Abstract
We succeeded in isolating a novel organic solute carrier from a human placenta cDNA library. The isolated cDNA consisted of 1137 base pairs that encoded a 379-amino acid protein, hOSCP1. Northern blot and reverse transcription PCR analyses revealed that the hOSCP1 mRNA is expressed in the placenta and testis and weakly expressed in the thymus and small intestine. When expressed in Xenopus laevis oocytes, hOSCP1 mediated the high affinity transport of p-aminohippurate (PAH) (K(m) = 35.0 +/- 7.5 microm) and tetraethylammonium (K(m) = 62.3 +/- 12.2 microm) in a sodium-independent manner. However, the hOSCP1-expressing oocyte did not mediate the transport of L-carnitine. The transport of PAH by hOSCP1 was sensitive to pH, but the tetraethylammonium was not transported at the high pH examined. hOSCP1 transported prostaglandin E(2), prostaglandin F(2alpha), estrone sulfate, glutarate, L-leucine, L-ascorbic acid, and tetracycline. Thus, hOSCP1 also showed broad substrate specificity. A wide range of structurally unrelated organic compounds inhibited the hOSCP1-mediated PAH uptake. Immunohistochemical analysis revealed that the hOSCP1 protein is localized in the basal membrane of the syncytiotrophoblast in the human placenta. Our results suggest that hOSCP1 is a novel polyspecific organic solute carrier protein responsible for drug clearance from the human placenta.
Highlights
The blood-placenta barrier is well known to exist in the human placenta
Our results indicate that human organic solute carrier protein 1 (hOSCP1) is a novel gene that mediates various kinds of organic solutes in a pH-dependent and sodium-independent manner on the basal membrane of the placenta
Two clones were identical to the phospholipid transfer protein (GenBankTM accession number AB076694); the remaining four clones had overlapping identical sequences and were identified as human organic solute carrier protein 1. hOSCP1 has 1137 bp with a single open reading frame encoding a 379-amino acid sequence with a calculated molecular mass of 43.3 kDa (GenBankTM accession number AB079075)
Summary
Materials—[14C]PAH (40.6 mCi/mmol) and [3H]valproate (55 Ci/mmol) were purchased from Moravek (Brea, CA). [14C]TEA (55 mCi/mmol), [3H]tetracycline (1 Ci/mmol), [14C]glutarate (55 mCi/ mmol), L-[14C]leucine (55 mCi/mmol), and L-[3H]carnitine hydrochloride (80 Ci/mmol) were purchased from ARC Inc. The nucleic acids were transferred onto a nylon membrane (Hybond Nϩ, Amersham Biosciences) These filters were hybridized at 42 °C overnight in a hybridization solution (50% formamide) with fulllength cDNA of hOSCP1, which was randomly labeled with [32P]dCTP as described above. Kinetic Study—Concentration-dependent uptake experiments of [14C]PAH or [14C]TEA in oocytes expressing hOSCP1 were performed with each compound at final concentrations of 1, 5, 10, 20, 50, and 100 M and 1, 10, 20, 50, 100, and 200 M, respectively. The compounds were incubated with expressing hOSCP1 oocytes for 2 h at room temperature, stopped with ice-cold ND96 solution, and washed five times as described above. Inhibition Study—For inhibition experiments, oocytes expressing hOSCP1 were incubated for 1 h in ND96 solution containing 10 M [14C]PAH in the presence or absence of various inhibitors at a final concentration of 1 mM, excluding PAH (2 mM). Comparisons of data measuring the initial rates of the uptake of radiolabeled substrates in the presence and absence of inhibitors were performed by the unpaired Student’s t test or one-way analysis of variance
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