Abstract
The choroid plexus uptake of [3H]cefadroxil was studied in peptide transporter 2 (PEPT2) wild-type and null mice as a function of temperature, transport inhibitors, pH, and saturability. At normal pH (7.4) and temperature (37°C), the uptake of 1 μM cefadroxil was reduced by 83% in PEPT2–/– mice as compared with PEPT2+/+ mice (p < 0.001). A further reduction was achieved in null animals by reducing the temperature to 4°C, or by adding saturating concentrations of unlabeled cefadroxil or p-aminohippurate (p < 0.05). Glycylsarcosine coadministration could inhibit the uptake of cefadroxil in PEPT2+/+ mice (p < 0.01) but not PEPT2–/– mice. Although a proton-stimulated uptake of cefadroxil was demonstrated in PEPT2+/+ mice (pH 6.5 versus pH 7.4; p < 0.01), no pH dependence was observed in PEPT2–/– mice. Kinetic parameters for cefadroxil (without p-aminohippurate) in wild-type mice were: Vmax = 5.4 pmol/mg/min, Km = 34 μM, and Kd = 0.0069 μl/mg/min; in the presence of p-aminohippurate, the parameters were: Vmax = 4.1 pmol/mg/min, Km = 27 μM, and Kd = 0.0064 μl/mg/min. In null animals, the kinetic parameters of cefadroxil (without p-aminohippurate) were: Vmax = 2.7 pmol/mg/min, Km = 110 μM, and Kd = 0.0084 μl/mg/min; in the presence of p-aminohippurate, only a Kd = 0.010 μl/mg/min was observed. Based on kinetic and inhibitor analyses, it was determined that (under linear conditions), 80 to 85% of cefadroxil's uptake in choroid plexus is mediated by PEPT2, 10 to 15% by organic anion transporter(s), and 5% by nonspecific mechanisms. These findings demonstrate that PEPT2 is the primary transporter responsible for cefadroxil uptake in the choroid plexus. Moreover, the data suggest a role for PEPT2 in the clearance of peptidomimetics from cerebrospinal fluid.
Footnotes
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This study was supported in part by Grants R01 GM035498 (to D.E.S.), and R01 NS034709 and P01 HL018575 (to R.F.K.) from the National Institutes of Health. S.M.O. was supported by an American Foundation for Pharmaceutical Education PreDoctoral Fellowship and the Pharmacological Sciences Training Program of the National Institutes of Health (Grant T32 GM007767).
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DOI: 10.1124/jpet.103.060400.
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ABBREVIATIONS: POT, proton-coupled oligopeptide transporter; PEPT, peptide transporter; PHT, peptide/histidine transporter; BCSFB, blood-cerebrospinal fluid barrier; CNS, central nervous system; aCSF, artificial cerebrospinal fluid; MES, 2-(N-morpholino)ethanesulfonic acid; OAT, organic anion transporter.
- Received September 22, 2003.
- Accepted October 30, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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