Contribution of ion pair complexation with bile salts to biliary excretion of organic cations in rats

Im-Sook Song, Suk-Jae Chung, and Chang-Koo Shim

Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul 151-742, Korea


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of this study was to examine whether ion pair complexation with endogenous bile salts in hepatocytes contributes to the preferential biliary excretion of organic cations (OCs). Tributylmethylammonium (TBuMA; mol wt 200) and triethylmethylammonium (TEMA; mol wt 116) were selected as model OCs that exhibit significant and negligible biliary excretion, respectively, in rats. The apparent lipophilicity of TBuMA, but not that of TEMA, was increased by the presence of either rat bile or specific bile salts, suggesting the formation of lipophilic ion pair complexes for TBuMA with bile salts in the liver. The uptake of TBuMA into canalicular liver plasma membrane (cLPM) vesicles, but not that of TEMA, was increased in the presence of bile salts, with a significant increase for both ATP-dependent transport and passive diffusion. The uptake of TBuMA in the presence of the bile salts was inhibited by representative P-glycoprotein (P-gp) substrates and vice versa, suggesting the involvement of P-gp in the canalicular excretion of TBuMA-bile salt complexes in vivo. Increased affinity toward P-gp is suggested as the mechanism responsible for the increased ATP-dependent transport for the ion pair complexes. We propose that ion pair formation with bile slats in hepatocytes may be responsible for the preferential biliary excretion of high-molecular-weight OCs including TBuMA.

canalicular liver plasma membrane vesicles; P-glycoprotein; tributylmethylammonium; triethylmethylammonium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PREFERENTIAL EXCRETION of high-molecular-weight (e.g., mol wt >200 ± 50) organic cations (OCs) into the bile was established in the 1970s (10, 11). For example, tributylmethylammonium (TBuMA; mol wt 200) is excreted to a significant degree (>40% of iv dose; 13 µmol/kg body wt) into the bile in rats, whereas triethylmethylammonium (TEMA; mol wt 116) is excreted only to a negligible extent (~0.3% of corresponding dose of TBuMA) (21). In a previous study (9), we demonstrated that canalicular membrane transport is primarily responsible for this difference; the in vivo excretion clearance across the canalicular membrane was found to be 188-fold greater for TBuMA than for TEMA. In another study (32), which involved the use of canalicular liver plasma membrane (cLPM) vesicles, we were able to show that TBuMA, but not TEMA, is preferentially transported via an ATP-dependent transport process and that P-glycoprotein (P-gp) appears to play a role in this transport. Therefore, the fact that the transport of TBuMA is ATP dependent whereas the transport of TEMA is not points out a major difference between the two compounds in relation to their hepatobiliary transport. However, the ATP-dependent in vitro intrinsic transport clearance of TBuMA (32) accounts for only 0.6% of the in vivo canalicular excretion clearance (9). Thus it is reasonable to assume that other transport mechanism(s) may be involved in the transport of TBuMA across the canalicular membrane. Currently, the underlying mechanisms for hepatobiliary transport are not fully understood.

In general, high-molecular-weight OCs are known to form lipophilic ion pair complexes in the presence of some organic anions, which results in an increase in the permeability of OCs across biological membranes (23). For example, isopropamide, a quaternary ammonium drug, forms ion pair complexes with organic anions such as trichloroacetate (13), salicylate (35), and bile salts (6), thereby resulting in an increase in gastrointestinal absorption and percutaneous penetration of the drug. Similar results have been reported for a variety of OCs, such as propantheline (16), bretylium (2), AMD-3100 (7), and a number of other drugs (23, 28). Of the counteranions that form ion pair complexes with OCs, bile salts represent the most widely examined compounds (6, 7, 16, 23, 28, 29).

On the basis of these reports, we hypothesize that the formation of lipophilic ion pair complexes with endogenous bile salts in the hepatocyte may be a contributing factor in the transport of some OCs across the canalicular membrane. The reason for this hypothesis is that bile salts are synthesized endogenously in the hepatocyte and hence would be expected to interact with OCs in the hepatocyte before the OCs undergo canalicular transport. Thus, in the present study, we report on a study of the effect of ion pair complexation with endogenous bile salts on the bile canalicular transport of OCs. Our findings show that TBuMA, but not TEMA, forms lipophilic ion pair complexes with bile salts, leading to a significantly increased level of P-gp-mediated active transport as well as the passive diffusion of the compound. Ion pair complexation with endogenous bile salts in the hepatocyte appears to play a critical role in the biliary excretion of OCs.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. [3H]TBuMA (0.2 Ci/mmol) and [3H]TEMA (0.2 Ci/mmol) were synthesized (9, 22, 32) by the reaction of an excess of tributylamine or triethylamine (Aldrich Chemical, Milwaukee, WI) with [3H]methyl iodide (85 Ci/mmol; Amersham, Arlington Heights, IL). [3H]taurocholate (3.7 Ci/mmol) and [3H]daunomycin (4.1 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). n-Octanol was purchased from Aldrich Chemical. Sucrose was purchased from Junsei Chemical (Tokyo, Japan). All other chemicals, including bile salts, were purchased from Sigma Chemical (St. Louis, MO).

Determination of the apparent partition coefficients of organic cations. The effect of rat bile or selected bile salts on the apparent partition coefficient (APC) of OCs between aqueous and organic phases was investigated. n-Octanol and a membrane suspension buffer (MSB, pH 7.4) that contained (in mM) 250 sucrose, 10 HEPES, 10 Tris, 10 MgCl2, and 0.2 CaCl2 were used as the organic and aqueous phases, respectively, in the partition study. n-Octanol and MSB were saturated with respect to one another before use. For the study on the effect of rat bile, the bile was collected from five Sprague-Dawley (SD) rats (250-270 g; housed in the Animal Center for Pharmaceutical Research, Seoul National University, Seoul, Korea) over a 1-h period through cannulation of the bile duct with polyethylene tubing (PE-10, Becton Dickinson, Sparks, MD). Collection for a longer period of time was not performed to minimize possible alteration in the composition of the bile due to obstructed enterohepatic recycling as a result of the cannulation. Total collected bile was pooled and diluted appropriately with MSB to yield 0, 20, 40, 60, 80, and 100% (vol/vol) of biological fluid. TBuMA or TEMA was then dissolved to a concentration of 0.1 mM. To 5 ml of each solution, [3H]TBuMA or [3H]TEMA (0.5 µCi each) was added. Five milliliters of the presaturated n-octanol was then added to each of the above-described aqueous phases (5 ml) in a screw-capped test tube, and the mixture was vortexed vigorously for 5 min followed by shaking for 2 h at 25°C in a temperature-controlled water bath. After standing for 30 min at 25°C in the water bath, the mixture was separated into two phases by centrifugation at 3,000 rpm for 10 min. The radioactivity of a 100-µl aliquot of aqueous or organic phase was measured using a Wallac 1409 liquid scintillation counter (Wallac, Gaithersburg, MD). APC was estimated from the concentration ratio of each compound between the organic phase and MSB.

For the study on the effect of bile salts, endogenous bile salts of rats were selected. The total amounts of bile acids in the liver tissue of rats are known to be 32.7 ± 3.85 nmol/100 mg of liver tissue. The major components are tauro-beta -muricholate (TMC, 36.2%), taurocholate (TC, 31.8%), and taurohyodeoxycholate (THDC, 13.7%) (26). Among these, TC was selected as a conjugated bile salt of a primary bile salt and TMC and THDC were not selected because they were not commercially available. Among minor components of rat bile salts, sodium cholate (CH) and sodium deoxycholate (DC), as representative primary and secondary bile salts, respectively, sodium glycocholate (GC) and sodium taurochenodeoxycholate (TCDC), as conjugated bile salts of primary bile acids, and sodium taurodeoxycholate (TDC) and sodium glycodeoxycholate (GDC), as conjugated bile salts of a secondary bile acid, were selected. The bile salts were dissolved in the MSB to yield concentrations in the range of 0.1-1.6 mM. TBuMA or TEMA was then dissolved at a concentration of 0.1 mM. To 5 ml of each solution, [3H]TBuMA or [3H]TEMA (0.5 µCi each) was added, and the aqueous phase was mixed with 5 ml of the presaturated n-octanol. Subsequent procedures were identical to those of the partition study on the effect of rat bile.

Estimation of true partition coefficients, formation constants, extraction constants, and concentration of ion pair complexes. The APC of an ionic compound (e.g., TBuMA in this study) varies depending on the concentration of the compound and counterions, thereby giving limited information on the lipophilicity of corresponding ion pair complexes. Thus some other parameters appear to be necessary to describe the ion pair complexation regardless of the concentration of ions involved in the complexation. The true partition coefficient (TPC), the formation constant (Kf) in an aqueous phase, and the extraction constant (Ke) between the aqueous and n-octanol phases represent these parameters (28). They were determined using equations developed previously by Shim et al. (28). For the TBuMA-TDC ion pair complex, for example, TPC, Kf, and Ke are defined as [TBuMA-TDC]o/[TBuMA-TDC]w, [TBuMA-TDC]w/([TBuMA]w × [TDC]w), and [TBuMA-TDC]o/([TBuMA]w × [TDC]w), respectively, where brackets indicate concentrations and subscripts o and w denote organic and aqueous phases, respectively. In this estimation, a 1:1 molar ratio formation of ion pair complexes between OCs and bile salts was assumed based on our previous report, in which a 1:1 formation of ion pair complexes of tetrabutylammonium and isopropamide, quaternary ammoniums, with TDC was found (28). The calculation of the concentration of the ion pair complex in the transport medium, i.e., [TBuMA-TDC]w, was made as follows. The [TBuMA-TDC]w-to-[TBuMA]w ratio can be calculated from the definition of Kf, provided that the Kf and [TDC]w values are available. Kf can be easily obtained by the method of Shim et al. (28), and [TDC]w can be approximated to be the initial concentration of TDC in the transport medium, so long as it is added in a large excess relative to TBuMA. The approximate [TBuMA-TDC]w can be estimated from the calculated [TBuMA-TDC]w-to-[TBuMA]w ratio and a measured total TBuMA concentration in the transport medium (i.e., sum of [TBuMA]w and [TBuMA-TDC]w). The concentration of the ion pair complex [TBuMA-TDC]w, as the result of the addition of 10 µM TBuMA and 100 µM TDC, for example, was calculated to be ~7.51 µM, indicating that the majority (~75%) of the TBuMA exists in the form of an ion pair complex in the transport medium under these conditions.

Preparation of cLPM Vesicles. cLPM vesicles were prepared from male SD rats (250-270 g) by the method of Inoue et al. (12) as described in our previous study (32). The activity of alkaline phosphatase (25), a marker enzyme for the canalicular membrane, was enriched ~52-fold in the vesicle preparation compared with that in crude liver homogenates. The protein concentration of the vesicle preparation was 0.14 ± 0.03 mg/g liver, as measured by the Lowry method (19) using bovine serum albumin as a standard. The inside-out proportion of the vesicles was in excess of 30% (i.e., 69.8 ± 8.6% for right-side-out vesicles; mean ± SD for 4 measurements) when determined by measurement of the exposed sialic acid concentration (34), which is consistent with previous reports (5). The functional activity of the vesicle preparation was confirmed by at least a fivefold higher uptake of [3H]TC in the presence of an ATP regenerating system (1.2 mM ATP, 3 mM phosphocreatine, and 3.6 µg/100 µl creatine phosphokinase) compared with the uptake in the absence of ATP (24). Immediately after the preparation, the cLPM vesicles were suspended in MSB to yield a protein concentration of 4-6 mg/ml. The suspension was stored at -70°C, for periods of up to 2 wk, until the transport studies were carried out.

Effect of bile salts on uptake of TBuMA and TEMA into cLPM vesicles in presence of ATP. The effect of TDC, a representative bile salt that forms a lipophilic ion pair complex with TBuMA, on the temporal uptake profiles of [3H]TBuMA and [3H]TEMA into cLPM vesicles in the presence and absence of ATP was measured by a rapid filtration technique (4). Briefly, a frozen vesicle suspension was quickly thawed by immersion in a 37°C water bath, revascularized by passing through a 25-gauge needle 20 times, and appropriately diluted with MSB to a concentration of 1-1.5 mg/ml protein. The diluted suspension (20 µl) was preincubated in a test tube at 37°C for 4 min, and the incubation buffer (80 µl), containing 10 µM [3H]TBuMA or [3H]TEMA (0.16 µCi each), 100 µM TDC, and the ATP regenerating system, was then added to the diluted vesicle suspension. At preselected times, the uptake was quenched by the addition of 4 ml of an ice-cold stop solution, i.e., MSB containing TBuMA or TEMA (1 mM each, pH 7.4). The entire sample was then rapidly filtered through a 0.45-µm (pore size) MF-MEMB 25-mm (diameter) filter (Seoul Science, Seoul, Korea), which had been presoaked for 2 h in the ice-cold stop solution. The tube was rinsed again with 4 ml of ice-cold stop solution and filtered. After being washed with 8 ml of ice-cold stop solution, the filter was dissolved in 4 ml of scintillation cocktail and the radioactivity of the mixture was measured. Presoaking and rinsing the filter with the ice-cold stop solution, which contained 1 mM TBuMA or TEMA in MSB (pH 7.4), resulted in a very small level of nonspecific binding of OCs to the filter (i.e., negligible radioactivity in the filter; data not shown). The binding of TEMA or TBuMA to the surface of the vesicles at equilibrium (i.e., 60 min) was ~15% of the peak uptake value, as estimated from the relationship between the uptake and the osmolarity of the incubation medium. Thus it is not likely that the estimated transport would be affected by the presence of surface-bound OCs.

Characteristics of ATP-dependent uptake of ion pair complexes between TBuMA and bile salts. The concentration dependence of the initial uptake rate of TBuMA-bile salt complexes was examined for various concentrations of [3H]TBuMA (5-1,000 µM, 0.16 µCi each) and a 10-fold molar excess of TDC, TCDC, GDC, and TC in the presence of an ATP regenerating system. The initial uptake rate was obtained from a linear portion (generally up to 30 s) of the temporal profiles of the ion pair complexes and then plotted against the initial concentration of substrates (i.e., ion pair complexes) in the medium. The concentration of the ion pair complex was estimated as described in Estimation of true partition coefficients, formation constants, extraction constants, and concentration of ion pair complexes. The resulting profile was fitted to eq. 1 to estimate the parameters for the mixed uptake processes involving saturable and linear kinetics. A nonlinear regression analysis was performed in the fitting with the WINNONLIN program (version 1.0; SCI Software, Lexington, KY).
V<SUB>o</SUB><IT>=V</IT><SUB>max</SUB><IT>·</IT>S<IT>/</IT>(K<SUB>m</SUB><IT>+</IT>S)<IT>+</IT>CL<SUB>linear</SUB><IT>·</IT>S (1)
where Vo is the initial uptake rate of the substrates (pmol/mg protein per 30 s) and S is the initial concentration of substrates in the medium (µM). Vmax and Km represent the maximum uptake rate and the medium concentration at one-half of the maximal uptake rate, respectively, and CLlinear represents the linear uptake clearance.

Uptake inhibition by various compounds. To elucidate the mechanism of increased uptake of TBuMA into the cLPM vesicles in the presence of TDC and ATP, the inhibitory effect of various compounds on the uptake of TBuMA was measured. To the transport medium, which contained 10 µM [3H]TBuMA (0.16 µCi), 100 µM TDC, and the ATP regenerating system, 100 µM of various compounds such as verapamil, daunomycin and cyclosporin A, TC, benzyl penicillin, and tetraethylammonium and TEMA was added, and the uptake of TBuMA into the cLPM vesicles was then measured for 30 s using the rapid filtration technique described in Effect of bile salts on uptake of TBuMA and TEMA into cLPM vesicles in presence of ATP.

The potential involvement of the multidrug resistance (MDR) transporter P-gp in the increased uptake of TBuMA in the presence of TDC and ATP was examined by measuring the inhibitory effect of various concentrations of TBuMA-TDC complex (prepared by using 1, 10, and 100 µM TBuMA and a 10-fold molar excess of TDC in the transport medium) on the 30-s uptake of 0.5 µM [3H]daunomycin (0.16 µCi) into the vesicles. The involvement of the bile salt export pump Bsep in the increase in the ATP-dependent uptake of TBuMA in the presence of TDC was also examined by measuring the effect of varying concentrations of TBuMA (0, 10, 100, and 1,000 µM) on the inhibitory effect of TDC (10 µM) on the ATP-dependent 30-s uptake of 1 µM [3H]TC (0.5 µCi).

The experimental conditions (e.g., the composition of the ATP regenerating system, buffer composition, etc.) in these studies were identical to those used for the TBuMA uptake study, except that TBuMA was replaced in the ice-cold stop solution by 50 µM daunomycin (for the P-gp study) or 1 mM TC (for the Bsep study).

Data analysis. All data are expressed as means ± SD. A two-way analysis of variance was performed to test the differences between transport conditions in the temporal uptake profiles. Student's t-test was used to test the inhibitory effect of various compounds on the mean uptake value. In all cases, P < 0.01 was accepted as denoting a statistical difference.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of bile and bile salts on partitioning behavior of TBuMA and TEMA. Figure 1 shows the APC profiles of TBuMA and TEMA between MSB (pH 7.4) and n-octanol in the presence of different volumes of rat bile (Fig. 1A) and different concentrations of bile salts (Fig. 1B). In general, the APC of TBuMA increased as a function of the concentration of bile (0-100% vol/vol) as well as bile salts (0.1-1.6 mM), suggesting the formation of lipophilic ion pair complexes of TBuMA in their presence. The components of MSB, such as HEPES and Tris, had no effect on the APC of TBuMA and TEMA (data not shown). Of the bile salts examined, the conjugated salts of deoxy bile acids (i.e., TDC, TCDC, and GDC) showed a larger increase in the APC of TBuMA compared with the other bile salts (i.e., CH, DC, GC, and TC), consistent with previously published findings (6, 16). This effect was the most prominent for TDC, followed by TCDC, GDC, TC, GC, DC, and CH. The conjugates of a C-7 (i.e., TDC or GDC) or a C-12 (i.e., TCDC) dehydroxylated bile salt exhibited significant increase in the APC of TBuMA, whereas the conjugates of a C-3, -7, -12 trihydroxylated bile salt (i.e., TC and GC) did not. This suggests that the conjugation of a carboxyl group on the corresponding bile acid as well as dehydroxylation at the C-7 and/or C-12 positions may enhance the APC of OCs by accelerating ion pair formation with the OCs. In this sense, THDC, which comprises 13.7% of total bile acids in the rat liver, is of interest because it is conjugated and dehydroxylated at the C-12 position. The contribution of THDC, however, was not investigated in the present study, because it was not commercially available. The preferential ion pair formation of TBuMA with some dehydroxylated bile salts is consistent with a previous suggestion that the extent of the hydrophobic interaction is dependent on the shape, size, and conformational flexibility of both ions involved in ion pair formation (33). In contrast to TBuMA, the APC of TEMA was not affected by the presence of bile and bile salts, even at their highest concentrations (i.e., at 100% vol/vol bile and 1.6 mM bile salts), suggesting the possibility that TEMA does not form lipophilic ion pair complexes with these components in the hepatocyte.


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Fig. 1.   Effect of various concentrations of rat bile (A) and bile salts (B) on the apparent partition coefficients (APC) of tributylmethylammonium (TBuMA, 0.1 mM; solid lines) and triethylmethylammonium (TEMA, 0.1 mM; broken lines) between n-octanol and membrane suspension buffer (MSB, pH 7.4). Each data point represents the mean ± SD of triplicate measurements. , Taurodeoxycholate (TDC); open circle , taurochenodeoxycholate (TCDC); black-down-triangle , glycodeoxycholate (GDC); down-triangle, taurocholate (TC); , glycocholate (GC); , deoxycholate (DC); black-lozenge , cholate (CH).

The APC of TBuMA represents the apparent partition of both ion pair complexes and TBuMA itself (28). Thus, to differentiate the partition characteristics of ion pair complexes from the apparent partition, the values of TPC, Ke, and Kf for the ion pair complexes were determined using the equations developed by Shim et al. (28), assuming a 1:1 (OCs-bile salts) formation of ion pair complexes (Ref. 28; Table 1). As expected from Fig. 1B, the largest TPC and Ke values were obtained for the TBuMA-TDC ion pair complex, followed by the TCDC, GDC, TC, GC, DC, and CH complexes of TBuMA. As a result, 69-, 48-, 38-, 24-, 12- and 8-fold increases in TPC, compared with TBuMA itself (0.12), were observed for the respective ion pair complexes of TBuMA. In contrast to TBuMA, no increases in any of the parameters (i.e., TPC, Kf, and Ke) were observed for TEMA by the addition of the bile and bile salts (data not included in Table 1). For the partition of TBuMA in the presence of rat bile (Fig. 1A), TPC, Kf, and Ke values could not be calculated because of a lack of molecular information on the component and the concentration of the ion-pairing organic anions (e.g., bile salts) in the bile.

                              
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Table 1.   True partition coefficients, extraction constants, and formation constants of ion pair complexes of TBuMA with various bile salts

Effect of bile and bile salts on temporal uptake of TBuMA and TEMA into cLPM vesicles. To deduce preliminary information on the interaction of OCs in the hepatocytes before being transported across the canalicular membrane, the effect of bile in the transport medium on the uptake of TBuMA and TEMA into cLPM vesicles was examined (Fig. 2A). As is obvious from Fig. 2A, bile (e.g., 20% vol/vol) increased the uptake of TBuMA, but not that of TEMA, in the presence of the ATP regenerating system. This observation is consistent with the fact that bile increased the APC of TBuMA but not that of TEMA (Fig. 1A). We attempted to elucidate which specific components of the bile contribute to the increase in the uptake of TBuMA. Several endogenous bile salts were examined for their contribution to the increased uptake of TBuMA in the present study. Among the bile salts examined, all of the conjugates of the deoxy form of the bile acids (i.e., TDC, TCDC, and GDC) significantly increased the uptake of TBuMA, but not that of TEMA, as exemplified by TDC (Fig. 2B). This increase was the most dramatic for TDC, followed by TCDC and GDC (data not included in Fig. 2), which is consistent with the formation of lipophilic ion pair complexes (Fig. 1B). Thus we conclude that bile salts in the bile, especially conjugated salts of deoxy bile acids, might be responsible for the increased uptake of TBuMA in the presence of bile (Fig. 2A). Figure 2B shows the temporal uptake of TBuMA or TEMA into cLPM vesicles in the absence and presence of TDC and the ATP regenerating system. In the absence of TDC, the ATP-dependent uptake of 10 µM TBuMA was increased, which is consistent with P-gp-mediated TBuMA uptake (32). In the presence of 100 µM TDC (a 10-fold higher concentration compared with TBuMA), in which TBuMA is thought to exist predominantly as an ion pair complex in the aqueous phase as explained in METHODS, the uptake of 10 µM TBuMA was increased significantly irrespective of the presence of ATP. The increase by TDC in the absence of ATP may well be related to the increased passive diffusion of the lipophilic TBuMA-TDC ion pair complex into the cLPM vesicles. The passive uptake rate of the ion pair complex (e.g., 87.3 pmol/mg protein at 1 min) was further increased by the addition of ATP (e.g., to 176.6 pmol/mg protein at 1 min), in which the ATP-dependent uptake component is more than 1.6-fold higher than that for TBuMA itself (i.e., 124.9 - 69.9 = 55.0 pmol/mg protein at 1 min), indicating a significantly greater active uptake for the ion pair complex than for the free TBuMA. Among the bile salts (100 µM each), TDC in the presence of ATP increased the uptake of 10 µM TBuMA the most (data not shown). The 30-s uptake rate of TBuMA (10 µM) in the presence of TDC increased as a function of TDC concentration (10-2,000 µM), exhibiting a maximal increase at a TDC concentration of ~100 µM (Fig. 3). In contrast to TBuMA, no appreciable increase in the uptake of TEMA by TDC (100 µM) was observed (Fig. 2B). This is consistent with the fact that the APC of TEMA is not influenced by the presence of bile salts (Fig. 1B) and that ATP-dependent transport is not involved in the uptake of TEMA.


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Fig. 2.   Effects of 20% (vol/vol) rat bile (A) or 100 µM TDC (B) on the temporal uptake of TBuMA (10 µM) and TEMA (10 µM) into cLPM vesicles at 37°C in the presence (solid lines) and absence (broken lines) of an ATP regenerating system. Each data point represents the mean ± SD of triplicate measurements. open circle , -Bile (A) or -TDC (B)/+ATP; , +bile (A) or +TDC (B) /+ATP; down-triangle, -bile (A) or -TDC (B)/-ATP; black-down-triangle , +bile (A) or +TDC (B)/-ATP. *Significantly different (P < 0.01) from down-triangle; **significantly different (P < 0.01) from open circle .



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Fig. 3.   Effects of TDC concentration on the initial uptake rate of TBuMA (10 µM) in the presence (solid line) and absence (dashed line) of ATP regenerating system at 37°C. Each data point represents the mean ± SD of triplicate measurements.

The increase in uptake, however, might be related to an increased permeability of the cLPM vesicle membranes, because both bile and bile salts are known to be surfactants. This possibility was examined as follows. A cLPM vesicle suspension (20 µl, 1-1.5 mg/ml protein) was pretreated by incubation with 80 µl of TDC (1.2 and 12 mM) for 10 min at 37°C. [14C]mannitol (10 µM, 20 µl) uptake for 1 min was then measured by a rapid filtration technique. The results showed that mannitol leakage was not affected by this pretreatment (control group 0.40 ± 0.02%, 1.2 mM TDC group 0.38 ± 0.02%, 12 mM TDC group 0.40 ± 0.04%; n = 3 for each group). Moreover, the uptake of TEMA was not changed by the presence of the bile or TDC (Fig. 2B). Therefore, it was concluded that the change in the permeability of cLPM vesicles is not involved in the increased uptake of the ion pair complexes.

Characteristics of ATP-dependent uptake of ion pair complexes. To characterize the ATP-dependent uptake of ion pair complexes of TBuMA, the 30-s uptake of TBuMA into cLPM vesicles was measured for transport medium containing varying concentrations of TBuMA-bile salt complexes (Fig. 4). In this experiment, the concentration of TBuMA in the medium was varied over the range of 5-1,000 µM and the concentrations of bile salts were maintained at 10-fold higher than those of TBuMA. The purpose of this study was to ensure that the majority of TBuMA in the medium existed as ion pair complexes, as explained in METHODS. In this study, the approximate concentrations of TBuMA-bile salt complexes in the transport medium could be calculated as described in METHODS and are plotted in Fig. 4. Figure 4 shows relationships between the apparent concentrations of each ion pair complex and the rate of TBuMA uptake for 30 s. A concentration dependence was found for the uptake rate of TDC (Fig. 4A), TCDC (Fig. 4B), and GDC (Fig. 4C) complexes of TBuMA. Eadie-Hofstee plots (insets in Fig. 4) for the uptake data indicated mixed uptake processes (i.e., saturable and linear kinetics) for the uptake of these ion pair complexes. Thus a kinetic analysis based on eq. 1 was performed for these complexes, and the resultant kinetic parameters (Km, Vmax, and CLlinear) are summarized in Table 2. Those parameters relative to the uptake of TBuMA alone (i.e., those in the absence of bile salts; Ref. 32) are also listed in Table 2 for comparison. The Vmax values for these ion pair complexes were comparable to that for TBuMA alone. However, the Km values of these complexes were greatly reduced (i.e., 1/47 for TDC, 1/31 for TCDC, and 1/17 for GDC) compared with that for TBuMA alone. As a result of the decrease in Km, intrinsic uptake clearances (CLint = Vmax/Km) for these ion pair complexes were dramatically increased compared with TBuMA alone, showing the largest increase for the TBuMA-TDC ion pair complex (45-fold), followed by the TBuMA-TCDC complex (23-fold) and the TBuMA-GDC complex (19-fold). In addition, a marked increase in CLlinear (5,792-, 1,554- and 1,654-fold increase for TDC, TCDC, and GDC complexes, respectively) was also obtained for the ion pair complexes compared with that for TBuMA (Table 2). Despite the greater fold increase in CLlinear compared with CLint as the result of the ion pair complexation, CLint still primarily accounts for the in vitro clearance (i.e., 77, 86, and 83% for TDC, TCDC, and GDC complexes of TBuMA, respectively).


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Fig. 4.   Concentration-dependent uptake of the TBuMA complexes of TDC (A), TCDC (B), GDC (C), and TC (D) into cLPM vesicles in the presence of ATP at 37°C. Uptake was measured under varying concentrations of TBuMA (5-1,000 µM) and bile salts (10-fold molar excess of TBuMA). The approximate concentration of each ion pair complex in the transport medium was calculated as described in METHODS. Each point represents the mean ± SD of triplicate measurements. Insets, Eadie-Hofstee transformation of corresponding data. V, uptake rate of TBuMA; C, concentration of ion-pair complex.


                              
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Table 2.   Kinetic parameters for ATP-dependent (Km and Vmax) and linear uptake (CLlinear) of TBuMA and TBuMA-bile salt ion pair complex into cLPM vesicles

For the uptake of TBuMA in the presence of TC (Fig. 4D), GC, DC, or CH (data not shown), such a concentration dependence was not observed, as evidenced by the linear profile of the graph for TC in Fig. 4D (as well as by the Eadie-Hofstee plot in the inset). These results suggest that the formation of highly lipophilic ion pair complexes is necessary for the complexes to be transported via the saturable kinetics (i.e., probably via a carrier-mediated transport system).

Inhibition study on the uptake into cLPM vesicles. The uptake rate for TBuMA (10 µM) into cLPM vesicles in the presence of TDC (100 µM) and the ATP regenerating system was cis-inhibited to a significant extent (P < 0.01) by representative P-gp substrates (i.e., 100 µM daunomycin, verapamil, and cyclosporin A; Fig. 5A). Because the majority (>75%, see METHODS for calculation) of the TBuMA is believed to exist in the form of a TBuMA-TDC ion pair complex in the transport medium under the experimental conditions used in this study (i.e., a 10-fold higher molar concentration of TDC compared with TBuMA concentration), the observations shown in Fig. 5A suggest that the P-gp transporter is also responsible for the ATP-dependent carrier-mediated uptake of the ion pair complexes of TBuMA (as exemplified by TBuMA-TDC) as well as for the uptake of TBuMA itself (32). On the other hand, such a significant inhibition was not observed by representative substrates of the other transporters on the canalicular membranes (i.e., tetraethylammonium and TEMA of the OC/proton exchanger and taurocholate of the bile salt export pump Bsep) and benzyl penicillin, an organic anion (Fig. 5A), suggesting that the ATP-dependent uptake of ion pair complexes is not related to these transporters on hepatocyte membranes.


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Fig. 5.   A: effect of 100 µM of daunomycin (DM), verapamil (VP) and cyclosporin A (CyA), taurocholate (TC), benzyl penicillin (BP), and TEMA and tetraethylammonium (TEA) on the ATP-dependent uptake of 10 µM [3H]TBuMA into cLPM vesicles in the presence of 100 µM TDC and an ATP regenerating system. B: effect of various concentrations of TBuMA-TDC (produced by 1, 10, and 100 µM TBuMA and 10-fold molar excess TDC) on the ATP-dependent uptake rates of 0.5 µM [3H]daunomycin into cLPM vesicles for the 30-s fraction. C: effect of 100 µM TBuMA or 10 µM TDC with varying concentrations of TBuMA (0, 10, 100 and 1,000 µM) on the ATP-dependent uptake rates of 1 µM [3H]taurocholate into cLPM vesicles for the 30-s fraction. The concentration of the ion pair complex was approximated as described in METHODS. Each data point represents the mean ± SD of triplicate measurements. * Significantly different (P < 0.01) from control.

The involvement of P-gp in the uptake of ion pair complexes was further examined by measuring the effect of the TBuMA-TDC complex on the ATP-dependent uptake rate of 0.5 µM daunomycin, a representative substrate for the transporter (Fig. 5B). The uptake rate of daunomycin was inhibited by the presence of TBuMA alone, supporting the conclusion that TBuMA is transported via the P-gp transporter (32). As expected, the rate of uptake was not inhibited by the presence of TDC alone, a substrate for Bsep (24). On the other hand, the rate of uptake of daunomycin was inhibited by the TBuMA-TDC complex, and the extent of inhibition increased as the concentration of the ion pair complex increased (i.e., from 1 to 10 to 100 µM for TBuMA with 10-fold molar excess for TDC; Fig. 5B). Thus lipophilic ion pair complexes of TBuMA with bile salts, such as the TBuMA-TDC complex, again appear to be transported via the P-gp transporter.

Because TBuMA forms ion pair complexes with bile salts, an ATP-dependent bile acid transport system in the canalicular membrane, Bsep (3, 24), might be involved in the uptake of bile salt complexes of TBuMA. To clarify this issue, the effect of TDC (100 µM) in the presence of varying concentrations of TBuMA (0, 10, 100, and 1,000 µM) on the ATP-dependent uptake of TC (1 µM), a representative substrate for Bsep, was examined (Fig. 5C). The uptake rate of TC was not inhibited by TBuMA (100 µM), indicating that Bsep is not involved in the uptake of TBuMA itself. As a matter of course, the uptake rate of TC was inhibited by the presence of 100 µM TDC, probably because of the competitive binding of the compounds to the transporter Bsep. The inhibition by TDC was decreased with increasing concentrations of TBuMA (0, 10, 100, and 1,000 µM; Fig. 5C), again suggesting a lack of involvement of Bsep in the uptake of ion pair complexes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was focused on the issue of whether TBuMA and TEMA are capable of forming lipophilic ion pair complexes with bile salts in hepatocytes and whether the complexation contributes to the bile canalicular transport of the OCs. A partition study between n-octanol and an aqueous phase (MSB, pH 7.4) revealed a significant increase in the APC of TBuMA but not of TEMA in the presence of the (Fig. 1A). The most important constituents of rat bile, besides water and inorganic salts, are bile salts (38 mM), phospholipids (0.53 mM as phosphatidylcholine), and cholesterol (0.58 mM) (14). The presence of phospholipids (such as phosphatidylcholine) and cholesterol did not lead to a significant increase in the APC of TBuMA at moderate concentrations (up to 1 mM, data not shown), whereas some bile salts clearly caused an increase in APC (Fig. 1B). This suggests that the formation of lipophilic ion pair complexes with some bile salts represents a likely mechanism for the increased APC of TBuMA in the presence of bile. The fact that some bile salts increased the partition of TBuMA but not TEMA suggests a molecular weight dependence for the partition of OCs in the presence of counterions such as bile salts.

The concentration of each bile salt in the liver was reported to be in the range of nanomoles per gram of liver in humans and rats (26, 27). Thus the formation of lipophilic ion pair complexes of TBuMA with some endogenous bile salts in the liver appears to be plausible. We examined the issue of whether the ion pair complexes contribute to the canalicular transport of the OCs by using a cLPM vesicle system. The uptake of TEMA into the vesicles was not increased by any of the bile salts examined. On the other hand, the uptake of TBuMA was increased in the presence of some bile salts, which increased the partition of TBuMA in their presence (Fig. 2A), suggesting a contribution by ion pair complexation to the canalicular transport of TBuMA. The mechanism of the increased uptake into the vesicles was also investigated. The uptake rate of TBuMA in the presence of some bile salts (i.e., TDC, TCDC, and GDC) exhibited an ATP (Figs. 2 and 3) and ion pair concentration dependence (Fig. 4). Nonlinear regression analysis for the uptake revealed a significant decrease in Km and an increase in CLlinear for the ion pair complexes (Table 2) compared with those found for TBuMA itself (32), indicating a significant increase in both passive diffusion (i.e., CLlinear) and ATP-dependent transport by the ion pair formation. The sum of the uptake clearances for the ion pair complexes of TBuMA was estimated to be 487 µl · min-1 · kg body wt-1 for the ATP-dependent component and 117 µl · min-1 · kg body wt-1 for the passive diffusion component (Table 2). The total in vitro clearance for these ion pair complexes (i.e., 604 µl · min-1 · kg body wt-1, calculated from Table 2) potentially accounts for 61.6% of the in vivo excretion clearance of TBuMA across the canalicular membrane (i.e., 980 µl · min-1 · kg body wt-1) (9), which is much higher than TBuMA itself (i.e., 0.6%) (32). Such an increase in CLint and CLlinear of TBuMA was not observed for other bile salts examined (data not shown), which is consistent with an unchanged partition of TBuMA in their presence (Fig. 1B). Thus the formation of lipophilic ion pair complexes appears to be necessary for the increased uptake of TBuMA into cLPM vesicles. The lipophilicity (i.e., TPC) and extent of extraction of an ion pair complex by an organic solvent (i.e., Ke, which represents Kf × TPC) appear to govern the magnitude of uptake clearance (i.e., sum of CLint and CLlinear). In fact, a linear relationship between the uptake clearances and TPC or Ke of the ion pair complexes was observed (Fig. 6), exhibiting a better correlation with Ke than with TPC (r2 = 0.8592 vs. 0.9763; P < 0.05). These data suggest that not only the lipophilicity of the ion pair complex (i.e., TPC) but also the degree of ion pair formation in the medium (Kf) contribute to the canalicular transport of the complex. It is noteworthy that the in vitro uptake clearance of ion pair complexes would be predicted from their physicochemical properties such as TPC or Ke. Despite the apparent contribution of ion pair complexation to the increased uptake of TBuMA, the possibility of transport of intact ion pair complex across biological membranes remains unknown. The simultaneous determination of the concentration of bile salts, as well as that of OCs, inside the cLPM vesicles may provide useful information.


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Fig. 6.   Relationship between CLtotal values [i.e., the sum of intrinsic clearance (CLint) and linear uptake clearance (CLlinear)] and true partition coefficient (TPC) (A) or extraction coefficient (Ke) (B) values for ion pair complexes of TBuMA with various bile salts and bile acids. Parameters were taken from Tables 1 and 2. The fitted equations are CLint = 2.68 × TPC - 1.96, (r2 = 0.8592; A) and CLint = 0.0007 × Ke + 1.14 (r2 = 0.9763; B). 1, CH; 2, DC; 3, GC; 4, TC; 5, GDC; 6, TCDC; 7, TDC complexes of TBuMA.

The uptake of TBuMA-TDC complex into cLPM vesicles, in the presence of ATP, was inhibited by some representative substrates of P-gp (Fig. 5A), and the uptake of daunomycin, a representative substrate for P-gp, was inhibited with increasing concentration of the TBuMA-TDC complex (Fig. 5B). Thus the involvement of P-gp in the uptake of the ion pair complexes was proposed. This conclusion is consistent with previous reports of a higher affinity of P-gp toward OCs with higher molecular weight (20) and lipophilicity (17). Increased lipophilicity of TBuMA via the formation of ion pair complexes would increase the binding of the OC to the transporter P-gp. A significant decrease in Km (Table 2) may represent such an increase in the affinity of TBuMA toward the transporter.

Because bile salts are incorporated into ion pair complexes, an ATP-dependent bile acid transporter in the canalicular membrane, Bsep, as well as P-gp, might be involved in the uptake of the ion pair complexes. As expected, TDC and TC appeared to share a common transporter, probably Bsep (Fig. 5C). The inhibitory effect of TDC on the uptake of TC was reduced by increasing concentrations of TBuMA. This observation is consistent with the hypothesis that the formation of an ion pair complex with TBuMA reduces the free concentration of TDC, thereby decreasing the competitive inhibitory effect of TDC on the uptake of TC through Bsep. Thus Bsep appears not to be involved in the uptake of TBuMA-bile salt complexes. The lack of involvement for Bsep is further supported by our preliminary experiment using LLC-PK1 cells (a kidney cell line that expresses P-gp but not Bsep; Ref. 15). Apical-to-basolateral transport across the cell monolayers was identical for TBuMA and TBuMA-TDC complexes, whereas the basal-to-apical transport of the complex was approximately threefold higher than that of TBuMA alone. These observations are consistent with the hypothesis that the canalicular transport of lipophilic ion pair complexes of OCs is mediated by P-gp and that Bsep may not be involved in the transport. In the literature, gene products of both mdr1a and -1b, which encode P-gp, appear to be involved in the transport of TBuMA across biological membranes (30, 31). Because the transport of TBuMA-bile salt complexes in the cLPM vesicles was inhibited in the presence of TBuMA, the transport of the complex may also be mediated by mdr1a and -1b gene products in the cLPM vesicles.

In contrast to the uptake into cLPM vesicles, the sinusoidal uptake of 10 µM TBuMA into hepatocytes was decreased 22% in the presence of 30 µM TDC in our preliminary experiment, consistent with Zhang et al. (36), who reported an increase in the affinity of type I OCs (such as TBuMA and TEMA) toward hOCT1, a sinusoidal transporter responsible for the hepatic uptake of the OCs as their lipophilicity increased. Therefore, the increased transport of the ion pair complexes appears to be specific for the canalicular membrane. This different contribution of ion pair formation to the sinusoidal and canalicular membranes of hepatocytes might be related to the protection of the liver from xenobiotics.

Recently, Lo and Huang (18) reported a decrease in the basolateral-to-apical flux of epirubicin, a P-gp substrate, across Caco-2 cell monolayers in the presence of DC. However, such an effect (i.e., P-gp inhibition) was not observed in any of the bile salts examined in the present study for either the vesicular uptake of TBuMA or the basolateral-to-apical transport of TBuMA across the LLC-PK1 monolayer (data not shown). These data suggest that the effect of DC might vary depending on the nature of the P-gp substrates. Elucidation of the underlying mechanism for the different effects of DC on epirubicin and TBuMA (including TBuMA-bile salt ion pair complexes) will likely contribute to our understanding of the role of bile salts in the biliary excretion of compounds that are substrates for the P-gp transporter.

Given the above findings, ion pair complexation with some endogenous bile in the hepatocyte appears to contribute to the canalicular excretion of many OCs, probably with high molecular weight (i.e., mol wt >200 ± 50), in vivo. The extent of the ion pair formation varies depending on the concentrations of OCs and bile salts in the liver. The concentration of TDC in the present study (i.e., 100 µM in Figs. 2 and 5) is obviously higher than the reported value for the rat liver (i.e., 13 µM; Ref. 26). However, the vesicular uptake of 10 µM TBuMA is increased even by the presence of 10 µM TDC (Fig. 3). In addition, the total concentration in the rat liver of TDC, TCDC, and THDC, the bile salts that are expected to form lipophilic ion pair complexes with TBuMA, exceeds 70 µM (26). Therefore, ion pair formation with some bile salts appears to contribute substantially to the in vivo canalicular excretion of some OCs. A firm conclusion for this hypothesis awaits extension of the present results to various OC drugs and bile salts, as well as quantitative analysis of bile salts in the liver.


    ACKNOWLEDGEMENTS

This study was supported by a grant (HMP-99-D-07-0004) from the Ministry of Health and Welfare of Korea.


    FOOTNOTES

Address for reprint requests and other correspondence: C.-K. Shim, Dept. of Pharmaceutics, College of Pharmacy, Seoul National Univ., San 56-1, Shinlim-dong, Kwanak-gu, Seoul 151-742, Korea (E-mail: shimck{at}plaza.snu.ac.kr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 June 2000; accepted in final form 18 April 2001.


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DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(2):G515-G525
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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