H]taurine in the rat retina was found to be 259 μL/(min radical dot g retina), supporting carrier-mediated influx transport of taurine at the BRB. [³H]Taurine uptake by TR-iBRB2 cells was Na⁺-, Cl⁻- and concentration-dependent with a K_m of 22.2 μM and inhibited by TauT inhibitors, such as β-alanine and hypotaurine. RT-PCR and immunoblot analyses demonstrated that TauT is expressed in TR-iBRB2 and primary cultured human retinal endothelial cells. The uptake of [³H]taurine and the expression of TauT mRNA in TR-iBRB2 cells increased under hypertonic conditions but decreased following pretreatment with excess taurine. In conclusion, TauT most likely mediates taurine transport and regulate taurine transport at the inner BRB.

Introduction

Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in the retina (12 μmol/g retina ≈ 12 mM in rats) and accounts for more than 50% of the free amino acid content in the rat retina (Pasantes-Morales et al., 1972). The taurine concentration in the retina is about 100 times greater than that in the serum (100–300 μM) (Dawson et al., 1999, Törnquist and Alm, 1986). Although cysteine sulfinic acid decarboxylase, a rate-limiting enzyme for taurine biosynthesis, is expressed in the retina (Lin et al., 1985), its activity in the rat retina is low in comparison with the abundance of retinal taurine (Heinamaki, 1988). Although abnormal electroretinograms and visual disturbances have been found in patients undergoing long-term parenteral nutrition lacking taurine, these abnormalities returned to normal following administration of taurine intravenously (Geggel et al., 1985, Vinton et al., 1990). This evidence prompts the hypothesis that the blood-to-retina transport system(s) of taurine plays a key role in maintaining the taurine concentration in the retina by supplying taurine.

The nutrient supply to the retina from the circulating blood is regulated by the blood–retinal barrier (BRB), which is composed of retinal capillary endothelial cells (inner BRB) and retinal pigment epithelial cells (RPE, outer BRB) (Cunha-Vaz et al., 1966, Hosoya and Tomi, 2005). The transport of β-amino acids, such as taurine and β-alanine, are known to be mediated by an Na⁺- and Cl⁻-dependent taurine transporter (TauT/Slc6a6) (Smith et al., 1992). TauT knockout mice are reported to exhibit a loss of vision due to severe retinal degeneration, in addition to having low taurine concentrations in a variety of tissues and reduced fertility (Heller-Stilb et al., 2002), suggesting that TauT is critical for normal retinal development and function. Using the retinal uptake index method, Törnquist and Alm (1986) demonstrated [³H]taurine uptake from the circulating blood to the retina across the BRB and its inhibition by an excess of taurine and β-alanine, suggesting that the BRB supports taurine transport process(es). Although immunohistochemical study revealed that the outer BRB expresses TauT (Bridges et al., 2001), our knowledge of the taurine transport mechanism at the inner BRB is still incomplete. The inner two thirds of the human retina are known to be nourished by a direct blood supply through the inner BRB (Harris et al., 2001). Moreover, the inner BRB equips several amino acid transport systems which were determined by using isolated retinal capillaries (Betz and Goldstein, 1980, Hjelle et al., 1978, Tomi and Hosoya, 2004). Therefore, it is important to investigate the molecular mechanism of taurine transport at the inner BRB as well as the outer BRB in order to clarify the whole system supplying taurine to the retina.

The purpose of the present study was, firstly, to clarify the transport mechanism of taurine at the inner BRB and, secondly, to investigate the regulation of taurine transport by hypertonicity and taurine pretreatment. The characteristics and functions of taurine transport at the inner BRB were examined by in vivo integration plot analysis and using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2).

Section snippets

Animals

Male Wistar rats, weighing 250–300 g, were purchased from SLC (Shizuoka, Japan). Rats were maintained on 12-h light/12-h dark cycles with food and water provided ad libitum and used to measure taurine transport near the middle of the light phase of their light–dark cycle in order to minimize the effect of light exposure on taurine uptake by the retina (Hillenkamp et al., 2006). The investigations using rats described in this report conformed to the provisions of the Animal Care Committee,

Blood-to-retina transport of taurine across the BRB

The in vivo blood-to-retina influx transport of taurine from the circulating blood to the retina through the BRB was evaluated and compared with the brain by means of integration plot analysis after intravenous administration of [³H]taurine to rats. The K_{in, retina} of [³H]taurine was determined to be 259 ± 39 μL/(min radical dot g retina) (mean ± SD) from the slope representing the apparent influx permeability clearance across the BRB (Fig. 1A). The K_{in, brain} of [³H]taurine was 9.07 ± 0.77 μL/(ming brain) (mean ±

Discussion

The present study demonstrates that [³H]taurine is transported against a concentration gradient from the circulating blood (100–300 μM in rat serum) (Dawson et al., 1999, Törnquist and Alm, 1986) to the retina (12 μmol/g retina ≈ 12 mM in rats) (Pasantes-Morales et al., 1972) across the inner and outer BRB (Fig. 1A). The apparent influx clearance (K_{in, retina}) of 259 μL/(min radical dot g retina) is far greater than that of [¹⁴C]sucrose and [³H]D-mannitol [0.26 and 0.75 μL/(ming retina), respectively] used as

Acknowledgments

This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and a grant for Research on Sensory and Communicative Disorders by the Ministry of Health, Labor, and Welfare, Japan and a grant from the Korea–Japan Basis Cooperation Program (F01-2005-000-10145-0) of the KOSEF and the SRC/ERC Program (R11-2005-017) of the KOSEF/MOST. The authors would like to thank Drs. Kazunori Katayama and Masanori Tachikawa for valuable

References (36)

A.L. Betz et al.
Transport of hexoses, potassium and neutral amino acids into capillaries isolated from bovine retina
Exp. Eye Res.
(1980)
R. Dawson
Effects of dietary taurine supplementation or deprivation in aged male Fischer 344 rats
Mech. Ageing Dev.
(1999)
J. Hillenkamp
Effect of taurine and apical potassium concentration on electrophysiologic parameters of bovine retinal pigment epithelium
Exp. Eye Res.
(2006)
K. Hosoya
Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat
Exp. Eye Res.
(2001)
G. Levi
Control of cerebral metabolite levels. I. Amino acid uptake and levels in various species
Arch. Biochem. Biophys.
(1967)
H. Pasantes-Morales
Free amino acids of chicken and rat retina
Brain Res.
(1972)
X. Qian
Molecular characterization of taurine transport in bovine aortic endothelial cells
Biochim. Biophys. Acta
(2000)
N.E. Vinton
Visual function in patients undergoing long-term total parenteral nutrition
Am. J. Clin. Nutr.
(1990)
J.S. Adorante et al.
Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na, K, Cl cotransport
J. Gen. Physiol.
(1990)
S. Bialek et al.
K⁺ and Cl⁻ transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition
J. Physiol.
(1994)

C.C. Bridges

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

Am. J. Physiol.: Cell Physiol.

(2001)

J.G. Cunha-Vaz

Studies on the permeability of the blood–retinal barrier. I. On the existence, development, and site of a blood–retinal barrier

Br. J. Ophthalmol.

(1966)

A. El-Sherbeny

Osmoregulation of taurine transporter function and expression in retinal pigment epithelial, ganglion, and Muller cells

Invest. Ophthalmol. Visual Sci.

(2004)

S.R. Ennis et al.

Sucrose permeability of the blood–retinal and blood–brain barriers. Effects of diabetes, hypertonicity, and iodate

Invest. Ophthalmol. Visual Sci.

(1986)

H.S. Geggel

Nutritional requirement for taurine in patients receiving long-term parenteral nutrition

N. Engl. J. Med.

(1985)

S.H. Hansen

The role of taurine in diabetes and the development of diabetic complications

Diabetes Metab. Res. Rev.

(2001)

A. Harris

Retinal and choroidal blood flow in health and disease

A.A. Heinamaki

Endogenous synthesis of taurine and GABA in rat ocular tissues

Acta. Chem. Scand. B.

(1988)

Cited by (56)

Plasma Metabolites Associated with OCT Features of Age-Related Macular Degeneration
2024, Ophthalmology Science
The most widely used classifications of age-related macular degeneration (AMD) and its severity stages still rely on color fundus photographs (CFPs). However, AMD has a wide phenotypic variability that remains poorly understood and is better characterized by OCT. We and others have shown that patients with AMD have a distinct plasma metabolomic profile compared with controls. However, all studies to date have been performed solely based on CFP classifications. This study aimed to assess if plasma metabolomic profiles are associated with OCT features commonly seen in AMD.
Prospectively designed, cross-sectional study.
Subjects with a diagnosis of AMD and a control group (> 50 years old) from Boston, United States, and Coimbra, Portugal.
All participants were imaged with CFP, used for AMD staging (Age-Related Eye Disease Study 2 classification scheme), and with spectral domain OCT (Spectralis, Heidelberg). OCT images were graded by 2 independent graders for the presence of characteristic AMD features, according to a predefined protocol. Fasting blood samples were collected for metabolomic profiling (using nontargeted high-resolution mass spectrometry by Metabolon Inc). Analyses were conducted using logistic regression models including the worst eye of each patient (AREDS2 classification) and adjusting for confounding factors. Each cohort (United States and Portugal) was analyzed separately and then results were combined by meta-analyses. False discovery rate (FDR) was used to account for multiple comparisons.
Plasma metabolite levels associated with OCT features.
We included data on 468 patients, 374 with AMD and 94 controls, and on 725 named endogenous metabolites. Meta-analysis identified significant associations (FDR < 0.05) between plasma metabolites and 3 OCT features: hyperreflective foci (6), atrophy (6), and ellipsoid zone disruption (3). Most associations were seen with amino acids, and all but 1 metabolite presented specific associations with the OCT features assessed.
To our knowledge, we show for the first time that plasma metabolites have associations with specific OCT features seen in AMD. Our results support that the wide spectrum of presentations of AMD likely include different pathophysiologic mechanisms by identifying specific pathways associated with each OCT feature.
Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
Selective drug delivery to the retinal cells: Biological barriers and avenues
2023, Journal of Controlled Release
Retinal drug delivery is a challenging, but important task, because most retinal diseases are still without any proper therapy. Drug delivery to the retina is hampered by the anatomical and physiological barriers resulting in minimal bioavailability after topical ocular and systemic administrations. Intravitreal injections are current method-of-choice in retinal delivery, but these injections show short duration of action for small molecules and low target bioavailability for many protein, gene based drugs and nanomedicines. State-of-art delivery systems are based on prolonged retention, controlled drug release and physical features (e.g. size and charge). However, drug delivery to the retina is not cell-specific and these approaches do not facilitate intracellular delivery of modern biological drugs (e.g. intracellular proteins, RNA based medicines, gene editing). In this focused review we highlight biological factors and mechanisms that form the basis for the selective retinal drug delivery systems in the future. Therefore, we are presenting current knowledge related to retinal membrane transporters, receptors and targeting ligands in relation to nanomedicines, conjugates, extracellular vesicles, and melanin binding. These issues are discussed in the light of retinal structure and cell types as well as future prospects in the field. Unlike in some other fields of targeted drug delivery (e.g. cancer research), selective delivery technologies have been rarely studied, even though cell targeted delivery may be even more feasible after local administration into the eye.
SLC6A and SLC16A family of transporters: Contribution to transport of creatine and creatine precursors in creatine biosynthesis and distribution
2022, Biochimica et Biophysica Acta - Biomembranes
Creatine (Cr) is needed to maintain high energy levels in cells. Since Cr plays reportedly a critical role in neurodevelopment and the immune system, Cr dynamics should be strictly regulated to control these physiological events. This review focuses on the role of transporters that recognize Cr and/or Cr precursors. Our previous studies revealed physiological roles of SLC6A and SLC16A family transporters in Cr dynamics. Creatine transporter (CRT/SLC6A8) contributes to the influx transport of Cr in Cr distribution. γ-Aminobutyric acid transporter 2 (GAT2/SLC6A13) mediates incorporation of guanidinoacetate (GAA), a Cr precursor, in the process of Cr biosynthesis. Monocarboxylate transporter 12 (MCT12/SLC16A12) functions as an efflux transporter for Cr and GAA, and contributes to the process of Cr biosynthesis. Accordingly, the SLC6A and SLC16A family of transporters play important roles in the process of Cr biosynthesis and distribution via permeation of Cr and Cr precursors across the plasma membrane.
A novel organic cation transporter involved in paeonol transport across the inner blood-retinal barrier and changes in uptake in high glucose conditions
2021, Experimental Eye Research
Paeonol exerts various pharmacological effects owing to its antiangiogenic, antioxidant, and antidiabetic activities. We aimed to investigate the transport mechanism of paeonol across the inner blood-retinal barrier both in vitro and in vivo. The carotid artery single injection method was used to investigate the retina uptake index of paeonol. The retina uptake index (RUI) value of [³H]paeonol was dependent on both concentration and pH. This value decreased significantly in the presence of imperatorin, tramadol, and pyrilamine when compared to the control. However, para-aminohippuric acid, choline, and taurine had no effect on the RUI value. Conditionally immortalized rat retina capillary endothelial cells (TR-iBRB cell lines) were used as an in vitro model of the inner blood-retinal barrier (iBRB). The uptake of [³H]paeonol by the TR-iBRB cell lines was found to be time-, concentration-, and pH-dependent. However, the uptake was unaffected by the absence of sodium or by membrane potential disruption. Moreover, in vitro structural analog studies revealed that [³H]paeonol uptake was inhibited in the presence of organic cationic compounds including imperatorin, clonidine and tramadol. This is consistent with the results obtained in vivo. In addition, transfections with OCTN1, 2 or plasma membrane monoamine transporter (PMAT) small interfering RNA did not affect paeonol uptake in TR-iBRB cell lines. Upon pre-incubation of these cell lines with high glucose (HG) media, [³H]paeonol uptake decreased and mRNA expression levels of angiogenetic factors, such as hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) increased. However, after the pretreatment of unlabeled paeonol in HG conditions, the mRNA levels of VEGF and HIF-1 were comparatively reduced, and the [³H]paeonol uptake rate was restored. After being exposed to inflammatory conditions induced by glutamate, TNF-α, and LPS, paeonol and propranolol pretreatment significantly increased the uptake of both [³H]paeonol and [³H]propranolol in TR-iBRB cell lines compared to their respective controls. Our results demonstrate that the transport of paeonol to the retina across the iBRB may involve the proton-coupled organic cation antiporter system, and the uptake of paeonol is changed by HG conditions.
Imidazole-4-acetic acid, a new lead structure for interaction with the taurine transporter in outer blood-retinal barrier cells
2017, European Journal of Pharmaceutical Sciences
Citation Excerpt :
TAUT is a high-affinity sodium dependent transporter playing a key role in the transport of taurine (Fig. 1) and also GABA at both the inner and the outer BRB (El-Sherbeny et al., 2004; Lambert et al., 2015; Tomi et al., 2007). It has previously been estimated that the transport of [3H]taurine from the circulating blood to the rat retina is almost 30 times higher than the corresponding value across the blood-brain barrier (BBB), which could indicate a higher expression of TauT in the rat retina compared to the brain (Tomi et al., 2007). Studies have shown TAUT to be expressed in ARPE-19 cells, a spontaneously arising human retinal pigment epithelial (RPE; outer BRB) cell line (El-Sherbeny et al., 2004).
Retinal diseases leading to impaired vision and ultimately blindness are mainly characterized by ischemic and hypoxic stress. Targeting the retinal ρ-containing γ-aminobutyric acid type A receptors (ρ GABA_ARs) and thereby decreasing the retinal neuronal activity has been proposed as a novel therapeutic approach. The taurine transporter (TAUT) plays a key role in the retinal transport of GABA and has been previously suggested to display a higher functional activity in the retina compared to the brain. TAUT would therefore stand as a suitable target for the selective delivery of ρ GABA_AR ligands into the retina. Consequently, an in vitro model of TAUT at the outer blood-retinal barrier (BRB) was developed and characterized using the ARPE-19 cell line. Furthermore, the structural requirements of GABA_AR ligands for interacting with TAUT at the BRB were investigated for a series of standard GABA_AR ligands by testing their ability to inhibit the TAUT-mediated influx of taurine in ARPE-19 cells. Results showed that taurine influx was seven-fold higher when the ARPE-19 cells were cultured under hyperosmotic conditions and was demonstrated to display saturable kinetics (K_m = 27.7 ± 2.2 μM and J_max = 24.2 ± 0.6 pmol/cm²·min). Furthermore, the taurine influx was significantly inhibited in a concentration-dependent manner by GABA and imidazole-4-acetic acid (IAA), which is a naturally occurring metabolite of histamine. These compounds display similar K_i values of 644.2 μM and 658.6 μM, respectively. Moreover, IAA demonstrated higher inhibitory properties than the other tested GABA analogs: 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP), muscimol, and thiomuscimol. These studies demonstrated that IAA interacts with TAUT, which makes IAA a new lead structure in the development of new compounds, which are not only interacting with TAUT but also potent ρ GABA_AR ligands.
Effects of simvastatin on CAT-1-mediated arginine transport and NO level under high glucose conditions in conditionally immortalized rat inner blood-retinal barrier cell lines (TR-iBRB)
2017, Microvascular Research
Citation Excerpt :
They were obtained from commercial sources. TR-iBRB cell lines (conditionally immortalized rat retinal capillary endothelial cell lines) (Tomi et al., 2007; Tomi et al., 2009; Lee et al., 2016) were cultured at 33°C in a 5% CO2/air humidified incubator on rat tail collagen type 1-coated tissue dishes (Iwaki, Tokyo, Japan). Because TR-iBRB cells were isolated from transgenic rat harboring a temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat) and ts SV 40 large T-antigen is inactivated at 37 °C (Hosoya et al., 2001; Terasaki and Hosoya, 2001).
Hyperglycemia causes the breakdown of the blood-retinal barrier by impairing endothelial nitric oxide synthase (eNOS) function. Statins have many pleiotropic effects such as improving endothelial barrier permeability and increasing eNOS mRNA stability. The objective of this study was to determine effect of simvastatin on l-arginine transport and NO production under high-glucose conditions in conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB).
Changes in l-arginine transport uptake and, expression levels of cationic amino acid transporter 1 (CAT-1) and eNOS mRNA were investigated after pre-treatment with simvastatin and NOS inhibitors (l-NMMA and l-NAME) under high-glucose conditions using TR-iBRB, an in vitro model of iBRB. The NO level released from TR-iBRB cells was examined using Griess reagents.
Under high glucose conditions, [³H]l-arginine uptake was decreased in TR-iBRB cells. Simvastatin pretreatment elevated [³H]l-arginine uptake, the expression levels of CAT-1 and eNOS mRNA, and NO production under high-glucose conditions. Moreover, the co-treatment with simvastatin and NOS inhibitors reduced [³H]l-arginine uptake compared to pretreatment with simvastatin alone.
Our results suggest that, in the presence of high-glucose levels, increased l-arginine uptake due to simvastatin treatment was associated with increased CAT-1 and eNOS mRNA levels, leading to higher NO production in TR-iBRB cells. Thus, simvastatin might be a good modulator for diabetic retinopathy therapy by increasing of the l-arginine uptake and improving endothelial function in retinal capillary endothelial cells.

View all citing articles on Scopus

View full text

Function and regulation of taurine transport at the inner blood–retinal barrier

Abstract

Introduction

Section snippets

Animals

Blood-to-retina transport of taurine across the BRB

Discussion

Acknowledgments

Exp. Eye Res.

Mech. Ageing Dev.

Exp. Eye Res.

Exp. Eye Res.

Arch. Biochem. Biophys.

Brain Res.

Biochim. Biophys. Acta

Am. J. Clin. Nutr.

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na, K, Cl cotransport

J. Gen. Physiol.

K+ and Cl− transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition

J. Physiol.

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

Am. J. Physiol.: Cell Physiol.

Studies on the permeability of the blood–retinal barrier. I. On the existence, development, and site of a blood–retinal barrier

Br. J. Ophthalmol.

Osmoregulation of taurine transporter function and expression in retinal pigment epithelial, ganglion, and Muller cells

Invest. Ophthalmol. Visual Sci.

Sucrose permeability of the blood–retinal and blood–brain barriers. Effects of diabetes, hypertonicity, and iodate

Invest. Ophthalmol. Visual Sci.

Nutritional requirement for taurine in patients receiving long-term parenteral nutrition

N. Engl. J. Med.

The role of taurine in diabetes and the development of diabetic complications

Diabetes Metab. Res. Rev.

Retinal and choroidal blood flow in health and disease

Endogenous synthesis of taurine and GABA in rat ocular tissues

Acta. Chem. Scand. B.

K⁺ and Cl⁻ transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition