Novel properties of hepatic canalicular reduced glutathione transport revealed by radiation inactivation

Aravind V. Mittur1,2, Neil Kaplowitz1, Ellis S. Kempner3, and Murad Ookhtens1

1 Liver Disease Research Center, Department of Medicine, and 2 Department of Biomedical Engineering, University of Southern California, Los Angeles, California 90033; and 3 Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transport of GSH at the canalicular pole of hepatocytes occurs by a facilitative carrier and can account for ~50% of total hepatocyte GSH efflux. A low-affinity unit with sigmoidal kinetics accounts for 90% of canalicular transport at physiological GSH concentrations. A low-capacity transporter with high affinity for GSH has also been reported. It is not known whether the same or different proteins mediate low- and high-affinity GSH transport, although they do differ in inhibitor specificity. The bile of rats with a mutation in the canalicular multispecific organic anion transporter (cMOAT or MRP-2, a 170-kDa protein) is deficient in GSH, implying that cMOAT may transport GSH. However, transport of GSH in canalicular membrane vesicles (CMV) from these mutant rats remains intact. We examined the functional size of the two kinetic components of GSH transport by radiation inactivation of GSH uptake in rat hepatic CMV. High-affinity transport of GSH was inactivated as a single exponential function of radiation dose, yielding a functional size of ~70 kDa. In contrast, low-affinity canalicular GSH transport exhibited a complex biexponential response to irradiation, characterized by an initial increase followed by a decrease in GSH transport. Inactivation analysis yielded a ~76-kDa size for the low-affinity transporter. The complex inactivation indicated that the low-affinity transporter is associated with a larger protein of ~141 kDa, which masked ~80% of the potential transport activity in CMV. Additional studies, using inactivation of leukotriene C4 transport, yielded a functional size of ~302 kDa for cMOAT, indicating that it functions as a dimer.

biliary transport; target analysis; canalicular multispecific organic anion transporter

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

REDUCED GLUTATHIONE (GSH), the most abundant endogenous thiol in virtually all cells, is maintained at millimolar (approximate micromoles per gram tissue) intracellular concentrations (24). It plays a major role in detoxification of xenobiotics and regulation of the thiol-disulfide status of cellular proteins. In addition it serves as a storage and transfer vehicle for cysteine. Hepatic release of GSH into the sinusoidal plasma and the canalicular bile undergoes a maturational change characterized by the biliary fraction rising from ~5 to ~50% of the total efflux (25).

Although sinusoidal efflux of GSH is well understood (16, 26, 28), the significance of canalicular efflux and its high concentration in bile (mM) is unclear. Biliary GSH has been hypothesized to be a secretagogue for bile acid independent bile flow (4), although the importance and purpose of bile acid independent bile flow are unknown. Cysteine in the biliary tract, liberated by the hydrolysis of GSH, may be reabsorbed from the intestine and utilized for the resynthesis of GSH. The intestine may also reabsorb intact GSH through a Na+-dependent transport mechanism (11). Thus the biliary efflux of GSH may have a role in hepato-bilio-enteric circuit similar to that of sinusoidal efflux in the hepatosystemic circulation (26, 28).

Studies of the kinetics of biliary GSH efflux in isolated perfused livers are limited because hepatic GSH concentrations cannot be raised more than two- to threefold above in vivo levels by typical treatments (17, 25). However, the canalicular membrane-enriched vesicle (CMV) preparation is a suitable model that has been used extensively to characterize the canalicular GSH transport system (2, 7, 9, 10, 14, 27). Canalicular transport of GSH is mediated by two kinetic components: one of high affinity with Km of ~100-200 µM and one of low affinity with Km ~14-17 mM for GSH. The high-affinity component is governed by Michaelis-Menten kinetics, whereas the low-affinity component is characterized by sigmoidal kinetics with a Hill coefficient of n ~2 (27, 21). The latter implies allosterism and positive cooperativity between multiple binding sites on a monomer or interactions between subunits in an oligomeric transport system.

In two mutant rat strains, TR- and EHBR, hyperbilirubinemia is characterized by the hepatic retention of certain organic anions and their conjugates due to deficient secretion. This defect has been attributed to the absence of canalicular multispecific organic anion transporter (cMOAT or MRP-2, a 170-kDa protein), from the plasma membrane, due to a point mutation in this protein (15, 29). Unlike normal rats, these mutant rats do not have GSH in their bile, implying that cMOAT may be the principal transporter for biliary GSH (15, 29). However, the transport of GSH in CMV from EHBR rats remains intact, suggesting the existence of a distinct GSH transporter (10). An initial attempt to clone this transporter was reported (35), but further observations have shown the clone to be artifactual and indicative of a bacterial protein.

The in situ functional size of the canalicular GSH transport system is unknown. Thus it is not known whether the functional size of the canalicular GSH transport system is similar to the molecular size of cMOAT (15, 29) or whether the GSH transport system associates with other proteins. It is also not known whether the two kinetic components of canalicular GSH transport (with distinct affinities and kinetic features) are mediated by common or different proteins.

We used the radiation inactivation technique and target size analysis (13) in the CMV model to estimate the functional size(s) of the protein(s) that mediates canalicular GSH transport. Additional studies, using a specific substrate of cMOAT, i.e., leukotriene (LT) C4, were done to compare the in situ characteristics of GSH transporter(s) and cMOAT and their estimated sizes in our experimental model. Radiation inactivation is a powerful tool that has been applied to determine the functional molecular sizes of enzymes, receptors, and transport systems (13, 19). The distinct characteristics of the kinetics of canalicular GSH transport, namely the negligible cross-contributions of high- and low-affinity components to total transport at the extremes of GSH concentrations, presented us with a unique opportunity to functionally differentiate and study the two kinetic components.

In this study, we have attempted to answer the following two questions. 1) What are the in situ functional sizes of the proteins responsible for the high- and low-affinity transport of GSH across the canalicular membrane? 2) How do the functional sizes of the GSH transport systems compare with the putative size of cMOAT?

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals and assays. All chemicals were purchased from Sigma (St. Louis, MO) or other reputable commercial sources. [35S]GSH (>100 Ci/mmol), [3H]taurocholic acid (>2 Ci/mmol), L-[3H]glutamate (>40 Ci/mmol) [3H]LTC4 (>110 Ci/mmol), and Aquasol scintillation cocktail were procured from NEN (Boston, MA). Membrane filters (HAWP 02500) were obtained from Millipore (Bedford, MA). The purity and molecular form of each lot of [35S]GSH were confirmed by HPLC (8), using a Shimadzu binary system (LC-10AT pumps and SPD-10A controller), equipped with a UV-VIS detector (SPD-10A) and an in-line beta -RAM radiodetector (IN-US Systems). Membrane filters were dissolved in 10 ml Aquasol, and the radioactivity was counted in a Beckman LSC6000 liquid scintillation spectrometer. Spectrophotometric assays were performed on a Beckman DU 7400 diode array spectrophotometer. An HPLC method was used to assay 5'-nucleotidase as described (1).

Animals. Mature (90-135 days old) male Sprague-Dawley rats weighing 380-450 g were purchased from Harlan Laboratories (San Diego, CA) and housed in a constant temperature and humidity environment with alternating 12:12-h light-dark cycles. The rats had free access to water and were fed Purina Rodent Chow ad libitum.

Isolation, characterization, and handling of membrane vesicles. CMV were prepared by the method of Meier et al. (23) with minor modifications. Purity and enrichment of membrane fractions were determined by marker enzymes as described previously (10). Vesicles were suspended in cryoprotective buffer (14% glycerol, 1.4% D-sorbitol, 150 mM KCl, 0.2 mM CaCl2, 0.2 mM MgCl2 in 10 mM HEPES-Tris buffer, pH 7.5). Aliquots of 200 µl were separated in glass ampoules (2 ml; 12011L-2, Kimble), rapidly frozen with liquid nitrogen, sealed with an O2-gas torch, and maintained at -80°C, except during irradiation at -135°C. Radiation dosimetry and sample handling were monitored and validated by inactivation of the apical enzyme, 5'-nucleotidase, as an internal control in every irradiated preparation of CMV (18).

Transport measurements. Transport of radiolabeled substrates was determined with a rapid filtration technique (22). Frozen canalicular membranes were quickly thawed at 37°C and vesiculated by 15 passages through a 25-gauge needle. The suspension was diluted in the cryoprotective buffer (2-3 mg protein/ml) and mixed by five additional passes through a 25-gauge needle.

Aliquots of CMV (20 µl, 30-50 µg protein) were preincubated (3 min, 25 or 37°C) and uptake of radiolabeled substrates was initiated by addition of 80 µl of incubation buffer containing the radiolabeled substrate and an appropriate driving force, when required (e.g., ATP for LTC4 and taurocholate). Uptake was terminated by the addition of 2 ml ice-cold stop buffer. Stop buffers were identical in composition to incubation buffers, without the substrate or driving force.

Transport of GSH (7, 10) and LTC4 was determined under voltage-clamped conditions at 25 and 37°C, respectively, similar to the uptake of L-glutamate and taurocholate. Tracer [35S]GSH was supplemented with unlabeled GSH to arrive at final concentrations of 0.02 µM (trace) to 50 mM GSH. Transport of LTC4 was studied at 1 and 5 µM. Initial rates of uptake were measured at 10 (GSH) and 15 s (LTC4) under zero-trans conditions by terminating the incubations with addition of 2 ml ice-cold stop solution. The selected sampling times were in the linear range of the time-dependent uptake of each compound. "Nonspecific" binding was estimated by the radioactivity bound to CMV at 4°C and 0 time, determined by adding a chilled aliquot of CMV to 2 ml ice-cold stop solution. The net initial rate of uptake (nmol · mg-1 · 10 s-1) was calculated by subtracting the nonspecific binding from the 10- and 15-s uptakes. Uptake of GSH was determined under voltage-clamped conditions in CMV resuspended at a final concentration of 75 mM K+ and treated with valinomycin dissolved in ethanol (10-12 µg/mg protein) for 1 h at 25°C. Uptake of [3H]taurocholate (~1 µM) was studied at 37°C under voltage-clamped conditions in an incubation buffer that contained a final concentration of 75 mM K+ and 5 mM ATP (5). The stop buffer also contained 75 mM K+. The ATP-dependent fraction in LTC4 transport was obtained from the difference of uptake in the absence and presence of 4 mM ATP in an ATP regenerating system.

The presence of significant ATP-dependent uptake of taurocholate and sodium-dependent uptake of glutamate was used as a benchmark for the functional integrity of CMV suspended in cryoprotective buffer. CMV preparations that failed to demonstrate adequate activity of these two transport systems were not used.

Radiation inactivation and target size analysis. Aliquots of CMV preparations were irradiated at -135°C with 10 MeV electrons from a linear accelerator (Armed Forces Radiobiology Research Institute, Bethesda, MD) as described (13). Analysis of the surviving activity in radiation inactivation studies has been described elsewhere (13, 20). The exponential decay in the activity with radiation dose (in rad), AD = A0 e-µD, is used for target analysis to establish the target or functional size of proteins using the following relationship
M = −17.9 × 10<SUP>5</SUP> &mgr;
where µ is the rate constant of the exponential representing the inactivation dose-response curve and M is the functional size of the protein in kilodaltons.

Postirradiation residual transport activity (V) was normalized to the transport activity in the unirradiated control (V0) and expressed as %V/V0. The rate constant of the inactivation curve [slope of ln(V/V0) against radiation dose, D] was estimated by fitting the data from both individual experiments separately and by fitting the mean of residual activities, statistically weighted by their respective variances in each group. In cases where a radiation-insensitive component (Z) of activity was observed, the data were analyzed as described (27) and fitted with
A<SUB>D</SUB> − Z = A<SUB> 0</SUB> <IT>e</IT><SUP>−&mgr;D</SUP>
In cases where a complex biphasic radiation response was observed, i.e., a rise and fall in surviving transport activities (see Fig. 4), the data were fitted with the difference of two exponentials (20)
<IT>V</IT> − Z = [A<SUB> 0</SUB> <IT>e</IT><SUP>−&mgr;D</SUP>] − [B<SUB>0</SUB> <IT>e</IT><SUP>−∂D</SUP>]
where V is transport activity measured in samples exposed to radiation dose D, A0 is total potential transport activity in unirradiated samples, and µ is rate constant of the terminal exponential decay of the inactivation curve, proportional to the functional mass of the transporter, as indicated above; Z is the same as above; B0 equals A0 - 100; partial  is the rate constant of the exponential defined by the difference between the terminal exponential and the data (i.e., A0 e-µD minus the observed transport at dose D).

Kinetic analysis and fitting of data. Kinetics of transport were analyzed using the sum of a Michaelis-Menten and a sigmoidal Hill (n ~2) component
<IT>V</IT> = <IT>V</IT><SUB>H</SUB> + <IT>V</IT><SUB>L</SUB>
where V is the total net transport rate of GSH by the entire transport system.
<IT>V</IT><SUB>H</SUB> = <IT>V</IT><SUB>max1</SUB> ⋅ [S] / (<IT>K</IT><SUB>m1</SUB> + [S]), high affinity component
<IT>V</IT><SUB>L</SUB> = <IT>V</IT><SUB>max2</SUB> ⋅ [S]<SUP><IT>n</IT></SUP> / ([<IT>K</IT><SUB>m2</SUB>]<SUP><IT>n</IT></SUP> + [S]<SUP><IT>n</IT></SUP>), low-affinity component
The fits to the kinetic as well as the irradiation data were obtained using the SAAM II program (version 1.0.3 for Macintosh) (32) and a nonlinear, least-squares error criteria. Data from individual experiments were fitted by weighting the points equally, whereas SEs were used to weight the mean values of the pooled data from each group.

    RESULTS
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Introduction
Materials & Methods
Results
Discussion
References

Enrichment, purity, and functional integrity of membrane vesicles. The CMV fractions were enriched 23-fold in Mg2+-ATPase and 55-fold in alkaline phosphatase but deenriched or not enriched in intracellular organelle markers (Table 1). Each CMV preparation was essentially free of cross-contamination by sinusoidal membranes, as indicated by the negligible activity of the basolateral marker Na+-K+-ATPase. All seven vesicle preparations showed the characteristic increase in the uptake of [3H]taurocholate driven by ATP (22), confirming the functional integrity of CMV (data not shown). The Na+-dependent transport of L-[3H]glutamate (2 µM and 50 mM) in the presence of a K+ gradient was used as a marker of passive permeability in three CMV preparations as an additional check for integrity of vesicles. Transport of glutamate (2 µM) in the absence of Na+ (3) was negligible and did not change with irradiation dose, further confirming the functional integrity of the CMV preparations (data not shown).

                              
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Table 1.   Enrichment of marker enzymes in the canalicular membrane fractions

Inactivation of 5'-nucleotidase. We estimated the functional size of 5'-nucleotidase as an internal control to monitor the dosimetry and consistency of irradiations in individual vesicle preparations. In seven independent preparations, the inactivation of this enzyme yielded a target size of 83 ± 2 (SE) kDa, in excellent agreement with the known structure and previous radiation inactivation analyses (18). The mean of residual activities pooled from seven individual preparations is shown in Fig. 1 along with the fitted single exponential that defines the decay.


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Fig. 1.   Inactivation of hepatic 5'-nucleotidase. Residual activity in 7 independent preparations were pooled, and mean values are shown (open circle ). Solid curve represents fit of monoexponential function to data weighted by SEs, represented by the bars (see target size in text).

Kinetics of GSH transport in unirradiated vesicles. We verified that the kinetics of GSH uptake in the presence of the cryoprotective buffer was similar to that observed in standard suspension buffers reported in past studies (2, 7, 9, 10, 14, 27). The kinetics of GSH uptake in four to seven independent CMV preparations suspended in the cryoprotective buffer is presented in Fig. 2. [Voltage-clamped and nonvoltage-clamped determinations of kinetics were indistinguishable (not shown).] The solid lines in Fig. 2 represent the fits to the data using the model presented in MATERIALS AND METHODS and are representative of high- (i.e., 150 µM) and low-affinity (i.e., 16 mM) average Km values reported in past studies (2, 7, 9, 10, 14, 27). As can be seen, the kinetics measured in the cryoprotective buffer was not different from that in noncryoprotective buffers. The broken lines in Fig. 2 show the contributions of the high- and low-affinity components to overall transport.


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Fig. 2.   Kinetics of GSH transport in canalicular membrane vesicles. Data represent means ± SD of uptake in 4-7 independent preparations suspended in cryoprotective buffer. Initial rates of [35S]GSH uptake (10 s) were measured under voltage-clamped conditions at concentrations ranging from 0.02 µM (trace) to 50 mM GSH. Solid lines indicate fit to overall transport obtained by the sum of 2 components, Michaelis-Menten (high affinity) and Hill (low affinity). B shows high-affinity region on expanded scale. Broken and dotted lines indicate contributions by high- and low-affinity components, respectively. Km values used were representative of those from past studies conducted in noncryoprotective buffers (see text). A: Km = 16.0 mM. B: Km = 0.15 mM. There were no significant differences between kinetics determined in either medium. Kinetics indicate that >99% of transport at trace and 0.02 mM GSH is by high-affinity component, whereas >95% of transport at 25 and 50 mM GSH is by low-affinity component.

Radiation inactivation of GSH transport. The kinetics of GSH transport in CMV (Fig. 2) indicates that >99% of total transport at trace (0.02 µM) and 0.02 mM GSH is mediated by the high-affinity component. Therefore, the functional size of this component was determined by radiation inactivation of uptake at these two concentrations of GSH. Radiation exposure caused a decay in residual transport down to ~10% of that in the unirradiated samples, but further exposure (>65 Mrad) had little effect on uptake (Fig. 3, A and B).


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Fig. 3.   Inactivation of high-affinity transport of GSH. Residual transports at trace GSH (A, open circle ) in 4 independent preparations and at 0.02 mM GSH (B, square ) in 7 independent preparations are shown. triangle , Inhibited values of residual GSH transport in presence of 5 mM disulfobromophthalein (mean of duplicate determinations from up to 2 preparations). Solid lines indicate fits obtained using monoexponential function plus constant value of 7% (see text) weighted by SEs, represented by the bars (see target sizes in text).

The radiation-insensitive residual transport is due to nonspecific factors, such as binding and/or passive diffusion. Experimental verification of this point was made with disulfobromophthalein (diBSP), an organic anion that inhibits canalicular GSH transport (Ref. 2 and Mittur and Ookhtens, unpublished observations). Addition of 5 mM diBSP reduced GSH uptake in both unirradiated and irradiated samples from the levels observed in the uninhibited controls down to a mean value of ~7% at both trace and 0.02 mM GSH (Fig. 3, A and B, triangle ). Thus nonspecific activity accounts for most of the residual transport at the highest radiation exposures. Therefore, the pooled data were fitted with the sum of an exponential plus a constant, representing the noninhibitable fraction equal to ~7% of the unirradiated transport. The functional sizes for the high-affinity GSH transport were estimated to be 73 ± 5 (SE) kDa at trace GSH and 69 ± 5 (SE) kDa at 0.02 mM GSH (fits to pooled data, weighted by SEs, shown with solid curves in Fig. 3, A and B). No differences in the estimated mean target sizes were found when the data from individual experiments were fitted separately (Table 2), compared with the estimates obtained by fitting the pooled data.

                              
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Table 2.   Functional size of canalicular GSH transport at various concentrations

The functional size of the low-affinity component of GSH transport was determined at 25 and 50 mM GSH, at which it accounts for >95% of total transport (see Fig. 2). At these concentrations, the effect of irradiation on GSH transport revealed the response shown in Fig. 4. Low doses of radiation (<36 Mrad) resulted in an activation of GSH uptake that peaked at ~200% of the rates measured in unirradiated samples. This rise was followed by a decay in transport at higher radiation doses (36-76 Mrad). At the highest radiation doses (>76 Mrad), residual transport reached a plateau ~20% of that in unirradiated samples. This ~20% residual fraction, insensitive to radiation, could again be attributed to nonspecific effects and/or passive diffusion. To verify this point, the inhibition of residual transport of GSH at 50 mM was studied in the presence of 5 mM diBSP. Uptake of GSH at all doses was inhibited by diBSP (data not shown), resulting in inhibited residual values equal to 24 ± 3% (mean ± SE, n = 3) at the highest radiation dose.


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Fig. 4.   Inactivation of low-affinity transport of GSH. Residual transport at 25 and 50 mM GSH from 4 (25 mM) and 7 (50 mM) independent preparations were pooled. Mean values (open circle ), weighted by SEs (bars), were fitted (solid line) with difference of 2 monoexponential functions plus a constant value of 24% (see text). Broken lines represent resultant 2 exponentials obtained by fitting inactivation data (see target sizes in text).

The data were therefore analyzed by fitting them with the difference of two exponentials plus a constant equal to 24% (Fig. 4). This analysis is based on a model of GSH transport that implies mediation of a smaller transport unit with a larger structure that blocks or masks the transporter. The estimated rate constant of the terminal (decaying) exponential, defining the inactivation phase, yields the target size of the transporter. The difference remaining after subtraction of the terminal exponential plus the constant from the data defines a second exponential that yields the target size of the larger "inhibitor" or "regulator" protein. Fitting the mean values of the data, pooled from 25 and 50 mM GSH transport experiments (weighted by SEs shown in Fig. 4), gave a target size of 76 ± 11 (SE) kDa for the low-affinity transporter and 141 ± 30 (SE) kDa for the inhibitory or regulatory structure. In this analysis, the exponential defining the inactivation of the low-affinity transporter extrapolates back to ~600% of transport in unirradiated vesicles, indicating that ~80% of potential transport activity remains inhibited in unirradiated samples. These estimated target sizes, obtained by fitting the mean values of pooled data, were similar to estimates obtained by fitting the data from individual experiments separately, calculated by averaging all individual estimates representing the sizes obtained with 25 and 50 mM GSH (Table 2).

Radiation inactivation of cMOAT. To compare the characteristics of inactivation and functional size of cMOAT with those of the GSH transporter, we measured the inactivation of ATP-dependent transport of LTC4 (a substrate with high affinity for cMOAT) in three additional independent vesicle preparations. In all three preparations transport of 1 µM LTC4 and in one additional preparation transport of 5 µM LTC4 were studied. Because cMOAT is a 170-kDa protein, the range of radiation doses in these studies was restricted to 0-35 Mrad. However, using 25 mM GSH, we confirmed that the activation of low-affinity uptake of GSH occurred in these vesicles at similar magnitude to those observed in the vesicles used to study transport of GSH (data not shown).

As can be seen (Fig. 5), in direct contrast to the low-affinity transport of GSH, irradiation resulted in a monoexponential decay of LTC4 transport down to an irradiation-insensitive fraction equal to ~17% of that in unirradiated controls. The functional size of cMOAT was estimated by fitting an exponential plus a constant to the residual ATP-dependent transport activities (fit shown in Fig. 5). Our analysis yielded an average functional size of 302 ± 32 (SE) kDa, which approximates a dimer of cMOAT.


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Fig. 5.   Inactivation of ATP-dependent leukotriene (LT) C4 uptake. Data represent residual ATP-dependent transport of LTC4 (bars are SE) from duplicate determinations in 3 independent vesicle preparations. Solid curve represents fit to data (see target size in text). Note that maximal irradiation dose in these experiments was 35 Mrad.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The kinetics of GSH transport in CMV exhibit two distinct components: 1) a high-affinity Michaelis-Menten element and 2) a low-affinity sigmoidal element with n ~2 (Fig. 2; Refs. 21 and 27). This feature made it possible to study the functional size(s) of the GSH transport system by inactivation of transport at two distinct ranges of concentrations, reflecting essentially the activity of each kinetic component separately. Radiation inactivation identifies the mass of the molecular components required for GSH transport.

Nine independent CMV preparations were irradiated. The activity of 5'-nucleotidase and the transport of GSH and LTC4 were assayed. Radiation target sizes were obtained by analyses of the data from individual experiments as well as from data pooled from multiple preparations. No significant differences were observed between the results with the two methods of analysis. The integral membrane enzyme 5'-nucleotidase served as an internal control, yielding a target size in excellent agreement with both published values from radiation analyses as well as the known structure of the protein. These results validate both sample treatment and radiation dosimetry in our studies.

The functional size of the high-affinity transporter was determined at trace and 0.02 mM GSH concentrations. Radiation inactivation indicated a single protein structure. The estimated mean target sizes obtained at these two concentrations, after accounting for the noninhibitable fraction, were indistinguishable, i.e., ~73 and ~69 kDa, respectively. The low-affinity transporter was studied at 25 and 50 mM GSH. In this case, radiation inactivation revealed a more complex system, characterized by a rise and fall of transport. The estimated size of the transporter from the pooled data at these high concentrations of GSH was ~76 kDa, very close to that found for the high-affinity transporter. The similarity of the functional sizes of the high- and low-affinity components raises the possibility, but does not prove, that the two kinetic components may be mediated by the same protein.

The complex radiation inactivation pattern of the low-affinity component indicated an initial "activation" of GSH transport at low radiation doses. [Note that concomitant radiation inactivation studies conducted on sinusoidal-enriched membrane vesicles did not reveal such an activation pattern (unpublished observations)]. Similar findings in other radiation studies have been interpreted as the destruction of a larger inhibitory or regulatory structure that masks the smaller transporter unit (12, 33, 34). With the combined data at 25 and 50 mM GSH, this larger structure was estimated to be ~141 kDa. Extrapolation to zero dose of radiation (y-axis intercept) of the exponential that defines the inactivation of the transporter unit indicated that >80% of potential GSH transport capacity in normal, unirradiated CMV is suppressed by this larger structure. This outcome indicates that the associated protein exerts a major inhibitory effect on the transport of GSH in CMV. Thus it is conceivable that factors that determine the secretion of GSH into bile may do so by transduction of signals to this regulator to enhance or decrease biliary GSH secretion. An alternative model might be considered, wherein GSH transport in CMV may be conducted by noncovalently associated homodimers (34). Because noncovalent bonds do not transmit radiation-induced damage (13), in theory, irradiation could potentially liberate constituent, fully active, monomeric transporters from homodimers. In this model, the homodimers would be less active in low-affinity transport of GSH, compared with monomers, which would be fully active. However, monomers are not released from many different irradiated homo-oligomers (Kempner, unpublished observations), making this homodimeric model very unlikely. The above two models differ in the characterization of the GSH transport system as a hetero-oligomer (former) vs. a homo-oligomer (latter). However, the radiation inactivation technique cannot discriminate between such detailed molecular characteristics in a membrane transport system in situ.

A transporter involved in the excretion of certain organic anions at the canalicular domain of hepatocytes has been characterized (15, 29). This 170-kDa protein termed cMOAT or MRP-2 is an ATP binding cassette (ABC) transporter that mediates the flux of certain anionic conjugates of GSH into bile (15, 29). Based on indirect evidence (see the introduction), cMOAT would appear to be the principal canalicular transporter for GSH (15, 29). However, many features of GSH transport are inconsistent with those of cMOAT, as enumerated elsewhere (21). For example, protein kinase C (PKC) activation enhances the secretion of organic anions into bile (31) but inhibits biliary GSH secretion (21, 30). On the other hand, the reverse effect was observed with PKC inhibitors (21, 30). These contrasting results of PKC modulation could represent opposite effects on two distinct transporters or two distinct functions of the same transporter. In both cases GSH efflux could be mediated by cMOAT in an ATP-independent manner (driven by electrogenic or electrochemical forces) and functionally regulated in a manner distinct from ATP-driven organic anion transport.

To investigate the relationship between GSH transport and cMOAT in our model, we studied the inactivation of LTC4 transport to compare the functional sizes of the GSH transport system and cMOAT. Our estimate of the functional size of cMOAT from the analysis of our data is ~302 kDa, approximating a dimer of 170 kDa (6, 29). Thus the minimal functional size required for GSH transport in CMV, i.e., 70-76 kDa, is distinctly different from that of cMOAT. These findings indicate that the canalicular GSH transporter is distinct from cMOAT. However, because radiation inactivation cannot identify the details of the molecular components of the GSH transport system, it is possible that the larger regulatory protein associated with low-affinity GSH transport may be cMOAT.

Two other possibilities, although less likely, cannot be unequivocally ruled out at this time. First low-affinity ATP-independent transport of GSH may be mediated by one of the two transmembrane domains of cMOAT. As is a characteristic of many ABC proteins, cMOAT is a single polypeptide containing two asymmetrical transmembrane domains and two nucleotide binding domains. The functional size of the GSH transporter would be consistent with cMOAT only if either transmembrane domain of cMOAT exists as an independent polypeptide (due to posttranslational modifications) and functions individually in the canalicular membrane. Second, canalicular plasma membrane fractions are invariably contaminated by certain intracellular organelles, such as endoplasmic reticulum. Whether or to what extent these may contribute to high-capacity GSH transport in the CMV preparations remains unknown and subject to future investigations. Thus the postirradiation activation of uptake in the CMV may reflect the characteristics of a high-capacity GSH carrier localized in an intracellular organelle. However, this possibility is unlikely because radiation inactivation of GSH transport in simultaneously isolated sinusoidal plasma membrane vesicles (unpublished data) with similar intracellular organelle distributions as CMV resulted in a distinct target size for low-affinity transport and did not exhibit an activation of high- or low-affinity GSH transport. Furthermore, the parallel operation of ATP-dependent transport of dinitrophenyl-glutathione (DNPSG; another substrate for cMOAT) has been found to stimulate GSH uptake in these CMV preparations (9, 10), presumably by transstimulation of GSH uptake by the rapidly accumulating intravesicular DNPSG. Irrespective of the mechanism of this phenomenon, it unequivocally demonstrates that the same membrane vesicles contain cMOAT and the GSH transporter.

    ACKNOWLEDGEMENTS

This study was supported by National Institutes of Health Grants R01-A607467 and R37-DK-30312. Rat liver plasma membrane vesicles were prepared by the Subcellular-Organelle Core of the University of Southern California Research Center for Liver Disease (RCLD; P30-DK-48522). Analysis and fitting of data were performed by the Kinetic and Mathematical Analysis-Modeling Services of LDRC.

    FOOTNOTES

Address for reprint requests: M. Ookhtens, Univ. of Southern California School of Medicine, 1333 San Pablo St., MMR-428, Los Angeles, CA 90033.

Received 6 August 1997; accepted in final form 15 January 1998.

    REFERENCES
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Materials & Methods
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Discussion
References

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AJP Gastroint Liver Physiol 274(5):G923-G930