Transport of the areca nut alkaloid arecaidine by the human proton-coupled amino acid transporter 1 (hPAT1).

OBJECTIVES: The pyridine alkaloid arecaidine is an ingredient of areca nut preparations. It is responsible for many physiological effects observed during areca nut chewing. However, the mechanism underlying its oral bioavailability has not yet been studied. We investigated whether the H⁺-coupled amino acid transporter 1 (PAT1, SLC36A1), which is expressed in the intestinal epithelium, accepts arecaidine, arecoline, isoguvacine and other derivatives as substrates. METHODS: Inhibition of L-[³H]proline uptake by arecaidine and derivatives was determined in Caco-2 cells expressing hPAT1 constitutively and in HeLa cells transiently transfected with hPAT1-cDNA. Transmembrane transport of arecaidine and derivatives was measured electrophysiologically in Xenopus laevis oocytes. KEY FINDINGS: Arecaidine, guvacine and isoguvacine but not arecoline strongly inhibited the uptake of L-[³H]proline into Caco-2 cells. Kinetic analyses revealed the competitive manner of L-proline uptake inhibition by arecaidine. In HeLa cells transfected with hPAT1-cDNA an affinity constant of 3.8 mm was obtained for arecaidine. Electrophysiological measurements at hPAT1-expressing X. laevis oocytes demonstrated that arecaidine, guvacine and isoguvacine are transported by hPAT1 in an electrogenic manner. CONCLUSION: We conclude that hPAT1 transports arecaidine, guvacine and isoguvacine across the apical membrane of enterocytes and that hPAT1 might be responsible for the intestinal absorption of these drug candidates.

Transport of L-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells.

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 µM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate L-[(3)H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H(+) gradient-stimulated uptake of L-[(3)H]proline. Uptake was rapid, independent of a Na(+) gradient but stimulated by an inwardly directed H(+) gradient with maximal uptake occurring at pH 6.0. L-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K(t)) of 130 ± 10 µM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min(-1 )mg of protein(-1). Glycine, L-alanine, and L-tryptophan strongly inhibited L-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H(+)-amino acid cotransport system PAT2.

Protocol for effective differentiation of 3T3-L1 cells to adipocytes.

In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 µM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10.

Three-dimensional quantitative structure-activity relationship analyses of substrates of the human proton-coupled amino acid transporter 1 (hPAT1).

The proton-coupled amino acid transporter hPAT1 has recently gained much interest due to its ability to transport small drugs thereby allowing their oral administration. A three-dimensional quantitative structure-activity relationship (3D QSAR) study has been performed on its natural and synthetic substrates employing comparative molecular similarity indices analysis (CoMSIA) to investigate the structural requirements for substrates and to derive a predictive model that may be used for the design of new prodrugs. The cross-validated CoMSIA models have been derived from a training set of 40 compounds and the predictive ability of the resulting models has been evaluated against a test set of 10 compounds. Despite the relatively narrow range of binding affinities (K(i) values) reliable statistical models with good predictive power have been obtained. The best CoMSIA model in terms of a proper balance of all statistical terms and the overall contribution of individual properties has been obtained by considering steric, hydrophobic, hydrogen bond donor and acceptor descriptors (q(cv)(2)=0.683, r(2)=0.958 and r(PRED)(2)=0.666). The 3D QSAR model provides insight in the interactions between substrates and hPAT1 on the molecular level and allows the prediction of affinity constants of new compounds. A pharmacophore model has been generated from the training set by means of the MOE (molecular operating environment) program. This model has been used as a query for virtual screening to retrieve potential new substrates from the small-molecule, 'lead-like' databases of MOE. The affinities of the compounds were predicted and 11 compounds were identified as possible high-affinity substrates. Two selected compounds strongly inhibited the hPAT mediated l-[(3)H]proline uptake into Caco-2 cells constitutively expressing the transport protein.

Transport of angiotensin-converting enzyme inhibitors by H+/peptide transporters revisited.

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1.

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.