Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets

The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions. This transport function is markedly diminished in mutant rats that lack the transport protein. To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity. The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted. The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methyfluorescein. The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane. Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane. The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference. These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane. The relevance of this phenomenon for the intact liver is discussed

Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells

Role of Breast Cancer Resistance Protein in the Adaptive Response to Cholestasis

ABSTRACT: Breast cancer resistance protein (Bcrp) is a member of the ATPbinding cassette membrane transporter family, which is expressed apically in liver, kidney, and intestine epithelium. Recent reports suggest that in addition to xenobiotics, porphyrins, and food toxins, Bcrp can also transport bile acids and, therefore, may participate in the adaptive response to cholestasis. Bile duct ligation (BDL), an experimental model of obstructive cholestasis, was performed in male wild-type (WT) and Bcrp knockout (KO) mice. An initial time course of 3, 7, and 14 days of BDL in WT mice revealed that Bcrp expression was significantly reduced in liver but increased in ileum by 7 days. Subsequent experiments using 7-day BDL in WT and Bcrp KO mice demonstrated that there was no difference in liver necrosis, serum glutamic pyruvate aminotransferase, bilirubin, or bile acid levels in serum, hepatic tissue, bile, urine, or feces between the two groups. Protein expression levels for liver organic solute transporter (Ost) ␣ and multidrug resistance protein 1 and kidney multidrug resistance-associated protein (Mrp) 2, Mrp3, and Mrp4 were significantly greater in the sham Bcrp KO versus sham WT mice. The expression of Mrp2 and Mrp4 in KO kidneys was further increased after BDL. In contrast, the adaptive response of transporters to BDL in the liver was similar in KO and WT BDL mice, including Ost␣ and Ost␤ expression, which increased in liver and kidney but decreased in the ileum. These findings suggest that Bcrp does not have a significant role in the adaptive response to cholestasis in the liver but may be more important for solute export in the kidney and intestine