Abstract
ABC transporter trafficking in rat liver induced by cAMP or taurocholate and [(35)S]methionine metabolic labeling followed by subcellular fractionation were used to identify and characterize intrahepatic pools of ABC transporters. ABC transporter trafficking induced by cAMP or taurocholate is a physiologic response to a temporal demand for increased bile secretion. Administration of cAMP or taurocholate to rats increased amounts of SPGP, MDR1, and MDR2 in the bile canalicular membrane by 3-fold; these effects abated after 6 h and were insensitive to prior treatment of rats with cycloheximide. Half-lives of ABC transporters were 5 days, which suggests cycling of ABC transporters between canalicular membrane and intrahepatic sites before degradation. In vivo [(35)S]methionine labeling of rats followed by immunoprecipitation of (sister of P-glycoprotein) (SPGP) from subcellular liver fractions revealed a steady state distribution after 20 h of SPGP between canalicular membrane and a combined endosomal fraction. After mobilization of transporters from intrahepatic sites with cAMP or taurocholate, a significant increase in the amount of ABC transporters in canalicular membrane vesicles was observed, whereas the decrease in the combined endosomal fraction remained below detection limits in Western blots. This observation is in accordance with relatively large intracellular ABC transporter pools compared with the amount present in the bile canalicular membrane. Furthermore, trafficking of newly synthesized SPGP through intrahepatic sites was accelerated by additional administration of cAMP but not by taurocholate, indicating two distinct intrahepatic pools. Our data indicate that ABC transporters cycle between the bile canaliculus and at least two large intrahepatic ABC transporter pools, one of which is mobilized to the canalicular membrane by cAMP and the other, by taurocholate. In parallel to regulation of other membrane transporters, we propose that the "cAMP-pool" in hepatocytes corresponds to a recycling endosome, whereas recruitment from the "taurocholate-pool" involves a hepatocyte-specific mechanism.
Highlights
ATP binding cassette (ABC) transporter trafficking in rat liver induced by cAMP or taurocholate and [35S]methionine metabolic labeling followed by subcellular fractionation were used to identify and characterize intrahepatic pools of ABC transporters
Groups of rats were injected with either Bt2cAMP (20 mol/kg) or TC (50 mol/kg) for various time periods, and canalicular amounts of ABC transporters were determined in canalicular membrane vesicles (CMV) by Western blotting (Fig. 1)
After treatment with Bt2cAMP, an increase in MDR1, MDR2, and sister of P-glycoprotein (SPGP) in CMVs was observed after 15 min; the effect peaked at 45 min to 4 h and disappeared after 6 h
Summary
ABC transporter trafficking in rat liver induced by cAMP or taurocholate and [35S]methionine metabolic labeling followed by subcellular fractionation were used to identify and characterize intrahepatic pools of ABC transporters. Intravenous administration to rats of dibutyryl-cAMP (Bt2cAMP) or taurocholate (TC) rapidly and selectively increased the functional activity and amount of each ABC transporter in the canalicular membrane; these effects were inhibited by prior administration of colchicine, which disrupts microtubules [11], and Wortmannin, which inhibits phosphatidylinositol 3-kinase [12] These observations indicate that an intracellular microtubule-dependent transport mechanism, which is sensitive to active phosphatidylinositol 3-kinase, is required for trafficking of ABC transporters to the canalicular membrane. The increase in the content of ABC transporters in the canalicular membrane after administration of Bt2cAMP or TC in vivo [11] and in isolated perfused rat liver [12, 13] occurred within minutes after the administration of the effectors These observations suggest that the increment in canalicular ABC transporters is likely to represent recruitment from pre-existing intrahepatic pools rather than from enhanced transcription or translation. These observations suggest that Bt2cAMP and TC recruit ABC transporters to the canalicular membrane from
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