Introduction

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The role of the "cation or hydrogen exchange mechanism" in the renal excretion of xenobiotic OCs was first proposed by Baer and colleagues (Baer et al., 1956). This hypothesis is based on observations that induction of aciduria results in an increased excretion of xenobiotic cations such as mecamylamine, nicotine and quinine in humans and dogs (Baer et al., 1956; Haag and Larson, 1942; Haag et al., 1943). Later in vitro studies on luminal membrane vesicles isolated from renal cortical tissue and on isolated perfused renal proximal tubules directly demonstrated OC transport by a proton exchange mechanism (for reviews, see Pritchard and Miller, 1993; Roch-Ramel et al., 1992). Indeed, several xenobiotic OCs that have been shown to be secreted in vivo such has amiloride, cimetidine, and mepiperphenidol are also countertransported for protons in isolated renal cortical luminal membrane vesicles (BBMV) (Rafizadeh et al., 1986; Takano et al., 1985; Wright and Wunz, 1989). Furthermore, functional integration of luminal OC/H⁺ exchange in net tubular secretion of xenobiotic OCs has been demonstrated in perfused proximal tubules. In that preparation, the transepithelial flux of procainamide and cell-to-lumen flux of tetraethylamonium (TEA) are stimulated by acidification of the tubule lumen (McKinney, 1984; Dantzler and Brokl, 1988). These findings suggest a role for secretory exchange of xenobiotic OCs for protons across the luminal membrane in renal tubular secretion in vivo.

Transport of OCs across the luminal membrane is of particular interest, as it is the rate-limiting step in the secretion of these compounds. Proton antiport is thought to be a primary means by which OCs are translocated across the luminal membrane. Although the existence of the luminal OC exchanger is well documented, the physiological role of this transporter in renal excretion of OCs, endobiotic OCs in particular, is not fully understood. Available data suggest that OC/H⁺ exchange may not be the sole means by which endobiotic OCs are transported across the luminal membrane. As demonstrated in rabbit BBMV, choline is poorly transported by the OC/H⁺ exchange mechanism, but is transported by electrogenic facilitated diffusion (Wright et al., 1992). The role of the luminal OC exchanger in secretion of NMN is also quesitonable. Although countertransport of NMN for protons and OCs has been demonstrated in BBMV of several mammalian species (Holohan and Ross, 1981; Griffiths et al., 1992; Ott et al., 1991; Wright, 1985), in isolated perfused proximal tubules of rabbit and snake, secretion of NMN is unaltered by changes in luminal concentrations of protons or OCs (Besseghir et al., 1990; Dantzler and Brokl, 1987). Studies on rabbit BBMV also suggest that guanidine shares a common proton exchange pathway with TEA; however, the kinetic parameters of guanidine/H⁺ exchange indicate that it may be transported by multiple carriers within the luminal membrane (Miyamoto et al., 1989). Other than guanidine, NMN, and choline, few endogenous OCs have been tested for transport by the luminal OC exchanger.

The objective of the present study was to elucidiate the role of the renal luminal OC exchanger in the transport of endobiotic OCs. To this end, the substrate specificity of the luminal OC exchanger for endobiotic and xenobiotic compounds was evaluated using an in vitro experimental model for renal luminal OC transport, avian BBMV (Villalobos and Braun, 1995). Endobiotic and xenobiotic OCs known to undergo net tubular secretion by the intact avian and mammalian kidneys (table 1; Rennick, 1981, Roch-Ramel et al., 1993, Wideman, 1988) were tested against a model substrate for the luminal OC exchanger, ¹⁴C-TEA, which is poorly transported by the luminal multidrug-resistance transporter. The criteria for classifying an OC as a transported substrate were: 1) cis inhibition and 2) trans stimulation of carrier-mediated transport of the model substrate by the test OC (Holohan and Ross, 1981; Wilbrandt and Rosenberg, 1961). Accordingly, each OC was tested for its ability to cis inhibit ¹⁴C-TEA/H⁺ exchange and trans-stimulate ¹⁴C-TEA efflux.

	Materials and Methods

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Animals. Six to 8-month-old White Leghorn hens, Gallus domesticus (1.5-2 kg), reared at the University of Arizona farm were used for all experiments. Before use, birds were given free access to water and commercial mash.

Brush-border membrane vesicle isolation. Kidneys were perfused in situ with ice-chilled homogenization buffer (50 mM mannitol, 20 mM Tris-HEPES, pH 7.4, excised, minced and weighed to the nearest 0.1 g. Kidney tissue from a single animal (8-20 g) was used for each BBMV preparation. Brush-border membranes were isolated from a crude renal homogenate by Ca⁺⁺ precipitation and subsequent serial differential centrifugation as described previously (Villalobos and Braun, 1995). The final membrane fraction was suspended and preequilibrated in vesicle buffer containing 100 mM KCl, 200 mM mannitol and 10 mM HEPES-KOH (pH 6.0 or 7.5) for 60 min at 4°C. Any modifications of the vesicle buffer are listed in the figure legends. Protein content of the final membrane fraction was determined using the BioRad protein assay and bovine plasma -globulin as a standard. The mean protein yield was 0.35 ± 0.05% of the total protein in the initial homogenate. In the final membrane fraction, the specific activity of the brush-border membrane enzyme alkaline phosphatase was enriched ~18-fold over that in the initial homogenate; the specific activities of the basolateral and mitochondrial enzymes, Na⁺-K⁺,ATPase and succinate dehydrogenase, were not significantly greater than those in the initial homogenate.

Transport assay. Uptake of ¹⁴C-TEA by BBMV was assayed by rapid filtration at room temperature (23-25°C) as outlined previously in detail (Villalobos and Braun, 1995). Vesicles were preincubated at room temperature for 20 min before the transport assay. In a 12 × 75 mm tube, uptake was initiated with the addition of 10 µl of vesicles to 90 µl of transport buffer containing 65 µM ¹⁴C-TEA. Transport buffer also contained 100 mM KCl, 195-200 mM mannitol, 10 mM HEPES-KOH (pH 7.5); concentrations of test compounds in the transport buffer are listed in the tables and figure legends. Uptake was terminated with the addition of 1 ml of ice-chilled "stop" buffer (200 mM KCl, 2 mM CaCl₂, 0.1 mM HgCl₂ and 20 mM Tris-HEPES, pH 7.8). Under vacuum, 1 ml of this mixture was collected on a prewetted nitrocellulose filter (Millipore, HAWP 024, 0.45 µm pore size) that was then washed with 4 ml of ice-cold stop buffer. The filter was placed in a scintillation vial with 10 ml of EcoLite (ICN Biomedicals, Inc.), and the radioactivity was determined by a liquid scintillation system (Beckman LS 5801 spectrometer). Correction was made for nonspecific binding of isotope to the filter at each time point. The mean protein content per 10 µl vesicles was 50.1 ± 1.6 µg. Uptake was expressed as picomoles ¹⁴C-TEA per mg vesicle protein. Unless otherwise stated, uptake was assayed at each time point in triplicate in at least three separate vesicle preparations (i.e., n = 3).

Chemicals. ¹⁴C-Tetraethylammonium bromide (56 mCi/mmol) was purchased from Wizard Labs (West Sacramento, CA). Mepiperphenidol (Darstine) was contributed by Merck Sharp and Dome Laboratories (Rahway, NJ). All other chemicals were of the highest purity and obtained from standard sources.

Statistical analysis. In experiments in which cis-inhibition of ¹⁴C-TEA uptake by OCs was tested, uptake was expressed as the absolute value of vesicular isotope content (pmol ¹⁴C-TEA^.mg vesicle protein¹), and data were compared by analysis of variance (ANOVA). During the testing of trans-stimulation of ¹⁴C-TEA efflux by OCs, it was determined that the absolute values for initial vesicular ¹⁴C-TEA content as assayed in the presence of inwardly directed gradients of several test compounds (e.g., amiloride, quinidine) were consistently greater than control values (i.e., no test compound). Therefore, control and experimental values for vesicular ¹⁴C-TEA content were converted to a percent of the initial ¹⁴C-TEA content as assayed in the absence and presence of a given test compound, respectively, i.e. the fraction of ¹⁴C-TEA retained. The percent of ¹⁴C-TEA retained at 5 sec and 15 sec in the presence of each test compound was statistically compared to corresponding control values by three-way analysis of variance. Differences were deemed significant when the probability values were less than .05.

	Results

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Cis-inhibition of TEA/H⁺ exchange by OCs. Transmembranal transport of a model OC, TEA in these studies, by the renal OC exchanger requires that the cation bind to the substrate site on the carrier. Thus, binding of TEA to the exchanger and its subsequent transport would be inhibited by a second OC present at the same face of the membrane. Impedence of transport of a model substrate by a second substrate in this manner, referred to as cis inhibition, is an index of carrier specificity or affinity (Holohan and Ross, 1981; Wilbrant and Rosenberg, 1961). Based on this principle, the affinity of the luminal OC exchanger for several test OCs was assessed. Five-second uptake of 65 µM ¹⁴C-TEA was assayed in the presence of an outwardly-directed proton gradient (pH_in 6.0: pH_out 7.5) and 100 µM of each endogenous and xenobiotic OCs, including unlabeled TEA, was assayed (fig. 1). Unlabeled TEA inhibited ¹⁴C-TEA uptake by 20%. The endogenous cations, ACh, choline and guanidine each failed to inhibit TEA uptake (P < .08; fig. 1A). However, thiamine and serotonin moderately inhibited TEA transport, decreasing uptake by ~60% and ~45%. Epinephrine and NMN were less effective, reducing uptake by ~20%. In contrast, xenobiotic cations were potent inhibitors of TEA/H⁺ exchange (fig. 1B). Amiloride and quinidine each inhibited ¹⁴C-TEA uptake at least 90%; however, mepiperphenidol (Darstine) inhibited uptake ~60%, and isoproterenol decreased uptake only 30%. Equilibrium vesicular content of ¹⁴C-TEA (2 hr; no inhibitor, 59.0 ± SE 1.1 pmol^.mg protein¹) was not altered by any test compound (e.g., ranitidine, 62.5 ± SE 2.4 pmol^.mg protein^1.2 hr¹). Excluding the endogenous OCs ACh, choline, and guanidine, the cis inhibitory potency of the OCs tested at 100 µM decreased in the order: quinidine > amiloride > quinine > cimetidine  ranitidine > Darstine > thiamine > procainamide > serotonin > isoproterenol > TEA  epinephrine  NMN.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Cis effects of 100 µM of (A) endogenous and (B) xenobiotic OCs on proton-driven TEA uptake by avian renal BBMV. Vesicles were pre-equilibrated in vesicle buffer (pH 6.0) for 60 min at 4°C. At 25°C, 5 sec uptake of 14C-TEA was assayed by diluting 10 µl BBMV with 90 µl isosmotic transport buffer (pH 7.5) with 65 µM 14C-TEA and 0 or 111.11 µM of a test OC. Vesicle and transport buffers each contained 100 mM KCl, 200 mM mannitol and 10 mM HEPES-KOH. Each OC was tested in triplicate in three separate BBMV preparations (i.e., n = 3). Data are displayed as a percent of uptake in the absence of inhibitor (Control; mean ± SE); however, absolute values of 14C-TEA uptake (pmol/mg protein-5 sec) were statistically analyzed by ANOVA. For Control vs. test OC: *P < .05. Control 14C-TEA uptake values at 5 sec and 2 hr were 172.7 ± SE 5.7 and 59.0 ± SE 1.1 pmol/mg protein. Acetylcholine, ACh; mepiperphenidol, Darstine; N1-methylnicotinamide, NMN.

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Trans-stimulation of TEA efflux by organic cations. Translocation of ions by an antiport or exchange mechanism involves physical coupling of the flux of one substrate to the counterdirected flux of a second. As demonstrated in this avian renal BBMV system, the OC exchanger mediates the exchange of TEA for either protons or OCs, TEA and NMN (Villalobos and Braun, 1995). Therefore, if a test OC were indeed a substrate for the OC exchanger, the mediated flux of TEA should be stimulated in the presence of a counterdirected transmembrane gradient of the test OC. Augmentation of mediated transport of a model substrate by a second substrate in this manner, or trans-stimulation, is an index of the carrier's transport capacity. Therefore, effectiveness of test OCs to trans-stimulate TEA efflux from BBMV in the absence of a pH gradient (pH_in = pH_out = 7.5) was examined. Vesicles were preloaded with 150 µM ¹⁴C-TEA, and then incubated with 0 or 1 mM of each test OC for 0, 5 and 15 sec (figs. 2 and 3). Under control conditions, vesicular ¹⁴C-TEA content decreased over time, such that 71.5% ± SE 5.2% was retained at 5 sec, and 54.8% ± SE 4.2% at 15 sec. External 1 mM unlabeled TEA produced further loss of isotope; roughly 45% and 40% of the initial ¹⁴C-TEA was retained at corresponding time points (fig. 3A). As a group, the endogenous OCs moderately stimulated time-dependent ¹⁴C-TEA efflux (fig. 2A). However, external thiamine failed to stimulate TEA efflux (i.e. the percent of ¹⁴C-TEA retained in BBMV was not significantly different than control; P < .07, fig. 2A). Although external ACh, choline and guanidine failed to inhibit TEA uptake, each stimulated TEA efflux, decreasing vesicular ¹⁴C-TEA content through 15 sec by an additional 10% below control (e.g., choline, 5-sec vesicular ¹⁴C-TEA retention, 55.4% ± SE 5.4%; fig. 2B). External epinephrine, NMN, and serotonin also stimulated TEA efflux, as indicated by the ~15-10% decrease in vesicular isotope content with time (fig. 2B). Other than unlabeled TEA, isoproterenol was the only test xenobiotic OC to significantly trans-stimulate ¹⁴C-TEA efflux, and like TEA, decreased 5 and 15 sec vesicular ¹⁴C-TEA content ~20% and ~10% below control (fig. 3A). The remaining xenobiotics trans-inhibited, rather than trans-stimulated TEA efflux such that the percent of ¹⁴C-TEA retained within BBMV was significantly greater than control. For example, in the presence of procainamide, 95.3% ± SE 5.3% of the initial vesicular TEA was retained at 5 sec (fig. 3B).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Trans effects of 1 mM endogenous OCs on time-dependent efflux of TEA from avian renal BBMV. Following preequilibration with vesicle buffer (pH 7.5) containing 150 µM 14C-TEA for 60 min at 4°C, 10 µl BBMV were diluted with 90 µl transport buffer (pH 7.5) containing 0 or 1.11 mM of a test OC. A, Trans effects of 1 mM epinepherine (Epi), N1-methylnicotinamide (NMN), serotonin and thiamine. B, Trans effects of 1 mM acetylchoine (ACh), choline and guanidine. Each OC was tested in triplicate in three separate BBMV preparations (n = 3). Retention of 14C-TEA by BBMV at 5 and 15 sec was expressed as a percent of the initial isotope content; data were analyzed by a three-way ANOVA. With the exception of thiamine, each test endogenous OC significantly decreased the fraction of 14C-TEA retained below control values (i.e., trans-stimulated efflux; P < .05).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Trans effects of 1 mM xenobiotic OCs on time-dependent efflux of TEA from avian renal BBMV. A, Trans effects of 1 mM TEA, amiloride, mepiperphenidol (Darstine) and isoproterenol. B, Trans effects of 1 mM procainamide, quinidine, quinine and ranitidine. Each OC was tested in triplicate in three separate BBMV preparations (n = 3). The assay for efflux of 14C-TEA and statistical analysis of data were conducted as described for figure 2. Unlabeled TEA and isoproterenol significantly decreased the fraction of 14C-TEA retained at 5 and 15 sec below control values (i.e., time-dependent efflux was trans-stimulated; P < .05). However, for each of the remaining test xenobiotic OCs, the fraction of 14C-TEA retained was significantly greater than control values (i.e., time-dependent efflux was trans-inhibited; P < .05).

	Discussion

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Extensive data from in vitro and in vivo experimental systems implicate a pH-dependent carrier system, presumably the luminal OC exchanger, in the renal secretion of many xenobiotic cations (e.g., Baer et al., 1956; Dantzler and Brokl, 1988). Although many vital endobiotic OCs, such as serotonin, ACh and thiamine, are also secreted by the renal tubules in vivo (Rennick et al., 1984; Roch-Ramel et al., 1992), few have been tested for transport efficacy by the renal luminal OC exchanger. Substrate specificity of the exchanger for these and other endobiotics was evaluated in avian luminal membrane vesicles (BBMV). Several endobiotic and xenobiotic OCs known to undergo net tubular secretion by the intact kidney (table 1) were tested for cis and trans effects on the transport of a model xenobiotic substrate, TEA. Although the data on cis inhibition of OC transport indicate that the relative affinity of the renal luminal OC exchanger was greater for xenobiotics (fig. 1), the data on trans stimulation indicate that xenobiotics were transported with lower relative transport capacity than were endobiotics (figs. 2 and 3).

Luminal OC exchanger's substrate affinity. A broad diversity in structural, physical, physiological, and pharmacological properties exists among the OCs secreted by the kidney. OCs that are known to undergeo net tubular secretion are all primary, secondary or tertiary amines or quarternary ammoniums that carry a positive charge on the amine nitrogen at physiological pH (Pritchard and Miller, 1993; Rennick, 1981; Roch-Ramel et al., 1992). Previous investigators have used several homologous series of structural analogs to systematically quantify relationships of the affinty of the luminal OC exchanger to the chemical and physical properties of OCs based on inhibitory potency of test compounds. In isolated membranes, Wright et al. (1995) observed positive correlations of carrier affinity with the relative lipophilicity and the length of alkyl chain of R-groups attached to the parent structure and a negative correlation with the presence of hydrophilic moities for a series of structurally related quaternary ammonium compounds. Using an in vivo renal microperfusion system, David et al. (1995) tested several series of structurally related amines and quarternary ammoniums and observed positive correlations of carrier affinity with the substrate's molecular size, hydrophobicity and basicity, i.e., pK_a values. Nevertheless, these correlations are general, considering the diversity of structural and chemical properties among OCs that inhibit or are transported by the OC exchanger. For the heterogenous series of OCs tested in the present study, a general relationship of affinity to molecular size was observed; larger compounds, such as amiloride and quinine, were more potent inhibitors of OC transport than were smaller compounds, such as NMN and guanidine (table 1, fig. 1). However, contrary to the previously observed correlation of affinity and pK_a values, compounds with lower pK_a values, such as amiloride and cimetidine, were more potent than compounds with pK_a values as great as 13, such as ACh and serotonin (table 1, fig. 1).

Transport of OCs by the luminal OC exchanger. In several in vivo and in vitro OC transport systems, the affinity of the luminal OC exchanger has been assessed based on relative cis-inhibitory potency of test compounds or inhibitor-induced changes in the K_m for a model substrate (David et al., 1995; Wright et al., 1995; fig. 1). However, the essence of an exchange or antiport mechanism is the physical coupling of the transmembrane flux of one substrate to the opposing flux of a second. Therefore, the relative transport capacity of the OC exchanger for a test substrate cannot be evaluated by cis-inhibitory interactions alone (Holohan and Ross, 1981; Wilbrandt and Rosenberg, 1961). Reorientation or turnover of the carrier is the rate-limiting event in mediated OC exchange. In theory, translocation of a model substrate should be stimulated in the presence of an opposing gradient of a second transported substrate. Thus, an OC transported by the luminal OC exchanger should not only cis-inhibit, but also trans-stimulate transport of the model substrate. The limited capacity of xenobiotics to trans-stimulate TEA transport indicated that exchanger's apparent transport capacity for these substrates is relatively low (fig. 3), despite the high affinity for such substrates (fig. 1B). Conversely, trans-stimlation of TEA transport by endobiotics indicated that they were transported with moderate transport capacity (fig. 2), despite modest affinity for these agents (fig. 1A). There were two exceptions. First, isoproterenol, a modest inhibitor of TEA uptake (fig. 1B), was the only xenobiotic to trans-stimulate TEA efflux, indicating it was transported by the OC exchanger (fig. 3A). Second, thiamine, a moderate inhibitor of TEA uptake (fig. 1), was the only endobiotic that did not trans stimulate TEA efflux, indicating it was a poorly transported substrate (fig. 2A). More specifically, for this series of OCs the ability to trans-stimulate OC transport was inversely related to the cis-inhibitory potency of a given OC. At the concentrations tested, OCs which cis-inhibited proton-driven TEA uptake by 40% or less (i.e., ACh, choline, epinephrine, isoproterenol, guanidine, NMN, serotonin, and unlabeled TEA) trans-stimulated TEA efflux. Conversely, OCs which cis-inhibited TEA uptake by 55% or more either failed to alter TEA efflux (i.e., thiamine) or trans-inhibited efflux (i.e., amiloride, cimetidine, mepiperphenidol, procainamide, quinidine, quinine, and ranitidine).

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