Introduction

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Biliary excretion of drugs or their usually more polar conjugates involves consecutive processes of net sinusoidal uptake, intracellular trafficking through the cytoplasm and excretion across the apical or canalicular membrane. At the sinusoidal membrane, specialized transport systems for bile acids (Hagenbuch et al., 1991; Hagenbuch and Meier, 1994), organic anions (Jacquemin et al., 1991, 1994; Kullak-Ublick et al., 1994) and cations (Bossuyt et al., 1996; Gründemann et al., 1994) have been discovered. Equally important is the host of canalicular transporters that contribute to the formation of concentrated bile. Although the canalicular membrane accounts for only 15% of the total surface area of the hepatocyte cell membrane (Evans, 1980), it is highly specialized for excretion, a process facilitated by the presence of specific transporters.

The canalicular transporters belong to a superfamily of proteins occurring in all cellular organisms and are characterized by an ABC in their primary structure. To date, three subfamilies have been identified of ABC transporters that mediate concentrative transport of endobiotics and xenobiotics on the canalicular membrane (for reviews, see Gatmaitan and Arias, 1995; Meier et al., 1997; Müller and Jansen, 1997). One subfamily of the ABC transporters, the P-glycoproteins, are coded by various multidrug-resistant genes in humans (MDR) and mice (Mdr) and are found to transport a wide variety of substrates, which are mostly lipophilic, neutral, or positively charged, including phosphatidylcholine and phospholipids (Lomri et al., 1996; Oude Elferink et al., 1997). A second ABC transporter that is responsible for ATP-dependent bile acid transport has been purified and functionally reconstituted (Müller et al., 1991; Ruetz et al., 1987; Sippel et al., 1990). A third distinct ABC system mediates the concentrative transport of a variety of non-bile acid organic anions (Büchler et al., 1996; Paulusma et al., 1996; Müller et al., 1996b). This transport system was named cMOAT. Separate and distinct sinusoidal and canalicular transport proteins have been implicated in the transport of the important tripeptide, GSH (Fernandez-Checa et al., 1992).

The molecular basis of cMOAT was disclosed recently. cMOAT bears sequence homology with an MRP found to be associated with lung cancer cells (Büchler et al., 1996) and appears to be an isoform found exclusively in the canalicular membrane of hepatocytes, denoted as cMrp (Büchler et al., 1996) or mrp2 (Müller et al., 1996b) in the rat and cMRP (Kartenbeck et al., 1996) or MRP2 (Müller et al., 1996b; Paulusma et al., 1996) in humans. Although MRP1 is present in many other tissues (Müller et al., 1996a), MRP2 is located on the lateral membrane of the liver (Mayer et al., 1995).

The presence of cMOAT was prompted by the serendipitous discovery of several strains of jaundiced mutant rats. They are the TR and GY rats originating from the Wistar strain (Jansen et al., 1985; Kuipers et al., 1988; Kitamura et al., 1990; Nishida et al., 1992) and the EHBR (EHBR/Eis) from the Sprague-Dawley strain (Hosokawa et al., 1992). In TR rats, the defect is due to a single-nucleotide deletion in the gene coding for cMRP, resulting in reduced mRNA levels and absence of the 190-kDa cMOAT protein in the canalicular membrane (Paulusma et al., 1996). A similar reduction in protein as well as mRNA levels were shown in EHBR (Ito et al., 1996; Kartenbeck et al., 1996), where the genetic defect occurs due to the substitution of one nucleotide, which results in the introduction of a premature stop codon (Ito et al., 1997). These mutant rats manifest predominantly conjugated hyperbilirubinemia and lack the hepatic excretion of various non-bile acid organic anions. It was recently shown that cMrp is absent in EHBR and TR rats (Kobayashi et al., 1990; Mayer et al., 1995; Müller et al., 1996b) and that cMRP was absent in a patient with Dubin-Johnson syndrome (Kartenbeck et al., 1996; Müller et al., 1996b). The phenotype of these mutant rats follows autosomal recessive inheritance and is similar to that of the mutant Corriedale sheep and human Dubin-Johnson syndrome.

There is dramatic impairment of biliary excretion of various organic anions in these mutant (TR, GY and EHBR) rat strains. The organic anions include the sulfate and glucuronides of bile acids (Jansen et al., 1985; Kuipers et al., 1988), bilirubin glucuronide (Nishida et al., 1992), indocyanine green (Hosokawa et al., 1992; Sathirakul et al., 1993), GSH and cysteine adducts (Oude Elferink et al., 1989 Kitamura et al., 1990; Geng et al., 1995b; Müller et al., 1996a) and drug glucuronide conjugates (Shimamura et al., 1994, 1996; Takenaka et al., 1995a, 1995b). BSPGSH has been used repeatedly as a model substrate for the study of this canalicular transporter because cleavage of the GSH moiety to the cysteinylglycinyl or cysteinyl adducts seldom occurs, and even if it does, the extent is only minimal (Sano et al., 1992; Snel et al., 1993; Sorrentino et al., 1989; Zhao et al., 1993). Avid excretion and an extremely high bile/liver concentration ratio of 1600, presumably due to the formation of micelles, have been reported for BSPGSH in liver perfusion experiments (Geng et al., 1995b). Saturable uptake and slow efflux as well as high plasma and tissue binding are other characteristics found to be associated with BSPGSH (Geng et al., 1995b).

In the present investigation, we examined the handling of BSPGSH by the perfused EHBR liver with the MID technique to assess quantitatively the transport processes for BSPGSH. Because the microcirculation and polarity are maintained in the perfused liver preparation, the kinetics of basolateral and canalicular transport are assessed without disruption of cellular integrity and bioenergetics, thus allowing evaluation of the impact of the mutation on the functionality of the organ. In addition, the MID technique exploits tracer methodology and enables investigation of the transport and removal processes to be studied simultaneously. Through analysis of the dilution profiles of [³H]BSPGSH and comparison with those of a set noneliminated reference indicators, we examined the uptake of BSPGSH in absence of excretion in the EHBR and compared this with the observations in the SDR (Geng et al., 1995b). The difference in tissue binding of BSPGSH between these two rat strains also was studied.

	Experimental Procedures

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Materials. Unlabeled bromosulfophthalein, reduced GSH, GST (rat) and bovine serum albumin (25% in Tyrode's buffer) were obtained from Sigma Chemical (St. Louis, MO). [³H]GSH (specific activity, 1.22 Ci/mmol) was obtained from DuPont Canada (Markham, Ontario, Canada). Unlabeled BSPGSH and [³H]BSPGSH were synthesized from GSH and [³H]GSH, respectively, as previously described (Snel et al., 1993; Whelan et al., 1970). After purification, the purity of BSPGSH, determined by HPLC (Snel et al., 1993), was >95%, whereas the radiochemical purity of [³H]BSPGSH was >96% as found by TLC (silica gel in a solvent system of 1-butanol/water/acetic acid, 75:25:10, v/v/v) and >93% as found by HPLC. All reagents used were of glass-distilled HPLC grade or the highest purity available (Fisher Scientific, Mississauga, Ontario, Canada).

Rat liver perfusion. Male EHBR (289 ± 19 g; liver weights, 14 ± 1 g) were provided by Eisai Co. Ltd. (Tsukuba City, Ibaraki, Japan) and were kept under artificial lighting on a 12:12-hr light/dark cycle. The animals were fed ad libitum (Purina Rat Chow) and allowed free access to water. Before surgery, the animals were anesthetized with intraperitoneal administration of sodium pentobarbital (50 mg/kg). The surgical procedure and the perfusion apparatus were identical to those described previously (Geng et al., 1995b). The artificial perfusate contained 20% washed outdated human RBC (Red Cross, Toronto, Canada), 1% bovine serum albumin, 3% dextran T-40 (Pharmacia Fine Chemicals, Piscataway, NJ) and 17 mM glucose (Travenol Labs, Deerpark, IL) in KHB solution buffered to pH 7.4. Because the perfusion medium was identical in composition to that used for SDR liver perfusion, the binding of BSPGSH to albumin should be identical to that observed previously (Geng et al., 1995); namely, there were two classes of binding sites: n₁ = 0.5, K_A1 = 1 × 10⁵ M¹; n₂ = 5, K_A2 = 3.5 × 10³ M¹, where n₁ and n₂ are the numbers of sites for classes I and II, and K_A1 and K_A2 are the corresponding binding association constants. The common bile duct was cannulated with polyethylene PE-50 tubing (Becton Dickinson, Parsippany, NJ), and the portal vein was cannulated with an intravenous catheter/needle unit (Vascular Access; Becton Dickinson, Sandy, UT). The perfusate entered the liver through the portal vein and exited through the hepatic vein at a rate of 12 ml/min (0.85 ± 0.07 ml·min¹·g¹) in a single-pass fashion (nonrecirculating); the hepatic artery was ligated.

MID study. Injection of the MID dose was carried out during steady state (at 90 or 120 min after the onset of perfusion), as described previously (Geng et al., 1995b). The injection mixture (0.23 ml) contained [³H]BSPGSH (0.64 ± 0.12 µCi), unlabeled BSPGSH (at the same concentration as in the perfusate), a vascular indicator (⁵¹Cr-labeled washed human RBC, 0.57 ± 0.25 µCi), interstitial indicators [¹²⁵I-labeled albumin (3.3 ± 0.8 µCi) and [¹⁴C]sucrose (0.6 ± 0.08 µCi)] and an accessible water space indicator (D₂O, 0.14 ± 0.012 ml), in a composition otherwise identical to that of the perfusate. This was rapidly introduced into the portal vein via an electronically controlled HPLC injection valve (Valco Instruments, Houston, TX). Simultaneously, serial outflow samples (at successive 1-, 2- and 3-sec intervals for a total of 180 sec) were collected using a homebuilt fraction collector. In all experiments, the Hct of the blood perfusate and the dose were determined by use of an Hct centrifuge (model MB Microhematocrit Centrifuge, IEC, Fisher Scientific).

Binding to intracellular proteins. The EHBR liver was perfused with ice-cold KHB for 3 to 5 min and then homogenized with 3 volumes of ice-cold KHB with a homogenizer (Ultra-turrax T25; Janke & Kunkel IKA-Labortechnik, Staufen im Breisgau, Germany). The liver homogenate was centrifuged at 9000 × g (M2-J Centrifuge; Beckman Canada, Mississauga, ON, Canada) for 20 min at 4°C. After centrifugation, the supernatant was removed, and bulk BSPGSH and [³H]BSPGSH were added to this. The final concentrations of BSPGSH in the supernatant (S9) varied from 3.5 to 526 µM. Tissue protein binding was examined through ultrafiltration (Centricon, Amicon; 10,000 molecular weight cutoff) of a 0.5-ml sample of S9 at 1000 × g (M2-J Centrifuge, Beckman) for 20 min at room temperature.

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Assays. Quantification of ⁵¹Cr and ¹²⁵I radiolabels in blood perfusate (25-100 µl) was carried out by two-channel gamma counting with crossover correction (Cobra II; Canberra-Packard Canada Ltd., Mississauga, Ontario, Canada). Assays of [¹⁴C]sucrose and [³H]BSPGSH were carried out according to Geng et al. (1995b). All plasma samples (50-200 µl) were made up to a constant volume of 200 µl with blank plasma for constant quench, and 200 µl of a saturated urea solution (1 g/ml) was added to the plasma sample for the release of [³H]BSPGSH from its tight binding to albumin. After the further addition of 800 µl of acetonitrile, the ³H and ¹⁴C radioactivities in 1 ml of supernatant were determined by triple-channel (³H, ¹²⁵I and ¹⁴C) counting (model 5801; Beckman Instruments, Brea, CA) in 5 ml of ReadyProtein (Beckman). Previous studies had demonstrated that ¹²⁵I-labeled albumin, normally precipitated with acetonitrile, persisted here as small ¹²⁵I-labeled peptide fragments due to the use of urea (Geng et al., 1995b; Snel et al., 1993). D₂O in plasma samples (80 µl) was assayed by Fourier transform infrared spectrometry (model 1600; Perkin Elmer Canada, Rexdale, Ontario, Canada) over the frequency interval from 2300 to 2700 cm¹, as previously described (Pang et al., 1991). Assay of the MID dose was performed in a similar fashion, after dilution of the blood or plasma dose 1:10 (v/v) with blank blood perfusate or blank plasma, respectively.

Unlabeled BSPGSH in plasma, S9 fraction and bile. The concentration of unlabeled BSPGSH in plasma samples was determined directly by UV spectroscopy at 580 nm after alkalinization of the plasma samples with 100 µl of 0.1 N NaOH. The bile samples were examined by HPLC (Snel et al., 1993). The concentration of BSPGSH in S9 was determined spectrophotometrically, after deproteinization with acetonitrile (Geng et al., 1995b).

Data treatment. The steady-state extraction ratio, E, was used to monitor the disappearance of bulk BSPGSH from plasma. E was calculated from:

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where C_In and C_Out are the steady-state input and output plasma concentrations of BSPGSH, respectively. For the EHBR which lacks the canalicular transporter for BSPGSH biliary excretion, E was found to be virtually zero.

Splining, transit times and superposition. For the MID portion of the experiment, the outflow radioactivity for each indicator was expressed as a fraction of radioactivity of injected dose per milliliter of blood. The concentration of radiolabels at the end of the collection (180 sec) was <0.1% of peak values. The resulting outflow profiles were approximated with cubic spline functions (De Boor, 1978). From these, recoveries were calculated as the product of the time integrals of the fractional recovery and blood flow. Integrals of the product of fractional recovery and time (AUMC) and fractional recovery (AUC) were calculated similarly (Geng et al., 1995b). The ratio AUMC/AUC gave the mean transit time.

Modeling. A scheme describing the kinetic events underlying the disposition of BSPGSH is shown in figure 1. As a starting point, the model adopted previously for interpretation of the uptake and excretion of BSPGSH in perfused liver studies of SDR (Geng et al., 1995b) was used (fig. 1A). The rate constants (k₁ and k₁), which are related to the cellular volume, V_cell, have been defined previously (table 1); alternatively, these are expressed as the rate coefficients (k₁₂ and k₂₁), which are defined with respect to the compartments from which the fluxes originate. The sequestration coefficient, k_seq or k₂₀, was set to zero to reflect the dysfunctional canalicular excretion of BSPGSH in EHBR. Although there is no binding of BSPGSH to RBC, binding of BSPGSH to proteins occurs within both the plasma and interstitial spaces where protein concentrations are assumed to be the same as are the unbound and bound concentrations of BSPGSH (Geng et al., 1995b). Because albumin is excluded from part of the Disse space (Goresky, 1964), the space of distribution for bound BSPGSH is diminished accordingly. The unbound BSPGSH in the Disse space exchanges with that in the hepatocellular compartment, denoted by the appropriate transfer coefficients for sinusoidal entry into (k₁₂) and efflux from (k₂₁) hepatocytes. Correction of the rate coefficients for binding to plasma and cellular proteins, respectively, and multiplication by the appropriate compartmental volumes will yield the permeability-surface area products for transport (P_inS or P_outS) (table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Schematic representation of BSPGSH uptake at the level of a single sinusoid for SDR (A), whose rate constants for influx (k1), efflux (k1) and sequestration (kseq) have been defined in reference to Vcell, the accessible cell water volume (see table 1), and for EHBR (B), for which excretion is absent and there was segregation of a shallow pool and a deep pool within the hepatocyte. The sinusoidal transfer coefficients are denoted by k12 and k21, respectively, and the rate constants between the cellular and deep pools by k23 and k32 (see table 1 for definition). However, elimination (denoted by dashed line) could hypothetically occur from the shallow pool (with rate constant k20 >0) or from the deep pool (with rate constant k30 >0). Equilibria are assumed between bound and unbound forms of BSPGSH in plasma and tissue. cp,u, cp,b, cD,u, cD,b, ch,u and ch,b are the unbound and bound concentrations of BSPGSH in the sinusoid, Disse space and cell, respectively.

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	Results

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Tissue binding of BSPGSH and tissue concentrations. The intracellular binding of BSPGSH was explored over tissue concentrations ranging from 3.5 to 526 µM, in duplicate. The optimized fitting indicated that there was only one class of binding sites for BSPGSH in S9 fraction (fig. 2A). The effective binding concentration, n[P_t], and the dissociation constant, K_D, were obtained from a nonlinear regression procedure. After correction for the dilution of S9 for the study of tissue binding, the resulting binding constants were: K_A, the binding association constant, or 1/K_D, was 2.4 × 10⁴ M¹, and the binding capacity, n[P_t], was 53 µM. These constants revealed a weaker binding of BSPGSH to EHBR liver S9 proteins in relation to those from SDR (Geng et al., 1995b). The unbound fraction of BSPGSH in EHBR liver homogenate (f_t = C_t,u/C_t) was found to increase from 0.15 to 0.56 for BSPGSH S9 concentrations of 3.5 to 526 µM. These unbound tissue fractions were much higher than those observed at comparable BSPGSH concentrations in the liver S9 fraction for the SDR (Geng et al., 1995b).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Tissue binding of BSPGSH to EHBR liver S9. Nonlinear regression of BSPGSH protein binding was performed, and binding conformed to one class of cytosolic binding site. The binding constants denote lesser binding of BSPGSH to EHBR liver proteins in comparison to controls (SDR).

Lack of removal of BSPGSH. Among the perfusion experiments where different bulk plasma concentrations of BSPGSH (26-257 µM) were used, the steady-state hepatic extraction ratios of unlabeled BSPGSH was ~0 (fig. 3). The radiolabel in the outflowing perfusate was mainly [³H]BSPGSH, as found by TLC. Only trace amounts of metabolites of BSPGSH were found in bile with HPLC. Cleavage products were not found in liver homogenates (data not shown). In contrast, the extraction ratio of [³H]BSPGSH, estimated as (1  integral recovery in plasma), was 0.26 ± 0.11 at the lower concentration of BSPGSH; the lack of complete recovery in outflow plasma was attributed to accumulation of [³H]BSPGSH in the liver, whose efflux into hepatic venous plasma was slow, as found earlier (Geng et al., 1995b).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   The steady state extraction ratios of BSPGSH in EHBR in the present single-pass studies (virtually zero in value) are compared with those in SDR (data of Geng et al., 1995b).

MID studies. Recoveries of radiolabeled ⁵¹Cr-RBC, ¹²⁵I-albumin, [¹⁴C]sucrose and D₂O in hepatic venous samples were complete, within experimental errors. Representative outflow profiles for the labeled substances injected into the portal vein of the liver, expressed as fractional recovery of dose per milliliter for each species, are shown in figure 4.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Fractional recoveries of [3H]BSPGSH and the noneliminated reference indicators, with different input concentrations of BSPGSH in linear (left) and semilogarithmic (right) presentation, with a lower (top) and higher (bottom) input concentration of BSPGSH. Note the presence of tailing of the outflow profile of [3H]BSPGSH at low BSPGSH bulk concentration. Saturation of the uptake process was evident because there was less separation between the [14C]sucrose and [3H]BSPGSH curves at the higher bulk concentrations of BSPGSH.

BSPGSH behavior. The initial upslopes in the outflow profiles for [³H]BSPGSH were slightly delayed with respect to those for the labeled RBC and albumin, reached a lower magnitude and decayed more slowly (fig. 4, right). A characteristic tailing curve pattern was observed at lower bulk BSPGSH concentrations, whereas at a higher bulk concentration of BSPGSH, the [³H]BSPGSH curve was of a higher magnitude and exhibited a downslope similar to that of sucrose (fig. 4, right), suggesting saturation of the process underlying uptake and an earlier return to the vascular space.

One- vs. two-pool model. The calculated BSPGSH outflow profile obtained through convolution of the liver transport function for BSPGSH, h_BSPGSH(t), for the one-pool model (equation A9, Appendix) with the outflow profile obtained from the apparatus in the absence of a liver, C_cath(t), failed to describe the late-in-time [³H]BSPGSH outflow data (fig. 5). In contrast, when the liver transport function for the two-pool model (equation A11, Appendix) was used, a much improved fit was obtained (fig. 5). The fitted curves, based on the two-pool model, were further segregated into the throughput and returning components (fig. 6). A greater throughput component (proportion of the dose traversing the organ without entering the liver cell) existed with the higher BSPGSH concentration (fig. 7). The concentration dependence observed for EHBR was similar to that observed for SDR.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Fits of the [3H]BSPGSH curve to the one-pool (see fig. 1A) and two-pool (see fig. 1B) with absence of sequestration. Note the superior fit of the data to the two-pool model.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Segregation of the [3H]BSPGSH fitted curve according to the throughput component, the portion of dose which never left the vasculature, and the returning component, the portion of dose which entered the hepatocyte and returned at a later time.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   The throughput component was higher with increasing logarithmic average plasma unbound concentration of BSPGSH, p,u, for EHBR. The trend was similar to that previously observed for SDR (data from Geng et al., 1995b) and suggests saturation of the uptake process.

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View larger version (15K): [in this window] [in a new window]   Fig. 8.   Plots of the influx permeability-surface area product (A) and the influx rate constant, k1 (B), as functions of the logarithmic average plasma unbound concentration of BSPGSH, p,u, for EHBR and SDR (data of Geng et al., 1995b).

Efflux of BSPGSH and tissue partitioning. The interpretation of the efflux permeability-surface area product, P_outS, depends on whether the deep pool shares the same space as the shallow pool or whether the deep pool represents some other distinct space. For this reason, two sets of efflux permeability-surface area products and tissue-partitioning ratios (P_inS/P_outS) were estimated with equations 3 and 4. Values for P_outS associated with the shallow and deep pools being a common space were similar to those for influx (P_inS), but these were substantially lower when the shallow and deep pools were viewed as separate spaces (table 3). The tissue-partitioning (P_inS/P_outS) values of BSPGSH for the former case varied from 2.3 to 0.03 with increasing BSPGSH concentration but were dramatically higher (from 29 to 0.2) when the deep pool was a separate, discrete space. The trend for higher values was similar to that observed for the ratios of the unbound tissue to unbound plasma concentration, which ranged from 43 to 1 (table 3).

	Discussion

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The handling of BSPGSH, a model substrate that enters and leaves the liver cell through carrier-mediated systems at both the sinusoidal and canalicular membranes, was presently studied in the EHBR perfused liver preparation. In bile, only trace amounts of BSPGSH or its cleavage metabolites were found by HPLC, confirming the impairment of ATP-dependent biliary transport of the GSH conjugates. Compared with normal SDR, an apparent steady state was reached only slowly in EHBR (after 2 hr at <150 µM and 1 hr at >200 µM BSPGSH). Although the apparent steady-state hepatic extraction ratio of unlabeled BSPGSH was almost equal to zero, an apparent loss of [³H]BSPGSH from plasma within the sampling time for MID was observed at the lower BSPGSH concentrations. The loss could be accounted for by the temporary storage in liver inasmuch as efflux of BSPGSH is known to be extremely slow (Geng et al., 1995b).

Apart from the lack of biliary excretion in EHBR rats, both similarities and differences in trans-sinusoidal transfer and hepatic distribution were found. Of note, the blood volume, Disse spaces for albumin and sucrose, Disse-sinusoidal space ratios () and accessible intracellular water space in the EHBR liver were found to be similar to those in controls (table 2). The t₀ was, however, smaller, and the reason for the difference is not apparent because there was no difference observed among the physiological volumes. The measured protein concentration in S9 (156 ± 23 mg/g liver, n = 10 livers) in EHBR liver was consistently lower than that in normal SDR (200-250 mg/g liver), and the binding of BSPGSH in EHBR liver tissue also differed from controls (Geng et al., 1995b). In EHBR, only one class of binding site was identified in the liver S9 fraction (K_A = 2.4 × 10⁴ M¹ and an effective binding concentration of 53 µM). In control SDR, tissue binding showed tighter binding of BSPGSH with two classes of binding sites (2.5 × 10⁵ and 4.8 × 10³ M¹; effective binding concentrations of 29 and 657 µM) (Geng et al., 1995b). The binding proteins likely represent GSH transferase B (ligandin or Y protein), and to a lesser extent, fatty acid-binding protein (Sorrentino et al., 1989). Because of lower binding capacity and affinity, the tissue unbound fractions of BSPGSH (0.15-0.56 for the tissue S9 concentration range of 3.5-526 µM) were significantly higher than those at comparable concentrations in SDR; the latter varied from 0.1 to 0.3 when the S9 BSPGSH concentrations ranged from 3 to 522 µM (Geng et al., 1995b). Reduced tissue binding of indocyanine green to ligandin was similarly observed in EHBR livers (Sathirakul et al., 1993), and changes in plasma protein binding also have been noted in vivo (Nadai et al., 1994). However, there was no change in the cytosolic binding of dibromosulfophthalein or for other sulfate/glucuronide conjugates (Takenaka et al., 1995a). The alteration in cytosolic binding appears to be substrate specific.

Modeling of the outflow dilution data of [³H]BSPGSH in EHBR with a one-pool system in the liver, as described previously for the MID data obtained from SDR liver perfusions (Geng et al., 1995b), was unable to fully describe the late-in-time data (fig. 5). The tail normally has been construed as a slowly returning component due to the presence of an extra deep pool (Schwab et al., 1990). The ratio k₂₃/k₃₂ was found to be 12 ± 8 (table 3), suggesting that this deep pool contained much higher BSPGSH concentrations than those in the cellular pool. The deep pool cannot be readily associated with liver-binding proteins because the effective binding concentration for BSPGSH (53 µM) was actually lower than that found for controls (Geng et al., 1995b). An equally plausible explanation of the data would be the slow intracellular diffusion (Luxon and Weisiger, 1993); however, this view is incompatible with relatively high unbound fractions inside the cells. Thus, the physiological meaning of this extra deep pool is not evident.

Similar parameters for P_inS and _rel were obtained for EHBR when data were fitted to the one-pool model, although the tail component was not fitted well (fig. 5). Alternatively, when the two-pool model was applied to fit the SDR data, good fits were obtained, and there was virtually no change in the values of P_inS and _rel between the one- and two-pool models (comparison not shown). In contrast, the incorporation of the additional deep pool fully described the outflow profile of [³H]BSPGSH in the EHBR perfused livers. Discrete patterns of influx and efflux of BSPGSH were obtained in EHBR rats in the absence of excretion. With appraisal of the outflow dilution profile of [³H]BSPGSH in relation to that for the theoretical reference, the influx parameters, characterized by the influx permeability-surface area product (or influx clearance/g of liver), P_inS (fig. 8A), or the influx rate constant, k₁ (fig. 8B), were similar to those for SDR (Geng et al., 1995b), and the pattern is characteristic of a carrier-mediated process (Geng et al., 1995a). The absence of change in the influx rate constant between SDR and EHBR livers was also observed for the dual thromboxane A₂ synthetase and 5-lipoxygenase inhibitor, 6-hydroxy-5,7-dimethyl-2-methyamino-4(3-pyridylmethylbenzothiazole), E3040 (Takenaka et al., 1995b).

The data for EHBR were, however, consistent with those for the SDR. In SDR, biliary excretion reduces the amount of labeled BSPGSH available for return to the vasculature. When the return rate of this material is very low, the tail was much smaller and was not consistently present. The presence of the deep pool will be concealed and the one-pool model was considered adequate to describe the SDR data (Geng et al., 1995b). If this hypothesis is correct, the only difference between the two systems should be excretion at the canalicular membrane. This is illustrated in figure 9, which depicts the influence of biliary excretion on the shape of a BSPGSH outflow profile for EHBR at low BSPGSH concentration. Keeping the parameters the same as for the EHBR curve, inclusion of excretion from the shallow pool (k₂₀ = 0.1 sec¹) in the two-pool system predicted an outflow profile with a much smaller tail, resembling the data observed for SDR. In contrast, the correspondence of the predicted outflow profile to SDR data was not as good if excretion was assumed to occur from the "deep pool" (k₃₀ = 0.1 sec¹). Hence, in the absence of the ATP-dependent biliary excretion mechanism, as is the situation in EHBR mutant rats, BSPGSH will accumulate in hepatocytes, revealing the presence of the deep pool.

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Plots of the[3H]BSPGSH outflow data and fitted curve based on a two-pool model for the EHBR against a background set of data obtained with SDR (Geng et al., 1995b) at comparable [3H]BSPGSH concentrations (25 µM) in single-pass perfused rat liver preparations (12 ml/min). The [3H]BSPGSH curve for EHBR was modified by the incorporation of excretion in the shallow (k20 = 0.1 sec1) or deep (k30 = 0.1 sec1) pool. Note the similarity in shapes between the SDR data and the predicted curve based on excretion from the shallow pool.

There existed remaining ambiguity in the assessment of BSPGSH efflux (k₁, k₂₁ or P_outS) and its equilibrium tissue partitioning for EHBR due to the uncertainty regarding the deep pool, whether this is physically part of, or separate from, the shallow pool. The values for P_outS would indeed differ between the one- and two-pool models (comparison not shown). Although the equilibrium partitioning between the deep and shallow pools (k₂₃/k₃₂) is known, the volume of the deep pool appears to be unattainable from the data. Values of P_outS associated with a common space for the shallow and deep pools are, however, too high and are incompatible with previous characteristics observed for BSPGSH, which is seldom detected in the hepatic venous blood/plasma after administration of bromosulfophthalein (Chen et al., 1984; Zhao et al., 1993). This has been attributed to the inherently slow efflux of BSPGSH (Geng et al., 1995b). Moreover, the partitioning of BSPGSH in S9 was high (see table 3), as was that for SDR (Geng et al., 1995b). For the above reasons, that separate spaces exist for the distribution of BSPGSH in liver appears to be a reasonable assumption.

In summary, the sinusoidal blood volume, Disse space and accessible cellular water space in EHBR livers were found to be similar to those for control rats (table 2). The fitting procedure evoked an additional pool that became apparent only in EHBR because biliary excretion of BSPGSH was absent. The deep pool appeared to be a space distinct from that for the shallow pool in view of the low level of return of BSPGSH and the tissue-to-plasma concentration ratios (table 3). The entry of BSPGSH into the liver cell of EHBR rats, given the scatter observed in the data, was found to be similar to that of controls. These findings confirm the notion that the mutation present in EHBR has only eliminated canalicular transport without causing other major effects on cellular function.