Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant ( Km) for the former (2.4 μM) than for the latter (5.7 μM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the Km values were 1.2 and 5.8 μM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.

The functional characteristics of human proton coupled folate transporter (hPCFT)/heme carrier protein (HCP) 1 were investigated. hPCFT/HCP1 expressed transiently in human embryonic kidney 293 cells mediated the transport of folate at an acidic extracellular pH of 5.5 in a manner independent of Na ϩ and insensitive to membrane potential, but its transport activity was absent at near-neutral pH. Folate transport mediated by hPCFT/hHCP1 at pH 5.5 was saturable with a K m of 1.67 M and extensively inhibited by reduced folates, such as folinate, 5-methyltetrahydrofolate, and methotrexate (MTX). Sulfobromophthalein and 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid were also found to be potent inhibitors of hPCFT/hHCP1, but hemin was found to exhibit only minimal inhibitory effect.When expressed stably as a protein fused with green fluorescent protein (GFP-hPCFT/HCP1) in MDCKII cells, GFP-hPCFT/ HCP1 was mainly localized at the apical membrane, and the cellular accumulation of MTX was higher from the apical side than from the basal side. These functional features of hPCFT/ HCP1 are consistent with those of the well characterized carrier-mediated folate transport system in the small intestine, suggesting that hPCFT/HCP1 is responsible for the intestinal absorption of folate and also MTX. We also found that sulfasalazine is a potent inhibitor of hPCFT/HCP1, which would interfere with the intestinal absorption of MTX when coadministered in therapy for rheumatoid arthritis as well as folate.

ABSTRACT:We have cloned and functionally characterized the rat ortholog of multidrug and toxin extrusion type transporter 1 (rMATE1). The mRNA of rMATE1 was strongly expressed in kidney and detectable in the various tissues such as brain, stomach, colon, lung, liver, spleen, skeletal muscle, and prostate. When stably expressed in HEK293 cells, rMATE1 could mediate the transport of tetraethylammonium (TEA) and cimetidine under the condition where the membrane potential was disrupted by a high concentration of potassium ion and intracellular pH was reduced by NH 4 Cl pretreatment. When extracellular pH was changed from 5.5 to 8.5, the transport of TEA by rMATE1 was greatest at pH 7.5. Kinetic analyses showed that the transports of TEA and cimetidine mediated by rMATE1 were both saturable with a K m of 260 ؎ 10 and 3.01 ؎ 0.21 M, respectively. It was found that cimetidine is the most potent inhibitor of rMATE1, and many other organic cations, such as 1-methyl-4-phenylpyridinium, amiloride, imipramine, and quinidine, are also effective as inhibitors. Pretreatment of the cells expressing rMATE1 with p-chloromercuribenzene sulfonate significantly reduced TEA transport, but this effect was totally reversed by subsequent treatment with dithiothreitol. These results indicate that the functional nature of rMATE1 is consistent with that of the hypothetical organic cation/H ؉ antiporter system in the brushborder membrane of the renal tubular epithelial cells. Accordingly, these results suggest that rMATE1 is an electroneutral and multispecific organic cation transporter energized by the trans-proton gradient, and plays a physiological role in renal secretion of organic cations, including clinically used cationic drugs.

Nifedipine is one of drugs that have been suggested to undergo significant first-pass metabolism by cytochrome P450 (CYP) 3A in the intestine, based mainly on pharmacokinetic analyses of in vivo observations. To further substantiate this suggestion, we examined the metabolic extraction of nifedipine from the rat small intestine, using intestine perfused in situ by a single-pass technique and microsomes in vitro. When the intestinal lumen was perfused with nifedipine solution (30 microM) at the flow rate of 0.15 mL/min and steady-state was achieved, the fraction that disappeared from the intestinal lumen (F(a)) and the fraction absorbed into the mesenteric venous blood (F(a,b)) was 0.26 and 0.13, respectively. Thus, F(a,b) was 50% smaller than F(a), indicating a significant extraction of nifedipine during passage through the intestinal mucosa. When ketoconazole (40 microM), a specific inhibitor of CYP3A, was added to the perfusion solution, F(a,b) was increased to a level comparable with F(a), while F(a) remained unchanged, suggesting the complete inhibition of metabolic extraction by CYP3A. A similar result was obtained for cyclosporin A (40 microM), another specific CYP3A inhibitor. In intestinal microsomes, the metabolic degradation of nifedipine (1 microM) was almost completely inhibited by ketoconazole (10 microM) and cyclosporin A (10 microM), consistent with the results in the perfused intestine. It was also found in intestinal microsomes that anti-rat CYP3A2 antibody can inhibit nifedipine metabolism completely. Thus, the present study demonstrates that nifedipine undergoes significant extraction during passage through the intestinal mucosa, and provides substantial evidence that CYP3A2 is responsible for that.

The mechanism of intestinal glycerol transport was investigated by using the in vitro everted sac method involving the rat small intestine. The uptake of glycerol into everted sacs was saturable with a Michaelis constant (K m ) of 0.77 mM and a maximum transport rate (J max ) of 11.5 nmol/min/100 mg wet tissue weight (wtw), suggesting the involvement of carrier-mediated transport, and was accompanied by unsaturable transport (passive transport) with a membrane permeability clearance (CL m,d ) of 4.9 m ml/min/100 mg wtw. The carrier-mediated uptake of glycerol was inhibited by the removal of Na ؉ and also by the addition of 2,4-dinitrophenol (DNP) and sodium azide (NaN 3 ), which are metabolic inhibitors. These results suggest that the carrier-mediated glycerol transport is Na ؉ -dependent and secondary active. Since glycerol uptake was also inhibited by p-chloromercuribenzene sulfonate (pCMBS), a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier. We further found that glycerol uptake was selectively inhibited by glycerol-3-phosphate, chloramphenicol and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol uptake included D-glucose and 5-fluorouracil, which are known to be transported by specific carriers, and none of the selective inhibitors of glycerol uptake inhibited the uptake of D-glucose and 5-fluorouracil. Therefore, the carriers for these two compounds do not seem to be involved in glycerol uptake. It is likely that the carrier-mediated transport system involved in glycerol uptake is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system might be involved in drug absorption and also that it might be used for oral drug delivery.