β-Alanine and l-histidine transport across the inner blood-retinal barrier: Potential involvement in l-carnosine supply

Highlights

• l-Carnosine is a bioactive dipeptide of β-alanine and l-histidine.

• TAUT is responsible for the influx transport of β-alanine across the inner BRB.

• LAT1 is responsible for the influx transport of l-histidine across the inner BRB.

• TAUT and LAT1 contribute to the l-carnosine supply to the retina.

Abstract

The supply of l-carnosine, a bioactive dipeptide of β-alanine and l-histidine, to the retina across the blood-retinal barrier (BRB) was studied. The in vivo and in vitro studies revealed low uptake activities for [³H]Gly-Sar, a representative dipeptide, suggesting that l-carnosine transport plays only a minor role at the BRB. The in vivo study using rats showed approximately 18- and 23-fold greater retinal uptake indexes (RUI) for [³H]β-alanine and [³H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [³H]β-alanine was taurine- and γ-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [³H]β-alanine uptake, suggesting that a carrier-mediated process was involved in β-alanine transport across the inner BRB. [³H]β-Alanine uptake was inhibited by taurine and β-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of β-alanine across the inner BRB. Regarding l-histidine, the l-leucine-sensitive RUI of [³H]l-histidine was identified, and the in vitro [³H]l-histidine uptake by TR-iBRB2 cells suggested that a carrier-mediated process was involved in l-histidine transport across the inner BRB. The inhibition profile suggested that L-type amino acid transporter (LAT1/SLC7A5) is responsible for the influx transport of l-histidine across the inner BRB. These results show that the influx transports of β-alanine and l-histidine across the inner BRB is carried out by TAUT and LAT1, respectively, suggesting that the retinal l-carnosine is supplied by enzymatic synthesis from two kinds of amino acids transported across the inner BRB.

Introduction

l-Carnosine (β-alanyl-l-histidine) is a naturally-occurring dipeptide consisting of β-alanine and l-histidine, and its immune-modulating, tissue-repairing and hypoglycemic effects are well known (Nagai et al., 1986, Nagai et al., 2003, Ansurudeen et al., 2012). Recently, it has been suggested that l-carnosine is of possible benefit in the treatment of ischemia and Alzheimer's disease since in vivo and in vitro studies suggest that l-carnosine is effective in scavenging reactive oxygen species in cerebral ischemia and in protecting neural cells from amyloid-β in Alzheimer's disease (Boldyrev et al., 2004, Rajanikant et al., 2007). Also, these pharmacological effects are derived from the neuroprotective action of l-carnosine, such as anti-oxidant, anti-glycation and metal ion-chelating effects (Babizhayev et al., 1994, Stvolinsky et al., 1999, Horning et al., 2000, Tiedje et al., 2010, Bellia et al., 2011).

l-Carnosine has also been detected in the retina (Margolis and Grillo, 1984, Sauerhöfer et al., 2007), and an in vivo study with streptozotocin-induced diabetic rats suggests that l-carnosine has a protective effect against the retinal vascular damage in diabetic retinopathy (Pfister et al., 2011). In addition, a study using the frog retina shows a possible role of l-carnosine in modulating the function of receptor molecules expressed in the retinal neural cells, such as photoreceptor cells and bipolar cells (Panzanelli et al., 1997). These reports support a significant role of l-carnosine in the retina, a neural tissue, in controlling vision, and suggest the importance of the physiological system for l-carnosine supply to the retina from the circulating blood.

The neural retina and the circulating blood are separated by the blood-retinal barrier (BRB) that consists of two structures, the inner and outer BRB formed by the retinal capillary epithelial cells and retinal pigment epithelial (RPE) cells, respectively. At the inner and outer BRB, paracellular solute transports were restricted by the tight junction structure formed by the retinal capillary epithelial cells and RPE cells, respectively, and the efficient supply of essential nutrients, such as sugars, amino acids and nucleoside, to the retina across the BRB is carried out by transporter molecules expressed at the plasma membrane of the retinal capillary epithelial cells and RPE cells (Stewart and Tuor, 1994, Cunha-Vaz, 2004, Hosoya et al., 2010, Hosoya et al., 2011). In particular, at the inner BRB that nourishes two-thirds of the retina, it has been reported that the retinal capillary endothelial cells express a variety of transporters, such as glucose transporter (GLUT1/SLC2A1), equilibrative nucleoside transporter 2 (ENT2/SLC29A2), taurine transporter (TAUT/SLC6A6), L (leucine-referring)-type amino acid transporter (LAT1/SLC7A5), neutral and basic amino acid transporter (y⁺LAT2/SLC7A6), cationic amino acid transporter 1 (CAT1/SLC7A1) and neutral amino acid transporter (ATA2/SLC38A2) (Takata et al., 1992, Kumagai et al., 1996, Tomi et al., 2005, Tomi et al., 2007, Tomi et al., 2009, Nagase et al., 2006, Yoneyama et al., 2010). The results of a functional study described the involvement of TAUT and LAT1 in the uptakes of taurine and l-leucine across the inner BRB, respectively (Tomi et al., 2005, Tomi et al., 2007), and this evidence supported their possible contribution to the retinal uptake of β-alanine and l-histidine that have been reported to be substrates of TAUT and LAT1, respectively (Liu et al., 1992, Kanai et al., 1998).

In transporter research, peptide transporter molecules have been shown to recognize l-carnosine as their transport substrate (Daniel and Kottra, 2004), and an earlier report showed the expression of peptide/histidine transporter 1 (PHT1/SLC15A4) at the outer BRB (Ocheltree et al., 2003). However, at the same time, this report strongly suggests the absence of a carrier-mediated process for dipeptide transport at the outer BRB while another report suggests the possible contribution of a carrier-mediated transport process to dipeptide transport across the BRB in a study using rabbits (Ocheltree et al., 2003, Atluri et al., 2004). Therefore, little is known about the detailed mechanism for the supply of l-carnosine to the retina, and its clarification will be helpful to maintain the healthy retina and treat a number of retinal diseases, such as diabetic retinopathy, age-related macular degeneration and glaucoma.

In the present study, to investigate the mechanism for the supply of l-carnosine to the retina, the influx transport of l-carnosine, β-alanine and l-histidine across the inner BRB was examined. In an in vivo study, the retinal uptake index (RUI) was measured to estimate the in vivo BRB permeability, and an in vitro uptake study using TR-iBRB2 cells and primary-cultured RPE cells was also performed. In particular, TR-iBRB2 cells are a conditionally immortalized cell line of retinal capillary endothelial cells, and are a useful in vitro cell line to estimate the in vivo BRB permeability of a number of compounds including nutrients and drugs (Hosoya et al., 2001a, Hosoya et al., 2001b, Hosoya and Tomi, 2005, Kubo et al., 2012).