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

Hosoya K-i, T. Kondo, Masatoshi Tomi, H. Takanaga, S. Ohtsuki, T. Terasaki

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

Purpose. 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°C, and L-lactic acid uptake was monitored by measuring [ 14C]L-lactic acid at 37°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-trifluoromethoxy-phenylhydrazone (protonophore), α-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 1 mRNA was expressed in TR-iBRB2 cells. Conclusion. L-Lactic acid transport at the iBRB is an H +-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.

Original languageEnglish
Pages (from-to)1669-1676
Number of pages8
JournalPharmaceutical Research
Volume18
Issue number12
DOIs
Publication statusPublished - 2001
Externally publishedYes

Fingerprint

Blood-Retinal Barrier
Retina
Lactic Acid
Blood
Acids
Pharmaceutical Preparations
Messenger RNA
Polymerase chain reaction
Transcription
Reverse Transcription
Real-Time Polymerase Chain Reaction
4-Chloromercuribenzenesulfonate
Dicarboxylic Acids
Sodium-Hydrogen Antiporter
Carrier transport
Salicylates
Endothelial cells
Valproic Acid
Rats
Cultured Cells

Keywords

  • Inner blood-retinal barrier
  • L-lactic acid transport
  • MCT1
  • Retinal capillary endothelial cell line

ASJC Scopus subject areas

  • Chemistry(all)
  • Pharmaceutical Science
  • Pharmacology

Cite this

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. / K-i, Hosoya; Kondo, T.; Tomi, Masatoshi; Takanaga, H.; Ohtsuki, S.; Terasaki, T.

In: Pharmaceutical Research, Vol. 18, No. 12, 2001, p. 1669-1676.

Research output: Contribution to journalArticle

@article{f29b148ff0eb463fa27994d2fe794718,
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 = "Purpose. 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°C, and L-lactic acid uptake was monitored by measuring [ 14C]L-lactic acid at 37°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-trifluoromethoxy-phenylhydrazone (protonophore), α-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 1 mRNA was expressed in TR-iBRB2 cells. Conclusion. L-Lactic acid transport at the iBRB is an H +-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.",
keywords = "Inner blood-retinal barrier, L-lactic acid transport, MCT1, Retinal capillary endothelial cell line",
author = "Hosoya K-i and T. Kondo and Masatoshi Tomi and H. Takanaga and S. Ohtsuki and T. Terasaki",
year = "2001",
doi = "10.1023/A:1013310210710",
language = "English",
volume = "18",
pages = "1669--1676",
journal = "Pharmaceutical Research",
issn = "0724-8741",
publisher = "Springer New York",
number = "12",

}

TY - JOUR

T1 - MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier

T2 - A possible route for delivery of monocarboxylic acid drugs to the retina

AU - K-i, Hosoya

AU - Kondo, T.

AU - Tomi, Masatoshi

AU - Takanaga, H.

AU - Ohtsuki, S.

AU - Terasaki, T.

PY - 2001

Y1 - 2001

N2 - Purpose. 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°C, and L-lactic acid uptake was monitored by measuring [ 14C]L-lactic acid at 37°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-trifluoromethoxy-phenylhydrazone (protonophore), α-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 1 mRNA was expressed in TR-iBRB2 cells. Conclusion. L-Lactic acid transport at the iBRB is an H +-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.

AB - Purpose. 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°C, and L-lactic acid uptake was monitored by measuring [ 14C]L-lactic acid at 37°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-trifluoromethoxy-phenylhydrazone (protonophore), α-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 1 mRNA was expressed in TR-iBRB2 cells. Conclusion. L-Lactic acid transport at the iBRB is an H +-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.

KW - Inner blood-retinal barrier

KW - L-lactic acid transport

KW - MCT1

KW - Retinal capillary endothelial cell line

UR - http://www.scopus.com/inward/record.url?scp=0035662969&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035662969&partnerID=8YFLogxK

U2 - 10.1023/A:1013310210710

DO - 10.1023/A:1013310210710

M3 - Article

C2 - 11785685

AN - SCOPUS:0035662969

VL - 18

SP - 1669

EP - 1676

JO - Pharmaceutical Research

JF - Pharmaceutical Research

SN - 0724-8741

IS - 12

ER -