Introduction

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Studies on the cellular basis of peptide transport have provided valuable information concerning the driving force for peptide uptake, the substrate specificity of peptide transporters, and the presence of multiple peptide transport systems. More recently, with the cloning of PepT1 (Fei et al., 1994; Liang et al., 1995; Saito et al., 1995; Miyamoto et al., 1996) and PepT2 (Boll et al., 1996; Liu et al., 1995; Saito et al., 1996), cellular studies were confirmed and then expanded with respect to the molecular and functional aspects of these novel, electrogenic and proton-coupled transporters. There have also been several reports on the structural requirements for substrate recognition by the intestinal and renal peptide carriers (Meredith and Boyd, 1995; Daniel, 1996; Leibach and Ganapathy, 1996; Daniel and Herget, 1997). In general, PepT2 shows some functional similarities to PepT1; however, distinct differences are present with respect to the pH optimum for transport, substrate affinity such that PepT2 is a high-affinity transporter while PepT1 is a low-affinity transporter and substrate specificity.

With respect to substrate specificity, di-, tripeptides and aminocephalosporins are accepted by both the renal and intestinal transporters. In contrast, ACE inhibitors (i.e., captopril and enalapril) lacking an -amino group are believed not to interact with the substrate binding site of the renal carrier protein PepT2 even though these same peptidomimetic drugs are transported by the intestinal peptide/H⁺ symporter PepT1 (Boll et al., 1994; Boll et al., 1996). Still, comparative studies were limited to a couple of ACE inhibitors and were performed in a single experimental system (i.e., cRNA-injected Xenopus oocytes).

Previous studies in isolated perfused rat kidneys (Kugler et al., 1996) and in vivo micropuncture experiments (Smith et al., 1995) suggested that quinapril (fig. 1), an ACE inhibitor peptidomimetic lacking an -amino group, may be reabsorbed in proximal tubules via the proton-coupled peptide transport system. Based on these initial results and conflicting data from other investigators (Boll et al., 1994, 1996), we examined the interaction between quinapril and the high-affinity peptide transporter, PepT2, in rabbit renal brush border membrane vesicles (BBMV). In contrast to other studies, we demonstrate for the first time that quinapril can inhibit glycylsarcosine transport by the renal peptide transporter, and in a noncompetitive manner.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structures of quinapril and glycylsarcosine (GlySar).

	Methods

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Materials. [¹⁴C]Glycylsarcosine (GlySar; 119 mCi/mmol) was purchased from Amersham (Chicago, IL). Quinapril and quinaprilat were gifts from Parke-Davis (Ann Arbor, MI). Enalapril and enalaprilat were gifts from Merck (Rahway, NJ). Additional ACE inhibitors (i.e., lisinopril), cephalosporins (i.e., cefadroxil, cephalexin, cephaloridine, cephalothin and cephapirin), dipeptides (i.e., glycylproline and glycylsarcosine), amino acids (i.e., glycine, proline and sarcosine) and organic acids and bases (i.e., SITS and tetraethylammonium, respectively) were obtained from Sigma Chemical (St. Louis, MO). Other chemicals were obtained from standard sources and were of the highest quality available.

Renal membrane vesicles. BBMVs were isolated using a divalent cation precipitation method described by Evers et al. (1978), as used by McKinney and Kunnemann (1985) and Griffiths et al. (1992), with minor modifications. A male New Zealand White rabbit (2-3 kg) was anesthetized with xylazine (10 mg/kg body weight, i.m.) and ketamine (44 mg/kg body weight, i.m.). Kidneys were perfused in situ via the renal artery with an ice cold solution containing 140 mM NaCl, 10 mM KCl and 1.5 mM CaCl₂. After the kidneys had blanched, they were excised, decapsulated and placed in an ice-cold perfusion solution. The whole cortex plus outer medulla were combined for membrane preparation since both regions possess peptide transport activity (Miyamoto et al., 1988). Typically, ~12 to 15 g of tissue per rabbit were obtained. The tissue was then minced and homogenized in 200 ml of homogenizing buffer #1 (2 mM Tris and 10 mM mannitol, pH 7.0) for 5 min using a Waring blender at speed setting 6. A 0.5 ml aliquot of the homogenate was saved at 4°C for protein and marker enzyme assays. The entire procedure was carried out on ice or at 4°C. A Sorvall RC-5C Plus refrigerated centrifuge (SS-34 fixed-angle rotor) was used for all centrifugations.

Marker enzyme and protein assays. Alkaline phosphatase (ALP), a marker for brush border membranes, was determined using a commercially available kit (Sigma Diagnostics, Kit #245, St. Louis, MO). Cross-contamination of BBMV with basolateral membranes was assessed by measuring the Na⁺/K⁺-ATPase activity according to Jørgensen and Skou (1971) with phosphate determined by the method of Fiske and Subbarow (1925). Protein was assayed according to the method of Bradford (1976) using -globulin as standard.

Uptake experiments. Uptake studies were conducted by a rapid filtration technique (Hopfer et al., 1973) using a 10-place filtering manifold (Hoefer Scientific Instruments, San Francisco, CA). All transport measurements were performed at 22°C. Typically, BBMV were suspended in loading buffer to give a final protein concentration of 5 to 10 mg/ml and incubated for 1 hr at room temperature before uptake measurements. In general, the reaction was initiated by adding 80 µl of uptake medium into 20 µl of the vesicle suspension. At appropriate times, the incubation was terminated by addition of 2 ml ice-cold stop solution (2 mM HEPES and 210 mM KCl, pH 7.5). The content was immediately filtered under vacuum through a prewetted Millipore filter (PHWP, 0.3 µm) and washed five times with 2 ml ice-cold stop solution. The radioactivity remaining on the filters was counted by standard liquid scintillation technique after dissolution in 8 ml scintillation cocktail (Ready Protein, Beckman Instruments, Fullerton, CA). Correction for nonspecific binding was performed by running a zero time in the presence of vesicles where radiolabeled substrate and stop solution were added simultaneously. This value was subtracted from the uptake data. Ganapathy et al. (1984) have reported that glycylsarcosine does not bind to the membrane vesicles, therefore radiolabeled GlySar retained on the filters after blank correction represents intravesicular drug. GlySar is also a model substrate due to its resistance to enzymatic hydrolysis in purified BBMV (Silbernagl et al., 1987).

Data analysis. Unless otherwise specified, data are reported as mean ± S.D. from at least 3 different membrane preparations, with each preparation conducted in triplicate. Statistical comparisons were performed using analysis of variance (ANOVA; SYSTAT v5.03, Systat, Inc., Evanston, IL). Pairwise comparisons were made using Tukey's test. A probability of P  0.05 was considered statistically significant. Nonlinear regression analysis was performed 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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Membrane purity. ALP activity from rabbit BBMV (whole cortex plus outer medulla) was enriched 13.3 ± 2.4-fold, whereas Na⁺,K⁺-ATPase activity was not enriched (0.40 ± 0.05-fold). These values compare favorably with those reported in the literature (Evers et al., 1978; McKinney and Kunnemann, 1985; Griffiths et al., 1992; Miyamoto et al., 1988) and demonstrate that the BBMV preparations were highly purified with negligible cross-contamination.

Concentration-dependent uptake studies. The initial rate uptake (at 10 sec) of radiolabeled GlySar (15 µM) was determined during an inwardly-directed H⁺ gradient, and as a function of increasing concentrations of unlabeled drug (0-5 mM). As shown in figure 2, these preliminary studies indicate the presence of two distinct peptide/H⁺ transport systems, one representing a low-affinity, high-capacity system (i.e., PepT1) and the other a high-affinity, low-capacity system (i.e., PepT2). As a result, subsequent studies were performed using GlySar concentrations of  500 µM, values at which transport is functionally dominated by the high-affinity renal peptide carrier.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Saturable uptake of GlySar in BBMV. Membrane vesicles were suspended in buffer, pH 8.3 (50 mM HEPES, 75 mM Tris and 100 mM K2SO4) and uptake was initiated in buffer, pH 6.0 (50 mM Mes, 50 mM HEPES, 25 mM Tris and 300 mM mannitol; final incubation pH 6.7). The 10-sec uptake of 15 µM [14C]GlySar was measured in the presence of increasing concentrations of unlabeled drug (0-5 mM). The inset shows a Woolf-Augustinsson-Hofstee transformation of the data [GlySar uptake, v (pmol/mg/10 sec) vs. GlySar uptake/concentration, v/S (µl/mg/10 sec)]. Data are mean ± S.E. from 5 separate membrane preparations.

Cis-inhibition studies. The interaction of selected ACE inhibitors, cephalosporins and dipeptides with the renal peptide transport system was investigated by determining the extent of cis-inhibition on H⁺-dependent GlySar uptake. Cis refers to the compound present on the same side of the membrane as radiolabeled GlySar. The 10-sec uptake of GlySar (15 µM) was determined in the presence of an inwardly-directed H⁺ gradient, and in the presence of 1 mM of test inhibitors. As shown in figure 3, quinapril was the only ACE inhibitor that significantly reduced the uptake of GlySar (i.e., 49% of control). Enalapril, enalaprilat, lisinopril and quinaprilat had no effect at the concentrations tested. While the aminocephalosporins cefadroxil and cephalexin inhibited GlySar uptake by 99% and 88%, respectively, cephalosporins without an -amino group (i.e., cephaloridine, cephalothin and cephapirin) had no effect. GlySar uptake was also markedly inhibited by dipeptides (i.e., glycylproline and glycylsarcosine) but not by amino acids (i.e., glycine, proline and sarcosine). These results are consistent with the fact that aminocephalosporins and dipeptides share a common transport system. Other compounds such as SITS (an anion exchange inhibitor) and tetraethylammonium (an organic cation transport substrate) showed no effect, serving as our negative controls. It should be appreciated that the inhibitory effects were specific since no compound altered the equilibrium value of GlySar uptake (as measured at 1 and 4 hr).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Cis-inhibition by ACE inhibitors, cephalosporins and dipeptides on GlySar uptake in BBMV. Membrane vesicles were suspended in buffer, pH 8.3 (50 mM HEPES, 75 mM Tris and 100 mM K2SO4) and uptake was initiated in buffer, pH 6.0 (50 mM Mes, 50 mM HEPES, 25 mM Tris and 300 mM mannitol; final incubation pH 6.7). The 10-sec uptake of 15 µM [14C]GlySar was measured in the absence (control) and presence of 1 mM various test inhibitors. Data are mean ± S.E. from 3-5 separate membrane preparations. SITS, 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid. *Significantly different from control, P < 0.0002.

Dose-response studies. The inhibitory effect of quinapril on H⁺-dependent GlySar uptake was evaluated by the 10-sec uptake of radiolabeled GlySar (15 µM) in the presence of increasing concentrations of quinapril (0.1-5 mM). For comparison, uptake was also measured with increasing concentrations of unlabeled GlySar (0-5 mM). As shown in figure 4, both compounds completely inhibited the uptake of GlySar and in a concentration-dependent manner. However, inhibitory potency of quinapril was less than that of unlabeled GlySar. While the IC50 of quinapril was 1.07 ± 0.05 mM (n = 3), the IC50 of GlySar was 0.186 ± 0.027 mM (n = 5). Therefore, quinapril was ~6 times less potent than GlySar. Slope factor estimates (s) were 1.52 ± 0.05 and unity for quinapril and GlySar, respectively. For all analyses, the coefficient of determination (r²) was  0.987. 

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Dose-response effect of quinapril and unlabeled glycylsarcosine on GlySar uptake in BBMV. Membrane vesicles were suspended in buffer, pH 8.3 (50 mM HEPES, 75 mM Tris and 100 mM K2SO4) and uptake was initiated in buffer, pH 6.0 (50 mM Mes, 50 mM HEPES, 25 mM Tris and 300 mM mannitol; final incubation pH 6.7). The 10-sec uptake of 15 µM [14C]GlySar was measured as a function of increasing concentrations of unlabeled drug (0-5 mM). Data are mean ± S.E. from 3-5 separate membrane preparations. Lines were generated using fitted mean parameters (see text), as determined by nonlinear regression analysis.

Kinetic studies. The dose-response studies demonstrated that the peptide transport system in kidney was significantly and completely inhibited by quinapril. To further investigate the nature of this interaction, the 10-sec uptake kinetics of increasing concentrations of GlySar (15-500 µM) were examined alone and in the presence of 0.5 and 1 mM quinapril. As shown by the Lineweaver-Burk plot in figure 5, a change was observed in the slope and y-intercept of the curve, but not x-intercept in the presence of inhibitor. Kinetic analysis (table 1) revealed a significant and dose-dependent decrease in the apparent V_max of GlySar (421 pmol/mg/10 sec for control vs. 307 and 202 pmol/mg/10 sec in the presence of 0.5 and 1 mM quinapril, respectively; P < .05). In contrast, the K_m values for GlySar were unaltered by the presence of quinapril (154 to 167 µM for all three treatments). Thus, the mechanism of inhibition was clearly of a noncompetitive type, suggesting that quinapril and GlySar do not share a common binding site on the carrier. Further evidence for a noncompetitive inhibition was provided by the Dixon plots in figure 6. As observed, the curves (1/uptake vs. inhibitor) intersect on the x-axis (left) and the slopes of these same curves vs. 1/GlySar have a y-intercept that is significant (right). K_i values, as determined by Lineweaver-Burk, Dixon and dose-response analyses, were in close agreement and approximated 1 mM (table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Lineweaver-Burk analysis of the effect of quinapril on GlySar uptake in BBMV. Membrane vesicles were suspended in buffer, pH 8.3 (50 mM HEPES, 75 mM Tris and 100 mM K2SO4) and uptake was initiated in buffer, pH 6.0 (50 mM Mes, 50 mM HEPES, 25 mM Tris and 300 mM mannitol; final incubation pH 6.7). The 10-sec uptake of increasing concentrations of [14C]GlySar was measured alone and in the presence of quinapril (0.5 and 1.0 mM). Data are mean ± S.E. from 4-5 separate membrane preparations. Lines were generated using fitted mean parameters (see table 1), as determined by linear regression analysis.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Dixon plots of the effect of quinapril on the kinetics of GlySar uptake in BBMV. Membrane vesicles were suspended in buffer, pH 8.3 (50 mM HEPES, 75 mM Tris and 100 mM K2SO4) and uptake was initiated in buffer, pH 6.0 (50 mM Mes, 50 mM HEPES, 25 mM Tris and 300 mM mannitol; final incubation pH 6.7). The 10-sec uptake of increasing concentrations of [14C]GlySar was measured alone and in the presence of quinapril (0.5 and 1.0 mM). Data are mean ± S.E. from 4-5 separate membrane preparations. Lines were generated using linear regression.

Trans-effect studies. Trans-stimulation is one of several criteria that demonstrates the involvement of a carrier or whether compounds share a common carrier. Trans refers to the side toward which the radiolabeled substrate is moving. In these studies, BBMV were preloaded with high concentrations of unlabeled GlySar (2, 5 and 10 mM) or quinapril (2 and 5 mM). The influx of radiolabeled GlySar was then measured in unloaded (control) and loaded vesicles under voltage-clamped conditions, with equal concentrations of K⁺ and H⁺ on both sides of the membrane. As shown in figure 7, unlabeled GlySar was capable of trans-stimulating itself. Moreover, the trans-effect occurred in a concentration-dependent manner with maximal uptake exceeding the equilibrium value (i.e., overshoot phenomenon). In contrast, loading vesicles with quinapril at 2 and 5 mM had no effect. Further loading of quinapril at 10 and 15 mM resulted in trans-inhibition (data not shown). These trans effects were specific since the equilibrium uptake of radiolabeled GlySar (as measured at 1 hr) was not affected when vesicles were preloaded with drug.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Trans-effect of unlabeled glycylsarcosine and quinapril on GlySar uptake in BBMV. Membrane vesicles were suspended in buffer, pH 6.0 (50 mM Mes, 50 mM HEPES, 25 mM Tris and 100 mM K2SO4) without loading compounds (control) or with unlabeled GlySar (2, 5 and 10 mM) and quinapril (2 and 5 mM). Valinomycin was added to the vesicles (6 µg/mg protein) 30 min prior to the uptake measurements. Uptake medium was of the same composition as loading buffer but without test compounds. Uptake was measured as a function of time by diluting vesicles 10-fold with uptake medium containing 15 µM [14C] GlySar. Data are mean ± S.E. from 3 separate membrane preparations.

	Discussion

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Using Xenopus oocytes injected with rabbit PepT1 cRNA (Boll et al., 1994), it was shown that 1 mM ³H-cefadroxil could be inhibited significantly by 10 mM of enalapril or captopril, (but not lisinopril) in pH 6.5 buffer. Electrophysiology experiments in cRNA-injected oocytes also demonstrated that 2 mM captopril was transported by rabbit PepT1 in an electrogenic fashion. From these results, the authors concluded that captopril and enalapril but not lisinopril are substrates for this transporter. In a subsequent investigation (Boll et al., 1996), a similar set of experiments was performed in Xenopus oocytes injected with rabbit PepT2 cRNA. In these studies, it was found that 5 mM of captopril or enalapril had no effect on the pH-gradient dependent uptake of 25 µM ³H-cefadroxil. Electrophysiology experiments in rabbit PepT2 cRNA-injected oocytes were not reported for ACE inhibitors. As a result, the authors concluded that, in contrast to the intestinal peptide/H⁺ symporter PepT1, ACE inhibitors (and other drugs lacking an -amino group) appear not to be transported by the renal carrier protein PepT2. However, in these PepT2 studies, only one concentration was tested for captopril and enalapril, while quinapril was not evaluated. Since quinapril was a more potent inhibitor than enalapril, the concentration being tested may not have been high enough for effective inhibition to occur.

In the present study, quinapril cis-inhibited GlySar uptake, suggesting an interaction with the renal peptide transporter, PepT2. The IC₅₀ and K_i values of quinapril (1 mM) were ~6 times greater than the K_m of GlySar (160 µM). Therefore, quinapril appears to be a low-affinity inhibitor of renal PepT2. Interestingly, kinetic studies revealed that the inhibition is of a noncompetitive type. This result suggests that quinapril interacts with PepT2 at a site distinct from that of GlySar. A similar observation was noted by Yuasa et al. (1994) in which enalapril noncompetitively inhibited the uptake of cephradine in rabbit intestinal BBMV. Although speculative, the authors proposed a model in which two classes of carriers were operative: one with only a substrate binding site, and one with a second enalapril-specific binding site in addition to a substrate binding site. Whether a similar explanation could apply to quinapril remains uncertain given the significant differences that exist between the intestinal (PepT1) and renal (PepT2) transporters in rabbit (Fei et al., 1994; Boll et al., 1996).

In membrane vesicle studies, the definitive criteria for showing a compound as a substrate for a specific transport system is the ability to both cis-inhibit and trans-stimulate a competitor (Holohan and Ross, 1980). In the present study, unlabeled GlySar trans-stimulated the uptake of [¹⁴C]GlySar, thereby confirming translocation across the membrane by the same transporter. In contrast, quinapril showed either no effect or trans-inhibition, suggesting a reduction of carrier availability on the outside of the membrane. Interestingly, enalapril, which noncompetitively inhibited cephradine uptake in intestinal BBMV, also showed a trans-inhibitory effect (Yuasa et al., 1994). The authors attributed this effect to immobilization of the carriers on the inside of the membrane. In general, carrier-mediated transport involves binding of the substrate at one face of the membrane, translocation of the loaded carrier from one face of the membrane to the other, dissociation of the substrate, and reorientation of the empty carrier back to the opposite face of the membrane (Sokol et al., 1987). Trans-stimulation assumes that the binding/dissociation steps are faster than the translocation step, and that the empty carrier migrates more slowly than the loaded carrier (Holohan and Ross, 1980). Therefore, the lack of trans-stimulation raises the possibility that quinapril may interact at the peptide transport site, but have a slow translocation rate. A similar scenario could arise due to a low affinity of quinapril for the transporter. Alternatively, quinapril may bind to the transporter at the inner face of the membrane but dissociate poorly at the outer face. It is also possible that quinapril may bind to the transporter but not be translocated. Regardless, it should be appreciated that the absence of trans-stimulation for a substrate does not necessarily mean that translocation does not occur. In this regard, counterflow studies have revealed a lack of trans-stimulation (Sokol and McKinney, 1990; Griffiths et al., 1992) or trans-inhibitory effects (Sokol et al., 1987; Steffens et al., 1989) for substrates that were known to be translocated by their respective transporters or found to do so in subsequent analyses (Lazaruk and Wright, 1990).

It should be appreciated that PepT1 and PepT2 are present in kidney and that renal BBMV preparations contain both transporters. However, GlySar transport (±quinapril) was dominated by renal PepT2 in our studies by optimizing our kinetic conditions and because of the relatively low expression levels of PepT1 in kidney (Fei et al., 1994; Leibach and Ganapathy, 1996; Daniel and Herget, 1997). In fact, under linear conditions, the high-affinity/low-capacity carrier accounted for over 90% of the flux for GlySar in rabbit renal BBMV (Lin and Smith, 1997). With this in mind, low substrate concentrations were applied to the cis-inhibition studies (fig. 3; GlySar at 15 µM), to the dose-response studies (fig. 4; GlySar at 15 µM), and to the Dixon plots (fig. 6; GlySar at 15-136 µM). Although somewhat higher concentrations were used in the Lineweaver-Burk plots (fig. 5; GlySar at 15-500 µM), reciprocal transformations inherent in this type of analysis (i.e., 1/v vs. 1/C) place greater weight on the low concentration data and less weight on the high concentration data. As displayed in this figure, the data are linear with no systematic deviations from the model. In addition, all methods resulted in essentially the same estimates of K_i as well as in the nature of inhibition for GlySar transport by quinapril (i.e., noncompetitive).

In summary, our studies in rabbit renal BBMV demonstrate for the first time that quinapril, an ACE inhibitor peptidomimetic, can interact with PepT2 and, as a result, cis-inhibit the H⁺-dependent uptake of GlySar. This represents a novel finding in that an -amino group appears not to be an absolute requirement for recognition by the renal peptide transporter PepT2. The interaction was also unique in that inhibition of PepT2 occurred in a noncompetitive manner with a K_i for quinapril of about 1 mM. These results suggest that quinapril is a low-affinity inhibitor of the renal peptide transporter and that it binds to a site distinct from that of the GlySar binding site.