January 2010
Volume 51, Issue 1
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Physiology and Pharmacology  |   January 2010
Involvement of OCTN2 in the Transport of Acetyl-l-Carnitine across the Inner Blood–Retinal Barrier
Author Affiliations & Notes
  • Masanori Tachikawa
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
  • Yoko Takeda
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
  • Masatoshi Tomi
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
  • Ken-ichi Hosoya
    From the Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
  • Corresponding author: Ken-ichi Hosoya, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan; hosoyak@pha.u-toyama.ac.jp
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 430-436. doi:10.1167/iovs.09-4080
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      Masanori Tachikawa, Yoko Takeda, Masatoshi Tomi, Ken-ichi Hosoya; Involvement of OCTN2 in the Transport of Acetyl-l-Carnitine across the Inner Blood–Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2010;51(1):430-436. doi: 10.1167/iovs.09-4080.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: To elucidate the mechanisms of acetyl-l-carnitine transport across the inner blood–retinal barrier (inner BRB).

Methods.: In vivo integration plot and retinal uptake index (RUI) analyses were used to examine acetyl-l-[3H]carnitine transport in the retina across the inner BRB in rats. RUI was determined from the ratio of acetyl-l-[3H]carnitine and [14C]n-butanol, a freely diffusible internal reference, in the retina divided by the same ratio in the solution injected in the carotid artery. The transport mechanism was characterized in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner BRB model.

Results.: The apparent influx permeability clearance (K in) per gram retina of acetyl-l-[3H]carnitine was found to be 2.31 μL/(minute · g retina). The K in of acetyl-l-[3H]carnitine was 3.7-fold greater than that of [3H]d-mannitol, a nonpermeable paracellular marker. Acetyl-l-[3H]carnitine uptake by the retina was found to be significantly inhibited by l-carnitine and acetyl-l-carnitine, supporting a carrier-mediated influx transport of acetyl-l-carnitine at the inner BRB. l-[3H]carnitine and acetyl-l-[3H]carnitine uptake by TR-iBRB2 cells was Na+- and concentration-dependent, with a K m of 29 and 26 μM, respectively. These forms of transport were significantly inhibited by organic cation/carnitine transporter (OCTN) substrates and inhibitors such as l-carnitine and acetyl-l-carnitine, tetraethylammonium, quinidine, and betaine. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was predominantly expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells.

Conclusions.: The findings suggest that OCTN2 is involved in the transport of acetyl-l-carnitine from the circulating blood to the retina across the inner BRB.

The small, water-soluble, quaternary amine l-carnitine (4-trimethylamino-3-hydroxybutyric acid) is essential for the translocation of acylcarnitine esters into mitochondria for β-oxidation of long-chain fatty acids and ATP generation in the liver, kidney, and muscles. 1,2 Although β-oxidation has a low activity in the neural tissues, l-carnitine accumulates in the retina and controls the level of the acetyl moiety in the neural retina. 3 Acetyl-l-carnitine serves as a source of neurotransmitters, such as acetylcholine, l-glutamate, and γ-aminobutyric acid, and also contributes to energy-producing reactions. 4,5 In nonvegetarians, approximately 75% of body l-carnitine is derived from the diet and 25% from de novo biosynthesis from lysine and methionine. 6 The concentrations of acetyl-l-carnitine and l-carnitine are higher in the retina (62 μM as acylcarnitines; 108 μM as l-carnitine) than that in the blood (18.4 μM as acylcarnitines; 48.9 μM as l-carnitine) in rabbits. 7 Several studies have demonstrated that acetyl-l-carnitine is effective in improving visual function in patients with early age-related macular degeneration, 8 and l-carnitine reduces retinal injury after ischemia-reperfusion in guinea pigs. 9 These pieces of evidence suggest that acetyl-l-carnitine is transported from the circulating blood to the retina across the blood–retinal barrier (BRB), which is composed of retinal capillary endothelial cells (inner BRB) and retinal pigment epithelial cells (RPE, outer BRB), via a specific transport process. 10  
There are several carnitine transporters known to be present in peripheral tissues. Recently, a new family of organic cation transporters, designated organic cation/carnitine transporters (OCTN), has been described, and novel members of this family—OCTN1, -2, and -3—can transport organic cations, l-carnitine, and acylcarnitines. 1114 OCTN1 is mainly expressed in the intestine, liver, and kidney and mediates the transport of a wide variety of organic cations in an Na+-independent and pH-dependent manner. OCTN2, which has been designated as carnitine transporter CT1, has been reported to be a high-affinity carnitine transporter (Michaelis constant: K m = 4.3 μM), and has been characterized as Na+-dependent and a transporter of acetyl-l-carnitine (K m = 9.0 μM). 11,15 OCTN2 also mediates organic cations in an Na+-independent manner. OCTN2 mRNA is expressed in kidney, skeletal muscle, heart, placenta, and brain in adult humans. 15 OCTN3, reported as a high-affinity carnitine transporter (K m = 3.0 μM), is characterized as Na+-independent, inhibited by acetyl-l-carnitine, and expressed mainly in testis. 14 Another carnitine transporter, carnitine transporter 2 (CT2), has been identified. 16 It is present in the epididymal epithelium, but is not expressed in other tissues. l-Carnitine is also transported in an Na+- and Cl-dependent manner by amino acid transporter B0+ (ATB0+) as a low-affinity transporter of l-carnitine (K m = 830 μM). 17 However, there is no information at all about the carnitine transport system at the inner BRB. It would be very useful to have more information about l-carnitine and acetyl-l-carnitine transport mechanisms at the inner BRB to understand the regulation of carnitine concentrations in the neural retina. 
The purpose of the present study was to elucidate the molecular mechanism of acetyl-l-carnitine transport at the inner BRB. The characteristics and functions of acetyl-l-carnitine and l-carnitine at the inner BRB were examined in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro model of the inner BRB, 18 with in vivo vascular injection techniques. The gene expression level of OCTN1 and -2 in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells was determined by quantitative real-time PCR analysis. 
Materials and Methods
Animals
Male Wistar rats, weighing 250 to 300 g, were purchased from Nippon SLC (Hamamatsu, Japan). The investigations using rats described in this report conformed to the provisions of the Animal Care Committee, University of Toyama (2008-P1), and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Blood-to-Retina Acetyl-l-[3H]carnitine Transport Studies
The apparent influx permeability clearance (K in) of [N-methyl-3H] acetyl-l-carnitine hydrochloride (acetyl-l-[3H]carnitine, 85 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) or d-[1-3H(N)]mannitol ([3H]d-mannitol, 14.2 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) in tissues was determined by integration plot analysis, as described elsewhere. 19 Briefly, the rats were anesthetized with an intramuscular injection of 1.22 mg xylazine and 125 mg ketamine/kg and then acetyl-l-[3H]carnitine or [3H]d-mannitol (10 μCi/head) was injected into the femoral vein. After blood samples were collected, the rats were decapitated, and the retinas and cerebrum were removed. The tissues were dissolved in 2 N NaOH and subsequently neutralized. The radioactivity was measured in a liquid scintillation counter (LSC-5000; Aloka, Tokyo, Japan). As an index of the tissue distribution characteristics of acetyl-l-[3H]carnitine or [3H]d-mannitol, the apparent retina-to-plasma concentration ratio (Vd) was used. This ratio [Vd(t)] (mL/g tissue) was defined as the amount of [3H] per gram tissue divided by that per milliliter plasma, calculated over the duration of the experiment (t). The K in, tissue [mL/(minute · g tissue)] can be described by the following equation:   where AUC(t) (dpm · minute/milliliter), C p(t) (dissociations per minute per milliliter [dpm/mL]), and V i (milliliters per gram retina) represent the area under the plasma concentration time curve of acetyl-l-[3H]carnitine or [3H]d-mannitol from time 0 to t, the plasma acetyl-l-[3H]carnitine or [3H]d-mannitol concentration at time t, and the rapidly equilibrated distribution volume of acetyl-l-[3H]carnitine or [3H]d-mannitol in tissue, respectively. V i is usually comparable with the vascular volume of tissue. The apparent influx permeability clearances of acetyl-l-[3H]carnitine or [3H]d-mannitol in retina (K in, retina) and brain (K in, brain) were determined. 
The inhibitory effect of l-carnitine and acetyl-l-carnitine on the blood-to-retina transport of acetyl-l-[3H]carnitine was evaluated by the retinal uptake index (RUI) method. 20,21 Briefly, the rats were anesthetized with an intramuscular injection of ketamine-xylazine, and then 200 μL of the injection solution was injected into the common carotid artery. The injection solution consisted of Ringer-HEPES buffer (141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 10 mM HEPES; pH 7.4) which contained both a test compound, 10 μCi acetyl-l-[3H]carnitine, and a freely diffusible internal reference, 0.1 μCi n-[1-14C]butanol ([14C]n-butanol, 2 mCi/mmol; American Radiolabeled Chemicals), in the presence or absence of inhibitors. The rats were decapitated 15 seconds after injection, and the retina and cerebrum were removed, dissolved in 2 N NaOH, and neutralized. The radioactivity was measured in a liquid scintillation counter (LSC-5000; Aloka). In this study, the RUI and brain uptake index (BUI) value was used as an index of the retinal and brain distribution characteristics of acetyl-l-[3H]carnitine and can be described by the following equation:    
Isolation of Rat Retinal Vascular Endothelial Cells (RVECs)
Magnetic beads coated with anti-rat CD31 antibodies were used to collect purified RVECs, as described previously. 22 Briefly, mouse anti-rat CD31 antibodies (Millipore Bioscience Research Reagents, Temecula, CA) were incubated with pan mouse IgG (Dynabeads; Invitrogen, Carlsbad, CA) overnight at 4°C to obtain magnetic beads coated with anti-rat CD31 antibodies. Rat retinas were minced and digested in 0.1% collagenase type I (Invitrogen) and 0.01% DNase I (Roche, Mannheim, Germany) in Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) for 30 minutes at 37°C with agitation. Digests were filtered through a 30-μm nylon mesh, and then centrifuged at 200 g for 10 minutes. The pellets were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Moregate Biotech, Bulimba, QLD, Australia) and incubated with magnetic beads coated with anti-rat CD31 antibodies for 1 hour at room temperature. RVECs labeled with the magnetic beads were positively selected by affinity binding to a magnet. 
Cell Culture
TR-iBRB2 cells possess endothelial markers and facilitative glucose transporter 1 (SLC2a1/GLUT1), P-glycoprotein (ABCB1a/mdr1a), creatine transporter (SLC6a8/CRT), L-type amino acid transporter 1 (SLC7a5/LAT1), taurine transporter (SLC6a6/TauT), reduced folate carrier 1 (SLC19a1/RFC1), and scavenger receptor class B, type1 (SR-BI) which are expressed at the inner BRB in vivo. 18,2327 Thus, TR-iBRB2 cells maintain certain in vivo functions and are a suitable in vitro model for the inner BRB. DMEM containing 100 U/mL benzylpenicillin potassium, 100 μg/mL streptomycin sulfate, and 10% FBS was used as the culture medium for the TR-iBRB2 cells. The cells (passages 27–38) were seeded onto rat tail collagen type I–coated tissue culture plates (BD Biosciences, Franklin Lakes, NJ) and cultured at 33°C in a humidified atmosphere of 5% CO2/air. The permissive-temperature for TR-iBRB2 cell culture is 33°C, due to the presence of temperature-sensitive SV 40 large T-antigen. 18  
Acetyl-l-[3H]carnitine and l-[3H]carnitine Uptake by TR-iBRB2 Cells
TR-iBRB2 cells (5 × 104 cells/cm2) were cultured at 33°C for 2 days on a rat tail collagen type I–coated 24-well plate (BD Biosciences) and washed with extracellular fluid (ECF) buffer consisting of 122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 10 mM d-glucose, and 10 mM HEPES (pH 7.4) at 37°C. Uptake was initiated by applying 200 μL ECF buffer containing 2.5 μCi acetyl-l-[3H]carnitine (147 nM) or 2.5 μCi l-[methyl-3H]carnitine hydrochloride (l-[3H]carnitine, 83 Ci/mmol; GE Healthcare, Piscataway, NJ) (150 nM) and 0.5 μCi d-[1-14C]mannitol (45 μM [14C]d-mannitol, 56 mCi/mmol; American Radiolabeled Chemicals) to estimate the volume of adhering water at 37°C in the presence or absence of inhibitors. Na+- and Cl-free uptake buffers were prepared by replacement with equimolar N-methyl-d-glucamine and gluconate, respectively. After a predetermined period, uptake was terminated by removing the solution, followed by immersing the cells in ice-cold uptake buffer to stop uptake. The cells were then solubilized in 1 N NaOH and subsequently neutralized with 1 N HCl. The cell-associated radioactivity and protein content were assayed by liquid scintillation counting (LSC-500; Aloka) and detergent compatible protein assay (DC protein assay kit; Bio-Rad; Hercules, CA) with bovine serum albumin as the standard. 
The uptake of acetyl-l-[3H]carnitine or l-[3H]carnitine by TR-iBRB2 cells was expressed as the cell-to-medium (cell/medium) ratio calculated by the following equation:   For kinetic studies, the Km, the maximum uptake rate (Vmax), and nonsaturable uptake rate (Kd) of acetyl-l-carnitine or l-carnitine uptake were calculated from the following equation, using the nonlinear least-squares regression analysis program, MULTI.28  where V and [S] are the uptake rate of acetyl-l-carnitine or l-carnitine for 10 minutes and the concentration of acetyl-l-carnitine or l-carnitine, respectively. 
Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Analysis
Total cellular RNA was prepared from phosphate-buffered saline (PBS)-washed cells (RNeasy Mini Kit; Qiagen, Hilden, Germany). Single-strand cDNA was made from 1 μg total RNA by reverse transcription (RT) using oligo dT primer. The polymerase chain reaction (PCR) was performed with a gene amplification system (GeneAmp PCR system 9700; Applied Biosystems, Inc. [ABI], Foster City, CA) with rat OCTN1, rat OCTN2, mouse ATB0+ and β-actin-specific primers through 40 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 1 minute. The sequences of the specific primers were as follows: the sense sequence was 5′-TGA TAG CCT TCC TGG GCG ATT GG-3′ and the antisense sequence was 5′-AAG GAG CCA CAG AGA ACG CCT AC-3′ for rat OCTN1 (SLC22a4, GenBank accession number NM_022270; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD); the sense sequence was 5′-CAG TGT CCT TCT CTT CAT ACA GCT G-3′ and the antisense sequence was 5′-TGT GCT CTT TAG GAC CGT TGG GCT-3′ for rat OCTN2 (SLC22a5, GenBank accession number AF110416); the sense sequence was 5′-GCT TCA TCC GAG AAC TTC CAT GTT G-3′ and the antisense sequence was 5′-TTA CTA TTG GTG TTC TGC TAC AGT TTT-3′ for mouse ATB0+ (SLC6a14, GenBank accession number NM_020049); and the sense sequence was 5′-TCA TGA AGT GTG ACG TTG ACA TCC GT-3′ and the antisense sequence was 5′-CCT AGA AGC ATT TGC GGT GCA CGA TG-3′ for the β-actin (GenBank accession number NM_031144). The PCR products were separated by electrophoresis on an agarose gel in the presence of ethidium bromide and visualized under ultraviolet light. The molecular identity of the resultant product was confirmed by sequence analysis with a DNA sequencer (Prism 310; ABI). 
Quantitative Real-Time PCR
Quantitative real-time PCR was performed with a sequence detector system (PRISM 7700 ABI) with 2× SYBR green PCR master mix (ABI) according to the manufacturer's protocol. The amount of specific mRNA in the samples was determined by a standard curve, generated for each run with the plasmid (pGEM-T Easy Vector; Promega, Madison, WI) containing the gene of interest. This enabled standardization of the initial mRNA content of cells relative to the amount of β-actin. The PCR was performed with rat OCTN1, rat OCTN2 or β-actin-specific primers. 
Data Analysis
Unless otherwise indicated, all data represent the mean ± SEM. An unpaired, two-tailed Student's t-test was used to determine the significance of differences between two groups. The statistical significance of differences among means of several groups was determined by one-way analysis of variance followed by the modified Fisher's least-squares method. 
Results
Blood-to-Retina Transport of Acetyl-l-Carnitine across the BRB
The in vivo blood-to-retina influx transport of acetyl-l-carnitine and d-mannitol across the BRB was evaluated and compared with that in other tissues by means integration plot analysis after intravenous administration of each of the radiolabeled compounds to the rats (Fig. 1). The K in, retina of acetyl-l-[3H]carnitine was determined to be 2.31 ± 0.25 μL/(minute · g retina) from the slope representing the apparent influx permeability clearance across the BRB by using equation 1, whereas the K in, retina of [3H]d-mannitol was 0.626 ± 0.099 μL/(minute · g retina) (Fig. 1A, Table 1). The K in, retina for acetyl-l-[3H]carnitine was 3.69-fold greater than that for [3H]d-mannitol. A similar difference was observed in the brain, and the ratio between the K in, brain of acetyl-l-[3H]carnitine and [3H]d-mannitol (Fig. 1B, Table 1) was 1.62. 
Figure 1.
 
Integration plot of the initial uptake of acetyl-l-[3H]carnitine (○) and [3H]d-mannitol (●) by the retina (A) and the brain (B) after intravenous administration. Acetyl-l-[3H]carnitine or [3H]d-mannitol (10 μCi/head) was injected into the femoral vein. Each point represents the mean ± SEM (n = 3–5).
Figure 1.
 
Integration plot of the initial uptake of acetyl-l-[3H]carnitine (○) and [3H]d-mannitol (●) by the retina (A) and the brain (B) after intravenous administration. Acetyl-l-[3H]carnitine or [3H]d-mannitol (10 μCi/head) was injected into the femoral vein. Each point represents the mean ± SEM (n = 3–5).
Table 1.
 
The Apparent K in per Gram Tissue and Volume of Interstitial Space in the Tissue
Table 1.
 
The Apparent K in per Gram Tissue and Volume of Interstitial Space in the Tissue
Acetyl-l-[3H]carnitine [3H]d-Mannitol
Retina Brain Retina Brain
K in [μL/(minute · g tissue)] 2.31 ± 0.25* 2.25 ± 0.19† 0.626 ± 0.099* 1.39 ± 0.17†
V i (μL/g tissue) 7.49 ± 11.23 36.7 ± 8.6 16.1 ± 3.3 30.4 ± 5.6
The influx transport of acetyl-l-carnitine across the BRB was also supported by the fact that the estimated RUI (18.0%) of acetyl-l-[3H]carnitine (Table 2) was greater than that of [3H]d-mannitol (11.6%) from our previous report. 21 Moreover, acetyl-l-carnitine and l-carnitine, at a concentration of 2 mM, significantly reduced the RUI of acetyl-l-[3H]carnitine to 81% and 83%, respectively, while the BUI (4.99%) was not affected in the presence of 2 mM acetyl-l-carnitine and l-carnitine (Table 2). These results confirm the carrier-mediated transport of acetyl-l-carnitine from the blood to the retina across the BRB. 
Table 2.
 
The RUI and BUI for Acetyl-l-[3H]carnitine in the Rat
Table 2.
 
The RUI and BUI for Acetyl-l-[3H]carnitine in the Rat
Inhibitor RUI (%) BUI (%)
Control 18.0 ± 1.3 4.99 ± 0.47
2 mM Acetyl-l-carnitine 14.6 ± 1.0* 4.74 ± 0.54
2 mM l-Carnitine 14.9 ± 1.7* 4.86 ± 0.94
Acetyl-l-[3H]carnitine and l-[3H]carnitine Uptake by TR-iBRB2 Cells
To elucidate the transport mechanism of acetyl-l-carnitine and l-carnitine, TR-iBRB2 cells were used as an in vitro rat inner BRB model. The acetyl-l-[3H]carnitine and l-[3H]carnitine uptakes by TR-iBRB2 cells exhibited time-dependent increases for at least 20 minutes, with an initial uptake rate of 479 and 320 nL/(minute · mg protein), respectively (Fig. 2). Under Na+-free conditions, the initial uptake rate of acetyl-l-[3H]carnitine and l-[3H]carnitine was 53.3 and 44.1 nL/(minute · mg protein) and was significantly reduced by 88.9% and 86.2%, respectively, while, under Cl-free conditions, the initial uptake rate of acetyl-l-[3H]carnitine and l-[3H]carnitine was 320 and 291 nL/(minute · mg protein), respectively, and was reduced by up to 33.2%. 
Figure 2.
 
Time-course of acetyl-l-[3H]carnitine (A) and l-[3H]carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi, 147 nM) (A) and l-[3H]carnitine (2.5 μCi, 150 nM) (B) was examined under the following conditions: (○) control (at 37°C in the presence of Na+ and Cl); (■) Na+-free at 37°C; (▴) Cl-free at 37°C. Each point represents the mean ± SEM (n = 4).
Figure 2.
 
Time-course of acetyl-l-[3H]carnitine (A) and l-[3H]carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi, 147 nM) (A) and l-[3H]carnitine (2.5 μCi, 150 nM) (B) was examined under the following conditions: (○) control (at 37°C in the presence of Na+ and Cl); (■) Na+-free at 37°C; (▴) Cl-free at 37°C. Each point represents the mean ± SEM (n = 4).
The acetyl-l-[3H]carnitine and l-[3H]carnitine uptake by TR-iBRB2 cells took place in a concentration-dependent manner with, respectively, a K m of 26.1 ± 7.7 and 29.0 ± 13.8 μM, a V max of 13.3 ± 2.1 and 4.63 ± 1.80 picomoles/(minute · mg protein), and a K d of 155 ± 5 and 72.3 ± 7.2 nL/(minute · mg protein) (mean ± SD; Fig. 3), indicating that acetyl-l-carnitine and l-carnitine are transported via an Na+-dependent carrier-mediated transport system in TR-iBRB2 cells. 
Figure 3.
 
Concentration-dependence of acetyl-l-carnitine (A) and l-carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi) (A) and l-[3H]carnitine (2.5 μCi) (B) took place at the indicated concentration for 5 minutes at 37°C. Data are expressed as the mean ± SEM (n = 4). Data were subjected to Michaelis-Menten and Eadie-Scatchard analyses (inset).
Figure 3.
 
Concentration-dependence of acetyl-l-carnitine (A) and l-carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi) (A) and l-[3H]carnitine (2.5 μCi) (B) took place at the indicated concentration for 5 minutes at 37°C. Data are expressed as the mean ± SEM (n = 4). Data were subjected to Michaelis-Menten and Eadie-Scatchard analyses (inset).
Inhibitory Effect of Several Compounds on Acetyl-l-[3H]carnitine and l-[3H]carnitine Uptake
The effect of substrates and inhibitors of carnitine transporters on acetyl-l-[3H]carnitine and l-[3H]carnitine uptake by TR-iBRB2 cells is summarized in Table 3. l-Carnitine and acetyl-l-carnitine at 1 mM caused marked inhibition (60%). Moreover, tetraethylammonium (TEA), quinidine, and betaine, substrates of OCTN2 at 1 mM, produced an inhibition of more than 20%, whereas the same concentration of l-arginine and l-leucine, substrates of ATB0+, did not have any significant effect. 
Table 3.
 
Inhibitory Effect of Several Compounds on l-[3H]carnitine and Acetyl-l-[3H]carnitine Uptake by TR-iBRB2 Cells
Table 3.
 
Inhibitory Effect of Several Compounds on l-[3H]carnitine and Acetyl-l-[3H]carnitine Uptake by TR-iBRB2 Cells
Inhibitors l-[3H]carnitine Acetyl-l-[3H]carnitine
Control 100 ± 4 100 ± 4
l-Carnitine 37.2 ± 1.1* 31.4 ± 5.0*
Acetyl-l-carnitine 40.0 ± 1.7* 31.8 ± 4.7*
TEA 57.7 ± 1.8* 76.9 ± 8.9†
Quinidine 27.4 ± 1.7* 34.1 ± 7.4*
Betaine 74.9 ± 4.9* 79.6 ± 13.8
l-Arginine 92.3 ± 4.4 115 ± 19
l-Leucine 88.0 ± 3.9 92.2 ± 3.6
Expression of OCTN1, OCTN2, and ATB0+ in TR-iBRB2 cells and RVECs
RT-PCR analysis was performed to examine the expression of OCTN1, OCTN2, and ATB0+ mRNA in TR-iBRB2 cells. The bands corresponding to the expected 454 and 433 bp for OCTN1 and -2 were amplified from TR-iBRB2 cells and rat kidney as a positive control, respectively (Fig. 4A). The nucleotide sequence of the bands of TR-iBRB2 cells was identical with that of rat OCTN1 and -2. ATB0+ mRNA was detected in mouse and rat lung as a positive control, but not in TR-iBRB2 cells. To determine the dominant gene for OCTN1 and -2 in TR-iBRB2 cells, quantitative real-time PCR analysis was performed to quantify the mRNA expression levels of OCTN1 and -2 (Fig. 4B). The expression of OCTN2 mRNA was 27.3-fold greater than OCTN1 mRNA in the TR-iBRB2 cells, supporting the hypothesis that OCTN2 mRNA is predominantly expressed in TR-iBRB2 cells. 
Figure 4.
 
(A) RT-PCR analysis of OCTN1, OCTN2, and ATB0+ in TR-iBRB2 cells in the presence (+) or absence (−) of reverse transcriptase. Rat kidney, mouse lung, and rat lung were used as positive controls. (B, C) Quantitative real-time PCR analysis of OCTN1 (■) and OCTN2 (□). (B) TR-iBRB2 cells; (C) freshly isolated RVEC and non-RVEC fractions.
Figure 4.
 
(A) RT-PCR analysis of OCTN1, OCTN2, and ATB0+ in TR-iBRB2 cells in the presence (+) or absence (−) of reverse transcriptase. Rat kidney, mouse lung, and rat lung were used as positive controls. (B, C) Quantitative real-time PCR analysis of OCTN1 (■) and OCTN2 (□). (B) TR-iBRB2 cells; (C) freshly isolated RVEC and non-RVEC fractions.
The mRNA expression at the in vivo inner BRB was determined by affinity purifying RVECs purified from rat retinal homogenate by using magnetic beads coated with antibodies against CD31, which is exclusively expressed on the membrane of endothelial cells. 22 The magnetically collected and noncollected cells were isolated as the RVECs and non-RVEC fractions, respectively. Quantitative real-time PCR analysis shows that the expression of OCTN2 mRNA was 45.9-fold greater than OCTN1 mRNA in RVECs, although there was only a 3.27-fold difference in non-RVECs (Fig. 4C). This result implies that OCTN2 is predominantly expressed at the inner BRB of the rat retina. 
Discussion
The present study demonstrated, for the first time, that acetyl-l-carnitine is transported from the circulating blood to the retina across the BRB and that OCTN2 is predominantly expressed in retinal capillary endothelial cells. The characteristics of acetyl-l-[3H]carnitine and l-[3H]carnitine uptake by TR-iBRB2 cells, used as an in vitro model of the inner BRB, support the hypothesis that OCTN2 is involved in acetyl-l-carnitine transport at the inner BRB. 
Acetyl-l-[3H]carnitine was transported from the blood to the retina across the BRB with a K in, retina of 2.31 ± 0.25 μL/(minute · g retina), which was 3.69-fold greater than that of [3H]d-mannitol [0.626 ± 0.099 μL/(minute · g retina)], which was used as a nonpermeable paracellular marker (Fig. 1, Table 1). The K in, retina of d-mannitol obtained by integration plot was in good agreement with that in a previous report [(0.75 μL/(minute · g retina)]. 29 Although it is conceivable that acetyl-l-carnitine is transported via some carrier-mediated transport process, rather than by passive diffusion at the BRB, we were able to use a single-arterial (e.g., intracarotid) injection technique to examine whether acetyl-l-carnitine transport across the BRB occurs via a carrier-mediated transport process. The RUI (18%) of acetyl-l-[3H]carnitine was also greater than that of [3H]d-mannitol (11%) from our previous report. 21 Moreover, acetyl-l-[3H]carnitine uptake into the retina was inhibited by acetyl-l-carnitine and l-carnitine (Table 2). This evidence suggests that acetyl-l-carnitine is transported via a carnitine transport process from the circulating blood to the retina. The BUI (5.0%) of acetyl-l-[3H]carnitine was also greater than that of [14C]d-mannitol (2.6%) and [14C]sucrose (2.4%) from previous reports. 30 Although no clear inhibition by acetyl-l-carnitine and l-carnitine was observed with the BUI method (Table 2), from both the integration plot and BUI analyses, the blood-to-brain acetyl-l-carnitine transport was found to be greater than that of nonpermeable markers, suggesting a carrier-mediated transport process of acetyl-l-carnitine across the BBB, rather than passive diffusion. In support of this notion, Kido et al. 30 reported that acetyl-l-[3H]carnitine transport across the BBB is greater than that of sucrose and suggested that OCTN2 is involved in the transport of acetyl-l-carnitine across the BBB. This report implies that OCTN2 also plays a role in acetyl-l-carnitine transport at the BBB. 
The characteristics of acetyl-l-[3H]carnitine and l-[3H]carnitine uptake by TR-iBRB2 cells indicate that the acetyl-l-carnitine and l-carnitine transport process is mediated by a secondary active transporter, since it is an Na+- and concentration-dependent process with a K m of 26.1 μM and 29.0 μM, respectively (Figs. 2 and 3). This Na+-gradient driven uptake of acetyl-l-[3H]carnitine at the inner BRB could explain the in vivo data showing that acetyl-l-[3H]carnitine is transported against a concentration gradient from the circulating blood to the retina. 7 To date, the Na+-dependent l-carnitine transporters have been identified as OCTN2 and ATB0+, which have different affinities for carnitine. The K m of acetyl-l-carnitine and l-carnitine uptake by TR-iBRB2 cells are comparable with those of acetyl-l-carnitine (K m = 4.3 μM for human OCTN2) and l-carnitine (K m = 25.4 μM for rat OCTN2, K m = 8.5 μM for human OCTN2), respectively. 12,31 As summarized in Table 3, inhibition profiles of acetyl-l-[3H]carnitine and l-[3H]carnitine uptake by TR-iBRB2 cells were almost identical, suggesting that acetyl-l-[3H]carnitine and l-[3H]carnitine are transported via a common transporter. Substrates of OCTN2, such as l-carnitine, acetyl-l-carnitine, TEA, quinidine, and betaine, 15,31,32 significantly inhibited acetyl-l-[3H]carnitine and l-[3H]carnitine uptake by TR-iBRB2 cells (Table 3). In contrast, substrates of ATB0+, such as l-arginine and l-leucine, did not have any significant effect (Table 3). These forms of inhibition in TR-iBRB2 cells are in agreement with those of OCTN2. Quantitative real-time PCR analysis using TR-iBRB2 cells and isolated RVECs revealed that OCTN2 mRNA was predominantly expressed in TR-iBRB2 cells and RVECs. These findings support the belief that OCTN2 is most likely involved in acetyl-l-carnitine transport across the inner BRB. 
The retina is the only tissue in which light is directly focused on cells and causes oxidative stress. Because acetyl-l-carnitine and l-carnitine have protective and therapeutic effects in the retinal diseases such as early age-related macular degeneration and retinal injury after ischemia-reperfusion, 8,9 exogenous administration of acetyl-l-carnitine and l-carnitine as supplements seems to be very effective. The present findings provide a pharmacokinetic basis for the supplementary administration of acetyl-l-carnitine and l-carnitine to the retina. The K m of acetyl-l-carnitine and l-carnitine uptake by TR-iBRB2 cells (26.1 and 29.0 μM, respectively, Fig. 3) are consistent with the blood concentration of acylcarnitines (18.4 μM), most of which is acetyl-l-carnitine, and l-carnitine (48.9 μM) in rabbits. 7 Assuming that the blood concentrations of acetyl-l-carnitine and l-carnitine in rabbits are similar to those in rats, the blood-to-retinal transport of acetyl-l-carnitine and l-carnitine transport at the rat inner BRB could be approximately 50% saturated by endogenous acetyl-l-carnitine and l-carnitine. This suggests that OCTN2 at the inner BRB still has the ability to carry out the blood-to-retinal transport of acetyl-l-carnitine and l-carnitine when their blood concentrations are increased. This explanation may elucidate why exogenous administration of acetyl-l-carnitine and l-carnitine is effective in increasing the retinal concentration of acetyl-l-carnitine and l-carnitine, which could lead to the improvement of several pathologic conditions in the retina. Therefore, OCTN2 at the inner BRB could be an important supplying pathway of acetyl-l-carnitine and l-carnitine to the retina in retinal disorders. A better understanding of the regulatory mechanisms of OCTN2 expression and function at the inner BRB will offer more rational therapy with acetyl-l-carnitine and l-carnitine for retinal diseases. 
Null mutations in the OCTN2 gene causes systemic carnitine deficiency (SCD) marked by cardiac and skeletal myopathy, encephalopathy, and acute liver failure. 33,34 Urban et al. have reported that although loss-of-function mutations in OCTN2 which causes SCD are likely to be rare, several variants of OCTN2 found in healthy populations contribute to variation in the disposition of carnitine. 35 Considering that several OCTN2 variants exhibit significant decreased function compared with the reference OCTN2, 35 it appears possible that OCTN2 function of acetyl-l-carnitine and l-carnitine transport at the inner BRB varies from person to person. In this regard, OCTN2 variants might affect the therapeutic efficacy of acetyl-l-carnitine and l-carnitine in patients with retinal diseases. It is thus intriguing to investigate the relationship between OCTN2 variants, the retinal distribution and the therapeutic efficacy of acetyl-l-carnitine and l-carnitine, and the frequency of retinal dysfunctions. 
In conclusion, OCTN2 most likely mediates acetyl-l-carnitine transport at the inner BRB. The functional role of OCTN2 at the inner BRB appears to involve the constant supply of acetyl-l-carnitine and l-carnitine from the circulating blood to the retina to protect the neural retina. Our present data may assist in the design of a suitable dosage regimen of acetyl-l-carnitine and l-carnitine for pharmacologic treatments of a variety of pathologic conditions in the retina. 
Footnotes
 Supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).
Footnotes
 Disclosure: M. Tachikawa, None; Y. Takeda, None; M. Tomi, None; K. Hosoya, None
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Figure 1.
 
Integration plot of the initial uptake of acetyl-l-[3H]carnitine (○) and [3H]d-mannitol (●) by the retina (A) and the brain (B) after intravenous administration. Acetyl-l-[3H]carnitine or [3H]d-mannitol (10 μCi/head) was injected into the femoral vein. Each point represents the mean ± SEM (n = 3–5).
Figure 1.
 
Integration plot of the initial uptake of acetyl-l-[3H]carnitine (○) and [3H]d-mannitol (●) by the retina (A) and the brain (B) after intravenous administration. Acetyl-l-[3H]carnitine or [3H]d-mannitol (10 μCi/head) was injected into the femoral vein. Each point represents the mean ± SEM (n = 3–5).
Figure 2.
 
Time-course of acetyl-l-[3H]carnitine (A) and l-[3H]carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi, 147 nM) (A) and l-[3H]carnitine (2.5 μCi, 150 nM) (B) was examined under the following conditions: (○) control (at 37°C in the presence of Na+ and Cl); (■) Na+-free at 37°C; (▴) Cl-free at 37°C. Each point represents the mean ± SEM (n = 4).
Figure 2.
 
Time-course of acetyl-l-[3H]carnitine (A) and l-[3H]carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi, 147 nM) (A) and l-[3H]carnitine (2.5 μCi, 150 nM) (B) was examined under the following conditions: (○) control (at 37°C in the presence of Na+ and Cl); (■) Na+-free at 37°C; (▴) Cl-free at 37°C. Each point represents the mean ± SEM (n = 4).
Figure 3.
 
Concentration-dependence of acetyl-l-carnitine (A) and l-carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi) (A) and l-[3H]carnitine (2.5 μCi) (B) took place at the indicated concentration for 5 minutes at 37°C. Data are expressed as the mean ± SEM (n = 4). Data were subjected to Michaelis-Menten and Eadie-Scatchard analyses (inset).
Figure 3.
 
Concentration-dependence of acetyl-l-carnitine (A) and l-carnitine (B) uptake by TR-iBRB2 cells. The uptake of acetyl-l-[3H]carnitine (2.5 μCi) (A) and l-[3H]carnitine (2.5 μCi) (B) took place at the indicated concentration for 5 minutes at 37°C. Data are expressed as the mean ± SEM (n = 4). Data were subjected to Michaelis-Menten and Eadie-Scatchard analyses (inset).
Figure 4.
 
(A) RT-PCR analysis of OCTN1, OCTN2, and ATB0+ in TR-iBRB2 cells in the presence (+) or absence (−) of reverse transcriptase. Rat kidney, mouse lung, and rat lung were used as positive controls. (B, C) Quantitative real-time PCR analysis of OCTN1 (■) and OCTN2 (□). (B) TR-iBRB2 cells; (C) freshly isolated RVEC and non-RVEC fractions.
Figure 4.
 
(A) RT-PCR analysis of OCTN1, OCTN2, and ATB0+ in TR-iBRB2 cells in the presence (+) or absence (−) of reverse transcriptase. Rat kidney, mouse lung, and rat lung were used as positive controls. (B, C) Quantitative real-time PCR analysis of OCTN1 (■) and OCTN2 (□). (B) TR-iBRB2 cells; (C) freshly isolated RVEC and non-RVEC fractions.
Table 1.
 
The Apparent K in per Gram Tissue and Volume of Interstitial Space in the Tissue
Table 1.
 
The Apparent K in per Gram Tissue and Volume of Interstitial Space in the Tissue
Acetyl-l-[3H]carnitine [3H]d-Mannitol
Retina Brain Retina Brain
K in [μL/(minute · g tissue)] 2.31 ± 0.25* 2.25 ± 0.19† 0.626 ± 0.099* 1.39 ± 0.17†
V i (μL/g tissue) 7.49 ± 11.23 36.7 ± 8.6 16.1 ± 3.3 30.4 ± 5.6
Table 2.
 
The RUI and BUI for Acetyl-l-[3H]carnitine in the Rat
Table 2.
 
The RUI and BUI for Acetyl-l-[3H]carnitine in the Rat
Inhibitor RUI (%) BUI (%)
Control 18.0 ± 1.3 4.99 ± 0.47
2 mM Acetyl-l-carnitine 14.6 ± 1.0* 4.74 ± 0.54
2 mM l-Carnitine 14.9 ± 1.7* 4.86 ± 0.94
Table 3.
 
Inhibitory Effect of Several Compounds on l-[3H]carnitine and Acetyl-l-[3H]carnitine Uptake by TR-iBRB2 Cells
Table 3.
 
Inhibitory Effect of Several Compounds on l-[3H]carnitine and Acetyl-l-[3H]carnitine Uptake by TR-iBRB2 Cells
Inhibitors l-[3H]carnitine Acetyl-l-[3H]carnitine
Control 100 ± 4 100 ± 4
l-Carnitine 37.2 ± 1.1* 31.4 ± 5.0*
Acetyl-l-carnitine 40.0 ± 1.7* 31.8 ± 4.7*
TEA 57.7 ± 1.8* 76.9 ± 8.9†
Quinidine 27.4 ± 1.7* 34.1 ± 7.4*
Betaine 74.9 ± 4.9* 79.6 ± 13.8
l-Arginine 92.3 ± 4.4 115 ± 19
l-Leucine 88.0 ± 3.9 92.2 ± 3.6
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