Experimental Procedures

Materials.

[³H]Enalapril was synthesized as described previously (Pang et al., 1998). Enalaprilat and unlabeled enalapril were obtained from Merck-Frosst (Montreal, PQ). [¹⁴C]Sucrose (specific activity, 0.05 mCi/ml) was purchased from DuPont (Boston, MA). Estrone-3-sulfate, harmol sulfate, DIDS (4,4′-diisothiocyanostilbene-2, 2′-disulfonic acid), ouabain, rotenone, BSA, and alanine-proline were purchased from Sigma Chemical Co. (St. Louis, MO). KCN was obtained from Aldrich Chemical Company Inc. (Milwaukee, WI). Collagenase A (clostridium histolyticum) was purchased from Boehringer Mannheim (Darmstadt, Germany). All other reagents and solvents were of HPLC grade.

Expression Systems for Oatp1, Oatp2, and OATP.

The transporter cDNA constructs and transfection of the cDNAs have been described in detail in a previous report (Cvetkovic et al., 1999). Transport studies in HeLa cells transfected with 1 μg of plasmid cDNA or the parental plasmid lacking any transporter sequence for the control were initiated by the addition of 0.4 ml/well of [³H]enalapril (5 μM). Uptake was allowed to occur at 37°C, then transport activity was stopped at 10 min as previously described (Cvetkovic et al., 1999). After lysing of the cells, the radioactivity associated with the cell lysate was counted by liquid scintillation spectrometry (Rackbeta model 1219; LKB Instruments, Gaithersburg, MD).

Isolation of Rat Hepatocytes.

Isolated rat hepatocytes were prepared by the method of Tan et al. (1999) from male Sprague-Dawley rats (275–375 g; Charles River, St. Constant, PQ). Rats were anesthetized by i.p. injection of pentobarbital (65 mg/kg) in accordance with protocols approved by the Animal Care Committee of the Department of Comparative Medicine at the University of Toronto. All buffers were pregassed with carbogen (95% O₂/5% CO₂; Matheson, Mississauga, ON). The portal vein was perfused with a Ca²⁺- free buffer [preperfusion buffer, consisting of Hanks' buffer (137 mM NaCl, 5.4 mM KCl, 0.5 mM NaH₂PO₄2H₂O, 0.42 mM Na₂HPO₄^.12 H₂O), plus 10 mM HEPES (pH 7.2), 0.5 mM EGTA, 4.2 mM NaHCO₃, 5 mM glucose, and 0.65% BSA] in a single pass fashion at 30 ml/min for 10 min. Hepatocytes from all zones of the liver were prepared by perfusion with collagenase (Hanks' buffer with 4 mM CaCl₂ and 0.05% collagenase), which recirculated through the liver for 7 to 9 min at the flow rate of 25 ml/min. Zonally isolated hepatocytes were harvested following the procedure of Lindros and Penttı̈la (1985) by zone-selective destruction with digitonin. The enrichment of hepatocyte populations was verified by marker enzymes (Tan et al., 1999): the activity of the PP marker enzyme alanine aminotransferase was found to be 2.13 ± 1.0 times higher in PP hepatocytes (433 ± 165 nmol/min/mg protein) than in PV hepatocytes (203 ± 58 nmol/min/mg protein). Conversely, glutamine synthetase, an enzyme expressed exclusively in the distal PV hepatocytes, exhibited significantly lower activity in PP hepatocytes (0.15 ± 0.1 nmol/min/mg protein) than in PV preparations (34.5 ± 10.6 nmol/min/mg protein) (ANOVAP < .05); the PP/PV ratio was 0.0043 ± 0.03.

The collagenase-digested liver was placed in a Petri dish filled with incubation buffer [137 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂^.6H₂O, 0.83 mM MgSO₄^.7H₂O, 0.5 mM NaH₂PO₄^.2H₂O, 0.42 mM Na₂HPO₄^.12H₂O, 4.2 mM NaHCO₃, 5 mM glucose, and 1 mM HEPES (pH 7.4)] and agitated to release isolated hepatocytes into the medium. The viability of the resulting hepatocyte suspension was >90%, as assessed by trypan blue exclusion.

Kinetic Uptake Studies.

Hepatocyte suspensions (1 ml of 2 × 10⁶cells/ml) were preincubated in a rotating water bath for 10 min at 37°C. Aliquots (500 μl) of the suspension were added to 100 μl of various solutions containing enalapril, tracer [³H]enalapril, and [¹⁴C]sucrose (an extracellular marker) in 1.15% KCl to result in enalapril concentrations of 1 to 750 μM in 1.67 × 10⁶ cells/ml. After admixture, samples (100 μl) were retrieved over 1 min and centrifuged immediately (9650g) for rapid filtration through a layer of silicon oil, separating the hepatocytes from the extracellular medium. Samples were taken from both the supernatant and cell pellet for liquid scintillation counting (Model LS 6800; Beckman Canada, Mississauga, ON).

Inhibitor Uptake Studies.

The ability of benzoic acid (100 μM), harmol sulfate (200 μM), estrone-3-sulfate (100 μM), or the dipeptides, alanine-proline (400 μM) and enalaprilat (400 μM), to cis-inhibit enalapril uptake was examined by individually adding the compounds to the enalapril solution before the addition to hepatocytes. The metabolic inhibitor KCN (2 mM), the anion transport inhibitor, DIDS (2 mM), and the Na⁺/K⁺ ATPase inhibitor, ouabain (1 mM), were added to the cell suspension during the last 2 min of the 10-min hepatocyte preincubation interval, whereas the metabolic inhibitor, rotenone (30 μM), was added to the hepatocyte suspensions for preincubation for 30 min before the addition of enalapril. An equivalent volume of buffer was added for the control samples. All control samples were preincubated for 10 min except for the controls for rotenone, which were preincubated for 30 min. In all cases, the final concentration in the incubation mixture was 1 μM enalapril and 1.67 × 10⁶ cells/ml. Data for the effects of treatment were expressed as percentages of the uptake rates of the controls.

Western Blot.

Cells were pelleted from zonal hepatocyte suspensions and frozen at −70°C until use. Cells were then extracted with Na₂CO₃ to remove the loosely associated membrane proteins according to a previously described method (Bergwerk et al., 1996), with modifications. Briefly, cell pellets were suspended in 28 ml of 0.1 mM Na₂CO₃ containing the following mixture of protease inhibitors: 20 μg/ml BAME (Nα-p-benzyl-l-arginine-methyl ester); 20 μg/ml soybean trypsin inhibitor; 20 μg/ml TAME (Nα-p-tosyl-l-arginine-methyl ester); 1 μg/ml leupeptin; 1 mM phenylmethylsulfonyl fluoride; 2 μg/ml aprotinin; 1 mM EDTA; and 5 mM EGTA. The suspension was rotated at 4°C for 15 min and then centrifuged at 4°C for 1 h at 100,000g. The resulting pellet was resuspended in 0.4 ml of PBS by mild sonication. Protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL) using BSA as standard. Immunoblot was performed using an antibody prepared to a synthetic peptide corresponding to a region of Oatp1 near the carboxy terminus as previously described (Bergwerk et al., 1996).

Kinetic Analysis.

The [¹⁴C]sucrose counts associated with the pellet accounted for the volume of extracellular medium entrapped by hepatocytes. The uptake of [³H]enalapril was thus corrected for the extracellular counts entrapped, and the specific activity of the radiolabeled enalapril was used to calculate the rate of enalapril uptake. The cumulative amounts taken up into cells (nmol/10⁶ cells), after correction for the entrapped extracellular components, were plotted against the incubation time. Linear regression of the data yielded the slope that represents the initial uptake rate (v). When v was plotted against the corresponding concentration [S], the kinetic constants were obtained by regression of v versus [S] according to the following equations:

For a system consisting of a single Michaelis-Menten component, Equation 1for a system with saturable and nonsaturable components, Equation 2and for a system consisting of two saturable components,v=Vmax,1[S]Km,1+[S]+Vmax,2[S]Km,2+[S] Equation 3where K_m represents the Michaelis-Menten parameter, V_max denotes the maximal velocity, and P_diff is the unidirectional transmembrane clearance at the sinusoidal membrane.

Fitting and Data Handling.

Data on the slopes (v) from each experiment were fitted individually to eq. 1, 2, or 3 by an iterative nonlinear least square procedure (Scientist v.2; Micromath Scientific Software, Salt Lake City, UT). Kinetic uptake data for each experiment were fit to all three equations. The Model Selection Criteria was used to determine the appropriateness of the equations: the greater the Model Selection Criteria value for a model, the better that model describes the data. A weighting scheme of 1/(observation)² was used because this provided the best fit. The goodness of fit was judged by the S.D. of the parameter estimate or the c.v. (S.D./parameter estimate), residual plots, and the residual sum of squares.

Statistics.

The data were presented as mean ± S.D. The means were compared by use of ANOVA or the paired t statistic accordingly, withP = .05 denoting the level of significance.

Results

Enalapril Uptake by Oatp1, Oatp2, and OATP.

The uptake of [³H]enalapril (5 μM) by Oatp2 and OATP was not significantly different from that of the control (Fig.1). By contrast, uptake was significantly higher for Oatp1 in comparison to the control, as found previously (Pang et al., 1998).

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Figure 1

Uptake of [³H]enalapril (5 μM) by Oatp1 (n = 4), Oatp2 (n = 4), and OATP (n = 4) in recombinant vaccinia expression systems.

Note the lack of uptake by Oatp2 and OATP because the uptake values are similar to that of the controls (n = 6). See text for details.

Enalapril Uptake into Hepatocytes.

Transport of enalapril among all hepatocyte preparations was linear over 1 min for all concentrations (see Fig.2 for representative study). Similar saturation curves for enalapril uptake were evident with rat hepatocytes isolated from whole liver, in the presence and absence of sodium ion (Fig. 3), and with PP and PV hepatocytes (Fig. 4). The best fit in all cases was eq. 1, which described a single saturable component. The fitted V_max value in the presence of sodium for hepatocytes from whole liver was 11 ± 1.5 nmol/min/10⁶ cells, and theK_m value was 344 ± 52 μM. These values did not differ from those obtained for the uptake of enalapril in absence of sodium ion (V_max value of 11.6 ± 4.2 nmol/min/10⁶ cells andK_m value of 446 ± 179 μM), and were similar to those for PP and PV cells (V_maxvalue of 9.5 ± 2.8 and 10.4 ± 2.0 nmol/min/10⁶ cells, andK_m values of 461 ± 177 and 411 ± 50 μM; P > .05, ANOVA, Table1).

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Figure 2

Time course of [³H]enalapril (1–750 μM) uptake by rat hepatocytes isolated from whole rat liver in a representative experiment in the presence of sodium over 1 min.

The initial uptake velocity (slope) obtained by linear regression of data was linear for all concentrations over 1 min.

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Figure 3

Uptake kinetics of enalapril by rat hepatocytes isolated from whole rat liver in the presence(●) or absence (○) of sodium ion.

Data points represent the mean ± S.D. (n = 4 or 5) of the uptake velocity with increasing concentrations of enalapril. The lines are based on the averaged fitted constants shown in Table 1for a single saturable uptake component.

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Figure 4

Uptake kinetics of enalapril by PP(●) and PV (○)hepatocytes isolated from the rat according to the methods of Lindros and Penttı̈la (1985) in the presence of sodium ion.

Data points represent the mean ± S.D. (n = 4) of the uptake velocity with increasing concentrations of enalapril. The lines are based on the averaged fitted constants shown in Table 1 for a single saturable uptake component.

Inhibitor Uptake Studies.

The uptake of 1 μM enalapril was unaffected by the metabolic inhibitors KCN (2 mM) and rotenone (30 μM) (P > .05, paired t test, Fig. 5). In addition, benzoic acid (100 μM), enalaprilat (400 μM), and alanine-proline (400 μM) failed to affect the uptake of enalapril (P > .05, paired t test, Fig. 5). However, the anion transport inhibitor DIDS (2 mM), temperature reduction, the Oatp1 substrate estrone-3-sulfate (100 μM) (Bossuyt et al., 1996), and surprisingly, harmol sulfate (200 μM), all significantly inhibited enalapril uptake (P < .05, pairedt test, Fig. 5). Unexpectedly, ouabain (1 mM), another Oatp1 substrate (Noé et al., 1997), failed to exert a significant effect on the uptake of enalapril. This may be due to the highK_m value of ouabain (1700–3000 μM) (Noé et al., 1997; Reichel et al., 1999) and to the variability in the data (Fig. 5).

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Figure 5

Effect of various compounds on enalapril(1 μM) uptake into rat hepatocytes prepared from whole liver, in the presence of sodium.

The ability for the Oatp1 substrates [estrone-3-sulfate (100 μM) and ouabain (1 mM)], harmol sulfate (200 μM), the dipeptides [alanine-proline (400 μM) and enalaprilat (400 μM)], the monocarboxylic acid benzoic acid (100 μM), the nonspecific anion transport inhibitor DIDS (2 mM), temperature reduction, and the metabolic inhibitors [KCN (2 mM) and rotenone (30 μM)] to inhibit enalapril uptake was examined over 1 min. Values were expressed as a percentage of control ± S.D. (n = 3–6). *, Denotes treatment values that are statistically different from controls, (P < .05, paired t test). Ala-Pro, alanine-proline dipeptide.

Western Blotting.

The expression of Oatp1 in PP and PV hepatocytes, quantified by Western blot analysis, was similar (Fig. 6); integration of the optical density of the immunoreactive proteins revealed similar intensities of 0.79 ± 0.13 and 0.88 ± 0.33 for the PP and PV regions, respectively (P > .05, ANOVA).

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Figure 6

Western blot analysis of Oatp1 in PP and PV hepatocytes.

Hepatocyte pellets were extracted with 0.1 mM Na₂CO₃ as described in Experimental Procedures. An aliquot (50 μg) of each pellet was applied to each lane. The odd-numbered lanes are extracts from PP hepatocytes and the even-numbered lanes are extracts from PV hepatocytes.

Discussion

Oatp1 and Oatp2 (Jacquemin et al., 1994; Noé et al., 1997), the sodium/taurocholate cotransporting polypeptide (Hagenbuch et al., 1991), and the organic anion transporter 2 (Oat2) (Sekine et al., 1998) are cloned proteins expressed on the rat hepatocyte basolateral membrane that are capable of transporting organic anions. Presently, we confirmed that sodium ion is not involved in the transport of enalapril in isolated rat hepatocytes (Table 1), excluding the role of sodium/taurocholate cotransporting polypeptide, as shown earlier (Pang et al., 1998). Moreover, uptake is not mediated by Oatp2, which is known to transport other anions such as the estrogen conjugates, estradiol 17β-glucuronide and estrone 3-sulfate (Noé et al., 1997), pravastatin (Tokui et al., 1999), bile acids, digoxin, and thyroxine (Kakyo et al., 1999; Reichel et al., 1999). Our observations conform to a recent observation on the lack of correlation of Oatp1 and Oatp2 substrate specificity (Reichel et al., 1999).

Structurally, enalapril is a modified dipeptide of alanine-proline. Both enalapril and alanine-proline have been reported to be substrates of the oligopeptide transporter PepT1 expressed in the rat intestine and kidney (Saito et al., 1995). A second isoform expressed in rat kidney, PepT2, however, does not transport enalapril (Boll et al., 1996). On examination of the inhibitory action of alanine-proline on enalapril uptake, none was found (Fig. 5). Enalaprilat, the active metabolite of enalapril and also an ala-pro analog, was unable to inhibit enalapril uptake into hepatocytes as well (Fig. 5). These observations are consistent with enalapril not being a substrate of PepT1 and PepT2, and that enalaprilat is neither a substrate of Oatp1 nor an inhibitor of enalapril transport in Oatp1-expressing HeLa cells (Pang et al., 1998). However, it was observed that enalaprilat was effective in inhibiting the transport of another ACE inhibitor, temocaprilat, into rat hepatocytes by processes that are mediated, in part, by Oatp1 (Ishizuka et al., 1998). As both enalaprilat and temocaprilat are dicarboxylic acids and enalapril is a monocarboxylic acid, it is plausible that enalapril and temocaprilat bind to different areas of Oatp1, explaining the differences in their susceptibility to enalaprilat inhibition.

To further explore the role of other transporters, we used substrates/inhibitors to effect cis-inhibition of enalapril transport into hepatocytes. There was expressed sensitivity toward the anion transport inhibitor, DIDS, which diminished enalapril uptake (Fig. 5), as expected for the behavior of an Oatp1 substrate (Pang et al., 1998; Tan et al., 1999). Estrone-3-sulfate, a known substrate of Oatp1 (Bossuyt et al., 1996) significantly reduced enalapril uptake (Fig. 5). Ouabain, another Oatp1 substrate (Noé et al., 1997), failed to affect the uptake of enalapril (Fig. 5), although ouabain (1 mM) was known to inhibit the activity of Na⁺/K⁺ ATPase at similar concentrations (Sillau et al., 1996). The lack of inhibition with ouabain was also observed for BSP in isolated hepatocytes (Schwenk et al., 1976) and Oatp1-cDNA transfected into Xenopus laevisoocytes (Kullak-Ublick et al., 1994), and for its glutathione conjugate (BSPGSH) (Schwarz et al., 1980), another Oatp1 substrate (Pang et al., 1998). Absence of inhibition may be due to the variability in the data and the relatively low affinity of ouabain for Oatp1 (K_m value of 1700–3000 μM) (Noé et al., 1997; Reichel et al., 1999) compared with that for enalapril (K_m value of 350–440 μM). Oatp2 will likely play a more important role than Oatp1 in the hepatic uptake of ouabain (Noé et al., 1997).

Although Oatp1 transport appears to be independent of ATP hydrolysis (Li et al., 1998), uptake may be affected when intracellular ATP is depleted. Uptake was further characterized with respect to temperature. An energy dependence was inferred because uptake was reduced drastically when the temperature was lowered (Fig. 5). However, the metabolic inhibitors rotenone (30 μM, preincubated for 30 min) and KCN (2 mM) were unable to reduce enalapril uptake in these studies. The lack of effect of rotenone or potassium cyanide on enalapril uptake is unexpected, because rotenone (30 μM) was known to reduce ATP levels by 90% when preincubated with hepatocytes over 30 min (Yamazaki et al., 1993). Indeed, conflicting results concerning the effects of cellular ATP depletion on Na⁺-independent organic anion uptake have been reported. Variations existed due to differences in the method used for ATP depletion and the time over which uptake was determined (Yamazaki et al., 1993). BSPGSH uptake was insensitive to the effects of rotenone (Schwarz et al., 1980). BSP uptake was found to be unaffected by the metabolic inhibitors antimycin A, rotenone, and carbonylcyanide m-chlorophenylhydrazone (Schwenk et al., 1976), although uptake was reduced when 2-deoxyglucose and sodium azide were used for ATP depletion via glycolysis and mitochondrial respiration in hepatocytes (Yamazaki et al., 1993) and Oatp1-expressing HeLa cells (Shi et al., 1995). Uptake of temocaprilat, measured over 90 s, was inhibited by the electron transport inhibitor, rotenone (Ishizuka et al., 1998). The discrepancies are possibly explained by the fact that uptake of both temocaprilat and BSP are only partly mediated by Oatp1 in hepatocytes (Hagenbuch et al., 1996; Ishizuka et al., 1998).

There exist other sodium-independent systems on the basolateral membrane of hepatocytes that could potentially transport enalapril. Benzoic acid, a putative substrate of the monocarboxylate transporter MCT2 (Garcia et al., 1995) and not of Oatp1 (Pang et al., 1998), failed to inhibit enalapril uptake (Fig. 5). The drug sulfate conjugate, harmol sulfate, which is transported into hepatocytes via an unknown mechanism, was inhibitory although harmol sulfate was not transported by Oatp1-expressing HeLa cells (Pang et al., 1998) and failed to inhibit the uptake of estrone sulfate (Tan et al., 1999). The recently cloned novel liver-specific transport protein OAT2 (Sekine et al., 1998) that affects the uptake of salicylate, PGE₂, dicarboxylates, andp-aminohippurate, is unlikely involved because there is a lack of inhibition of Oat2 transport by enalapril, and there is no known overlapping substrate specificity between Oat2 and Oatp1 (Kullak-Ublick et al., 1994; Sekine et al., 1998).

Inasmuch as Oatp1 is involved in the uptake of various pharmacologically important peptides and steroids, it becomes important to characterize its zonal expression and acinar function. No difference was observed for uptake between hepatocytes prepared from the PP and PV regions and whole rat liver (Fig. 4, Table 1). Additionally, Western blot analysis revealed a lack of quantifiable difference between Oatp1 protein expression in PP and PV hepatocytes (Fig. 6). This observation provides quantitative evidence for homogeneity of protein expression and is consistent with prior observations of the lack of noticeable unevenness in protein expression, qualitatively observed under confocal microscopy (Bergwerk et al., 1996), and findings of a lack of lobular heterogeneity for Oatp1 mRNA as measured by in situ hybridization (Dubuisson et al., 1996; Angeletti et al., 1998). By contrast, an enriched midzonal-PV distribution of Oatp2 was suggested recently (Kakyo et al., 1999; Reichel et al., 1999), and Oatp2 is not involved in enalapril uptake (Fig. 1). Because activities in zonal hepatocytes and Oatp1 are both homogeneously expressed across the acinus for the uptake of enalapril, and because similar K_mvalues are obtained for both systems, it appears that Oatp1 is likely the only transporter involved in the uptake of enalapril. Inasmuch as the transport of enalapril into rat hepatocytes is an early and mandatory process in the overall hydrolysis and excretion of both enalapril and enalaprilat, which subsequently return to the venous circulation, the PV preponderance in enalapril hydrolysis observed in the intact liver (Pang et al., 1991) may now be attributed to an enrichment in intracellular metabolic activity and not transport in the PV region. The PV preponderance of hydrolysis of enalapril by the 9000g supernatant fraction of hepatocyte homogenates has recently been verified in incubation experiments (Abu-Zahra and Pang, 2000).

In conclusion, we showed that enalapril uptake by isolated rat hepatocytes is sodium-independent and is best described by a single saturable component model with a V_max value of 9.5 to 11.6 nmol/min/10⁶ cells and aK_m value of 344 to 461 μM. The uptake of enalapril and the expression of Oatp1 were both found to be homogeneous across the liver acinus. Uptake of enalapril by isolated rat hepatocytes is consistent with Oatp1-mediated transport.