Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions (original) (raw)
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AJP: Cell Physiology, 2002
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AJP: Cell Physiology, 2002
The physiological relevance of the absorption of glucose from bile by cholangiocytes remains unclear. The aim of this study was to test the hypothesis that absorbed glucose drives aquaporin (AQP)-mediated water transport by biliary epithelia and is thus involved in ductal bile formation. Glucose absorption and water transport by biliary epithelia were studied in vitro by microperfusing intrahepatic bile duct units (IBDUs) isolated from rat liver. In a separate set of in vivo experiments, bile flow and absorption of biliary glucose were measured after intraportal infusion of d-glucose or phlorizin. IBDUs absorbedd-glucose in a dose- and phlorizin-dependent manner with an absorption maximum of 92.8 ± 6.2 pmol · min−1 · mm−1. Absorption of d-glucose by microperfused IBDUs resulted in an increase of water absorption ( J v = 3−10 nl · min−1 · mm−1, P f = 40 × 10−3 cm/sec). Glucose-driven water absorption by IBDUs was inhibited by HgCl2, suggesting that water passively follows absorbed d-...
Hepatic uptake and biliary secretion of bile acids in the perfused rat liver
Pharmacological Research, 1992
Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and, among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r=-0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.
Hepatology, 1997
cAMP concentrations increased both Na / -dependent fluidity and alkalinity absorbing and/or secreting fluid and Na / -independent Cl 0 /HCO 0 3 exchange activity. Stimand electrolytes, particularly HCO 0 3 and Cl 0 . Mechaulation of Cl 0 /HCO 0 3 exchange activity was not prenisms responsible for transepithelial H / /HCO 0 3 secretion vented by the Cl 0 channel inhibitor 5-nitro-2(2)-phein human cholangiocytes are largely unknown. Human nylpropyl-amino-benzoate(NPPB). In conclusion, hucholangiocytes isolated by enzymatic digestion and imman cholangiocytes possess two acid extruders (Na / /H / munomagnetic purification from normal liver tissue obexchanger and Na / -dependent Cl 0 /HCO 0 3 exchange) and tained from reduced grafts used for pediatric liver transan acid loader (Cl 0 /HCO 0 3 exchange), whereas no eviplantation were cultured in the presence of human dence was found for cAMP activated H / -ATPase. Bicarhepatocyte growth factor. Maintenance of cholangiocyte bonate influx is thus mainly mediated by Na-dependent phenotypic features was assessed using markers such Cl 0 /HCO 0 3 exchange, whereas Na / :HCO 0 3 cotransport is as cytokeratin 19, g-glutamyltranspeptidase, vimentin, not active in the physiological range of pHi. Stimulation factor VIII-related antigen, desmin, epithelial memof Na / -independent Cl 0 /HCO 0 3 exchanger by cAMP does brane antigen (EMA), and human epithelial antigen not require activation of Cl 0 conductances. These mech-(HEA) 125. Intracellular pH (pHi) transients were meaanisms may underlay hormone-regulated biliary HCO 0 3 sured microfluorimetrically 27-Bis(2-carboxyethyl)-5,6, secretion in the human biliary tree. (HEPATOLOGY carboxyfluorescein-acetossimethylester (BCECF). In 1997;25:976-985.) the absence of HCO 0 3 , pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na / -dependent and amiloride-inhibitable. No Na / -independent Interest in cholangiocyte physiology and pathophysiology recovery was recorded even after stimulation with has been stimulated by the observation that the biliary epiagents raising intracellular cyclic adenosine monophosthelium is the primary target in several chronic cholestatic phate (cAMP) concentrations. In the presence of liver disorders, including primary biliary cirrhosis, primary HCO 0 3 , recovery from an intracellular acid load required sclerosing cholangitis, and liver allograft rejection. 1-5 Inter-Na / , but was only partly inhibited by amiloride. In these estingly, in cystic fibrosis, a disease of ion transporting epiconditions H / extrusion was inhibited by 4,4,-diisothiothelia, hepatobiliary complications occur in approximately cyan atostilben-2,2,-disulfonic acid (DIDS) and by intra-30% of cases 6 and ultrastructural bile duct cell damage can cellular Cl 0 depletion. Acute removal of extracellular Cl 0 be shown well before hepatocellular damage. 7 In this condiinduced a pHi alkalinization that was inhibited by DIDS. tion, low rates of ductular HCO 0 3 and Cl 0 secretion may lead pHi recovery from an intracellular alkaline load (isohyto increased viscosity of bile and ''ductular'' cholestasis. 7,8 dric CO 2 changes) was Cl 0 -dependent and DIDS-inhibit-Hepatocellular bile is, in fact, extensively modified while flowing through the biliary tree. The epithelial cells lining the intrahepatic bile ducts (cholangiocytes) modulate bile fluidity Abbreviations: cAMP, cyclic adenosine monophosphate; NPPB, 5-nitro-2(2)-phenylproand alkalinity absorbing or secreting fluid and electrolytes, pyl-amino-benzoate; Hepes, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; DIDS, 4,4,-diisothiocyanatostilben-2,2,-disulfonic acid; DBcAMP, N 6 ,2-O-dibutyryladenosine particularly HCO 0 3 and Cl 0 , under the influence of digestive 3:5-cyclic monophosphate; IBMX, 3-isobutyl-1-methyl-xanthine; BCECF-AM, 2:7-Bis(2-hormones such as secretin and somatostatin. 1,3-5,9,10 Studies carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester; HEA, human epithelial antigen; in short-term cultured intrahepatic rat cholangiocytes have g-GT, g-glutamyltranspeptidase; pHi, intracellular measurements; Bi, intrinsic buffering characterized a number of membrane carriers and ion chanpower; KRB, bicarbonate buffered ringer. From the HCO 0 3 exchange, whereas HCO 0 3 entry through the basolat-Sponsored by grant 94.02908.CT04 and ''Progetto Nazionale Invecchiamento'' from Consiglio Nazionale delle Ricerche (Italy) and by ''Centro per lo studio dell'invecchiamento'', eral membrane was mediated by the electrogenic Na / : University of Padova. The financial support of Telethon-Italy (Grant MD.E-430) is gratefully HCO 0 3 cotransport. 11 On the other hand, studies in pig cholacknowledged. Dr. À kos Zsembery was a recipient of an International Fellowship for the angiocytes suggested a role for cyclic adenosine mono-University of Padova. phosphate (cAMP)-activated H / -ATPase in the generation of Part of this work has been presented at the 95th Annual Meeting of the American Gastroenterological Association (New Orleans, LA, 1994) and published in Abstract form intracellular HCO 0 3 . in Gastroenterology 106:A990, 1994. Because of the presence of a number of species-specific vari-Address reprint requests to: Mario Strazzabosco, M.D., Institute of Internal Medicine, ations in bile physiology 13,14 the relevance of the above trans-
Bile salt-associated electrolyte secretion
Experimental and Toxicologic Pathology, 1992
The mechanisms involved in bile salt-induced choleresis are poorly known. To give an insight in this physiological process, bile salt-associated electrolyte secretion was studied following relief of a short-term (2 h) biliary .obstruction in the rat, an experimental model that shows an important diminution of bile salt choleretic efficiency. For this purpose, biliary excretion of total bile salts and electrolytes (sodium, chloride and bicarbonate) were studied in such a model during taurocholate infusion at increasing rates. The results showed that bile flow, bile salt output and electrolyte secretion stimulated by taurocholate,administration were decreased in the rats that were subjected to biliary obstruction. Besides, the choleretic efficiency of the excreted bile salts, as estimated by the slope of the regression line of bile flow vs. bile salt output, was diminished by 46 % (p < 0.005). Multiple regression analysis of bile flow vs. bile salt and electrolyte outputs allowed to detect a selective diminution of the fraction of bile flow related to bile salt-associated electrolyte secretion ("secretory fraction" of the choleretic efficiency of bile salts) (3.2 ± 0.3 vs. 2.5 ± 0.2L1mol, p < 0.05) whereas the "osmotic fraction" of the choleretic efficiency of bile salts was not modified by the treatment (5. a ± 0.4 vs. 5.1 ± 0.3 Llmol, p> 0.05). Since both chloride and bicarbonate biliary concentrations in the volume of bile stimulated by taurocholate were reduced by 53 % and 52 % respectively, a role of these anions in the generation of bile salt-induced choleresis was suggested. Possible mechanisms involved in such a process and in its early impairment during cholestasis are discussed.
Journal of Clinical Investigation, 1997
Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [ 3 H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na ϩ -dependent transcellular transport of [ 3 H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na ϩ -dependent uptake of [ 3 H]taurocholate, with apparent K m and V max values of 209 Ϯ 45 M and 1.23 Ϯ 0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na ϩdependent taurocholate-cotransporting polypeptide and rat ileal apical Na ϩ -dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids. ( J. Clin. Invest. 1997. 100:2714-2721.) Key words: biliary epithelia • taurocholate • transport • liver • plasma membrane vesicles Preliminary portions of this work were presented at the 47th meeting of the American Association for the Study of Liver Diseases, and have been published in abstract form (1996. Hepatology. 94:897 a ).
Transport of drugs in isolated hepatocytes the influence of bile salts
Biochemical Pharmacology, 1978
The influence of bile salts on hepatic transport of drugs was studied using isolated hepatocyte suspensions. Upra~e of the organic anions, dibromosulphthalein (DBSP), indcrcyanine green (ICG) and an organic cation, N4-acetyi procainamide ethobromide (APAEB) was measured. Afte; 60 min incubation the amount of DBSP, ICG and APAEB nresent in the cells was 17. 41 and 4.5 ner cent of the added amount respectively. The release of DBSP, ICG and APAEB from the hepatocytes preincubated with the agents under study, after 60 min incubation in fresh medium was 80.5, 12.5 and 48.9 per cent of the amount initially present respectively. The presence of bile canalicular membranes in the isolated hepatocytes was demonstrated by enzymehistochemistry: 5'nucleotidase activity showed sharp branched bands over the cell surface. When bile salts were present in the incubation medium. the cellular content of DBSP, ICG and APAEB was diminished. The taurocholate concentration which caused 50 per cent of the maximal effect was 0.07mM. O.lOmM and 0.06mM in experiments with DBSP, ICG and APAEB respectively. Pharmacokinetic analysis revealed that the influence of bile salts on cellular content of the three compounds was due to inhibition of the uptake into the isolated hepatocytes, rather than stimulation of release from the cells. The hypothesis, that stimulation of biliary output of organic anions in viw is due to a modifying effect of bile salts on the canalicular membranes. instead of being the result of the increased bile flow, is not supported by this study.