TY - JOUR
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
N1 - Publisher Copyright:
© 2018 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2018/3
Y1 - 2018/3
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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U2 - 10.1124/mol.117.110445
DO - 10.1124/mol.117.110445
M3 - Article
C2 - 29326243
AN - SCOPUS:85041691264
SN - 0026-895X
VL - 93
SP - 208
EP - 215
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 3
ER -