Concentrative transport of antifolates mediated by the proton-coupled folate transporter (slc46a1); Augmentation by a hepes buffer

Rongbao Zhao, Mitra Najmi, Srinivas Aluri, David C. Spray, I. David Goldman

Research output: Contribution to journalArticle

Abstract

The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folatespecific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-bufferedmedium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na1/H1 exchanger(s); it was eliminated by inhibitors of Na1/H1 exchanger (s) or Na1/K1 ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.

Original languageEnglish (US)
Pages (from-to)208-215
Number of pages8
JournalMolecular Pharmacology
Volume93
Issue number3
DOIs
StatePublished - Mar 1 2018

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Proton-Coupled Folate Transporter
Folic Acid Antagonists
Buffers
HEPES
Tumor Microenvironment
Bicarbonates
Membrane Potentials
Pemetrexed
Polyglutamic Acid

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Concentrative transport of antifolates mediated by the proton-coupled folate transporter (slc46a1); Augmentation by a hepes buffer",
abstract = "The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folatespecific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-bufferedmedium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na1/H1 exchanger(s); it was eliminated by inhibitors of Na1/H1 exchanger (s) or Na1/K1 ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.",
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T1 - Concentrative transport of antifolates mediated by the proton-coupled folate transporter (slc46a1); Augmentation by a hepes buffer

AU - Zhao, Rongbao

AU - Najmi, Mitra

AU - Aluri, Srinivas

AU - Spray, David C.

AU - Goldman, I. David

PY - 2018/3/1

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N2 - The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folatespecific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-bufferedmedium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na1/H1 exchanger(s); it was eliminated by inhibitors of Na1/H1 exchanger (s) or Na1/K1 ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.

AB - The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folatespecific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-bufferedmedium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na1/H1 exchanger(s); it was eliminated by inhibitors of Na1/H1 exchanger (s) or Na1/K1 ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.

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