PURPOSE: To evaluate the expression and subcellular distribution of proton-coupled monocarboxylate transporters (MCTs) in human RPE in vivo and determine whether ARPE-19 cells retain the ability to express and differentially polarize these transporters. METHODS: Total RNA was prepared from human donor eyes and from ARPE-19 cell cultures. Expression of MCT transcripts was evaluated by RT-PCR amplification. Expression of MCT proteins in human RPE and ARPE-19 cells was evaluated by immunolocalization and Western blot analysis with isoform-specific anti-peptide antibodies. RESULTS: The expression of MCTs in human RPE was investigated by immunofluorescence analysis on frozen sections of human donor eyes. MCT1 antibody labeled the apical membrane of the RPE intensely, whereas MCT3 labeling was restricted to the basolateral membrane. MCT4 was detected in the neural retina but not in the RPE. ARPE-19 cells constitutively expressed MCT1 and MCT4 mRNAs. Expression of MCT3 mRNA increased over time as ARPE-19 cells established a differentiated phenotype. Western blot analysis revealed that ARPE-19 cells expressed high levels of MCT1 and MCT4 but very little MCT3 protein. Sections of differentiated ARPE-19 cells were labeled with MCT1, MCT4, and glucose transporter-1 antibodies. MCT1 was polarized to the apical membrane and MCT4 to the basolateral membrane, whereas GLUT1 was expressed in both membrane domains. CD147, which is necessary for targeting MCTs to the plasma membrane, was detected in the apical and basolateral membranes of human RPE in situ and ARPE-19 cells. CONCLUSIONS: These studies demonstrate for the first time that human RPE expresses two proton-coupled monocarboxylate transporters: MCT1 in the apical membrane and MCT3 in the basolateral membrane. The coordinated activities of these two transporters could facilitate the flux of lactate from the retina to the choroid. ARPE-19 cells express two MCT isoforms, polarized to different membrane domains: MCT1 to the apical membrane and MCT4 to the basolateral membrane. The polarized expression of MCTs in ARPE-19 demonstrates that these cells retain the cellular machinery necessary for transepithelial transport of lactate.
PURPOSE: To evaluate the expression and subcellular distribution of proton-coupled monocarboxylate transporters (MCTs) in humanRPE in vivo and determine whether ARPE-19 cells retain the ability to express and differentially polarize these transporters. METHODS: Total RNA was prepared from humandonoreyes and from ARPE-19 cell cultures. Expression of MCT transcripts was evaluated by RT-PCR amplification. Expression of MCT proteins in humanRPE and ARPE-19 cells was evaluated by immunolocalization and Western blot analysis with isoform-specific anti-peptide antibodies. RESULTS: The expression of MCTs in humanRPE was investigated by immunofluorescence analysis on frozen sections of humandonoreyes. MCT1 antibody labeled the apical membrane of the RPE intensely, whereas MCT3 labeling was restricted to the basolateral membrane. MCT4 was detected in the neural retina but not in the RPE. ARPE-19 cells constitutively expressed MCT1 and MCT4 mRNAs. Expression of MCT3 mRNA increased over time as ARPE-19 cells established a differentiated phenotype. Western blot analysis revealed that ARPE-19 cells expressed high levels of MCT1 and MCT4 but very little MCT3 protein. Sections of differentiated ARPE-19 cells were labeled with MCT1, MCT4, and glucose transporter-1 antibodies. MCT1 was polarized to the apical membrane and MCT4 to the basolateral membrane, whereas GLUT1 was expressed in both membrane domains. CD147, which is necessary for targeting MCTs to the plasma membrane, was detected in the apical and basolateral membranes of humanRPE in situ and ARPE-19 cells. CONCLUSIONS: These studies demonstrate for the first time that humanRPE expresses two proton-coupled monocarboxylate transporters: MCT1 in the apical membrane and MCT3 in the basolateral membrane. The coordinated activities of these two transporters could facilitate the flux of lactate from the retina to the choroid. ARPE-19 cells express two MCT isoforms, polarized to different membrane domains: MCT1 to the apical membrane and MCT4 to the basolateral membrane. The polarized expression of MCTs in ARPE-19 demonstrates that these cells retain the cellular machinery necessary for transepithelial transport of lactate.
Authors: Annette E Allen; Elizabeth A Martin; Katherine Greenwood; Claire Grant; Peter Vince; Robert J Lucas; William S Redfern Journal: Br J Pharmacol Date: 2020-09-13 Impact factor: 8.739