Presence and Localization of Three Lactic Acid Transporters (MCT1, -2, and -4) in Separated Human Granulocytes, Lymphocytes, and Monocytes
Environmental and Toxicologic Pathology Department, Armed Forces Institute of Pathology, Washington, DC
Correspondence to: William N. Fishbein, MD, PhD, Biochemical Pathology Div., Rm. M093C, Armed Forces Institute of Pathology, Washington, DC 20306-6000. E-mail: fishbein{at}afip.osd.mil or merezhin{at}afip.osd.mil
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Summary |
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Key Words: lactate transporters monocarboxylate transporters granulocytes monocytes lymphocytes platelets human leukocytes immunohistochemistry RT-PCR Western blotting
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Introduction |
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Whereas MCTs 1, 2, and 4 serve general lactate/pyruvate transport, other members have been tailored to more specific function or location. Thus, MCT3, with kinetics similar to MCT1, is present primarily in the retinal pigment epithelium in vertebrates (Philp et al. 2001), and MCT8 has recently been shown to exhibit remarkable substrate specificity for the transport of iodothyronines (Friesema et al. 2003
). This latter finding, parenthetically, clouds the distinction between the MCT family and that of the
-amino acid transporters. Several MCTs can be expressed in the same cell or tissue, and the pattern of expression differs from one species to another. The dissociation constants (Km) of lactate and pyruvate increase roughly fivefold from MCT2 to 1 and again from MCT1 to 4, suggesting a simple kinetic reason why it may be useful to express all three in the same cell if it must function at times under moderately to extremely hypoxic conditions, as must leukocytes. We have recently evaluated the relative distribution of MCT1, MCT2, and MCT4 in a panel of human tissues, and with more detail in muscle fibers, using specific antibodies (Fishbein et al. 2002
). Here we extend the investigation of MCT expression to fractionated white blood cells, about which there is very little information in the literature.
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Materials and Methods |
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The mononuclear cells at the interface were harvested, washed, and spun for 10 min at 200 x g at 24C to separate them from most of the platelets. Monocytes were separated from lymphocytes by selective adsorption onto anti-CD14coupled magnetic microbeads (Miltenyi Biotech; Auburn, CA). Cells were resuspended in 500 µl of ice-cold buffer A, and anti-CD14 microbeads were added to the cells (100 µl beads per 5 x 107 mononuclear cells). After incubation for 15 min at 40C, the cells were washed in buffer A and spun down at 300 x g for 10 min at 20C. An MS(+) positive selection column was placed into the magnetic field of a MACS separator attached to a metal stand and washed with 500 µl of degassed buffer A. Then 500 µl of cell suspension was applied to the column and the unbound flow-through fraction containing lymphocytes was collected. The column was rinsed with 500 µl of degassed buffer A four times and then removed from the magnetic field. One ml of buffer A was added to the column, and monocytes were eluted from the column with the aid of a plunger. The separated lymphocytes and monocytes were each washed with buffer A, resuspended in 500 µl PBS, used to prepare droplet and smear slides, then aliquotted and stored at 80C.
RNA Isolation and RT-PCR for Evaluation of Transcripts
Total RNA from the separated granulocytes, lymphocytes, and monocytes was obtained using RNAzolB (TEL-TEST; Friendswood, TX). Briefly, cells were lysed in RNAzolB and chloroform was added. After vigorous shaking, incubation on ice, and centrifugation, the colorless upper aqueous phase containing RNA was precipitated by treatment with isopropanol, washed, air dried, and dissolved in 1 mM EDTA, pH 7.0. The mRNA was isolated from 50 µl of total RNA using the QuickPrep Micro mRNA Purification Kit (Pharmacia; Piscataway, NJ) as recommended by the manufacturer. RT-PCR of MCT1, -2, and -4 was carried out using First Strand cDNA Synthesis Kit (Pharmacia) followed by PCR. The first strand synthesis reaction and PCR amplification were performed using 8 µl of mRNA and the primers listed in Table 2, synthesized by Midland Certified Reagents (Midland, TX). AmpliTaq polymerase (PE Applied Biosystems; Foster City, CA) with 2 µM primers and 2 mM MgCl2 was used for the PCR reaction. The reaction mixture was overlaid with mineral oil and heat denatured for 3 min at 95C, followed by 40 cycles of 15-sec denaturation (95C), 30 sec of annealing (60C), and 36+6 sec/cycle of elongation (72C). After a final 10-min polishing at 72C, the PCR products were electrophoresed in agarose gels. PCR product bands of expected size were excised from agarose gels and extracted using Gene Clean (BIO101; Carlsbad, CA) as recommended by the manufacturer. The PCR amplimers were sequenced using a 3730 DNA Analyzer (Applied Biosystems) and compared with the nucleotide NCBI database.
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Immunohistochemistry and Comparison of Fixatives
To evaluate fixatives, 6-µm sequential cryostat sections of unfixed frozen human skeletal muscle were air dried on microscope slides and immersed in 4C solutions of methanol, ethanol, acetone, or 10% formalin (3.7% formaldehyde) in PBS for 30 min. They were rinsed in PBS, air dried, and treated as described below, using anti-MCT1 as primary antibody, along with a sequential slide that remained unfixed, and compared at x100200 magnification.
Freshly separated white blood cells that had been drop dried or smeared on glass slides and fixed with 100% methanol for 30 min at 4C were used for antibody (Ab) staining. This was performed using Elite Rabbit IgG Vectastain ABC kit (Vector; Burlingame, CA) with 3,3'-diaminobenzidine substrate (DAB), after quenching with 0.3% H2O2 and blocking with normal goat serum. After 30 min with primary MCT Ab and washing, the biotinylated secondary Ab was added for 30 min, washed, then followed by preformed avidin DH-biotinylated horseradish peroxidase H complex for 30 min. Slides were then overlaid with DAB for 9 min, rinsed, dried, mounted, and coverslipped.
Protein concentrations were measured using the Lowry procedure (Lowry et al. 1951) modified to 0.8 ml final volume to triple the sensitivity; BSA provided the reference standard. For rabbit antisera purification, UV absorption was measured and calculated using the known specific absorption of human IgG. Standard Wright-Giemsa staining (Raber and Buckner 1994
) was used to evaluate the purity of the three white cell fractions, and the reverse ATPase reaction was used to evaluate the three muscle fiber types in frozen sections (Gregory and Griffin 1994
). Preparation of frozen sections for fluorescence microscopy and instrumentation used were the same as previously described (Fishbein et al. 2002
), and statistical analysis employed the GraphPad Prism 4 software (San Diego, CA).
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Results |
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RT-PCR of MCT1, MCT2, and MCT4
RT-PCR was performed to evaluate transcription of MCTs, using two or three primer pairs for each of three MCTs and the mRNA isolated from each of the three cell types. The primer pairs used are listed in Table 2. The PCR product amplimers were run on agarose gels and visualized by ethidium bromide staining. An example is shown in Figure 1
, in which three amplimers of MCT1 were obtained for granulocytes, lymphocytes, and monocytes, although the granulocyte bands are quite weak for two of the three PCR products. Figure 2
shows another agarose gel in which an amplimer for each of the three MCTs is present, in this instance from a monocyte isolate. At least two different bands of expected size for each MCT were cut out from the various gels, extracted, and sequenced. All of the sequences provided perfect matches to the database sequences of the corresponding MCTs, thus confirming the transcription of all three MCTs in each of the three white cell isolates. The number of base pairs sequenced for each amplimer are listed in Table 3.
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The results are shown in Figure 4
. In the four upper segments (Figures 4A4D), the best fixatives, methanol (Figure 4C) and acetone (Figure 4D), are compared with the unfixed stained section (Figure 4A), and a companion unfixed (and non-serial) section stained with omission of the first Ab (Figure 4B), which yields a barely detectable image. Although the photographic fields of Figures 4A, 4C, and 4D are not identical, the asterisks mark three fibers that are identical, so that the overlapping areas are easily compared. The methanol fixation mimics faithfully the unfixed section, emphasizing the sarcolemma and located in the same fibers, which were differentiated with the reverse ATPase stain (not shown). The fibers lining the vascular channel are primarily type 2b, which show minimal or no staining with MCT1 (Fishbein et al. 2002). Although acetone fixation also gives good preservation and staining, it is not as accurate for identification, because the cytoplasm is stained quite strongly and is not infrequently discordant with the sarcolemmal staining.
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Figure 5 shows, in composite, the full range of ICC performed. Figure 5M is at x700; all others are at x415. Although the Wright-Giemsastained samples show that the smears (Figures 5A, 5F, and 5K) detail the structure of the mononuclear cells more distinctly than do the droplets (Figures 5B, 5G, and 5L), it was very hard to find enough cells to evaluate the frequency of each feature, so the droplet method was used for ICC. The granulocytes stained strongly, with MCT2 > MCT1 >> MCT4. A feature of MCT1 staining was a dark nuclear membrane, based on our criterion of a line, following part of the nuclear envelope (or a fold in it), which was darker than the nucleoplasm and cytoplasm on either side. Arrows in Figure 5C show two examples. This feature was found in only a subset of cells, whereas cytoplasmic and plasma membrane stainings were almost universal and were occasionally distinctly granular. MCT2 stained the cytoplasm so darkly that our criteria for nuclear membrane staining were difficult to satisfy, but Figure 6 , which shows selected cells from each cell fraction at x1615, presents examples we consider convincing. In general, MCT2 stained the plasmalemma while sparing the nucleoplasm. The arrowheads in Figure 5D point to unstained red cells as a negative control, because these cells express only MCT1. MCT4 staining was weaker but evident in the plasma membrane and cytosol, which again were sometimes granular. We could not find convincing examples of nuclear envelope staining by MCT4 in any of the three cell types.
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Lymphocytes expressed staining patterns similar to those of granulocytes, with MCT2 > MCT1 >>> MCT4. Uniquely in this cell type, MCT2 stained nucleoplasm darker than cytosol and plasmalemma; yet in a number of cells, evidence of nuclear envelope staining could still be identified (see arrows in Figures 5N and 6F). MCT2 also stained the residual platelets in the isolate, emphasizing their plasma membrane (arrowheads in the same figure panels). MCT1 stained both plasma membrane and cytoplasm but not the nucleoplasm. Convincing staining of the nuclear envelope could often be detected, as shown by the arrows in Figures 5M and 6C (these panels are, respectively, x700 and x1615). MCT4 expression was very weak in lymphocytes and was limited to the plasma membrane.
Platelets present in the preparation of lymphocytes showed membrane staining only with MCT2, as noted above, and in all experiments, no staining was present if the primary Ab was omitted from the staining regimen. In all preparations from all three donors, virtually all of the leukocytes in each fraction showed some staining with each of the MCT antibodies, but the intensity of staining in various locations, i.e., nucleoplasm vs cytoplasm vs nuclear envelope vs plasmalemma varied considerably, so that only approximate and semiquantitative estimates are appropriate for these ICC studies. In addition, the ICC data indicate that attempts to reliably quantify Western blots are compromised by the many cell compartments that may be variably contributing MCTs to the SDS extracts (as well as to the cells themselves), unlike the situation encountered in most other tissues (Fishbein et al. 2002).
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Discussion |
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Four fixatives were evaluated in comparison to the unfixed frozen section, selecting the same microscopic fields. In summary, the staining pattern of the unfixed specimen = methanol fix acetone fix > formalin fix >>> ethanol fix. This satisfied our requirements, because methanol was so effective, but more fixatives and reactivation techniques could be added. In addition, we were able to quantify the decay of antigen activity during formalin fixation by automated photomicrography exposure times using the same field of view.
There has been little study of MCTs in leukocytes and none in separated cell types. This is somewhat surprising when one considers that these cells are among the most dependent on glycolysis of all the cells in the body. They must function in moderately to markedly hypoxic environments, such as abscesses, and can undertake such elaborate functions as phagocytosis under such stress, fueled purely by glycolysis. This has been demonstrated for monocytes and macrophages (Cline and Lehrer 1968; Cohn 1968
), neutrophils (Karnovsky 1962
), and eosinophils (Cline et al. 1968
). We would therefore presume that, as in contracting skeletal muscle, leukocytes would also express lactic acid transporters to facilitate its efflux in such states, so as to decrease intracellular acidosis.
Although studies are limited, MCT4 expression in mixed white blood cell populations has been observed at both the mRNA and the protein level. Price et al. (1998) found MCT4 mRNA in leukocytes by Northern blotting analysis, and the protein antigen was identified by Western blotting analysis by Wilson et al. (1998)
. Data on MCT1 and MCT2 expression have not been consistent. Hahn et al. (2000)
observed MCT1 mRNA and protein expression in peritoneal macrophages, whereas both Price et al. (1998)
and Lin et al. (1998)
failed to find MCT1 mRNA in mixed leukocytes by Northern blot, although the latter investigators did find it expressed in lymph nodes. MCT2 mRNA was identified in leukocytes by Lin et al. (1998)
, whereas Price et al. (1998)
reported it to be absent. The inconsistency may be explained by the lower sensitivity of the Northern blotting analysis compared with RT-PCR. Hahn et al. (2000)
could not detect any MCT1 mRNA in a macrophage cell line by Northern blotting, yet the protein antigen was expressed in the same cells, as identified by specific antibodies.
Using RT-PCR, we were able to verify two or three different sequences of each MCT cDNA in each of the three cell types, thus confirming transcriptional expression. Because each isolate contained <10% contamination by the other white cell types, it is unlikely that any of the Western blots are confounded by a false-positive, and this judgment is supported by the direct staining of the individual cells by ICC. Lymphocyte and monocyte extracts gave discrete bands with specific antibodies to each of the three MCTs on Western blotting analysis, with the same mobility in the leukocytes as in the tissue used as positive control. Granulocyte extracts, save for a rare weak band of 45 kD for MCT4, gave weak bands of greater mobility, which were in the 2832-kD range for both MCT1 and MCT2. However, these lower-MW bands were competed out by the corresponding specific peptide antigen just as effectively as were the full-size bands of the mononuclear cell extracts, and are therefore considered to be proteolytic fragments of the MCT1 and MCT2 protein antigens. Proteolysis is very pronounced in granulocytes because of their large stock of enzymes committed to degradation of foreign agents and damaged host cells and macromolecules.
High expression of all three MCTs was observed in monocytes, but they might have been in a stimulated state, because they were isolated by Ab binding to their CD14 receptor site. The entire separation procedure, through slide preparation and fixation, was completed within a matter of hours, so it is quite unlikely that a true activated state could have been attained. It would be necessary to compare positive with negative (i.e., depletion) selection to decipher the possible contribution of stimulation to MCT expression.
Brooks et al. (1999) presented strong evidence for the presence of MCT1 in mitochondria of cardiac and skeletal muscle; so it would seem reasonable to expect it to be expressed in the mitochondria of other cells and tissues as well. However, we see cytoplasmic staining with all three MCTs in all three leukocyte isolates (save for MCT4 in lymphocytes). Whether this represents mitochondria, leukocyte granules (i.e., lysosomes), or other intracellular compartments will require subcellular fractionation and/or electron microscopy to answer. Similarly, nuclear envelope staining seems convincing to us for MCT1 and MCT2 in all three isolates, although this is not definitive. The isolation of intact nuclei would permit a much improved test specimen that could still be evaluated by light microscopy and combined with subcellular compartment enzymology. We point out that Hanu et al. (2000)
observed strong punctate cytosolic labeling of cultured rat astrocytes with both MCT1 and MCT2. In addition, MCT1 labeled aster-like structures near the nuclei, and MCT2 labeled both the nuclei and the trans-Golgi network in their study.
It appears that for certain nuclear functions, even aggregated mitochondria at the nuclear envelope are insufficiently proximate to provide timely levels of ATP via oxidative phosphorylation, unless macromolecular shuttling by creatine kinase or adenylate kinase is also operating in a bucket-brigade fashion (Bessman and Carpenter 1985; Dzeja et al. 1996
,2002
). Indeed, this is further evidence of the inadequacy of simple micromolecular diffusion to satisfy cell requirements. Note that MCT Ab staining of mitochondrial aggregates on or at the nuclear membrane is also another possible explanation for the apparent nuclear membrane staining, which often appears granular or stippled. Otherwise, the presence of MCTs in the nuclear membrane indicates that facilitated transport of some carboxylic acid must be important and that it may not be lactic acid, because it is unlikely that glycolysis is more active in nucleoplasm than in cytoplasm.
The expression of three lactate transporters (with approximate respective Km values of 1 mM, 5 mM, and 25 mM for lactate) in all three major leukocyte fractions means that the export of lactic acid will be at maximal efficiency throughout the range of expected physiological accumulation, i.e., 40 mM to <1 mM. In addition, because the three MCTs are coded by separate genes on separate chromosomes (Garcia et al. 1994
; Lin et al. 1998
; Halestrap and Price 1999
), marked redundancy is present. The loss of one gene would not eliminate facilitated diffusion, although it may be slowed, and the loss of two genes would be extremely improbable.
On the basis of this work and our previous report (Fishbein et al. 2002), the presence of an inactivating mutation in any one of these MCTs should cause loss of expression of that MCT in muscle and in all blood cells. One might not expect the patient to have notable muscle symptoms except under conditions of extreme exercise or comparable stress, such as activity at high environmental temperatures, or in the presence of anemia, or anoxemia from working in a low-O2 environment. Under such conditions, the otherwise healthy person might develop rhabdomyolysis or, less threatening, muscle cramping and elevated creatine kinase, as was found in a military drill sergeant with defective erythrocyte lactate efflux (Fishbein 1986a
). Later, two other military personnel with repeated bouts of muscle cramping and elevated creatine kinase levels were also found to have erythrocyte lactate transport less than half the normal rate. On genetic analysis, all three were found to be heterozygous for a missense mutation in MCT1 that was rare in our normal population (Merezhinskaya et al. 2000
). Whether this was the cause of their symptoms or simply a surprising coincidence is still uncertain, and there has been no further investigation of this question to date.
We know now that these patients should also have the mutation in their white blood cells as well, although there was no clinical information to arouse suspicion. Perhaps homozygosity for the mutation is necessary here, and even then these patients might only manifest a slower than normal recovery from bacillary infections and poor athletic stamina, and thus be cursed with the label of being constitutionally inadequate. The lactic acid transporters therefore, like myo-adenylate deaminase, may be "perquisitory" catalysts, providing perquisites for maximal performance, rather than essential functions, and mutations would cause "diseases of healthy people" (Fishbein 1986b). Only further studies can settle this question, but it would be a grave mistake to disregard "perquisitory catalysts" and "diseases of healthy people." Imagine the number of "constitutional inadequates" there would be in a world without eyeglasses and contact lenses.
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Footnotes |
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Literature Cited |
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Bessman SP, Carpenter CL (1985) The creatinecreatine phosphate energy shuttle. Annu Rev Biochem 54:831862[CrossRef][Medline]
Brooks GH, Brown MA, Butz CE, Sicurello JP, Dubochaud H (1999) Cardiac and skeletal muscle mitochondria have a monocarboxylate transporter MCT1. J Appl Physiol 87:17131718
Cline MJ, Hanifin J, Lehrer RI (1968) Phagocytosis by human eosinophils. Blood 32:922934[Medline]
Cline MJ, Lehrer RI (1968) Phagocytosis by human monocytes. Blood 32:423435[Medline]
Cohn ZA (1968) The structure and function of monocytes and macrophages. Adv Immunol 9:163214[Medline]
Dzeja PP, Bortolon R, Perez-Terzic C, Holmuhamedov EL, Terzic A (2002) Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer. Proc Natl Acad Sci USA 99:1015610161
Dzeja PP, Zeleznikar RJ, Goldberg ND (1996) Suppression of creatine kinase-catalyzed phosphotransfer results in increased phosphoryl transfer by adenylate kinase in intact skeletal muscle. J Biol Chem 271:1284712851
Fishbein WN (1986a) Lactate transporter defect: a new disease of muscle. Science 234:12541256[Medline]
Fishbein WN (1986b) Myoadenylate deaminase deficiency. In Engel AG, Banker BQ, eds. Myology. Vol 2. New York, McGraw-Hill, 17451762
Fishbein WN, Merezhinskaya N, Foellmer J (2002) Relative distribution of three major lactate transporters in frozen human tissues and their localization in unfixed skeletal muscle. Muscle Nerve 26:101112[CrossRef][Medline]
Friesema ECH, Ganguly S, Abdalla A, Manning Fox JE, Halestrap AP, Visser TJ (2003) Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J Biol Chem 278:4012840135
Garcia CK, Li X, Luna J, Francke U (1994) cDNA cloning of the human monocarboxylate transporter 1 and chromosomal localization of the SLC16A1 locus to 1p13.2-p12. Genomics 23:500503[CrossRef][Medline]
Gregory CE, Griffin JL (1994) Enzyme histochemistry of skeletal muscle. In Mikel UV, ed. Advanced Laboratory Methods in Histology and Pathology. Washington, DC, Armed Forces Institute of Pathology, American Registry of Pathology, 161207
Grollman EF, Philp NJ, McPhie P, Ward RD, Sauer B (2000) Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast. Biochemistry 39:93519357[CrossRef][Medline]
Hahn EL, Halestrap AP, Gamelli R (2000) Expression of the lactate transporter MCT1 in macrophages. Shock 13:253260[Medline]
Halestrap AP, Price NT (1999) The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 343:281299[CrossRef][Medline]
Hanu R, McKenna M, O'Heill A, Resneck WG, Bloch R (2000) Monocarboxylic acid transporters, MCT1 and MCT2, in cortical astrocytes in vitro and in vivo. Am J Physiol 278:C921930
Juel C (2001) Current aspects of lactate exchange: lactate/H+ transport in human skeletal muscle. Eur J Appl Physiol 86:1216[Medline]
Juel C, Halestrap AP (1999) Lactate transport in skeletal muscle role and regulation of the monocarboxylate transporter. J Physiol 517:633642
Karnovsky ML (1962) Metabolic basis of phagocytic activity. Physiol Rev 42:143168
Kim-Garcia C, Goldstein JL, Pathak RK, Anderson RGW, Brown MS (1994) Molecular characterization of a membrane transporter for lactate, pyruvate and other monocarboxylates: implications for the Cori cycle. Cell 76:865873[Medline]
Lin RY, Vera JC, Chaganti RS, Golde DW (1998) Human monocarboxylate transporter 2 (MCT2) is a high affinity pyruvate transporter. J Biol Chem 273:2895928965
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265275
Merezhinskaya N, Fishbein WN (2001) Monocarboxylate transporters. In Wiley Encyclopedia of Molecular Medicine. Vol 2. New York, John Wiley, 21192123
Merezhinskaya N, Fishbein WN, Davis JI, Foellmer JW (2000) Mutations in MCT1 cDNA in patients with symptomatic deficiency in lactate transport. Muscle Nerve 23:9097[CrossRef][Medline]
Montero C (2003) The antigen-antibody reaction in immunohistochemistry. J Histochem Cytochem 51:14
Philp NJ, Yoon H, Lombardi L (2001) Mouse MCT3 gene is expressed preferentially in retinal pigment and choroid plexus epithelia. Am J Physiol 280:C13191326
Pilegaard H, Terzis G, Halestrap A, Juel C (1999) Distribution of the lactate/H+ transporter isoforms MCT1 and MCT4 in human skeletal muscle. Am J Physiol 276:E843E848[Medline]
Price NT, Jackson VN, Halestrap AP (1998) Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past. Biochem J 329:321328[Medline]
Raber TL, Buckner L III (1994) Cytopathology techniques. In Mikel UV, ed. Advanced Laboratory Methods in Histology and Pathology. Washington, DC, Armed Forces Institute of Pathology, American Registry of Pathology, 230231
Wilson MC, Jackson VN, Neddle C, Price NT, Pilegaard H, Juel C, Bonen A, et al. (1998) Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3. J Biol Chem. 273:1592015926