Expression of monocarboxylate transporters in rat ocular tissues

Glyn Chidlow,1 John P. M. Wood,1 Mark Graham,2 and Neville N. Osborne1

1Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford; and 2AstraZeneca R&D Charnwood, Loughborough, United Kingdom

Submitted 20 January 2004 ; accepted in final form 22 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study was to determine the distribution of monocarboxylate transporter (MCT) subtypes 1-4 in the various structures of the rat eye by using a combination of conventional and real-time RT-PCR, immunoblotting, and immunohistochemistry. Retinal samples expressed mRNAs encoding all four MCTs. MCT1 immunoreactivity was observed in photoreceptor inner segments, Müller cells, retinal capillaries, and the two plexiform layers. MCT2 labeling was concentrated in the inner and outer plexiform layers. MCT4 immunolabeling was present only in the inner retina, particularly in putative Müller cells, and the plexiform layers. No MCT3 labeling could be observed. The retinal pigment epithelium (RPE)/choroid expressed high levels of MCT1 and MCT3 mRNAs but lower levels of MCT2 and MCT4 mRNAs. MCT1 was localized to the apical and MCT3 to the basal membrane of the RPE, whereas MCT2 staining was faint. Although MCT1-MCT4 mRNAs were all detectable in iris and ciliary body samples, only MCT1 and MCT2 proteins were expressed. These were present in the iris epithelium and the nonpigmented epithelium of the ciliary processes. MCT4 was localized to the smooth muscle lining of large vessels in the iris-ciliary body and choroid. In the cornea, MCT1 and MCT2 mRNAs and proteins were detectable in the epithelium and endothelium, whereas evidence was found for the presence of MCT4 and, to a lesser extent, MCT1 in the lens epithelium. The unique distribution of MCT subtypes in the eye is indicative of the pivotal role that these transporters play in the maintenance of ocular function.

retina; eye; immunohistochemistry; polymerase chain reaction


MONOCARBOXYLATES, which include pyruvate, lactate, and ketone bodies, play a major role in the metabolism of cells. They are thought to be the principal energy substrates for certain cell types and are essential fuels when glucose availability is low. Of all the monocarboxylates, lactate is quantitatively the most important oxidizable substrate and is considered to be the preferred substrate of photoreceptor inner segments (23). Lactate is also the end product of glycolysis, and in cells with high rates of glycolysis, such as photoreceptor outer segments, lactate must be rapidly exported to prevent intracellular acidosis. The retina produces considerable quantities of lactate aerobically, and the retinal pigment epithelium (RPE) performs a vital role in transporting excess lactate from the subretinal space into the choroidal circulation. However, the retina is not the only ocular tissue to produce substantial amounts of lactate. The lens and cornea are avascular tissues that are largely mitochondria free and depend on anaerobic glycolysis to meet the majority of their energy needs. As a consequence, they produce and export significant amounts of lactate, and this explains why lactate levels in the aqueous humor are considerably higher than in plasma (9).

Monocarboxylates are transported across membranes via a family of proton-coupled carrier proteins known as monocarboxylate transporters (MCTs). Multiple MCT-related sequences have been identified, although only MCT1-MCT4 have been functionally characterized (15). To date, MCT1 is the best-characterized isoform and is found in the majority of tissues analyzed in the rat (15). In contrast, MCT2 has a more limited distribution in the rat and may well exist in several alternatively spliced forms (16). Rat MCT3 has been localized exclusively in the RPE (21), whereas rat MCT4 is particularly evident in tissues with high glycolytic rates, such as skeletal muscle (17). For clarification of the energy utilization requirements of the various ocular tissues, information as to the distribution of the different MCT subtypes in the eye is warranted. Despite the fact that lactate plays a central role in the metabolism of various ocular tissues, clear knowledge of the precise locations of the different MCT isoforms in the eye is lacking. Studies carried out to date have focused exclusively on the RPE (21) and retina (3, 12) and, with respect to the retina, have generated results that do not always correlate.

The distribution of the MCT1-MCT4 isoforms in the retina has thus far been studied using immunohistochemistry and immunoblotting. MCT1 is expressed throughout the retina, being particularly abundant in photoreceptor inner segments, Müller cells, and blood vessels (3, 12, 20). In contrast, data generated on MCT2 are contradictory: Gerhart et al. (12) showed MCT2-like staining to be associated with Müller cells and synaptic and nuclear layers of the retina, yet Bergersen et al. (3) found no evidence for MCT2-like immunoreactivity in the retina. Evidence to date indicates that MCT3 is not expressed in the retina (20), but some immunoreactivity for MCT4 has been reported (3).

In the rat RPE, Philp et al. (21) provided convincing data indicating that high levels of MCT 1 and MCT3 are polarized to the apical and basolateral membranes, respectively, of these cells. The localization of MCT1, as well as the absence of MCT2, was subsequently confirmed by the work of both Gerhart and Bergersen (3, 12). The latter group also concluded that a low level of MCT4-like immunoreactivity is associated with the RPE.

To our knowledge, no reports exist on the types of MCTs that are associated with the various structures of the anterior segment of the eye, yet there are valid reasons to warrant an investigation. Although historically it has been assumed that lactate diffuses out of the lens and cornea, it is more likely that lens epithelial cells and corneal epithelial or endothelial cells actively extrude lactate via one or more of the MCT isoforms. This idea is given significant weight by the finding in rabbit cornea of lactate transport mechanisms that are both carrier mediated and proton coupled (5, 13). With regard to the iris and ciliary epithelia, these tissues are derived from the same embryological structure as the RPE and, as a consequence, share a number of common properties. Indeed, iris pigment epithelium cells in culture can acquire many RPE properties (26). It is therefore of interest to determine whether a pattern of MCT expression exists in the iris and ciliary epithelium that is similar to that in the RPE.

The aim of the present study was to delineate the types of MCTs associated with the various tissues of the rat eye by using more sensitive technologies than previously employed. In particular, the objective was to provide information on the types of MCTs expressed by the different structures of the anterior uvea. To achieve these objectives, we used a combination of conventional and real-time RT-PCR in concert with immunoblotting and immunohistochemistry.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment of Animals

All experiments conformed to the Association for Research in Vision and Ophthalmology "Statement for the Use of Animals in Ophthalmic and Vision Research." Adult Wistar rats and Royal College of Surgeons (RCS) rats (200–250 g) were housed in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle and provided with food and water ad libitum. For bright light-induced injury, Wistar rats were exposed to evenly distributed bright light (~2,000 lux) for 48 h and returned to their normal housing for 5 days before being killed. For kainate-induced injury, Wistar rats were injected intravitreally in one eye with 5 nmol of kainate (5 µl in sterile water) and returned to their normal housing for 3 days before being killed.

Culture of Transformed Müller Cells

The RMC-1 transformed Müller cell line was obtained from Dr. V. Sarthy (Dept. of Ophthalmology, Northwestern Univ., Chicago, IL). These cells were grown in MEM supplemented with glucose (final concentration, 25 mM), 2.5 mg/ml amphotericin B, 100 µg/ml gentamicin, and 10% FBS. Cells were passaged at a ratio of 1:3 every 48–72 h and used for the outlined PCR studies when confluent, after being scraped and centrifuged to pellets.

PCR Analysis

For RT-PCR studies, retina, RPE, iris, ciliary body, whole cornea, corneal epithelium, corneal endothelium, lens, heart, testis, and skeletal muscle were carefully dissected and homogenized directly in TRI reagent (Sigma). Total RNA was isolated according to the manufacturer’s instructions, and first-strand cDNA synthesis was performed on 2 µg of DNase-treated RNA as described previously (19).

For conventional RT-PCR, the individual cDNA species were amplified in a reaction containing the cDNA equivalent of 25 ng of total RNA, PCR buffer, MgCl2 (3.5 mM for MCT4, 4 mM for all other primers), dNTPs, the relevant sense and antisense primers, and AmpliTaq Gold (Applied Biosystems, Foster City, CA). Reactions were initiated by incubating at 94°C for 10 min, and PCRs (94°C for 15 s; 52, 53, or 55°C for 30 s; and 72°C for 30 s) were performed for a suitable number of cycles (Table 1), followed by a final extension at 72°C for 3 min. The PCR products of cyclophilin, rhodopsin, and MCT1-MCT4 yielded single bands corresponding to the molecular masses expected on the basis of the relevant sequence (see Table 1). The oligonucleotide primer sequences and their annealing temperatures are also shown in Table 1. PCR reaction products were separated on 1.5% agarose gels with the use of ethidium bromide for visualization.


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Table 1. RT-PCR primer sequences for mRNAs amplified

 
Real-time RT-PCR reactions were carried out in 96-well optical reaction plates by using the cDNA equivalent of 50 ng of total RNA for each sample in a total volume of 25 µl with the use of the TaqMan universal PCR master mix (Applied Biosystems). The thermal cycling conditions of PCR were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of amplification comprising 95°C for 15 s and 60°C for 1 min. The PCR assay was performed using the ABI Prism 7700 sequence detector (Applied Biosystems). MCT primers and probes (Table 2) were selected from GenBank and designed using the primer design software Primer Express (Applied Biosystems), whereas GAPDH primers and probe were obtained from TaqMan rodent GAPDH control reagents (Applied Biosystems). Amplification of GAPDH mRNA was performed as the internal control gene to account for variations in RNA levels between different samples. To allow a comparison to be made between the relative levels of expression of the different MCT isoforms in the various rat ocular tissues examined and between light-damaged and normal retinas, results obtained from the real-time PCR experiments were quantified using the comparative threshold () method (18). This method essentially comprises two steps: first, the amount of target gene (MCT) in a given tissue is normalized to the levels of an endogenous housekeeping gene (GAPDH), and second, the normalized amount of target gene in the tissue is then expressed relative to the normalized amount of target gene in a calibrator tissue. The threshold cycles (CT) were calculated using ABI Prism 7000 SDS software (Applied Biosystems).


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Table 2. TaqMan primer and probe sequences for mRNAs amplified

 
Antibodies

Rabbit anti-rat MCT1 (1:1,000) and rabbit anti-rat MCT2 (1:1,000) were both generous gifts from Luc Pellerin (Geneva, Switzerland). For detection of MCT3, two distinct antisera were employed: rabbit anti-rat MCT3 from Chemicon (1:200) and rabbit anti-rat MCT3 from ADI (1:200). Rabbit anti-rat MCT4 (1:200) and mouse anti-chicken actin (1:2,000) were also obtained from Chemicon.

Laser Capture Microdissection

After enucleation, retinas were removed and snap frozen, and 10-µm-thick sections were produced. Slides were fixed in 70% ethanol for 1 min and then dehydrated and cleared in xylene. Cells were isolated from the inner and outer nuclear layers of retinal sections by using a PixCell I laser capture microdissection system (Arcturus Engineering, Harpenden, UK). The cells attached to the capture film were removed from the film with lysis buffer, and total RNA was then extracted as previously described.

Immunohistochemistry

Rats were anesthetized with pentobarbital sodium and transcardially perfused with phosphate-buffered saline (PBS) and, subsequently, with Davidson’s fixative. After enucleation, whole eyes were fixed in Davidson’s fixative for 24 h, transferred to industrial methylated spirits, and processed for routine paraffin-embedded sections on an automated tissue processor. The eyes were embedded sagitally, and 5-µm serial sections were cut using a rotary microtome. Tissue sections were deparaffinized, rehydrated into 70% ethanol, and treated for 10 min with 0.5% H2O2 in absolute methanol to block endogenous peroxidases before being taken to deionized water. Where necessary, antigen retrieval was achieved by incubating the slides in boiling 10 mM citrate buffer (pH 6.0) at a pressure of 5 lb/in.2 for 5 min. After being washed in PBS-T (PBS containing 0.1% Triton X-100), tissue sections were then equilibrated for in PBS-T containing primary antibody. Sections analyzed for immunohistochemical localization of MCT1 were incubated with primary antisera for 30 min at 37°C, whereas detection of MCT3 and MCT4 was undertaken with overnight antisera incubations at 4°C. MCT2 was analyzed using both methodologies. Furthermore, the antigen retrieval procedure was not employed for detection of MCT3 or MCT4, because this was noted to diminish antisera labeling efficiency. After incubation with primary antisera, sections were washed in PBS-T and incubated in a solution of PBS-T containing appropriate secondary antibodies linked to biotin (1:100; Vector Labs, Peterborough, UK) and horse serum (1:100) for 30 min. After a further wash with PBS-T, sections were developed using a standard avidin-peroxidase detection system (Vector) with 0.1% (vol/vol) H2O2 and 0.1% (wt/vol) 3',3'-diaminobenzidine in PBS-T. Sections were mounted in PBS-glycerol and visualized using light microscopy.

Immunoblotting

Tissue and RPE culture samples were sonicated in homogenization buffer (20 mM Tris·HCl, pH 7.4, containing 2 mM EDTA, 0.5 mM EGTA, 1% SDS, 0.1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50 µg/ml leupeptin, and 50 µg/ml pepstatin A). The protein contents of the samples were then measured using standard methodology, and samples were approximately equalized. An equal volume of sample buffer (62.5 mM Tris·HCl, pH 7.4, containing 4% SDS, 10% glycerol, 10% {beta}-mercaptoethanol, and 0.005% bromphenol blue) was added, and the samples were left at room temperature overnight. Electrophoresis of samples was performed using 10% polyacrylamide gels containing 0.1% SDS. Proteins were then blotted onto nitrocellulose. The blots were incubated with MCT or actin antisera at the appropriate dilutions (see above) for 3–4 h at room temperature, and secondary antibodies conjugated to horseradish peroxidase were subsequently employed (Sigma). Nitrocellulose blots were developed with a 0.016% (wt/vol) solution of 3-amino-9-ethylcarbazole in 50 mM sodium acetate (pH 5.0) containing 0.05% (vol/vol) Tween 20 and 0.03% (vol/vol) H2O2.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Specificity of MCT2 and MCT4 Antibodies

Localization of MCT2 has proved problematic in the past. To verify the specificity of the MCT2 and MCT4 antibodies for use with ocular sections, we performed preliminary staining using testis and skeletal muscle sections. These tissues are known to display discrete patterns of MCT2 (testis) and MCT4 (skeletal muscle) staining (14, 28). The MCT2 antibody stained specifically in sperm tails throughout the epididymis (Fig. 1A) but failed to label skeletal muscle (Fig. 1D), whereas incubation with the MCT4 antibody yielded strong staining in the sarcolemma of extraocular skeletal muscle (Fig. 1C) but no staining in the testis (Fig. 1B).



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Fig. 1. Positive control immunoreactivity for monocarboxylate transporters MCT2 and MCT4. Transverse sections through rat testis show clear labeling for MCT2 (A) in spermatozoan tails (arrows) but not for MCT4 (B). In transverse sections through rat extraocular skeletal muscle, however, MCT4 was clearly detectable (C) in the sarcolemma of myofibrillae (arrowheads). In such sections, no MCT2 was demonstrated (D). Scale bar, 50 µm.

 
Validation of RT-PCR

To detect the presence of MCT1–4 mRNAs in rat samples, we first performed RT-PCR by using primers specific to the published sequence for each of the isoforms. To verify the efficiency of each of the primer pairs for amplification of the relevant mRNAs, we included a positive control cDNA sample appropriate for each MCT isoform with the ocular samples (see Fig. 2). For each of the mRNAs analyzed, amplification of samples resulted in a single detectable product whose size was equivalent to that predicted from the relevant sequence (see Table 1). Synthesis of each product was cycle dependent, with saturation of signal achieved by ~40 cycles (data not shown), and contamination with genomic DNA was excluded by performing RT-PCR in the absence of reverse transcriptase (negative controls; see Fig. 2). In addition, for each mRNA amplified, nonspecific contamination was excluded by performing RT-PCR with the use of water instead of the cDNA sample (data not shown).



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Fig. 2. Expression of mRNAs encoding cyclophilin (A) and MCT1-MCT4 (B–E, respectively) by rat retina and retinal pigment epithelium (RPE) samples and cultured rat Müller cells, as determined by RT-PCR. Each gel shows the results for retina (lanes 3 and 4), RPE (lanes 5 and 6) and cultured Müller cells (lanes 7 and 8). A positive control is shown for each mRNA in lane 1 as follows: cyclophilin (brain; A), MCT1 (heart; B), MCT2 (testis; C), MCT3 (RPE; D), and MCT4 (skeletal muscle; E). A negative control is displayed in lane 2. For each reaction, amplification yielded a single band whose molecular mass was equivalent to that predicted from the relevant sequence (see Table 1). A 123-bp ladder (M) is shown alongside.

 
Localization of MCT1-MCT4 in Rat Ocular Tissues

Retina. Initial RT-PCR experiments showed clear signals for all four MCT mRNAs in normal retinal samples (Fig. 2). Real-time PCR experiments confirmed these results and demonstrated that the level of MCT1 mRNA is substantially higher than the levels of MCT2-4 mRNAs in the retina (Fig. 3). The presence of MCT3 mRNA in retinal samples was surprising, and to exclude the possibility of contamination from RPE, we utilized laser capture microdissection (LCM). Cells isolated from the ganglion cell layer and the inner and outer nuclear layers of the retina were pooled, the RNA was extracted, and real-time PCR was performed. The levels of all four MCT transcripts in the LCM sample were found to be similar to those measured in retinas dissected by conventional means (Fig. 4). Further information regarding the subretinal localization of the mRNAs encoding MCT1-MCT4 was provided by selectively killing certain cell types in the retina and examining whether the amounts of the MCT transcripts were altered. In 60-day-old RCS rats and in normal Wistar rats that had undergone exposure to intense light, there were marked losses of photoreceptors, which were verified by substantial reductions in the mRNA for the photoreceptor-specific marker rhodopsin (Table 3). Comparison of the abundance of the MCT mRNAs in these samples with that in age-matched control retinas revealed that in both groups of photoreceptor-deficient rats, there was a significant decrease in the level of MCT1 mRNA and considerable increases in the levels of MCT2, MCT3, and MCT4 mRNAs (Table 3). Conversely, in the retinas of rats given an intraocular injection of kainate, which kills cells in the inner retina but spares the outer retina, there was a decrease in the levels of MCT2, MCT3, and MCT4 but no change in the level of MCT1 (Table 3). Finally, cDNA samples were prepared from an immortalized rat Müller cell culture. Amplification of these samples resulted in a strong signal for MCT1 mRNA, weak signals for MCT2 and MCT4 mRNAs, and little or no signal for MCT3 mRNA (Fig. 2).



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Fig. 3. Detection of MCT1 (A), MCT2 (B), MCT3 (C), and MCT4 (D) mRNA expression in various ocular tissues using real-time RT-PCR analysis. Each value is the mean ± SE of 3 (RPE, ciliary body), 4 (iris, cornea, lens), or 7 samples (retina) and is normalized to the level of GAPDH mRNA. A positive control sample is included for MCT1 (heart), MCT2 (testis), and MCT4 (skeletal muscle) to provide a comparison with other tissues. Sk mus, skeletal muscle.

 


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Fig. 4. Detection of MCT1-MCT4 mRNA expression in total retina and laser capture microdissected (LCM) retina samples using real-time RT-PCR analysis. Each value is the mean ± SE of 3 (retina) or 1 sample(s) (LCM retina) and is normalized to the level of GAPDH mRNA.

 

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Table 3. Effect of degeneration of either photoreceptors or inner retinal neurons on total retinal levels of mRNAs encoding MCT1-MCT4

 
Antibodies to MCT1, MCT2, and MCT4 labeled single bands in immunoblots prepared from rat retina (Fig. 5). The MCT3 antibody failed to give a positive band (Fig. 5). In tissue sections, immunoreactivity for MCT1 was observed in photoreceptor inner segments, putative Müller cells, and retinal capillaries and was also evident in the two plexiform layers of the retina (Fig. 6, A and B). MCT2 staining in the retina was less abundant than MCT1 and tended to be concentrated in the inner retina, where immunoreactive bands could be clearly delineated in the plexiform layers (Fig. 6C). MCT4 immunolabeling was present in the inner retina, particularly in putative Müller cells, and in the two plexiform layers, but was absent from the photoreceptors and RPE (Fig. 6E). Strong staining for MCT4 was also apparent in the smooth muscle lining of blood vessels in the choroid (Fig. 6, E and F). No labeling could be observed in the retina with the MCT3 antibody (Fig. 6D).



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Fig. 5. Detection of MCT1-MCT4 in various rat tissue extracts using electrophoresis and immunoblotting. Samples were prepared as outlined in text. Lane 1, positive control rat tissue sample (heart for MCT1, testis for MCT2, empty lane for MCT3 where RPE acts as a positive control, and skeletal muscle for MCT4); lane 2, retina; lane 3, iris-ciliary body; lane 4, fresh, scraped RPE. MCT1-MCT4 were immunodetected at the molecular masses indicated.

 


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Fig. 6. Immunohistochemical localization of MCT1 (A and B), MCT2 (C), MCT3 (D), and MCT4 (E and F) in transverse sections through the rat retina. MCT1 was clearly localized to retinal photoreceptor inner segments (A, arrow) and Müller cell processes (B, arrows). MCT2 appeared to be present throughout the inner retina, particularly forming 2 distinct strata within the inner plexiform layer (C, arrow). MCT3 was not found in the retina (D), being confined to the RPE. MCT4 was present throughout the inner retina (inner and outer plexiform layers; E) but was not found in photoreceptor segments. MCT4 was also observed in the smooth muscle of large choroidal vessels (E and F, arrowheads). Scale bars, 50 µm. Note that photomicrographs shown in A, C, D, and E are all of the same magnification (representative scale bar shown in E). Numbers in D refer to retinal layers and are applicable to all sections shown (A–F): 1, ganglion cell layer; 2, inner plexiform layer; 3, inner nuclear layer; 4, outer plexiform layer; 5, outer nuclear layer; 6, rod inner segments; 7, rod outer segments; 8, retinal pigment epithelium; 9, choroid.

 
Immunohistochemical localization of MCTs in photoreceptor-deficient rats and kainate-injected rats correlated well with the PCR results. Immunoreactivities for MCT2 (Fig. 7) and MCT4 (Fig. 8), which are abundant in the inner retina, were reduced in kainate-injected rats but unaffected in photoreceptor-deficient retinas. Relative to total cell number in the retina, therefore, MCT2 and MCT4 immunoreactivities increased in photoreceptor-deficient retinas. In contrast, MCT1 immunoreactivity, which is abundant in photoreceptor inner segments but less concentrated in the inner retina, was largely unaffected by kainate injection (Fig. 7) but was obviously reduced in retinas lacking photoreceptors (Figs. 7 and 8).



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Fig. 7. Immunohistochemical localization of MCT1 (A, C, and E) and MCT2 (B, D, and F) in transverse sections through normal rat retina (A and B), kainate-injected rat retina (C and D), and bright light-treated rat retina (E and F). In the normal retina, MCT1 was abundant in retinal photoreceptor inner segments (A, arrowhead) but less concentrated in the inner retina (A, arrow). In the kainate-injected retina, the inner retina was reduced in thickness (C, arrow), but the photoreceptor inner segments were unaffected (C, arrowhead). In the bright light-treated retina, the inner retina appeared normal (E, arrow), but the photoreceptors and their associated MCT1 immunoreactivity were completely absent (E, arrowhead). In the normal retina, MCT2 was abundant in the inner retina (B, arrow) but largely absent from the photoreceptors (B, arrowhead). In the kainate-injected retina, the inner retina was reduced in thickness and the associated MCT2 immunoreactivity was decreased (D, arrow), whereas the photoreceptors were unaffected (D, arrowhead). In the bright light-treated retina, the inner retina appeared normal with abundant MCT2 immunoreactivity (F, arrow), but the photoreceptors were completely absent (F, arrowhead). Scale bar, 50 µm.

 


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Fig. 8. Immunohistochemical localization of MCT1 (A and C) and MCT4 (B and D) in transverse sections through 18- (predegeneration) and 40-day-old (middegeneration) Royal College of Surgeons (RCS) rat retina. In the predegeneration RCS retina, MCT1 was abundant in photoreceptor inner segments (A, arrow). In the middegeneration RCS retina, the photoreceptor inner segments were lost (C, arrow) and MCT1 staining was reduced. In the predegeneration RCS retina, MCT4 was abundant in the inner retina but largely absent from photoreceptors (B, arrowhead). In the middegeneration RCS retina, the photoreceptor layer was reduced in thickness (D, arrowhead), and thus MCT4 staining increased relative to total cell number in the retina. Scale bar, 50 µm.

 
Retinal pigment epithelium. Conventional and real-time RT-PCR analysis of freshly dissected RPE/choroid revealed the presence of all four MCT mRNAs (Figs. 2 and 3), although the levels of MCT1 and MCT3 were considerably higher than the levels of MCT2 and MCT4. Immunoblotting (Fig. 5) and immunohistochemistry experiments revealed that the RPE in situ expresses immunodetectable protein for MCT1-MCT3 but not for MCT4. MCT1 (Fig. 9A) was localized to the apical membrane and MCT3 (Fig. 9C) to the basal membrane of this cell layer, whereas MCT2 staining (Fig. 9B) was very faint and did not appear to be polarized.



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Fig. 9. Immunohistochemical localization of MCT1-MCT4 in rat RPE cells in situ. MCT1 was clearly present in the apical membrane of the RPE cellular monolayer (A, arrow), whereas MCT3 was confined to the basolateral surfaces of these cells (C, arrow). Diffuse, low-intensity labeling for MCT2 was present throughout the RPE (B), but MCT4 could not be demonstrated in these cells (D). Scale bar, 50 µm.

 
Iris and ciliary body. Amplification of cDNAs from distinct iris and ciliary body samples with the use of primers specific for the mRNAs encoding MCT1-MCT4 revealed the presence of all four MCT transcripts in both tissues (Fig. 10). The relative abundance was explored by using real-time PCR, and the results are shown in Fig. 3. It can be seen that the iris expresses a similarly high amount of MCT1 mRNA to the retina and RPE, yet unlike these latter tissues, it also expresses a high level of MCT2 mRNA. The ciliary body also expresses appreciable amounts of MCT1 and MCT2 mRNA, although the level of MCT2 is somewhat lower than in the iris. As was the case in the retina, significant levels of MCT3 mRNA were detected in iris and ciliary body samples. With regard to MCT4, the transcript was present at lower levels than the other MCT mRNAs in both tissues. In general, the immunoblot (Fig. 5) and immunohistochemical findings corroborated the PCR results. MCT1 and MCT2 antibodies labeled single bands in immunoblots prepared from iris-ciliary body tissue and stained iris and ciliary body tissue sections specifically. The pattern of labeling in the iris and ciliary body was similar for MCT1 and MCT2, with both antibodies yielding strong immunoreactivity in the epithelium of the iris and the nonpigmented epithelium of the ciliary processes (Fig. 11, A and B, respectively). MCT1, but not MCT2, immunoreactivity also was evident in the remainder of the iris and ciliary body tissue, albeit at a lower intensity. Despite the presence of MCT3 mRNA, no labeling was observed in either tissue with the use of the MCT3 antibody (Fig. 11C). MCT4 immunoreactivity was absent from the iris and ciliary body but, as in the retina, was clearly localized to the smooth muscle of the vasculature in these tissues, particularly to the short posterior ciliary arteries and/or arterioles (Fig. 11, D and E).



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Fig. 10. Expression of mRNAs encoding cyclophilin (A) and MCT1-MCT4 (B–E, respectively) by rat ocular samples from the anterior uvea, as determined by RT-PCR. Each gel shows the results for iris (lanes 1 and 2), ciliary body (lanes 3–5), lens (lanes 6–8), and cornea (lanes 9–11). For each reaction, amplification yielded a single band of the expected molecular weight, predicted from the relevant sequence (see Table 1). A 123-bp ladder (M) is shown alongside. Samples shown were run simultaneously with samples displayed in Fig. 2, and thus the positive and negative controls used for each transcript, which are displayed in Fig. 2, are also valid for these samples.

 


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Fig. 11. Localization of MCT subtypes in rat iris, ciliary body and lens. MCT1 (A, arrows) and MCT2 (B, arrows) were similarly localized to the posterior iris epithelium and the nonpigmented ciliary epithelium. MCT3 was not present in either the iris or the ciliary body (C), but MCT4 was localized to the smooth muscle of the vasculature in these tissues (D and inset, arrows). This is shown in detail in E, with MCT4 demonstrated surrounding the short posterior ciliary artery/arterioles (arrow). In the lens, labeling of MCT1 (F) and MCT4 (H) but not MCT2 (G) or MCT3 (data not shown) was detectable in epithelial cells. MCT1 immunoreactivity was present on all surfaces of the cell membrane (F, arrowheads), whereas MCT4 tended to be concentrated on the cell surface facing the aqueous humor (H, arrowhead). Scale bars, 50 µm.

 
Cornea. Positive signals for all four MCT mRNAs were evident in at least one of the cornea samples analyzed using RT-PCR, although the bands obtained for MCT1 and particularly MCT2 appeared to be brighter than for MCT3 and MCT4 (Fig. 10). Real-time PCR experiments substantiated these findings and revealed that the levels of MCT2 were not only dramatically higher than for the other MCTs but also considerably higher than in the other ocular samples analyzed (Fig. 3). The PCR data correlated quite well with the results obtained from immunohistochemistry experiments. MCT1 immunoreactivity was present in numerous cells within the corneal epithelium and also in the monolayer of cells that comprise the corneal endothelium (Fig. 12A). Interestingly, the MCT2 antibody yielded slightly different patterns of staining, depending on the temperature of incubation. At 37°C, staining was apparent throughout the corneal epithelium and endothelium (Fig. 12D). However, at 4°C, labeling was only observed to any significant degree around the margins of the basal cells of the epithelium (Fig. 12C). With regard to MCT3, light staining was detectable in the basolateral membrane of the basal epithelial cells with the use of the MCT3 antibody from Chemicon (Fig. 12E), but no specific labeling was discernible with the use of the MCT3 antibody from ADI (Fig. 12F). Although light staining for MCT4 was observed in the corneal epithelium, this was revealed to be nonspecific in nature, because an identical pattern of staining was apparent in the corresponding negative control section (Fig. 12, G and H). None of the antibodies labeled the stromal tissue.



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Fig. 12. Localization of MCT isoforms in rat cornea. MCT1 immunoreactivity was present in the corneal epithelium, where it was concentrated toward the stroma (A), and also in the corneal endothelium (A, arrow and inset). MCT2 immunoreactivity was likewise found in the epithelium (arrowheads) and endothelium (arrows) when the antiserum was applied for 30 min at 37°C (D). However, incubation of sections overnight at 4°C with the same MCT2 antiserum yielded a slightly different pattern of staining in which the posterior layer of columnar cells in the epithelium was particularly prominent but the endothelium was not labeled (C). Negative control labeling (nonimmune rabbit serum) for antisera shown in A, C, and D revealed no staining (B). MCT3 immunoreactivity was detected in the basal membrane of the basal epithelial cells with the Chemicon anti-MCT3 antiserum (E), but no labeling was observable after incubation with the ADI anti-MCT3 antiserum (F). Diffuse, low-intensity labeling for MCT4 was detected throughout the corneal epithelium (G), but negative control sections (affinity-purified rabbit IgG) showed a similar pattern of staining, signifying that the immunoreactivity was nonspecific (H). Scale bar, 50 µm.

 
Lens. RT-PCR analysis of lens samples with the use of primers specific for the mRNAs encoding MCT1-MCT3 yielded only faint (MCT1 and MCT3) or undetectable (MCT2) signals (Fig. 10). Real-time PCR confirmed these results and showed that the MCT1-MCT3 transcripts are not expressed to any significant degree in the lens. In contrast, RT-PCR analysis provided clearer evidence for expression of MCT4 mRNA in the lens, and examination of the real-time PCR data allows two quantitative deductions to be made. First, the expression of MCT4 mRNA is indeed higher than for the other MCT RNAs in the lens, and second, the relative level of MCT4 mRNA in the lens is higher than in the retina or RPE (Fig. 2). Immunohistochemical detection of MCT1-MCT4 demonstrated labeling for MCT1 (Fig. 11F) and MCT4 (Fig. 11H) in lens epithelial cells but no specific staining for MCT2 (Fig. 11G) and MCT3 (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retina

The results presented in this study demonstrate that the retina expresses MCT1, MCT2, and MCT4. MCT1 was localized throughout the retina but was particularly abundant in the outer retina, whereas MCT2 and MCT4 were located preferentially in the plexiform layers.

In agreement with the results of previous studies, we found MCT1 immunoreactivity in the rat retina to be strongly associated with photoreceptor inner segments (3, 12). The abundance of MCT1 at this site is illustrated by the substantial reduction of retinal mRNA content in the retinas of photoreceptor-deficient rats. Lactate is thought to be an important energy substrate of photoreceptor inner segments (23), and MCT1 probably facilitates lactate uptake for oxidative phosphorylation. Studies performed with retinal preparations in vitro have identified the Müller cells as the source of the lactate used by photoreceptors (23, 29). Astrocytes play a similar role in the brain, exporting lactate for use by neurons (8), and recent work indicated that the MCT1 and MCT4 isoforms facilitate this release of lactate (10). The MCT isoform responsible for lactate efflux from Müller cells has not, however, been definitively established. The current study sheds some light on the subject of which MCT isoforms are expressed by retinal Müller cells. Müller cells are identified by their processes, which insinuate themselves between neuronal cell bodies in the nuclear layers, ramify laterally in the plexiform layers, and terminate proximally and distally as end-feet in the nerve fiber layer and outer limiting membrane, respectively. In retinal sections, MCT1, MCT2, and MCT4 immunoreactivities were all clearly detectable in the nerve fiber layer and in both plexiform layers, and it may be inferred therefore that all three isoforms are localized to Müller cells. However, the presence of MCT isoforms in the nerve fiber layer may reflect expression by astrocytes rather than by Müller cell end-feet, and many classes of neurons extend processes into the plexiform layers. Thus, to state with any real confidence that a protein is expressed by Müller cells, it is important to examine the nuclear layers and outer limiting membrane, as well. MCT1 labeling was apparent throughout both nuclear layers and at the outer limiting membrane, and MCT4 labeling was present throughout both nuclear layers and at the outer limiting membrane but was less abundant than MCT1, whereas MCT2 labeling was not seen within either nuclear layer or distally. In cultured, transformed Müller cells, MCT1 was the only MCT transcript to be strongly expressed. The combined results suggest that MCT1 is the dominant MCT isoform found in Müller cells, with a limited expression of MCT4.

As mentioned in the Introduction, contradictory results have been reported regarding the localization of MCT2 in the retina: Gerhart et al. (12) showed MCT2-like staining to be associated with Müller cells and synaptic and nuclear layers of the retina, yet Bergersen et al. (3) found no evidence for MCT2-like immunoreactivity in the tissue. Indeed, in a recent paper, Bergersen et al. (4) suggested that the antibody used by Gerhart may recognize MCT4 rather than MCT2 and, by implication, that MCT2 may not be present in the retina. In this study, we used an MCT2 antibody that was previously successful for localization of MCT2 in mouse (10) and rat tissue (2). To verify its suitability for use in rats, we incubated testis and skeletal muscle sections with this MCT2 antibody. Specific staining was observed in sperm tails throughout the epididymis, but no immunolabeling was detectable in the sarcolemma of the extraocular skeletal muscle. Under the conditions employed in our study, therefore, the antibody is specific for MCT2. The results of our study show that MCT2 is present on the processes of neurons in the inner retina rather than in Müller cells.

With regard to MCT4, the combined PCR and immunohistochemistry results indicate that this isoform is likewise concentrated in the inner retina, specifically in the two plexiform layers. The lack of MCT4 in the photoreceptors is in agreement with the data of Bergersen et al. (3), whereas the presence of this isoform in the inner retina concurs with the unpublished data of Philp et al. in the mouse (see Ref. 20). We suggest that MCT4 is expressed by Müller cells, albeit at lower levels than MCT1, but that it is also localized to neuronal processes within the inner retina.

The finding in this study that the retina expresses significant amounts of MCT3 mRNA was surprising because previous work has shown this isoform to be absent from the neural retinas of rats (21) and mice (20, 22). Initially, we considered that the positive signals might have resulted from amplification of mRNA emanating from a few RPE cells that had inadvertently contaminated the retinal samples during the dissection process. However, through the use of LCM, we were able to eliminate this possibility. In previous studies, detection of MCT3 mRNA in the retina was not attempted using PCR but with the very much less sensitive technique of Northern blot analysis. Because the levels of MCT3 transcript are substantially less in the retina than in the RPE, they may well be below the level detectable by Northern blot analysis. Nevertheless, although we detected MCT3 mRNA in the retina, immunohistochemistry experiments failed to identify the corresponding protein. These results are in agreement with those of Philp and colleagues (20–22) and point to one of two explanations for the discrepancy: either the MCT3 antibodies used are not sensitive enough to detect low levels of the protein, or the MCT3 mRNA is, for some unknown reason, not translated into immunodetectable protein.

Retinal Pigment Epithelium

Previous work has shown that two MCT isoforms are expressed in significant levels by the adult rat RPE: MCT1 is polarized to the apical membrane, whereas MCT3 is restricted to the basal membrane (3, 21). The coordinated activity of these two transporters is suggested to allow the rapid transport of lactate from the subretinal space across the RPE and into the blood. The results of the current study are in complete agreement with these reported findings. The present study also provides tentative evidence that the MCT2 subtype is expressed at low levels in this cell type. The reason for the inability of previous investigators (3, 12) to detect MCT2 in the RPE is unclear but, as previously mentioned, is probably related to the particular antibodies used in these studies.

Iris and Ciliary Body

The present study shows that the iris expresses the mRNAs encoding all four MCT isoforms. The levels of the MCT1 and MCT2 transcripts were higher than those of MCT3 and MCT4, and immunohistochemistry was only able to show staining for MCT1 and MCT2 in iris tissue. The most prominent immunolabeling for both isoforms was seen in the posterior epithelium. Although little is known about transport processes across the iris, experiments have indicated that up to 45% of the total lactate content of the aqueous humor is lost to the blood vessels of the surrounding iris (24). Historically, this process has been considered to occur via passive diffusion, but in the light of these results, roles for MCT1 and MCT2 in the transport of lactate across the tissue and into the blood vessels the must be considered.

The results obtained with the ciliary body were broadly similar to those found in the iris. Ciliary body samples expressed mRNAs encoding all four MCTs, yet immunolocalization experiments were only able to show staining for MCT1 and MCT2. As in the iris, MCT1 and MCT2 immunolabeling was most intense in the epithelium of the tissue. The ciliary processes consist of a highly vascular zed stromal core and a specialized double layer of epithelium, which is responsible for the secretion of aqueous humor. The inner nonpigmented epithelium is directly adjacent to the posterior chamber, whereas the outer pigmented epithelium rests on the stromal. The patterns of MCT1 and MCT2 staining indicate that it is the nonpigmented cells that express these transporters. Formation of aqueous humor is a highly active process, and the ciliary epithelium meets its considerable energy demands partly through aerobic glycolysis. In fact, the tissue produces lactate in vitro at a rate similar to that in the cornea (25), and the majority of the lactate is generated by the more active nonpigmented epithelium. Lactate produced by the ciliary epithelium can be lost directly to the blood, unlike the situation in the cornea, but experiments have shown that >50% of the lactate produced by the ciliary body is released into the aqueous humor (7, 24). Transport of lactate between these cell layers and the blood and aqueous humor may well be facilitated by MCT1 and MCT2.

The finding that significant levels of MCT3 mRNA but not MCT3 protein are present in the iris and ciliary body is curious. Embryologically, the pigmented ciliary epithelium and the posterior pigmented iris epithelium are the anterior continuation of the RPE. These cell types share many similarities, and indeed, the iris epithelium offers potential as a candidate for transplantation in cases of RPE degeneration (26). Thus it is not surprising that the cells express a similar complement of mRNAs. Aside from the RPE, the only other tissue that expresses functional MCT3 is the structurally and functionally similar choroid plexus (22). Interestingly, compared with the RPE, choroid plexus expresses a similarly high level of MCT3 transcript yet substantially lower amounts of MCT3 protein. Philp et al (22) suggested that expression of MCT3 is regulated at the posttranscriptional level, and it appears that this is true for the iris and ciliary body.

Smooth Muscle

The present study demonstrates that, in the eye, the MCT4 isoform is not only localized to the sarcolemma of extraocular skeletal muscle but also strongly expressed by the smooth muscle lining of larger blood vessels within the iris, ciliary body, and choroid. Of particular note is the expression by vessels comprising the short posterior ciliary artery system. To our knowledge, this is the first demonstration in any tissue of MCT4 expression by vascular smooth muscle cells. Interestingly, MCT4 was not identified in the dilator or sphincter smooth muscle fibers of the iris or in any of the three types of smooth muscle fibers in the ciliary body. An explanation for the presence of MCT4 in ocular vessels is currently a matter for speculation, but given that such large quantities of lactate are produced by virtually all of the ocular tissues, it would be logical if MCT4 were to facilitate the rapid transfer of lactate from these tissues into the vascular system and thereby its removal from the eye. Future work is needed to determine whether MCT4 is expressed by vascular smooth muscle of nonocular tissues.

Cornea

The corneal epithelium is a highly glycolytic tissue that generates significant quantities of lactate. Generated lactate cannot pass into the ocular tear film because of the presence of numerous tight junctions, and kinetic studies have indicated that proton-coupled facilitated transport mechanisms exist for the passage of lactate from the corneal epithelium to the stroma (5) and from the stroma across the corneal endothelium to the aqueous humor (13). The results presented in this article provide evidence for the presence of both the MCT1 and MCT2 isoforms on corneal epithelium and corneal endothelium. The abundance of the MCT2 transcript in the cornea suggests that this isoform plays a key role in the transport of lactate, and it appears to be the dominant isoform for removal of lactate from the basal cells of the corneal epithelium. However, the remarkably high levels of MCT2 may reflect the fact that this isoform has a much lower substrate capacity than the other MCTs (15) and that higher levels are needed to ensure that lactate does not accumulate intracellularly and result in intracellular acidosis, fluid buildup, and light scatter.

The cornea, like the iris, ciliary body, and retina, expressed significant amounts of MCT3 mRNA. Unlike these latter tissues, however, tentative evidence was found to indicate that the mRNA is translated into immunodetectable protein. Using the Chemicon MCT3 antiserum, we detected some specific labeling in the basolateral membrane of the basal cells of the epithelial layer. However, we observed no similar staining with the ADI MCT3 antiserum, despite the fact that both MCT3 antisera labeled the RPE intensely. Future work is needed to examine in more detail than possible here whether the cornea expresses a physiologically relevant amount of MCT3. The finding is potentially interesting, because the cornea appears to be only the third structure after the RPE and choroid plexus to express MCT3. We had anticipated that the MCT4 isoform might be the predominant MCT isoform in the cornea because of the highly glycolytic nature of the tissue, but scant evidence could be found to support this idea.

Lens

To fulfill its major function of refracting light, the lens must remain transparent. As a consequence, it is both avascular and devoid of major organelles and meets the bulk of its energy demands through anaerobic metabolism of glucose supplied by the aqueous and vitreous humors. The major end product, lactate, has traditionally been thought to diffuse out of the lens, but indirect evidence generated using rabbit and bovine lenses points toward a hydrogen ion-coupled mechanism of lactate extrusion (1, 27). The combined PCR and immunohistochemistry results generated in this study indicate that MCT1 and MCT4 are located in this tissue. PCR analysis provided clear evidence for expression of MCT4 mRNA by the lens and more ambiguous evidence for expression of MCT1 mRNA. The immunohistochemical results show that both MCT1 and MCT4 immunoreactivities are associated with the membrane of lens epithelial cells. The MCT4 isoform has an extremely high capacity for lactate and has a marked preference for lactate over pyruvate as a physiological substrate (11). These features specialize MCT4 for the release of lactate from highly glycolytic cell types such as skeletal muscle (6). The expression of MCT4 by lens epithelial cells thus would be entirely consistent with this function.


    ACKNOWLEDGMENTS
 
We are extremely grateful to Dr. Philip Kerry and Emma Wightman of AstraZeneca for expert technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Chidlow, Nuffield Laboratory of Ophthalmology, Univ. of Oxford, Walton St., Oxford OX2 6AW, UK (E-mail: glyn.chidlow{at}eye.ox.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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