
   1. Michele Visentin,
   2. Min-Hwang Chang,
   3. Michael F. Romero,
   4. Rongbao Zhao and
   5. I. David Goldman

   1.
      Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy (M.V.); Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester (M.-H.C., M.F.R.); and Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York (R.Z., I.D.G.)

   1. Address correspondence to:
      I. David Goldman, Albert Einstein Cancer Center, Chanin 2, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: i.david.goldman{at}einstein.yu.edu

Abstract

Human organic anion-transporting polypeptide (OATP) 2B1 (OATP-B; SLCO2B1) is expressed in the apical membrane of the small intestine and the hepatocyte basolateral membrane and transports structurally diverse organic anions with a wide spectrum of pH sensitivities. This article describes highly pH-dependent OATP2B1-mediated antifolate transport and compares this property with that of sulfobromophthalein (BSP), a preferred OATP2B1 substrate. At pH 5.5 and low substrate concentrations (∼2.5 μM), only [3H]pemetrexed influx [in contrast to methotrexate (MTX), folic acid, and reduced folates] could be detected in OATP2B1-transfected HeLa R1-11 cells that lack endogenous folate-specific transporters. Influx was optimal at pH 4.5 to 5.5, falling precipitously with an increase in pH >6.0; BSP influx was independent of pH. Influx of both substrates at low pH was markedly inhibited by the proton ionophore 4-(trifluoromethoxy)phenylhydrazone; BSP influx was also suppressed at pH 7.4. At 300 μM MTX, influx was one-third that of pemetrexed; influx of folic acid, (6S)5-methyltetrahydrofolate, or (6S)5-formyltetrahydrofolate was not detected. There were similar findings in OATP2B1-expressing Xenopus laevis oocytes. The pemetrexed influx Km was ∼300 μM; the raltitrexed influx Ki was ∼70 μM at pH 5.5. Stable expression of OAPT2B1 in HeLa R1-11 cells resulted in substantial raltitrexed, but modest pemetrexed, growth inhibition consistent with their affinities for this carrier. Hence, OATP2B1 represents a low-affinity transport route for antifolates (relative affinities: raltitrexed > pemetrexed > MTX) at low pH. In contrast, the high affinity of this transporter for BSP relative to antifolates seems to be intrinsic to its binding site and independent of the proton concentration.
Footnotes

    *

      This work was supported in part by the National Institutes of Health National Cancer Institute [Grant CA82621] (to I.D.G.); the National Institutes of Health National Eye Institute [Grant EY017732] (to M.F.R.); the National Institutes of Health National Institute of Diabetes and Kidney and Digestive Diseases [Grants DK083007, DK090728] (to M.F.R.); and the American Heart Association [Scientist Development Grant] (to M.H.C.).
    *

      Data in this study contributed to the following dissertation: Visentin M (2011) Organic anion transporting polypeptide 2B1 (OATP2B1) as a new pH-dependent low-affinity folate transporter, Ph.D. thesis, University of Udine Graduation School of Medical Sciences, Udine, Italy.
    *

      Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

      http://dx.doi.org/10.1124/mol.111.074823.
    *

      ABBREVIATIONS:

      MTX
          methotrexate (4-amino-10-methyl-pteroylglutamic acid)
      BSP
          sulfobromophthalein
      RFC
          reduced folate carrier
      FCCP
          (4-(trifluoromethoxy)phenylhydrazone)
      OATP
          organic anion-transporting polypeptide
      PCR
          polymerase chain reaction
      PCFT
          proton-coupled folate transporter
      SLC
          solute carrier family
      (6S)5-formylTHF
          (6S)5-formyltetrahydrofolate
      (6S)5-methylTHF
          (6S)5-methyltetrahydrofolate
      MES
          2-(N-morpholino)ethanesulfonic acid
      ORF
          open-reading frame
      IPMX
          pemetrexed current
      E3S
          estrone-3-sulfate
      I-V
          current-voltage.

    * Received July 16, 2011.
    * Accepted October 21, 2011.
