Introduction

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The peptide transporters expressed in the brush border membrane of the small intestinal and renal epithelial cells are responsible for the absorption of small peptides that consist of two or three amino acids (Leibach and Ganapathy, 1996). Molecular cloning studies have resulted in the identification of two distinct peptide transporters in rabbit (Boll et al., 1994, 1996; Fei et al., 1994), rat (Saito et al., 1995, 1996; Miyamoto et al., 1996), human (Liang et al., 1995; Liu et al., 1995), and more recently mouse (Fei et al., 2000; Rubio-Aliaga et al., 2000). PEPT1 is expressed in intestine and to a smaller extent in kidney, whereas PEPT2 is expressed in kidney but not intestine (Shen et al., 1999). PEPT2 shows some functional similarities to PEPT1; however, PEPT2 shows a higher affinity for the same substrates and different substrate specificity compared with PEPT1. Moreover, several therapeutic agents are accepted as substrates by intestinal and renal peptide transporters, including aminocephalosporins and penicillins (Leibach and Ganapathy, 1996; Daniel and Herget, 1997), some angiotensin-converting enzyme inhibitors (Zhu et al., 2000), the antineoplastic agent bestatin (Saito and Inui, 1993), and two antiviral nucleoside prodrugs (Han et al., 1998; Ganapathy et al., 1998).

ACE¹ inhibitors are important therapeutic agents for treating patients with hypertension and cardiovascular diseases. Most ACE inhibitors are cleared by the kidney via glomerular filtration and tubular secretion. However, little is known about their reabsorption potential. It is generally believed that, in contrast to the intestinal transporter PEPT1, ACE inhibitors without an -amino side chain are not substrates for the renal homolog PEPT2. This belief was based on inhibition and electrophysiology studies in Xenopus oocytes expressing rabbit PEPT1 or PEPT2 (Boll et al., 1994). Subsequent to these experiments, studies in rabbit BBMV have demonstrated that a wide variety of ACE inhibitors can interact with the high-affinity peptide carrier in kidney (Akarawut et al., 1998; Lin et al., 1999). It was also observed that while quinapril was a noncompetitive inhibitor of GlySar, enalapril inhibited the uptake of GlySar in a competitive manner. These findings were confirmed by the electrogenic uptake of captopril and enalapril, but not quinapril, in oocytes expressing rat PEPT1 or PEPT2 (Zhu et al., 2000).

In one BBMV study (Lin et al., 1999), a highly significant correlation was observed between ACE inhibitor affinities for the renal peptide transporter (as assessed by GlySar inhibition) and their experimentally determined lipophilicities. Fosinopril, a phosphinic acid ester prodrug, was the most lipophilic and had the highest potency of the ACE inhibitors tested (micromolar as opposed to millimolar values for IC₅₀). However, these inhibition studies were limited because they did not determine whether or not fosinopril was actually transported by the renal peptide carrier PEPT2.

In view of these findings and since peptide transporters are present in both the intestine and kidney, the mechanism of dipeptide inhibition and direct transepithelial transport of fosinopril were studied in two model cell lines (i.e., Caco-2 for PEPT1 and SKPT for PEPT2). We demonstrate, for the first time, that an ACE inhibitor (i.e., fosinopril) can be transported by PEPT2 and PEPT1, with high affinity and in a concentration-dependent fashion.

	Materials and Methods

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Reagents. [¹⁴C]Glycylsarcosine (GlySar; 106 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Fosinopril sodium and [¹⁴C]fosinopril sodium (3.65 µCi/mg; purity 98.5%) were a gift from Bristol-Myers Squibb Co. (Princeton, NJ). Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA) and SKPT-0193 Cl.1 epithelial cells (Woost et al., 1996) were provided by Dr. Ulrich Hopfer (Case Western Reserve University, Cleveland, OH). Other chemicals were obtained from standard sources and were of the highest quality available.

Cell Cultures. Caco-2 and SKPT cells were cultured in minimal essential medium and in Dulbecco's modified Eagle's/F-12 (1:1) medium, respectively, as described previously (Brandsch et al., 1994, 1995). Culture medium was changed every other day, and the cells were passed every 3 to 5 days by digesting cells with 0.05% trypsin and 0.53 mM EDTA at 37°C.

GlySar Uptake. Each cell line was subcultured in 35-mm disposable plastic dishes. Uptake was measured 4 days after seeding. GlySar uptake was measured in cells using buffer in which the composition was 25 mM Mes/Tris (pH 6.0) or 25 mM HEPES/Tris (pH 7.4), 140 mM choline chloride, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose (Brandsch et al., 1994, 1995).

Fosinopril Intracellular Accumulation and Transepithelial Transport. Each cell line was seeded onto collagen-coated membrane filters (3-µm pores, 4 cm² for Caco-2 cells; 0.4-µm pores, 4 cm² for SKPT cells) inside Transwell cell culture chambers (Costar Plastics, Cambridge, MA) at a cell density of 3 × 10⁵ cells/filter, as previously described (Inui et al., 1992). The cell monolayers were given fresh complete medium every other day and were used between 18 and 21 days after seeding. To evaluate the integrity of the monolayers, transepithelial electrical resistance measurements were performed before experimentation using a Millicell-ERS (Millipore Corporation, Bedford, MA).

Protein Assay. The protein content of the solubilized cell monolayers was determined by the method of Bradford (1976) using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard.

Immunoblot Analysis. Apical membrane vesicles from SKPT cells were prepared as described previously for rabbit renal BBMV (Akarawut et al., 1998; Lin et al., 1999). Membrane proteins were then suspended in SDS sample buffer (1% SDS, 50 mM Tris-HCl, pH 7.0, 20% glycerol, 5% mercaptoethanol, and 0.01 mg/ml bromphenol blue). Samples were subjected to 7.5% SDS-polyacrylamide gel electrophoresis, and resolved proteins were transferred to nitrocellulose membranes and subjected to immunoblot analyses. Antibodies against PEPT1 and PEPT2 were generated by immunization of rabbits with the keyhole limpet hemocyanin-conjugated synthetic peptides (Shen et al., 1999). After incubation with 6% nonfat dry milk in TBS-T (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature, the membranes were incubated with polyclonal antibody (1:1000 dilution in blocking buffer) for 1.5 h at room temperature. The membranes were washed and incubated with the second antibody (peroxidase-conjugated goat anti-rabbit IgG, 1:5000). PEPT1 or PEPT2 protein in the apical membrane was detected by an enhanced chemiluminescence system (ECL Plus; Amersham Pharmacia Biotech).

Fosinopril Stability. The stability of fosinopril was evaluated during its intracellular accumulation and transepithelial transport in cell culture studies. Caco-2 and SKPT cells were incubated in apical buffer, pH 6.0, or basolateral buffer, pH 7.4, for 15 min in the presence of 1 mM [¹⁴C]fosinopril. At the end of incubation, the apical and basal media were aspirated and saved for analysis. The cell monolayers were washed 4 times with the ice-cold uptake buffer, and 1 ml of ice-cold Milli-Q water (Millipore Corporation) was then added. Cells were scraped off the support and sonicated for 10 min. Cell lysate was treated with acetonitrile, vortexed for 5 s, sonicated for 5 min, and centrifuged for 5 min at 4°C. The supernatant was then analyzed by high-performance liquid chromatography, and the concentrations of radiolabeled fosinopril and fosinoprilat were determined. The stability of fosinopril was determined by its recovery and the appearance of fosinoprilat following a 15-min incubation at 37°C (Fig. 1, for structures). Results were evaluated from three separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structures of fosinopril and fosinoprilat.

Drug Assay. Fosinopril prodrug and metabolite were detected using a high-performance liquid chromatography system consisting of a pump (model 510; Waters, Milford, MA), a reversed-phase column (Ultracarbon, 3 µm, C₁₈, 4.6 × 150 mm; Phenomenex, Inc., Torrance, CA), and a radiochromatography detector (FLO-ONE 500TR; Packard Instrument Co., Meriden, CT). The mobile phase was composed of 0.01 M phosphate buffer (pH 6.0) and methanol in a ratio of 20:80 (v/v), and isocratically pumped at 1 ml/min. Retention times for fosinoprilat and fosinopril were 2 and 4.2 min, respectively, under ambient conditions.

Data Analysis. Data are reported as mean ± S.D. of three independent experiments (unless otherwise indicated), with data from each experiment being determined in duplicate or triplicate. Statistical differences among treatment groups were evaluated by analysis of variance and pairwise comparisons were made using Tukey's test ( = 0.05). All statistical computations were performed using SYSTAT (v8.0, Systat, Inc., Evanston, IL). Nonlinear and linear regression analyses were conducted using Scientist (v2.01, MicroMath Scientific Software, Salt Lake City, UT), and a weighting factor of unity. The quality of the fit was determined by evaluating the coefficient of determination (r²), the standard error of parameter estimates, and by visual inspection of the residuals.

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	Results

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Interaction of Fosinopril with PEPT1 and PEPT2. The inhibitory effect of fosinopril on H⁺-dependent GlySar uptake was initially evaluated by the 10-min uptake of radiolabeled GlySar in the presence of increasing concentrations of fosinopril (1-1000 µM) in Caco-2 and SKPT cells. As shown in Fig. 2, A and B, fosinopril substantially inhibited the uptake of GlySar in a concentration-dependent manner in both cell lines. The IC₅₀ values of fosinopril in Caco-2 and SKPT cells were 35.2 ± 1.1 and 29.5 ± 2.4 µM, respectively, indicating a high-affinity interaction with PEPT1 and PEPT2 in these cell cultures. Slope factors (s values) were 1.2 ± 0.1 and 1.4 ± 0.1, respectively, in Caco-2 and SKPT cells.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Dose-response effect of fosinopril on GlySar uptake in Caco-2 (panel A) and SKPT cells (panel B) in pH 6.0 buffer at 37°C. The 10-min uptake of [14C]GlySar (10 µM for Caco-2; 5 µM for SKPT) was measured in cell monolayers as a function of increasing concentrations of unlabeled fosinopril (0-1.0 mM). All data were corrected for nonsaturable uptake and were expressed as mean ± S.E. from three separate experiments. Lines were generated using fitted mean parameters (see text), as determined by nonlinear regression analysis.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Dixon plots of the effect of fosinopril on the kinetics of GlySar uptake in Caco-2 (A, B) and SKPT cells (C, D) in pH 6.0 buffer at 37°C. The 10-min uptake of [14C]GlySar (20-100 µM for Caco-2; 5-20 µM for SKPT) was measured in cell monolayers as a function of increasing concentrations of unlabeled fosinopril (0-100 µM). All data were corrected for nonsaturable uptake and were expressed as mean ± S.E. from three separate experiments. Lines were generated using fitted mean parameters (see Table 1), as determined by linear regression analysis.

Intracellular Accumulation and Transepithelial Transport of Fosinopril. The intracellular accumulation and transepithelial flux of radiolabeled fosinopril were evaluated in Caco-2 cells (Fig. 4, A and B) and SKPT cells (Fig. 4, C and D) using apical buffer, pH 6.0, and basolateral buffer, pH 7.4. In both cell lines, fosinopril accumulation was far more rapid when introduced from the apical as opposed to basolateral surface of the membrane. In addition, the apical-to-basolateral transport of fosinopril was significantly faster than that of drug in the reverse direction (i.e., 3-fold difference for Caco-2; 4-fold difference for SKPT). These findings indicate the existence of distinct transport systems in the apical and basolateral membranes of both cell lines. Moreover, fosinopril is a transportable substrate, and taken together, these systems mediate a unidirectional transcellular transport that corresponds to intestinal absorption (Caco-2 cells) and renal reabsorption (SKPT cells).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Intracellular accumulation and transepithelial transport of fosinopril in Caco-2 (A, B) and SKPT cells (C, D). Monolayers grown in Transwell chambers were preincubated apically and basolaterally for 10 min at 37°C with 2 ml of incubation medium (pH 7.4). Medium was removed and appropriate volumes of uptake buffer containing [14C]fosinopril (10 µM for Caco-2; 5 µM for SKPT) were added to the apical (pH 6.0) or the basolateral side (pH 7.4); control buffer (i.e., no fosinopril) was added to the opposite side. Incubation proceeded for the specified period of time at 37°C. All data were corrected for nonsaturable uptake and were expressed as mean ± S.E. from three separate experiments.

Concentration-Dependent Uptake of Fosinopril. To compare the affinity of fosinopril for peptide transport systems in Caco-2 and SKPT cells, the concentration dependence of the drug's cellular accumulation was evaluated from either the apical or basolateral side of the membrane. As shown in Fig. 5, A and B (Caco-2 cells), K_m values were 154 ± 3 and 458 ± 6 µM when fosinopril uptake was measured from the apical and basal membranes, respectively (p < 0.001). V_max values were 12.8 ± 0.4 and 9.9 ± 0.1 nmol/mg/15 min, respectively, for Caco-2 apical and basal membranes (p < 0.003). In contrast, K_m values were 22 ± 1 and 104 ± 4 µM for fosinopril uptake in SKPT cells when measured from the apical and basal membranes, respectively (p < 0.001) (Fig. 5, C and D). The respective apical and basal V_max values were 0.9 ± 0.1 and 0.4 ± 0.1 nmol/mg/15 min in SKPT cells (p < 0.003). Fosinopril interacted with a single specific transporter at each membrane surface for both cell lines, as demonstrated by the linear relationship of the transformed data (Fig. 5, A-D, inserts). The kinetic data are consistent with the high-affinity, low-capacity properties of PEPT2 in SKPT cells and the low-affinity, high-capacity properties of PEPT1 in Caco-2 cells. These results also corroborate the accumulation and flux findings under the previous subsection, in which different transport systems exist in the apical and basolateral membranes of each cell line.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent uptake of fosinopril in Caco-2 (A, B) and SKPT cells (C, D). The 15-min uptake of [14C]fosinopril (0-1.0 mM) was measured in monolayers grown in Transwell chambers incubated apically (pH 6.0) or basolaterally (pH 7.4) at 37°C. All data were corrected for nonsaturable uptake and were expressed as mean ± S.E. from three separate experiments. Lines were generated using fitted mean parameters (see text), as determined by nonlinear regression analysis. Insets are Woolf-Augustinsson-Hofstee plots of the transformed data (v, nmol/mg/15 min versus v/S, µl/mg/15 min).

Selective Expression of PEPT2 in SKPT Cells. To establish unequivocally that SKPT cell cultures express PEPT2 protein, we performed immunoblot analysis of apical membrane vesicles prepared from these cells. Protein was also extracted from renal and intestinal brush border membrane vesicles for use as positive controls for PEPT2 and PEPT1, respectively. As shown in Fig. 6A, a primary hybridization band of about 85 kDa was detected in SKPT cells using PEPT2 antisera, as was a broad band of similar mass for rat kidney. In contrast, PEPT1 antisera failed to detect a brush border antigen in SKPT cells (Fig. 6B); a strong signal, however, was observed at about 90 kDa for rat intestine. Specificity was assured by preincubation of antisera with the appropriate immunizing synthetic peptide, as reported previously (Shen et al., 1999).

View larger version (60K): [in this window] [in a new window]   Fig. 6.   Immunoblot analyses for PEPT2 (A) and PEPT1 (B) in SKPT cells (100 µg of protein). As positive controls, brush border membrane vesicles (20 µg of protein) were analyzed from kidney (A) and small intestine (B). PEPT2 is observed as a band at 85 kDa and PEPT1 as a band at 90 kDa.

Fosinopril Stability. The stability of fosinopril was determined in the donor, receiver, and intracellular compartments of Caco-2 and SKPT cells. Regardless of whether the drug was introduced at the apical or basolateral membrane surface, significant hydrolysis of fosinopril was not evident in any of the samples being tested. Following incubation, fosinopril to fosinoprilat hydrolysis was <2 and <9% in the intracellular compartment of Caco-2 and SKPT cells, respectively. In both cell lines, 100% of the donor and receiver compartments were composed of parent drug. As a result, it is clear that fosinopril remains overwhelmingly intact during intracellular accumulation and during transepithelial transport across the cell.

	Discussion

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Previous findings demonstrate that fosinopril, an ACE inhibitor prodrug, interacts with the high-affinity peptide transporter in renal brush border membrane vesicles (Lin et al., 1999). This interaction is unique because fosinopril lacks a peptide bond and an -amino side chain. It also inhibits dipeptide uptake at micromolar rather than millimolar concentrations, unlike other ACE inhibitors (Lin et al., 1999; Zhu et al., 2000). However, it is unknown whether fosinopril is a substrate, in addition to being an inhibitor, of PEPT2. Furthermore, since both PEPT1 and PEPT2 are present in kidney, the role of each peptide transporter in the cellular uptake and transepithelial flux of this important drug is unclear. As a result, fosinopril's inhibitory and transport mechanisms were studied in cultured intestinal and renal cell lines. Caco-2 cells, which are of human intestinal origin, are known to express the low-affinity peptide transporter PEPT1, as demonstrated by functional studies (Brandsch et al., 1994) and immunoblot analysis (Fei et al., 1997). In contrast, SKPT cells, which are of rat kidney origin, have been shown to constitutively express the high-affinity peptide transporter PEPT2, according to dipeptide kinetic data and mRNA expression (Brandsch et al., 1995; Ganapathy et al., 1995). Our immunoblot studies have extended these findings and provide definitive evidence that PEPT2, but not PEPT1, protein is expressed in SKPT cell cultures.

Lin and coworkers (1999) observed a strong correlation between the lipophilicity of ACE inhibitors and their affinity for the high-affinity peptide transporter in kidney. In particular, the IC₅₀ values for fosinopril and zofenopril were 55 and 81 µM, respectively, whereas other ACE inhibitors had IC₅₀ values ranging from about 1 to 50 mM. Given the high-affinity of fosinopril (relative to the other drugs) and its availability as radiolabel, this ACE inhibitor was studied further. In the present study, fosinopril was shown to competitively inhibit the uptake of GlySar in Caco-2 and SKPT cells, thereby suggesting that fosinopril may be a transportable substrate of PEPT1 and PEPT2. The direct intracellular accumulation and flux of fosinopril confirmed this previous suggestion. In this regard, the uptake process was proton-stimulated, saturable, and of high-affinity (apical K_m values of 154 and 22 µM for Caco-2 and SKPT, respectively). Furthermore, the preference for apical-to-basal flux of fosinopril in both cell lines is consistent with the vectorial transport of drug from the lumen to blood during intestinal (PEPT1) absorption and renal (PEPT2) reabsorption.

Several lines of evidence point to distinct peptide transporters being present in the basolateral and apical membranes of mammalian intestine and kidney. First, transporter affinities for fosinopril are significantly different in intestinal Caco-2 (K_m values of 154 and 458 µM from apical and basal sides, respectively; p < 0.001) and renal SKPT cells (K_m values of 22.2 and 104 µM from apical and basal sides, respectively; p < 0.001). Second, fosinopril uptake by the basolateral peptide transporter is less sensitive to changes in medium pH than the apical peptide transporter in both cell lines (Fig. 7). Third, Caco-2 cells express PEPT1 (but not PEPT2), whereas SKPT cells express PEPT2 (but not PEPT1). Immunolocalization studies further demonstrate that both intestinal PEPT1 and renal PEPT1 and PEPT2 proteins are found in apical membranes alone (Shen et al., 1999). Thus, functional and molecular expression data support the contention of a distinct peptide transporter being present for efflux of peptides/mimetics from the cell to blood.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   pH dependent accumulation of fosinopril in Caco-2 (A) and SKPT cells (B). The 15-min uptake of [14C]fosinopril (10 µM for Caco-2; 5 µM for SKPT) was measured in monolayers grown in Transwell chambers containing buffer at pH 5.5 to 8.0 versus pH 7.4 for donor versus receiver compartments, respectively, at 37°C. All data were corrected for nonsaturable uptake and were expressed as mean ± S.E. from three separate experiments.

The nature of this basolateral transporter(s) is currently unclear. In this regard, Terada and coworkers (1999) suggested that a single facilitative peptide transporter was expressed at the basolateral membrane of Caco-2 cells with PEPT1 being present in the apical membrane. This conclusion is based, in part, on the intracellular to extracellular concentration ratios of GlySar, estimated from specific apical and basolateral uptake studies at equilibrium (i.e., 12.9 and 1.25, respectively). If we use the same Caco-2 cellular volume of 3.66 µl/mg of protein as these authors did, then the fosinopril intracellular to extracellular concentration ratios are approximately 32 and 10, when determined from the apical and basal membrane surfaces, respectively. As a result, both processes are concentrative and fosinopril transepithelial flux seems to be mediated in the intestine by two different active transport systems acting in concert. Although this finding differs from that of Terada et al. (1999), it is consistent with the results of Thwaites and coworkers (1993a,b), in which transepithelial GlySar transport in intestinal Caco-2 cells was reported to occur by H⁺-coupled carriers at both the apical and basal membranes.

Even less functional and molecular information is available on basolateral transport mechanisms of peptides and peptidomimetic drugs in kidney. Recently, the functional expression of a peptide transporter was reported in the renal basolateral membrane of MDCK cells. In these studies, Terada and coworkers (2000) characterized a high-affinity basal transporter in which GlySar (K_m of 71 µM) was inhibited by di- and tripeptides, but activity was maximally stimulated at neutral pH. In concluding, these authors indicated that the functional properties of the renal basolateral transporter were different from those of known peptide transporters (PEPT1 and PEPT2) and the intestinal basolateral peptide transporter. Still, the relevance of this transporter is unclear since MDCK cells display features of distal tubules or collecting ducts (Handler, 1986) as opposed to proximal tubules where peptide reabsorption occurs (Shen et al., 1999). Moreover, although MDCK cells express a H⁺-peptide cotransporter at the apical membrane, its kinetic characteristics are that of PEPT1 and not PEPT2 (Brandsch et al., 1995). For these reasons, we chose to study SKPT cell cultures as a model for PEPT2-mediated transport in the kidney. Based on our kinetic data for fosinopril inhibition, uptake, and flux, as well as our immunoblot analyses, it seems that PEPT2 mediates the H⁺-coupled apical uptake of peptides/mimetics from the tubular lumen into the renal cell. The differential affinities of fosinopril and pH-dependent uptake studies also indicate that the basolateral transporter is functionally distinct from that of PEPT2. Since we are unaware of the intracellular volume of SKPT cells, it is difficult to determine (as we did for Caco-2 cells) whether apical and basal uptakes are concentrative or equilibrative. However, if we assume that SKPT and Caco-2 cells have similar volumes (i.e., 3.66 µl/mg of protein), then the cell to medium ratios would be approximately 20 and 6 for the respective apical and basolateral uptakes. These estimates would suggest that SKPT cells (like Caco-2 cells) transport fosinopril in a concentrative manner and by two different active transport processes. For both cell lines, efflux across the basolateral membrane would be rate-limiting due to its lower affinity compared with apical uptake. Ultimately, the precise nature of the basolateral peptide transporter remains to be determined and when cloned should help to elucidate this issue.

Our findings are potentially very important for the design of newer drug candidates that can target peptide transporters in the intestine and kidney. In comparing fosinopril with other ACE inhibitors, it seems that increased lipophilicity can lead to high-affinity interactions with PEPT1 and PEPT2 (Zhu et al., 2000) and to the direct uptake and transepithelial transport of intact fosinopril (this study). As observed for fosinopril here, and valacyclovir previously (Han et al., 1998; Ganapathy et al., 1998), a peptide bond is not a prerequisite for substrate recognition by the intestinal and renal peptide transporters. It is reasonable that fosinopril absorption would be favored by having a high affinity for PEPT1. However, the clinical relevance of having a high affinity for PEPT2 is not obvious since fosinopril is hydrolyzed extensively to its active moiety, fosinoprilat, in the gut and liver during presystemic metabolism (Singhvi et al., 1988; Morrison et al., 1990). Notwithstanding this uncertainty, renal PEPT2 may still be valuable in reabsorbing unhydrolyzed fosinopril in the urine and recirculating the prodrug to hepatic and extrahepatic sites, including the kidney. In doing so, PEPT2 would allow for the efficient conversion of all intact drug and thereby increase the systemic exposure of the pharmacologically active species. It should also be appreciated that brain PEPT2 may be important for drug delivery and targeting since this protein is expressed and functionally active in choroid plexus (Berger and Hediger, 1999; Novotny et al., 2000; Teuscher et al., 2000). Although speculative, PEPT2-mediated transport of ACE inhibitors may occur in brain since, after a single oral dose to rats, fosinopril produced an immediate inhibition of brain ACE that lasted for at least 4 days (Cushman et al., 1989). A peptide carrier system present at the blood-brain barrier and/or blood-CSF barrier may be responsible for ACE inhibitor penetration into the brain.

In conclusion, this is the first demonstration that an ACE inhibitor (i.e., fosinopril) can be transported intact by PEPT2 and PEPT1 with high affinity and by a proton-coupled, saturable process. Our findings further suggest that the basolateral peptide transporter is distinct from the apical peptide transporter and that each plays an important role in modulating the intestinal absorption and renal reabsorption of peptides and peptide-like drugs.