Prevention of Ornithine Cytotoxicity by Nonpolar Side Chain Amino Acids in Retinal Pigment Epithelial Cells

Abstract

purpose. To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-δ-aminotransferase (OAT)–deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina.

methods. RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using dl-[¹⁴C]ornithine.

results. An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of dl -[¹⁴C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y⁺LAT1, and 4F2hc were expressed in RPE cells.

conclusions. The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.

Gyrate atrophy (GA) of the choroid and retina is a rare inborn defect of ornithine metabolism that is transmitted as an autosomal recessive trait. ¹ Patients with GA usually exhibit progressive night blindness and loss of vision leading to blindness, usually by the fifth decade of life, with numerous sharply demarcated circular depigmented areas observed in the peripheral to midperipheral retina that gradually spread and become connected, resulting in characteristic findings of GA during a fundus examination. In 1973, Simell and Takki ² discovered that the biochemical abnormalities of this disorder are hyperornithinemia and overflow ornithinuria. Further, the finding of an association between hyperornithinemia and GA led to the discovery of an enzyme defect, a deficiency of the mitochondrial matrix enzyme ornithine-δ-aminotransferase (OAT). ^{3 4 5}  

We have reported that inactivation of OAT in human retinal pigment epithelial (RPE) cells by 5-fluoromethylornithine (5-FMOrn), a specific irreversible inhibitor of OAT, ⁶ makes them susceptible to ornithine, leading to cell death. ⁷ A human hepatoma cell line, HepG2, and a fibroblast cell line, WI-38, which possess OAT activity comparable to that of RPE cells, are not affected by ornithine when OAT is inactivated by 5-FMOrn, suggesting that ornithine cytotoxicity toward 5-FMOrn–treated RPE can be used as an in vitro model of GA. We have also demonstrated that proline prevents ornithine-induced cell death. Mammalian cells have two biosynthetic pathways for proline and share a common intermediate, l-Δ¹-pyrroline-5-carboxylic acid (P5C), which is formed from ornithine by OAT and from glutamic acid by P5C synthase. In a previous study, ornithine almost completely blocked P5C synthase and decreased the incorporation of proline derived from glutamic acid into protein in fibroblasts cultured from patients with GA ⁸ ; however, the mechanisms of ornithine cytotoxicity in OAT-deficient RPE cells and cytoprotection by proline remain to be elucidated. 

In the course of our earlier experiments, we noticed that 5-FMOrn–treated RPE cells are damaged by ornithine when cultured in Ham F12 medium, whereas they are not affected by ornithine in Dulbecco’s modified Eagle’s (DME) medium devoid of proline. Those unexpected results prompted us to investigate the effect of other amino acids on ornithine cytotoxicity and incorporation of ornithine in human RPE cells to clarify the mechanisms of ornithine cytotoxicity. From the present results, we report that most of the nonpolar and uncharged polar side chain amino acids tested prevented ornithine cytotoxicity through L-type amino acid transporters. 

Material and Methods

Cell Culture

A human RPE cell line, hTERT-RPE, previously established by gene transfer of human telomerase reverse transcriptase cDNA, ⁹ was kindly provided by Donald J. Zack (Wilmer Ophthalmological Institute, Johns Hopkins University, Baltimore, MD). This RPE cell line has been reported to have several characteristics of other normal RPE cells, such as an expression of Rpe65 and in vitro differentiation capacity. ^{10 11} Cells were cultured in Ham F12 medium (Invitrogen-Gibco, Grand Island, NY) or DME medium (Nissui, Tokyo, Japan), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated in an atmosphere of 5% CO₂-95% air at 37°C in a 75-cm² flask and were supplied with fresh medium every 3 days. When the cells became confluent, they were subcultured at a split ratio of 1:3. 

Induction of Ornithine Cytotoxicity in OAT-Deficient RPE Cells

An irreversible specific OAT inhibitor, 5-FMOrn (0.5 mM), ⁶ generously provided by the Marion-Merrell Dow Research Institute (Strasbourg, France), was added to cells plated onto 24-well culture plates (Falcon, Franklin Lakes, NJ) at 1 × 10⁵ cells per well. After 30 minutes, 10 mM ornithine was added to the medium and, after defined periods, ornithine cytotoxicity was evaluated morphologically using microphotographs taken with a digital camera (SPOT; Diagnostic Instruments, Sterling Heights, MI) through an inverted confocal microscope (IX70; Olympus, Tokyo, Japan), and cellular viability was quantitatively determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, which is an established test for cytotoxicity. ¹² Briefly, MTT was incorporated into the cells and degraded by dehydrogenase on the mitochondrial internal membrane. The output of the product, formazan, correlated with the number of living cells. 

Effect of Amino Acids on Ornithine Cytotoxicity

Twenty l-amino acids, not including l-cysteine (Cys) and l-tyrosine (Tyr), were dissolved in phosphate-buffered saline (PBS) at pH 7.4 and, each was separately added to 5-FMOrn–treated hTERT-RPE cells at a final concentration of 20 mM, together with 10 mM ornithine. The cells were then incubated in F12 medium, and cytotoxicity was evaluated morphologically or colorimetrically 24 and 72 hours after the addition of ornithine. For the MTT colorimetric assay, hTERT-RPE cells were plated on 96-well culture plates (Corning Glass Co., Corning, NY) at 5 × 10³ cells per well in 200 μL of F12 medium supplemented with 10% FBS. After 72 hours of incubation, a 10-μL MTT solution (5 mg/mL) was added, and incubation was continued for another 4 hours. The cells were then lysed in dimethyl sulfoxide after three washes with PBS and absorbance was measured at 540 nm with a spectrophotometer (Multiskan MS; Labsystems, Helsinki, Finland). Data were normalized by the percentage of surviving cells compared with the untreated control. All amino acids used in the present study were purchased from Wako Pure Chemical Industries (Osaka, Japan). 

Effect of Amino Acid Transporter Inhibitor on Ornithine Cytotoxicity

BCH was used as a specific inhibitor of the conventional amino acid transport system L. ¹³ After incubation of hTERT-RPE cells with 0.5 mM 5-FMOrn for 30 minutes, we replaced the medium with fresh medium containing 10 mM ornithine and 0.1, 1, or 10 mM BCH (Sigma-Aldrich, St. Louis, MO). After 24 and 72 hours of incubation, ornithine cytotoxicity was evaluated by morphologic deterioration. 

Ornithine Incorporation Assay

The incorporation of ornithine into RPE cells was determined using dl-[¹⁴C]ornithine (Amersham Life Sciences, Tokyo, Japan). hTERT-RPE cells were plated onto culture plates (24 wells; Falcon Labware; BD Biosciences, Bedford, MA) at 1 × 10⁵ cells per well, and dl-[¹⁴C]ornithine (0.1 μCi/0.4 mL/well) and 20 mM Leu were added to the serum-free culture medium. After 24 and 48 hours of incubation, the cells were quickly washed twice with ice-cold PBS and lysed with 0.5 mL of 0.5 M NaOH. Aliquots of the lysates were neutralized with 0.5 mL of 0.5 M HCl and added to vials containing scintillation fluid to measure the radioactivity. Data were normalized to percent of radioactivity compared with the Leu-free control. 

Reverse Transcription–Polymerase Chain Reaction

Total RNA was isolated with extraction reagent (TRIzol; Invitrogen-Life Technologies, Carlsbad, CA) and total RNA (1 μg) was reverse transcribed into cDNA with a first-strand synthesis system for RT-PCR (SuperScript; Invitrogen). The first-strand cDNA was amplified with Taq polymerase (Takara, Shiga, Japan) and anti-Taq antibody (anti-Taq high; Toyobo, Osaka, Japan), with oligonucleotide primers specific for each component of the amino acid transporters. The oligonucleotide primer sequences used for the PCR reaction were: forward 5′-TGAATGAGTTAGAGCCCGAGAAGCA-3′ and reverse 5′-TTGTCGCG AGCTACCGGCGCAGAAGA-3′ for 4F2hc (309 bp); forward 5′-ATTATACAGCGG CCTCTTTGCCTAT-3′ and reverse 5′-AATGGGTCCCTGTTCACATCCTCCA-3′ for LAT1 (305 bp); forward 5′-TGTGGGAGCCCTCTGCTATGCTGAA-3′ and reverse 5′-TGGGTCAACTGTTCCAGTGTGCGGT-3′ for LAT2 (284 bp); and forward 5′-ACTGTGCCTATGTCAAATGGGGAAA-3′ and reverse 5′-GCCCATTGTCACCATCATCTATATC-3′ for y⁺LAT1 (308 bp). PCR conditions were: hold at 94°C for 30 seconds, 40 cycles of amplification (94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute), and a final extension at 72°C for 5 minutes, with a commercial PCR system (iCycler; BioRad, Hercules, CA). The presence of the corresponding PCR products was determined by electrophoresis on a 2% agarose gel in 1× Tris-acetate-EDTA buffer. 

Statistics

Data are expressed as the mean ± SD of three separate experiments. ANOVA was used for statistical analysis, with P < 0.05 considered statistically significant. 

Results

Disappearance of Ornithine Cytotoxicity in RPE Cells in DME Medium

In our previous report, ⁷ a 5-FMOrn–treated human RPE cell line 240 showed severe cell damage caused by ornithine when cultured in F12 medium, but it failed to grow because of repetitive subcultures. In the present study, 5-FMOrn–treated hTERT-RPE cells were markedly damaged by 10 mM ornithine after incubation for 24 and 72 hours (Figs. 1A 1B) , compared with control cells without ornithine (Fig. 1C) , when cultured in F12 medium. However, cells pretreated with 5-FMOrn were not affected by 10 mM ornithine when cultured in DME medium for 24 or 72 hours (Figs. 1D 1E) , compared with the control (Fig. 1F) . 

Prevention of RPE Cell Death by Nonpolar Side Chain Amino Acids

Table 1 shows a comparison of amino acid components between incubations in F12 and DME medium. DME medium contains a greater number of amino acids in higher concentrations than F12 medium, with a total concentration of 10.6 mM in the former, compared with 3.76 mM in the later. F12 medium contains Ala, Asn, Asp, and Glu at 0.1 mM, as well as Pro at 0.3 mM, which are not in DME medium. To clarify which amino acid(s) prevented ornithine cytotoxicity in 5-FMOrn–treated RPE cells, we added each separately to cells being cultured in F12 medium. Figure 2 shows the morphology of hTERT-RPE cells at 24 hours after addition of 20 mM of each amino acid. Compared with untreated cells (Fig. 2A) , 5-FMOrn–treated hTERT-RPE cells deteriorated morphologically and became partially detached from the substrate 24 hours after the addition of 10 mM ornithine (Fig. 2B) . Further, the cytotoxicity was more severe at 72 hours (data not shown). In addition, the nonpolar side chain amino acids Leu, Ile, Val, Met, Ala, Pro, Phe, and Gly and the uncharged polar side chain amino acids Gln, Asn, Ser, and Thr prevented RPE cell death by ornithine up to 24 hours (Figs. 2C 2D 2E 2F 2G 2H 2I 2J 2K 2L 2M 2N) . However, Trp as well as the acidic amino acids Asp and Glu, did not prevent ornithine cytotoxicity, as assessed by cellular morphology at 24 hours (Figs. 2O 2P 2Q) , and cell damage was apparently more severe after the addition of the basic amino acids Lys, Arg, and His (Figs. 2R 2S 2T) . 

For quantitative examination of the prevention of ornithine cytotoxicity by amino acids, we used an MTT colorimetric assay to detect the survival of hTERT-RPE cells 72 hours after addition of each. Neither 10 mM ornithine nor 0.5 mM 5FMOrn alone significantly affected the survival ratio of hTERT-RPE cells; however, ornithine at 10 mM reduced the survival rate of 5-FMOrn–treated cells to 15% at 72 hours after its addition (Fig. 3) . Consistent with the morphologic change findings at 24 hours (Fig. 2) , the nonpolar side chain amino acids Leu, Ile, Val, Met, Ala, Pro, and Phe and the uncharged polar side chain amino acids Gln, Asn, Thr, and Ser prevented ornithine cytotoxicity in RPE cells for up to 72 hours, whereas Gly considerably attenuated the cytotoxicity. In contrast, the acidic amino acids Glu and Asp did not prevent ornithine cytotoxicity. It was difficult to evaluate cell viability with an MTT colorimetric assay for cells treated with Trp and the basic amino acids, Lys, His, and Arg, because of severe cytotoxicity and of cells from the bottom of the dishes during the washing procedure (data not shown). 

Involvement of Amino Acid Transport in Ornithine Cytotoxicity

To clarify whether amino acid transport by the LAT system was involved in ornithine cytotoxicity, we examined the effect of BCH, an inhibitor of this specific amino acid transport system, on the viability of 5-FMOrn–treated hTERT-RPE cells. BCH at 1 and 10 mM, but not at 0.1 mM, prevented morphologic deterioration by ornithine in PRE cells at 24 and 72 hours (Fig. 4) . Cells incubated with BCH alone at concentrations up to 10 mM did not show any morphologic changes up to 72 hours (Figs. 4I 4J 4K 4L) . 

To examine functional interactions between ornithine and amino acids through the l-amino acid transport system, we determined the accumulation of ornithine into RPE cells using Leu, because, among the amino acids, it exhibited cytoprotection against ornithine cytotoxicity most effectively. Leu at 20 mM decreased dl-[¹⁴C]ornithine accumulation in hTERT-RPE cells to 77.2% and 79.3% of the control at 24 and 48 hours, respectively (Fig. 5) . 

Gene Expression of Amino Acid Transporters in hTERT-RPE Cells

To confirm the involvement of the amino acid transport system L in ornithine incorporation in hTERT-RPE cells, we examined mRNA expression of heterodimeric amino acid transporters by RT-PCR. As shown in Figure 6 , three light subunits of the transporters, LAT1, LAT2, and y⁺LAT1, and their common heavy subunit, 4F2hc, were detected as RT-PCR products with expected sizes in hTERT-RPE cells. 

Discussion

In another study, we have reported that ornithine exhibits severe cytotoxicity in cultured human RPE cells when OAT is inactivated by the specific inhibitor 5-FMOrn and also have demonstrated the protective effect of proline, a secondary metabolite of ornithine catalyzed by OAT, in OAT-deficient RPE cells. ⁷ However, we also have noticed that ornithine cytotoxicity does not occur in OAT-deficient RPE cells when proline-free DME medium is used, which contains high concentrations of amino acids, compared with Ham F12 medium. 

In the present study, most of the nonpolar side chain amino acids tested prevented ornithine cytotoxicity (Figs. 2 3) , whereas involvement of the LAT systems in ornithine cytotoxicity in 5-FMOrn–treated RPE cells was suggested by BCH, an inhibitor of the amino acid transporter system L (Fig. 4) . In fact, accumulation of dl-[¹⁴C]ornithine into RPE cells was inhibited by Leu at 24 and 48 hours (Fig. 5) . Further, the prevention of ornithine cytotoxicity profile by the amino acids (Figs. 2 3) correlated well with the substrate specificity of LAT2. Amino acid transport activity by LAT2 shows a broad specificity for small and large zwitterionic amino acids as well as the bulky analogue BCH; however, not for acidic and basic amino acids. ¹⁴ In contrast, whereas y⁺LAT1 mediates Na⁺-dependent uptake of large neutral amino acids such as Leu, it also transports cationic amino acids such as Lys, Arg, and His, as well as ornithine, regardless of the presence of Na⁺. ¹⁵ Because of this peculiar cation dependence, y⁺LAT1 is thought to mediate a heteroexchange, wherein the influx of large neutral amino acids is accompanied by the efflux of basic amino acids. The involvement of this transport system may explain the enhancement of ornithine cytotoxicity by the basic amino acids Arg and Lys. Although His uptake occurs through LAT2, His instead of ornithine can be heteroexchanged through y⁺LAT1, which creates a futile cycle between LAT2 and y⁺LAT1. This may explain the lack of cytoprotective effect of His. 

LAT2 and y⁺LAT1 both interact with the common heavy chain 4F2hc, which is necessary for their trafficking to the plasma membrane, and the high expression levels of LAT2 and y⁺LAT1 mRNAs found in the kidney suggest that these transporters contribute to renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells. ^{14 15} In the present study, we demonstrated the expression of LAT2, y⁺LAT1, and 4F2hc in hTERT-RPE cells (Fig. 6) . Functional coupling of LAT2 and y⁺LAT1 may participate in transepithelial amino acid transport and the secretion of basic amino acids in many cell types, including RPE cells. It should be noted that y⁺LAT1 is not expressed in the liver, ¹⁵ which may correlate with our previous finding that the human hepatoma cell line HepG2 is not susceptible to ornithine. 

The observations that neither ornithine nor 5-FMOrn alone was cytotoxic (Fig. 3) and BCH prevented ornithine cytotoxicity in OAT-deficient RPE cells (Fig. 4) suggest that ornithine exerts its cytotoxic effect in the intracellular space after incorporation into cells through amino acid transporters, and not on the cell surface. Nonpolar side chain amino acids have been said to exert cytoprotective effects by competitive inhibition of ornithine accumulation in RPE cells, but incorporation of proline into cells does not depend on either LAT2 or y⁺LAT1. ^{14 15} Yoo et al. ¹⁶ reported that a collagen synthesis inhibitor, 4-cis-hydroxyproline, causes deterioration of cellular morphology and also inhibits the proliferation and migration of bovine RPE cells in vitro, from which they concluded that collagen synthesis could be essential for RPE cells. 

A chronic deficiency of proline in RPE cells may be one of the causes of changes in collagen synthesis resulting in RPE degeneration in patients with GA. We have reported that collagen synthesis inhibitors diminish such a cytoprotective effect by proline in an epithelioid phenotype. ¹⁷ Proline is formed from ornithine by OAT and from glutamate by P5C synthase through P5C, and ornithine is known to inhibit P5C synthase activity. Therefore, it is likely that the cytoprotective effect of proline does not serve as a competitive inhibitor of amino acid transporters, but rather as a substrate for collagen synthesis after incorporation into RPE cells. However, the link between cytoprotection by proline and collagen synthesis requires further investigation. 

The major clinical symptom in GA is a slow, progressive loss of vision leading to blindness over approximately 50 years. GA is biochemically characterized by hyperornithinemia due to a deficiency of OAT. Plasma ornithine levels in GA range from 0.4 to 1.4 mM and a chronic reduction of plasma ornithine, and an arginine-restricted diet has been shown to slow or stop chorioretinal degradation. ¹⁸ This correlation between plasma ornithine level and progression of the disease implies an involvement of amino acid transporter systems in GA. Thus, excretion of ornithine by RPE cells in GA through amino acid transport systems may protect against and delay the onset of chorioretinal degeneration. It is possible that the numerous sharply demarcated, circular depigmented areas observed in the peripheral retina during the initial phase of GA reflect the expression of amino acid transport systems; however, that interesting issue remains to be studied. 

The present results demonstrated the expression of LAT1 in hTERT-RPE cells (Fig. 6) . LAT1 is thought to play a critical role in the blood–brain barrier, ^{19 20 21} and therefore amino acid transporters may also play crucial roles at the tight junction between RPE cells, resulting in prevention of ornithine cytotoxicity by reinforcement of the barrier. Although further investigation is needed to clarify the intracellular mechanisms of ornithine cytotoxicity and cytoprotection by other amino acids, our findings may provide further rationale for an arginine-restricted diet for treatment of patients with GA, whereas the amino acid transport system in RPE cells may be a good target for a new therapy against chorioretinal degeneration in GA. 

 Supported in part by grants-in-aid for Scientific Research in Priority Areas and for Creative Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; grants-in-aid for Scientific Research (B) from the Japan Society for the Promotion of Science; and grants from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.

 Submitted for publication April 23, 2003; revised July 7, 2003; accepted August 7, 2003.

 Disclosure: T. Nakauchi, None; A. Ando, None; M. Ueda-Yamada, None; Y. Yamazaki, None; M. Uyama, None; M. Matsumura, None; S. Ito, Marion-Merrell Dow Research Institute (F)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Difference in susceptibility of hTERT-RPE cells to ornithine between culture media. hTERT-RPE cells (1 × 10⁵ cells/well) were cultured for 24 hours in 24-well dishes in F12 medium (A–C) and DME medium (D–F) supplemented with 10% FBS and then incubated for 30 minutes with 0.5 mM 5-FMOrn to inactivate OAT activity. 5-FMOrn–treated cells were then cultured for 24 (A, D) or 72 (B, C, E, F) hours in the presence (A, B, D, E) or absence (C, F) of 10 mM ornithine. Original magnification, ×100.

Table 1.

 

Comparison of Amino Acid Components between DME and F12 Medium

Constituent	DME Medium (mM)	F12 Medium (mM)
l-Alanine (Ala)	—	0.10
l-Arginine (Arg)	0.40	1.00
l-Asparagine (Asn)	—	0.10
l-Aspartic acid (Asp)	—	0.10
l-Cysteine (Cys)	0.20	0.10
l-Glutamic acid (Glu)	—	0.10
Glycine (Gly)	0.40	0.10
l-Histidine (His)	0.20	0.10
l-Isoleucine (Ile)	0.80	0.03
l-Leucine (Leu)	0.80	0.10
l-Lysine (Lys)	0.80	0.20
l-Methionine (Met)	0.20	0.03
l-Phenylalanine (Phe)	0.40	0.03
l-Proline (Pro)	—	0.30
l-Serine (Ser)	0.40	0.10
l-Threonine (Thr)	0.80	0.10
l-Tryptophan (Trp)	0.08	0.01
l-Tyrosine (Tyr)	0.34	0.03
l-Valine (Val)	0.80	0.10
Total	10.60	3.76

Figure 2.

 

Prevention of ornithine cytotoxicity in hTERT-RPE cells by amino acids after 24 hours. hTERT-RPE cells were treated with 0.5 mM 5FMOrn for 30 minutes and incubated for 24 hours without (A) or with (B) 10 mM ornithine and 20 mM Leu (C), Ile (D), Val (E), Met (F), Ala (G), Pro (H), Phe (I), Gly (J), Gln (K), Asn (L), Ser (M), Thr (N), Trp (O), Asp (P), Glu (Q), Lys (R), Arg (S), or His (T). Original magnification, ×100.

Figure 3.

 

Inhibition of ornithine cytotoxicity by various amino acids. Cells were incubated for 72 hours with 10 mM ornithine alone (□), 0.5 mM 5-FMOrn alone ( ), or 0.5 mM 5-FMOrn and 10 mM ornithine in the absence ( ) or presence (▪) of 20 mM Leu (L), Ile (I), Val (V), Met (M), Ala (A), Pro (P), Phe (F), Gln (Q), Asn (N), Thr (T), Ser (S), Glu (E), or Asp (D). *P < 0.01 versus 5-FMOrn+ornithine.

Figure 4.

 

Effect of BCH on ornithine cytotoxicity in 5-FMOrn–treated hTERT-RPE cells. hTERT-RPE cells were incubated with 0.5 mM 5-FMOrn for 24 hours (A–D) or 72 hours (E–H) with 10.0 mM ornithine and in the absence (A, E) or presence of 0.1 mM (B, F), 1 mM (C, G), or 10.0 mM (D, H) of BCH. The cells were incubated for 72 hours without (I) or with 0.1 (J), 1.0 (K) or 10.0 mM (L) of BCH. Original magnification, × 100.

Figure 5.

 

Ornithine accumulation in hTERT-RPE cells. 5-FMOrn–treated hTERT-RPE cells (1 × 10⁵ cells/well) were cultured in 24-well dishes and incubated with dl-[¹⁴C]ornithine (0.1 μCi/well) in the presence ( ) or absence (□) of 20 mM Leu for 24 and 48 hours. The incorporation of dl -[¹⁴C]ornithine in the cells in the absence of Leu was 362.3 and 730.1 dpm at 24 and 48 hours, respectively, and these values were taken as 100% incorporation. *P < 0.01 versus controls.

Figure 6.

 

Expression of l-amino acid transporter mRNAs in hTERT-RPE cells. Shown are results for 4F2hc (lane 1, 309 bp), LAT1 (lane 2, 305 bp), LAT2 (lane 3, 284 bp), and y⁺LAT1 (lane 4, 308 bp).