title: MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: a possible route for delivery of monocarboxylic acid drugs to the retina.
abstract: OBJECTIVE
  The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB.
  
  
  METHODS
  TR-iBRB2 cells were cultured at 33 degrees C, and L-lactic acid uptake was monitored by measuring [14C]L-lactic acid at 37 degrees C. The expression and mRNA level of monocarboxylate transporter (MCT)1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively.
  
  
  RESULTS
  The [14C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [14C]L-lactic acid uptake at pH 6.0 was both temperature- and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxyphenylhydrazone (protonophore), alpha-cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H+-coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H+ gradient. [14C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT2 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [14C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na+/H+ exchanger I mRNA was expressed in TR-iBRB2 cells.
  
  
  CONCLUSIONS
  L-Lactic acid transport at the iBRB is an H-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Hosoya
authors.forename: T
authors.initials: T
authors.lastname: Kondo
authors.forename: M
authors.initials: M
authors.lastname: Tomi
authors.forename: H
authors.initials: H
authors.lastname: Takanaga
authors.forename: S
authors.initials: S
authors.lastname: Ohtsuki
authors.forename: T
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001813:Blood-Retinal Barrier / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000493:pharmacokinetics*; D008297:Male; D008954:Models, Biological; D027501:Monocarboxylic Acid Transporters / Q000378:metabolism*; D009930:Organic Chemicals / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000378:metabolism*; D000099233:Monocarboxylate Transport Protein 1
chemical_list: D019344:Lactic Acid; D027501:Monocarboxylic Acid Transporters; D009930:Organic Chemicals; D012333:RNA, Messenger; D027981:Symporters; D000099233:Monocarboxylate Transport Protein 1