Secretin induces the apical insertion of aquaporin-1  water channels in rat cholangiocytes

Aquaporin-1 (AQP1) water channels are present in the apical and basolateral plasma membrane domains of bile duct epithelial cells, or cholangiocytes, and mediate the transport of water in these cells. We previously reported that secretin, a hormone known to stimulate ductal bile secretion, increases cholangiocyte osmotic water permeability and stimulates the redistribution of AQP1 from an intracellular vesicular pool to the cholangiocyte plasma membrane. Nevertheless, the target plasma membrane domain (i.e., basolateral or apical) for secretin-regulated trafficking of AQP1 in cholangiocytes is unknown, as is the functional significance of this process for the secretion of ductal bile. In this study, we used primarily an in vivo model (i.e., rats with cholangiocyte hyperplasia induced by bile duct ligation) to address these issues. AQP1 was quantitated by immunoblotting in apical and basolateral plasma membranes prepared from cholangiocytes isolated from rats 20 min after intravenous infusion of secretin. Secretin increased bile flow (78%, P < 0.01) as well as the amount of AQP1 in the apical cholangiocyte plasma membrane (127%, P < 0.05). In contrast, the amount of AQP1 in the basolateral cholangiocyte membrane and the specific activity of an apical cholangiocyte marker enzyme (i.e., γ-glutamyltranspeptidase) were unaffected by secretin. Similar observations were made when freshly isolated cholangiocytes were directly exposed to secretin. Immunohistochemistry for AQP1 in liver sections from secretin-treated rats showed intensified staining at the apical region of cholangiocytes. Pretreatment of rats with colchicine (but not with its inactive analog β-lumicolchicine) inhibited both the increases of AQP1 in the cholangiocyte plasma membrane (94%, P < 0.05) and the bile flow induced by secretin (54%, P < 0.05). Our results in vivo indicate that secretin induces the microtubule-dependent insertion of AQP1 exclusively into the secretory pole (i.e., apical membrane domain) of rat cholangiocytes, a process that likely accounts for the ability of secretin to stimulate ductal bile secretion.

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Expression, Distribution, and Activity of Na+,K+-ATPase in Normal and Cholestatic Rat Liver

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549804600315 ◽

1998 ◽

Vol 46 (3) ◽

pp. 405-410 ◽

Cited By ~ 6

Author(s):

Lukas Landmann ◽

Sabine Angermüller ◽

Christoph Rahner ◽

Bruno Stieger

Keyword(s):

Enzyme Activity ◽

Plasma Membrane ◽

Driving Force ◽

Bile Secretion ◽

Transport Systems ◽

Apical Plasma Membrane ◽

Membrane Domain ◽

Expression Domain ◽

Duct Ligation ◽

Plasma Membrane Domain

Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+,K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (~10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis.

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Vitamin D inhibits development of liver fibrosis in an animal model but cannot ameliorate established cirrhosis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00132.2013 ◽

2015 ◽

Vol 308 (2) ◽

pp. G112-G120 ◽

Cited By ~ 37

Author(s):

Shirley Abramovitch ◽

Efrat Sharvit ◽

Yosef Weisman ◽

Amir Bentov ◽

Eli Brazowski ◽

...

Keyword(s):

Vitamin D ◽

Growth Factor ◽

Bile Duct ◽

Liver Fibrosis ◽

Bile Duct Ligation ◽

Active Form ◽

Preventive Treatment ◽

Antifibrotic Effect ◽

Duct Ligation

1,25(OH)2D3, the active form of vitamin D, has an antiproliferative and antifibrotic effect on hepatic stellate cells. Our aim was to investigate the potential of 1,25(OH)2D3 to inhibit the development of liver fibrosis and to ameliorate established fibrosis in vivo. The antifibrotic effect of 1,25(OH)2D3 was investigated in a thioacetamide (TAA) model (as a preventive treatment and as a remedial treatment) and in a bile duct ligation model. In the preventive model, rats received simultaneously intraperitoneum injection of TAA and/or 1,25(OH)2D3 for 10 wk. In the remedial model, rats were treated with TAA for 10 wk and then received 1,25(OH)2D3 or saline for 8 wk. Fibrotic score was determined by Masson staining. Collagen I, α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase-1 (TIMP1), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β) expression were measured by Western blot analysis and real-time PCR. Hypercalemia was detected by chemistry measurements. Preventive treatment of 1,25(OH)2D3 significantly suppressed liver fibrosis both macroscopically and microscopically and significantly lowered the fibrotic score of the TAA + 1,25(OH)2D3 group compared with the TAA group. 1,25(OH)2D3 significantly inhibited expression of PDGF and TGF-β by ∼50% and suppressed the expression of collagen Iα1, TIMP1, and α-SMA by approximately three-, two-, and threefold, respectively. In contrast, 1,25(OH)2D3 was inefficient in amelioration of established liver fibrosis. Administration of 1,25(OH)2D3 to bile duct ligation rats led to a high mortality rate probably caused by hypercalcemia. We conclude that 1,25(OH)2D3 may be considered as a potential preventive treatment in an in vivo model but failed to ameliorate established cirrhosis.

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Peripheral vascular neuroeffector mechanisms in experimental cholestasis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.3.g579 ◽

1993 ◽

Vol 265 (3) ◽

pp. G579-G586 ◽

Cited By ~ 1

Author(s):

G. Jacob ◽

O. Said ◽

J. Finberg ◽

A. Bomzon

Keyword(s):

Electrical Stimulation ◽

Bile Duct ◽

Vascular Reactivity ◽

Bile Duct Ligation ◽

Norepinephrine Uptake ◽

Pithed Rats ◽

Duct Ligation ◽

Adrenoceptor Agonists

Jaundiced patients have systemic hypotension and are more susceptible to hemorrhagic shock than nonjaundiced individuals. We have hypothesized that the mechanism whereby these cardiovascular complications arise is linked to a disturbance of the vascular neuroeffector process in the cardiovascular system. With the use of 3-day bile duct-manipulated (sham-operated) and bile duct-ligated rats, we have evaluated alpha-adrenoceptor function and amine uptake using in vivo and in vitro techniques. Blunted pressor responsiveness to norepinephrine, electrical stimulation, and the alpha 1-adrenoceptor agonists, methoxamine and phenylephrine, was observed in the bile duct-ligated pithed rats. In contrast, normal responsiveness to BHT-933 and clonidine, the alpha 2-adrenoceptor agonists, was seen in these animals. The uptake 1 blocker, cocaine, caused potentiation of equal magnitudes of the pressor responsiveness to electrical stimulation and norepinephrine in the sham-operated and bile duct-ligated pithed rats. In aortic rings prepared from the bile duct-ligated rats, blunted in vitro vascular reactivity to norepinephrine and the same alpha 1-adrenoceptor agonists was seen. Bile duct ligation had no effect on norepinephrine uptake or its kinetics in stressed and unstressed arterial rings and portal veins. We have thus concluded that bile duct ligation induces a defect in the functional expression of cardiovascular alpha 1-adrenoceptors without any effects on the activity of alpha 2-adrenoceptors or norepinephrine uptake.

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Differential alteration of cytochrome P450 isoenzymes in two experimental models of cirrhosis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y00-066 ◽

2000 ◽

Vol 78 (11) ◽

pp. 912-919 ◽

Cited By ~ 25

Author(s):

Marie-Claude Bastien ◽

François Leblond ◽

Vincent Pichette ◽

Jean-Pierre Villeneuve

Keyword(s):

Cytochrome P450 ◽

Carbon Tetrachloride ◽

Bile Duct ◽

Serum Albumin ◽

Breath Test ◽

Bile Duct Ligation ◽

Biliary Cirrhosis ◽

Breath Tests ◽

Duct Ligation

Liver diseases are associated with a decrease in hepatic drug elimination, but there is evidence that cirrhosis does not result in uniform changes of cytochrome P450 (CYP) isoenzymes. The objective of this study was to determine the content and activity of four CYP isoenzymes in the bile duct ligation and carbon tetrachloride (CCl4)-induced models of cirrhosis. The hepatic content of CYP1A, CYP2C, CYP2E1, and CYP3A was measured by Western blot analysis. CYP activity in vivo was evaluated with breath tests using substrates specific for different isoenzymes: caffeine (CYP1A2), aminopyrine (CYP2C11), nitrosodimethylamine (CYP2E1), and erythromycin (CYP3A). Bile duct ligation resulted in biliary cirrhosis; CYP1A, CYP2C and CYP3A content was decreased and the caffeine, aminopyrine, and erythromycin breath tests were reduced whereas CYP2E1 content and the nitrosodimethylamine breath test were unchanged compared with controls. CCl4 treatment resulted in cirrhosis of varying severity as assessed from the decrease in liver weight and serum albumin. In rats with mild cirrhosis, CYP content was comparable with controls except for a decrease in CYP2C. The activity of CYPs was also unchanged except for an increase in CYP2E1 activity. In rats with more severe cirrhosis, the content of all four CYP isoenzymes and the caffeine, aminopyrine, and erythromycin breath tests were reduced whereas the nitrosodimethylamine breath test was unchanged. In both models of cirrhosis, there was a significant correlation between the breath tests results and the severity of cirrhosis as assessed from serum albumin levels. These results indicate that content and the catalytic activity of individual CYP enzymes are differentially altered by cirrhosis in the rat and also suggest that drug probes could be useful to assess hepatic functional reserve.Key words: breath test, cirrhosis, cytochrome P450, bile duct ligation, carbon tetrachloride.

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Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

Canadian Journal of Biochemistry ◽

10.1139/o77-129 ◽

1977 ◽

Vol 55 (8) ◽

pp. 876-885 ◽

Cited By ~ 4

Author(s):

Patricia L. Chang ◽

John R. Riordan ◽

Mario A. Moscarello ◽

Jennifer M. Sturgess

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Rat Liver ◽

Plasma Membranes ◽

Specific Activity ◽

Specific Radioactivity ◽

Marker Enzyme ◽

Golgi Membranes ◽

Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

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Comparative studies on bile flow and biliary lipid excretion after bile-acid loading in normal and partially hepatectomized rats

Biochemical Journal ◽

10.1042/bj3050367 ◽

1995 ◽

Vol 305 (2) ◽

pp. 367-371 ◽

Cited By ~ 13

Author(s):

M Hoshino ◽

A Hirano ◽

T Hayakawa ◽

Y Kamiya ◽

T Ohiwa ◽

...

Keyword(s):

Bile Acid ◽

Rat Liver ◽

Bile Flow ◽

Acid Output ◽

Bile Secretion ◽

Biliary Lipid ◽

Isolated Perfusion ◽

Acid Loading ◽

Second Peak

This study was performed to investigate sequential changes in bile secretion and biliary lipids after taurocholic acid (TCA) loading of regenerating rat liver. TCA was administered intravenously at stepwise-increasing doses to groups of non-operated control and partially hepatectomized rats, 24, 72 and 168 h after surgery. Bile flow, bile-acid output (BAO) and phospholipid output (PLO) (expressed per gram of liver) in partially hepatectomized rats increased more than in the controls. Using an isolated perfusion rat-liver system, TCA infusion was also carried out on groups of non-operated control and hepatectomized rats 72 h after operation. Again bile flow, BAO and PLO (expressed per gram of liver) were significantly higher in the partial hepatectomy case, mirroring the results obtained in vivo. When horseradish peroxidase (HRP) was pulse-loaded in isolated perfusion preparations, the second peak of biliary HRP secretion in hepatectomized rats was significantly higher than in controls. We conclude that increased bile-acid flow in partially hepatectomized rats is dependent upon acceleration of vesicular transport accompanying or following proliferation in regenerating livers.

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Regulation of cholesterol and bile acid homoeostasis in bile-obstructed rats

Biochemical Journal ◽

10.1042/bj2800373 ◽

1991 ◽

Vol 280 (2) ◽

pp. 373-377 ◽

Cited By ~ 82

Author(s):

S Dueland ◽

J Reichen ◽

G T Everson ◽

R A Davis

Keyword(s):

Bile Acid ◽

Bile Duct ◽

Liver Function ◽

Urinary Excretion ◽

Bile Acids ◽

Bile Duct Ligation ◽

Microsomal Cytochrome ◽

Duct Ligation ◽

Cytochrome P 450

We examined how total blockage of biliary excretion, the major pathway through which cholesterol and bile acids are removed from the body, affects liver function, cholesterol and bile acid metabolism and homoeostasis. After 4 weeks of bile-duct ligation, rats showed impaired liver function, as documented by elevations in serum bilirubin and alkaline phosphatase activity. Moreover, bile-duct ligation decreased by about 30% both the amount of microsomal cytochrome P-450 in the liver and the elimination of aminopyrine in vivo, a reliable index in vivo of microsomal mixed-function oxidase activity. Cholesterol and bile acid contents in livers of bile-duct-ligated rats were doubled compared with sham-operated controls. Despite the increase in the contents of cholesterol and bile acids in liver, activities of the respective rate-limiting enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase, were doubled. Serum concentrations of bile acids and free cholesterol increased 25- and 4-fold respectively. The large increase in serum bile acids was associated with a 380-fold increase in the urinary excretion of bile acids. Although there is a general decrease in cytochrome P-450 content and drug metabolism involving cytochrome P-450-containing hydroxylases, the activity of cholesterol 7 alpha-hydroxylase, also a cytochrome P-450-containing enzyme, is actually increased. These data show that complete obstruction of the bile duct results in the selective impairment of microsomal cytochrome P-450. Increased activity of 7 alpha-hydroxylase, bile acid synthesis and urinary excretion provides an alternative excretory pathway that helps to maintain cholesterol homoeostasis when the biliary excretory pathway is eliminated.

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Deficiency of PI3K enhances inflammation and fibrosis in experimental non alcoholic steatohepatitis but attenuates bile duct ligation induced fibrosis in vivo

Zeitschrift für Gastroenterologie ◽

10.1055/s-0030-1263825 ◽

2010 ◽

Vol 48 (08) ◽

Author(s):

K Dostert ◽

R Wiest ◽

J Schölmerich ◽

C Hellerbrand ◽

E Gäbele

Keyword(s):

Bile Duct ◽

Bile Duct Ligation ◽

Alcoholic Steatohepatitis ◽

Duct Ligation

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Glucose transport and transporters in muscle giant vesicles: differential effects of insulin and contractions

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1993.264.2.e270 ◽

1993 ◽

Vol 264 (2) ◽

pp. E270-E278 ◽

Cited By ~ 18

Author(s):

T. Ploug ◽

J. Wojtaszewski ◽

S. Kristiansen ◽

P. Hespel ◽

H. Galbo ◽

...

Keyword(s):

Plasma Membrane ◽

Glucose Transport ◽

Maximum Velocity ◽

Enzyme Analysis ◽

Marker Enzyme ◽

Giant Vesicles ◽

Western Blots ◽

Glut 1 ◽

Glut 4

Collagenase treatment of skeletal muscle results in the formation of large spheres of membranes (3–30 microns diam). A procedure is described for purification and concentration of these giant membrane vesicles prepared from rat muscle. Morphological observations, marker enzyme analysis, and immunoblotting demonstrate that the vesicles are of plasma membrane origin and that sarcoplasmic reticulum, T-tubules, and mitochondrial inner membranes are absent from the preparation. Western blots demonstrate that the vesicles contain GLUT-4 glucose transporters, whereas GLUT-1 could not be detected. Vesicles prepared from control muscle display specific transport of D-glucose with a maximum velocity (Vmax) for glucose influx of approximately 2,500 pmol.mg plasma membrane protein-1.s-1 and an apparent Michaelis constant (Km) of 16 mM measured at zero-trans conditions at room temperature. Muscle contractions in vivo doubled the Vmax of vesicle glucose transport and membrane GLUT-4 content but did not change Km. In contrast, in vivo administration of insulin did not affect vesicle glucose transport or membrane GLUT-4 content. The combination of insulin and contractions caused similar changes as did contractions alone. It is concluded that the present vesicle population contains membrane components almost exclusively derived from the plasma membrane and contains very little if any GLUT-1 but substantial amounts of GLUT-4. Thus the preparation allows the study of transport kinetics of pure GLUT-4 transporters. The procedure for preparing vesicles probably results in activation of the glucose transport system similar to the activation by insulin but not by contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

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