Abstract
In cholestasis, the accumulation of organic anions in hepatocytes is reduced by transporters (multidrug resistance-associated proteins and OSTalpha-OSTbeta) able to extrude them across the basolateral membrane. Here we investigated whether organic anion-transporting polypeptides (OATPs) may contribute to this function. Xenopus laevis oocytes expressing human carboxylesterase-1 efficiently loaded cholic acid (CA) methyl ester, which was cleaved to CA and exported. Expression of OATP8/1B3 enhanced CA efflux, which was trans-activated by taurocholate but trans-inhibited by reduced (GSH) and oxidized (GSSG) glutathione. Moreover, taurocholate and estradiol 17beta-D-glucuronide, but not bicarbonate and glutamate, cis-inhibited OATP8/1B3-mediated bile acid transport, whereas glutathione cis-stimulated this process, which involved the transport of glutathione itself with a stoichiometry of 2:1 (GSH/bile acid). No cis-activation by glutathione of OATP-C/1B1 was found. Using real time quantitative reverse transcription-PCR, the absolute abundance of OATP-A/1A2, OATP-C/1B1, and OATP8/1B3 mRNA in human liver biopsies was measured. In healthy liver, expression levels of OATP-C/1B1 were approximately 5-fold those of OATP8/1B3 and >100-fold those of OATP-A/1A2. This situation was not substantially modified in several cholestatic liver diseases studied here. In conclusion, although both OATP-C/1B1 and OATP8/1B3 are highly expressed, and able to transport bile acids, their mechanisms of action are different. OATP-C/1B1 may be involved in uptake processes, whereas OATP8/1B3 may mediate the extrusion of organic anions by symporting with glutathione as a normal route of exporting metabolites produced by hepatocytes or preventing their intracellular accumulation when their vectorial traffic toward the bile is impaired.
Highlights
The removal from blood of endogenous and xenobiotic cholephilic organic anions, such as bile acids, bilirubin, and steroid hormones, is a major function carried out by the liver
Radiolabeled substrates were obtained from American Radiolabeled Chemicals (ITISA Biomedica, Madrid, Spain) or PerkinElmer Life Sciences, except 22,23-3H-labeled bile acids, [3H]taurochenodeoxycholic acid (TCDCA; 0.37 TBq/mmol), [3H]taurodeoxycholic acid (TDCA; 1.11 TBq/mmol), and [3H]tauroursodeoxycholic acid (TUDCA; 0.37 TBq/mmol), which were prepared by reductive tritiation of their corresponding ⌬22,23 precursors, as reported previously [21]. [14C]Cholic acid (CA) methyl ester (CAME) was synthesized by reaction of CA dissolved in methanol/ chloroform (1:2, v/v) with an excess of freshly distilled diazomethane in diethyl ether at room temperature for 12 h [22]
Because in preliminary experiments no significant difference between oocytes injected with TE buffer and noninjected oocytes regarding their ability to take up bile acids or E217G was found, in this study, noninjected oocytes were used to determine nonspecific uptake
Summary
Radiolabeled substrates were obtained from American Radiolabeled Chemicals (ITISA Biomedica, Madrid, Spain) or PerkinElmer Life Sciences, except 22,23-3H-labeled bile acids, [3H]taurochenodeoxycholic acid (TCDCA; 0.37 TBq/mmol), [3H]taurodeoxycholic acid (TDCA; 1.11 TBq/mmol), and [3H]tauroursodeoxycholic acid (TUDCA; 0.37 TBq/mmol), which were prepared by reductive tritiation of their corresponding ⌬22,23 precursors, as reported previously [21]. [14C]Cholic acid (CA) methyl ester (CAME) was synthesized by reaction of CA dissolved in methanol/ chloroform (1:2, v/v) with an excess of freshly distilled diazomethane in diethyl ether at room temperature for 12 h [22]. Cis-activation of the Efflux from Oocytes Loaded by Incubation—The oocytes were incubated with 10 M [14C]taurocholic acid in the absence of GSH at 25 °C for 120 min or in the presence of 20 mM [3H]GSH at 25 °C for 60 min The efflux of both compounds was determined at different time points after transferring the oocytes to radioactive substrate-free medium at 25 °C. Cis-activation of the Efflux from Oocytes Loaded by Microinjection—The oocytes were directly loaded with 50 nl of U medium containing 300 M [14C] taurocholic acid alone or with 20 mM [3H]GSH The efflux of both compounds was determined at different time points after immediately transferring the oocytes to radioactive substrate-free U medium at 25 °C. The primer oligonucleotides sequences and conditions for measuring the absolute abundances of OATP-A/1A2, OATP-C/1B1, and OATP8/ 1B3 have been reported elsewhere [8]
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