Characterization of an L-Carnitine Transport System in Murine Photoreceptor Cell Line

Abstract

While it is well known that L-carnitine [3-hydroxy-4-(trimethylazaniumyl)-butanoate] is an essential molecule for β-oxidation, it provides anti-oxidative effects as well. Since these effects have been observed in photoreceptor cells, the carnitine’s intracellular concentration is considered to play a protective role against oxidative damage to those cells. However, even though its high hydrophilicity makes it likely that carnitine import is accomplished via a dedicated host transport system, the specific uptake process into those cells is currently unknown. Therefore, in this study, we sought to identify and characterize photoreceptor cell carnitine uptake transporter(s) utilizing 661W cells as a photoreceptor cell model. The results of our uptake assays showed that carnitine was transported into 661W cells in a saturable manner (K_m=5.5 mM), and that the activity was susceptible to extracellular pH and Na⁺. While these data suggest the involvement of a transporter in 661W cell carnitine uptake, the observed transport profile did not correspond to any of the currently known carnitine transporters such as organic cation/carnitine transporter 1 (Octn1), Octn2, Octn3, B^0,+ and Ct2. In fact, in our experiments, the mRNA expressions for such carnitine transporters in 661W cells were consistently very low and the carnitine transporter substrates did not inhibit the uptake activities. Taken as a whole, our results indicate that carnitine is transported into 661W cells in a carrier-mediated manner. However, since its transport modes cannot be fully explained by known carnitine transporters, it is highly likely that photoreceptor cells utilize a unique molecularly-based carnitine uptake system.

The retina is a light-sensitive tissue structure on the inner surface of the eye that plays a vital role in vision. It consists of a pigmented epithelial cell layer and three neural cell layers formed by photoreceptor, bipolar, and retinal ganglion cells. Of these, photoreceptor cells are unique neurons that directly receive light, and then convert it into nerve pulse signals for transmission to the brain via the optic nerve.

It has long been known that functional impairments to photoreceptor cells will lead to vision loss, which is often associated with age-related macular degeneration (AMD) or retinitis pigmentosa (RP). Based on an examination of several proposed mechanisms underlying photoreceptor damage that have been reported to date,^1–3) it is believed that oxidative stress is likely to play a significant role in the pathogenesis of both AMD and RP.^4,5) Therefore, in order to prevent development of retinal diseases such as AMD by minimizing photoreceptor cell damage, it is necessary to identify the molecules that counteract oxidative stress and to determine how their roles are regulated in photoreceptor cells.

L-Carnitine (carnitine, [3-hydroxy-4-(trimethylazaniumyl)butanoate]) is a well-known molecule that is essential to mitochondrial β-oxidation and can be found throughout the body.⁶⁾ It has been reported that carnitine can reduce oxidative stress via its free radical scavenger property⁷⁾ and it is highly safe for intake as dietary supplement.⁸⁾ Furthermore, since the anti-oxidant effects of carnitine have been actually observed in retinal neuronal cells^9–11) and the carnitine concentration level is about twice higher in retina (108 µM) than in the blood (48.9 µM) in rabbits,¹²⁾ it is plausible to hypothesize that carnitine not only plays important roles in energy homeostasis, but it also helps to protect photoreceptor cells against oxidative damage. Human retinal carnitine concentration is speculated to be about 100 µM because the serum carnitine concentration after oral administration of carnitine at 60 mg/kg is 43.9 µM.¹³⁾

To fulfill the protective action of carnitine, photoreceptor cells must first uptake extracellular carnitine from the circulatory system. The primary sources of carnitine are the diet and biosynthesis in the liver and kidney.¹⁴⁾ Since carnitine is highly hydrophilic because of its polar zwitterionic property, a specific transport system is necessary to convey it across plasma membranes.

To date, five types of L-carnitine transporters have been identified. The first identified carnitine transporter is an organic cation/carnitine transporter 1 (OCTN1, SLC22A4),¹⁵⁾ which shows a low-affinity and Na⁺-dependent transport profile.¹⁶⁾ Subsequent studies have identified that ergothioneine is a preferable substrate.¹⁷⁾ OCTN2/CT1 (SLC22A5) and Octn3 (Slc22a21, identified only in mice) are well characterized high-affinity carnitine transporters that convey the compound in a Na⁺-dependent and independent manner, respectively.^16,18,19) In addition, the amino acid transporter B^0,+ (SLC6A14) is also capable of transporting carnitine in a low-affinity and Na⁺-dependent manner,²⁰⁾ and finally, carnitine transporter 2 (CT2, SLC22A16) is a testis-specific carnitine transporter with high-affinity and Na⁺-independent properties.²¹⁾

Furthermore, carnitine uptake that cannot be explained by any of these identified transporters has been observed in rat brain and liver specimens.^6,22) Therefore, it is possible that molecular-based, as yet unidentified carnitine transporter(s) exist in certain tissues.

In the retina, OCTN2 is reported to be expressed at the inner blood–retinal barrier, where it transports carnitine from the blood to the retinal tissue.²³⁾ However, since the carnitine transport process from the retinal tissue interstitial fluids into the photoreceptor cells has yet to be characterized, the responsible transporter remains unknown. Molecular identification of the carnitine transporter in the retina is important because gene mutations or expression downregulation of such transporter(s) might result in carnitine insufficiency, thereby hampering its effectiveness against oxidative stress in photoreceptor cells, which could eventually contribute to the onset or progression of AMD or RP. It should also be noted that compounds that upregulate or activate the photoreceptor cell carnitine transporter have potential as therapeutic agents in AMD and RP treatment regimes.

With this background in mind, this study sought to identify and characterize photoreceptor cell carnitine uptake transporter(s) using 661W cells, which is a murine cone photoreceptor-derived cell line.

MATERIALS AND METHODS

Materials

[³H]Carnitine (1 mCi/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.), while betaine monohydrate, 4-aminobutyric acid (GABA), L-lysine, choline, L-phenylalanine and L-arginine were purchased from Nacalai Tesque (Kyoto, Japan), and γ-butyrobetaine, and 1-methyl-4-phenylpyridinium (MPP⁺) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). L-Glycine was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and (±)-acetylcarnitine chloride (acetylcarnitine) was from Abcam (Cambridge, MA, U.S.A.). L-Ergothioneine (ergothioneine) was from Enzo (Farmingdale, NY, U.S.A.).

Cell Culture

The murine photoreceptor-derived 661W cells were graciously provided by Dr. Al-Ubaidi (University of Houston, Houston, TX, U.S.A.) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C and 5% CO₂. The cells were used at 14-17 passages.

[³H]Carnitine Uptake Assay

The 661W cells were seeded onto poly-D-lysine coated 24-well culture plates at a density of 1×10⁵ cells/well and then incubated for 24 h, after which the medium was replaced with serum-free Hanks’ balanced salt solution (HBSS). After 10 min incubation, carnitine uptake assays (30 nM or 10 µM) were performed based on the previously-described method.²⁴⁾

In our examination of Na⁺ or Cl⁻ dependency of carnitine uptake process, Na⁺ or Cl⁻-free HBSSs were prepared by replacing Na⁺ or Cl⁻ with equimolar lithium or gluconate, respectively. Similarly, in our examination of the pH dependency of carnitine uptake, the HBSS pH levels were adjusted to 5.4, 6.4, 7.4, and 8.4 with 1 M NaOH, and then used in the uptake assays.

The cellular radioactivity levels derived from transported carnitine were determined using the β-scintillation counter (LS6500, Beckman Coulter, Fullerton, CA, U.S.A.) and the values were expressed as those per unit cellular protein content (mg). The carrier-mediated carnitine uptake levels were calculated by subtracting the values obtained on ice from those obtained at 37°C.

In the kinetic analysis, carnitine uptake activities were determined at various concentrations (from 0.1 to 30 mM) and fitted with Michaelis–Menten equation,   

where [S] is the substrate concentration in the medium (mM), v is the uptake rate of the substrate (nmol/min/mg of protein), K_m is the Michaelis–Menten constant (mM), and V_max is the maximum uptake rate (nmol/min/mg protein). Eadie–Hofstee plot was developed based on the subtracted curve, and then K_m and V_max were calculated using the plot.

Carnitine Transport Inhibition Assays

Inhibition assays against carnitine transport activity in 661W cells were performed using OCTN substrates (acetylcarnitine, betaine, γ-butyrobetaine, and ergothioneine), B^0,+ substrates (glycine, phenylalanine, lysine and arginine), an organic cation transporter (OCT) substrate (MPP⁺), an OCT and choline transporter substrate (choline), and a GABA transporter substrate (GABA). The transport assay procedure was the same as described above. The B^0,+ inhibitor concentrations were set at 2.5 mM, whereas other inhibitors were set at 30 µM, based on the following estimated IC₅₀ values: 5–50 µM and <5 µM of acetylcarnitine or γ-butyrobetaine for OCTN2 and OCTN3,^16,18) respectively, 5–50 µM of acetylcarnitine and 5 µM of betaine for CT2, respectively,²¹⁾ <2.5 mM of glycine, phenylalanine, lysine or arginine for B^0,+.²⁰⁾ As for OCTN1, no IC₅₀ data has been reported, but the K_m value of main substrate (ergothioneine) is 21 µM.¹⁷⁾

RT-PCR Analysis

Total RNA was prepared from 661W cells using ISOGEN II (Nippon Gene, Tokyo, Japan). Single-strand cDNA was synthesized from 0.5 µg total RNA by reverse transcription (RT) with random primers and oligo dT primers using ReverTra Ace qPCR RT Master Mix with genomic DNA Remover (Toyobo, Tokyo, Japan). PCR was performed through 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min using the primers shown in Table 1. Mouse tissue cDNAs were used as positive controls (kidney for Octn1 and Octn2, lung for B^0,+, and testis for Octn3 or Ct2).

Table 1. Primers Used in Studies of Carnitine Transporter Gene Expression

Gene		Primers
Mouse Octn1	Slc22a4	Forward	5′-GAG TGG AAT CTG GTG TGT GAG-3′
		Reverse	5′-GGC TTT CTG GAT GAT CTG TTC-3′
Mouse Octn2	Slc22a5	Forward	5′-TGA CAC TCC TAA CTT GCA TGG-3′
		Reverse	5′-GCA TCT GGT CAA TGG TAT CTG-3′
Mouse Octn3	Slc22a21	Forward	5′-TTT GGA ATC ACC TCT GCC TAC-3′
		Reverse	5′-TGT TGT CGG TCA CTG ACA GTG-3′
Mouse B0,+	Slc6a14	Forward	5′-CTT CAG AGG TCA AGT GGA ATG-3′
		Reverse	5′-AGA GCT TCA GGA TAG GCA ATG-3′
Mouse Ct2	Slc22a16	Forward	5′-ATA AGT CCA GCT CCT GTG ACC-3′
		Reverse	5′-ATA AGT CCA GCT CCT GTG ACC-3′

Statistical Analysis

Statistical comparisons were made using a two-tailed Student’s t test or Dunnett’s test. The values (p) below 0.05 were regarded as statistically significant differences.

RESULTS

Time Course of Carnitine Uptake by 661W Cells

The characterization of carnitine uptake by 661W cells began with an examination of its time-dependency (Fig. 1). The results showed that the uptake level increased linearly for at least 30 min.

Fig. 1. Time Course of L-Carnitine Uptake by 661W Cells

The uptake of L-carnitine (10 nM) by 661W cells was measured in the Na⁺- and Cl⁻-containing HBSS buffer (pH 7.4) at 37°C. The uptake experiment was conducted twice, each time in quadruplicate, and the representative result is shown. The values obtained are presented as the mean±standard deviation (S.D.) of a representative experiment.

Concentration-Dependency of Carnitine Uptake by 661W Cells

Next, the concentration dependencies of carnitine uptake by 661W cells were determined in HBSS buffer (pH 7.4) containing Na⁺ and Cl⁻ by subtracting the values obtained on-ice condition from those obtained at 37°C. This is because active transport processes generally work at 37°C, but does not on ice-cold condition. The results showed that the subtracted carnitine uptake curve was saturable (Fig. 2a). The Eadie–Hofstee plot, which was calculated from the subtracted curve, provided a straight line and yielded the tentative K_m of 5.5±0.9 mM and the tentative V_max of 1.6±0.7 nmol/mg/min (Fig. 2b).

Fig. 2. Concentration-Dependency of L-Carnitine Uptake by 661W Cells

(a) The uptake of L-carnitine by 661W cells was measured for 10 min in Na⁺- and Cl⁻-containing HBSS buffer (pH 7.4). The open circle with dashed line and open circle with dotted line represent the L-carnitine uptake amount performed at 37°C and on ice, respectively. The solid line represents the L-carnitine uptake after subtraction of the uptake performed on ice from the uptake performed at 37°C. (b) L-Carnitine uptake by 661W cells were analyzed by Eadie–Hofstee plot. In (a) and (b), the uptake experiment was conducted three times, each in duplicate. The values obtained are presented as the mean±S.D.

Inhibitory Effects of Known Carnitine Transporter Substrates on Carnitine Transport Activity of 661W Cells

As previously mentioned, Octn1, Octn2, Octn3, B^0,+ and Ct2 have been identified as carnitine transporters in mice. Therefore, inhibitory assays toward carnitine transport activity were examined in 661W cells using acetylcarntine, betaine and γ-butyrobetaine (Octn2, Octn3 and Ct2 substrates), and ergothioneine (an Octn1 substrate) as respective substrates. As shown in Fig. 3a, neither of these substrates had any significant influence on the amount of carnitine uptake. Moreover, glycine, phenylalanine, lysine and arginine (B^0,+ substrates) were also not effective in blocking carnitine uptake (Fig. 3b).

Fig. 3. L-Carnitine Transport Inhibition Assay

Inhibition assays against L-carnitine transport activity in 661W cells were performed using (a) OCTN substrates (acetylcarnitine, betaine, γ-butyrobetaine, and ergothioneine), (b) B^0,+ substrates (glycine, phenylalanine, lysine and arginine), (c) an OCT substrate (MPP⁺), an OCT and choline transporter substrate (choline) and a GABA transporter substrate (GABA). The uptake of L-carnitine by 661W cells was measured for 10 min in Na⁺- and Cl⁻-containing HBSS buffer (pH 7.4) at 37°C. The concentrations of the B^0,+ inhibitors were set at 2.5 mM with L-carnitine at 10 µM, whereas other inhibitors at 30 µM with L-carnitine at 30 nM. The uptake experiment was conducted three times, each in triplicate or duplicate. The obtained values are presented as the mean±S.D. * p<0.05 versus no inhibitor group (Dunnet’s test).

Next, in order to investigate the possible involvement of other transporter types, experiments were conducted with MPP⁺ (an OCT substrate), choline (an OCT and choline transporter substrate), and GABA (a GABA transporter substrate) (Fig. 3c). Of these, only choline showed even a mild potential inhibition property against carnitine uptake by 661W cells (65.4%, Fig. 3c).

mRNA Expression Profiles of Previously-Identified L-Carnitine Transporters

To examine whether or not the previously-identified carnitine transporters were expressed in 661W cells, RT-PCR was performed. As shown in Fig. 4, each band of positive controls was detected around 500 bp, indicating that the primers work properly. On the other hand, no expression of the previously-identified carnitine transporters mRNA was found in 661W cells.

Fig. 4. RT-PCR Analysis of the Previously-Identified L-Carnitine Transporters

Mouse Octn1, Octn2, Octn3, B^0,+ and Ct2 mRNA expression were analyzed by RT-PCR. Total RNA was prepared from 661W cells. Mouse tissue cDNAs were used as positive controls (kidney for Octn1 and Octn2; lung for B^0,+; testis for Octn3 and Ct2). The PCR primer sequences used in this study were designed to yield PCR products between 450 and 550 bp.

Na⁺ and Cl⁻-Dependency of Carnitine Uptake by 661W Cells

Since it is well known that Na⁺ and Cl⁻ often work as a co-transport partner during the substrate transport processes of several SLC transporters, the possibility of their involvement of these ions in the carnitine transport system was tested in 661W cells. The transport assays were carried out using an Na⁺-free or Cl⁻-free reaction buffer, in which Na⁺ or Cl⁻ was replaced by Li⁺ or gluconate, respectively. As shown in Fig. 5, carnitine uptake in Na⁺-free buffer was reduced to 58.3±12.2% of that obtained in the Na⁺-containing buffer. On the other hand, the depletion of Cl⁻ in the reaction buffer did not significantly affect the uptake activity.

Fig. 5. Na⁺ and Cl⁻ Dependency of the L-Carnitine Uptake by 661W Cells

The uptake of L-carnitine (30 nM) by 661W cells was measured for 10 min in transport buffer (pH 7.4) at 37°C. The Na⁺ or Cl⁻-free uptake HBSS was prepared by replacement of Na⁺ or Cl⁻ with equimolar lithium or gluconate, respectively. The data points were normalized by setting the value in Na⁺- and Cl⁻-containing HBSS (normal HBSS) as a control to 100%. The uptake experiment was conducted three times, each time in triplicate. The obtained values are presented as the mean±S.D. * p<0.05 versus NaCl group (t-test).

Extracellular pH Dependency of the Carnitine Uptake by 661W Cells

Finally, we examined the pH dependency of the carnitine transport into 661W cells. The results show that acidification of the transport buffer to pH 5.4 and pH 6.4 led to a decrease in the carnitine uptake levels to 30.0±9.1% and 68.0±6.6%, respectively, as that determined at pH 7.4, whereas alkalization to pH 8.4 resulted in the carnitine uptake to 174.1±30.8% (Fig. 6). Based on the pK_a (=3.8) of the carboxylic acid of carnitine, the ratios of its zwitterion state in pH 5.4, 6.4, 7.4 and 8.4 were calculated as 97.6, 99.8, 99.9%, and over 99.9%, respectively. Therefore, it is assumed that the changes in the carnitine uptake result from the variations in the carnitine transporter activity at various pH conditions rather than the changes in the ionic state of carnitine.

Fig. 6. Extracellular pH Dependency of the L-Carnitine Uptake by 661W Cells

The uptake of L-carnitine (30 nM) by 661W cells was measured for 10 min at 37°C in Na⁺- and Cl⁻-containing HBSS at different pH. The pH of the HBSS was adjusted to 5.4, 6.4, 7.4 and 8.4 with 1 M NaOH, and used in the uptake assay. The data points were normalized by setting the value at pH 7.4 (normal HBSS) as a control to 100%. The uptake experiment was conducted three times, and each was performed in triplicate. The values obtained are presented as the mean±S.D. * p<0.05, and ** p<0.01 versus pH 7.4 group (t-test).

DISCUSSION

In this study, we investigated carnitine uptake profile of 661W cells, which is a murine photoreceptor cell line, and found that carnitine is transported in a saturable manner. The 661W cells have the potential to convey carnitine by an unidentified carnitine transporter.

In the kinetic study, there appears to be a small saturable component of the carnitine uptake even under the on ice-cold condition, whereas the uptake curve at 37°C is obviously saturable. As exemplified by human erythrocyte nucleoside transporter ENT1 that has been reported to work even at very low temperatures near 0°C,²⁵⁾ the carnitine uptake system in 661W cells also has a potential to somewhat work even under the ice-cold state. Thus, the calculated K_m value obtained from the carnitine uptake study in 661W cells is designated as a tentative value. Nevertheless, considering that the functional uptake level in ice-cold condition seems to be marginal, it is unlikely that the tentative K_m value could be drastically different from its intrinsic value.

The tentative K_m value (5.5 mM) was found to be significantly higher than those of known transporters (Octn1, Octn2, Octn3, B^0,+, Ct2; <1 mM), and carnitine appears to be transported in a partly Na⁺-dependent manner in 661W cells, which is inconsistent with the action modes of the previously reported carnitine transporters.^15,18–21)

Moreover, we found that neither of the compounds that are known as the substrates as carnitine transporters inhibited the carnitine uptake by 661W cells, and that no mRNA expression of such known carnitine transporters in 661W cells was detected. Therefore, our results do not support the hypothesis that a previously identified carnitine transporter would work in 661W cells.

Furthermore, it is also unlikely that cationic amino acid transporters, OCTs, or choline transporters are involved in the uptake of carnitine, since MPP⁺, arginine, lysine, and GABA did not inhibit the uptake activity. It should also be noted that, even though choline showed a mild inhibitory effect on the carnitine uptake in the present study, the Cl⁻-independent uptake profile and sensitivity profile to extracellular pH were not consistent with the properties of choline transporters²⁶⁾ and choline transporter-like proteins,²⁷⁾ respectively.

Given that the known transporters cannot explain its uptake by 661W cells, it is reasonable to assume that at least one previously unidentified carnitine transporter is expressed in 661W cells, which raises the question as to what type of transporter it could be. To summarize, the unknown transport system in 661W cells is characterized by high K_m, pH-dependency, and partial Na⁺-dependency. From a K_m value viewpoint, it is worth noting that a low-affinity carnitine uptake (K_m: 2.9 mM) has been reported in rat cerebral cortical slices.²²⁾ Accordingly, since photoreceptor cells belong to the neuronal cell category, it was thought that the 661W cell carnitine transporter might be related to the one reported in the rat brain. However, that possibility was confounded by differences in the inhibition profiles between these uptake systems (ref. 22 and Fig. 3).

On the other hand, the results of pH- and Na⁺-dependency profiles do provide some mechanistic insights into the carnitine uptake system of 661W cells. First, since extracellular pH increase accelerates carnitine uptake and vice versa, a possibility arises that proton (H⁺) or hydroxyl anion (OH⁻) transport may occur simultaneously during the carnitine transport in 661W cells. In addition, it has been reported that, although molecular entity remains unknown, clonidine uptake by mouse brain microvascular endothelial cells show a pH-dependent profile, suggesting a potential involvement of H⁺ antiport.²⁸⁾ In other cases, it has been shown that SLC4A11 has Na⁺-dependent OH⁻ cotransport activity.^29,30)

Furthermore, considering that L-carnitine is likely to be transported mostly in a zwitterion state, Na⁺ might be used to electronically neutralize carnitine/OH⁻ symport, or to offset electrogenesis during carnitine/OH⁻ symport or carnitine/H⁺ antiport. If either is actually the case, it would not be surprising to see that, even though Na⁺ would be preferable, it could be interchangeable with other cations (which would result in a partial Na⁺-dependency profile).

Taken together, it is suggested carnitine uptake by 661W cells may be accompanied by OH⁻ symport or H⁺ antiport by utilizing Na⁺ (or another cation) as a co-substrate, although more extensive studies are needed to confirm the notion. As far as we know, no transport system/molecule with this characteristic has been reported previously, so detailed characterization will be necessary to identify the molecular entity of this presumably new transporter. Even though the molecular entity remains unknown, our findings are an important step forward to its identification. The identification of the presumed molecular entity would pave the way for new studies aimed at understanding how its activity and expression levels can be altered, and/or whether there are genetic variations that cause loss of functions.

In conclusion, our results first show that 661W cells, which is a photoreceptor cell line, have a unique low-affinity type carnitine transport system with pH and Na⁺-sensitive uptake profiles. Furthermore, while identification of the responsible molecule must await further investigation, the results can be expected to shed light on a novel pathophysiological and pharmacological role of carnitine in photoreceptor cells.

Acknowledgment

The authors would like to extend their sincere thanks to Dr. Muayyed R Al-Ubaidi (University of Houston, Houston, TX, U.S.A.) for graciously providing the 661W cells used in this study. Y.O. thanks Yasuhiro Watanabe M.D. (Chiba University Graduate School of Medicine, Japan) for help with technical assistance. This study was supported in part by Grants from the Japan Society for the Promotion of Science (JSPS KAKENHI 26461258 (N.A.)), Strategic Research Foundation Grant-aided Project for Private Universities (S1412001), and Gout Research Foundation of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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