Irwin Arias - Academia.edu (original) (raw)

Papers by Irwin Arias

Research paper thumbnail of Submitted: 05/10/11

polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway. Proc Natl Acad Sci USA 2011; 108:1403–8; ... more polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway. Proc Natl Acad Sci USA 2011; 108:1403–8; PMID:21220320;

Research paper thumbnail of Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes

Journal of Biological Chemistry

Research paper thumbnail of Uptake of bilirubin glucuronides by isolated rat hepatocytes

Gastroenterologia Japonica

The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in ord... more The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in order to characterize the mechanism by which bile pigments are transported by the liver. The BDG uptake by hepatocytes was saturable. The uptake was inhibited by bilirubin, sulfobromophthalein, and bilirubin monoglucuronide, but not by taurocholate. The uptake was not affected by replacement of sodium with other cations except for choline. Only when sodium was replaced with choline, was significant decrease in uptake observed. When chloride was replaced with nitrate, BDG uptake decreased, but it was not changed by replacement with sulfate. Metabolic inhibitors did not affect BDG uptake significantly. Thus bile pigments share a common sodium-independent and electrogenic potential-dependent transporter in liver cell membranes. A high concentration of albumin interferes with BDG uptake.

Research paper thumbnail of The Role of Albumin in the Hepatic Transport of Bilirubin: Studies in Mutant Analbuminemic Rats1

The Journal of Biochemistry

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hep... more Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.

Research paper thumbnail of Hepatic Uptake of Bilirubin Diglucuronide: Analysis by Using Sinusoidal Plasma Membrane Vesicles1

The Journal of Biochemistry, 1990

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of biliru... more In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.

Research paper thumbnail of AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

PloS one, 2016

Mitochondrial damage is the major factor underlying drug-induced liver disease but whether condit... more Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitoc...

Research paper thumbnail of Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo

Hepatology (Baltimore, Md.), Oct 11, 2016

Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play criti... more Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), ...

Research paper thumbnail of The Mechanism of Biliary Secretion of Reduced Glutathione. Analysis of Transport Process in Isolated Rat-Liver Canalicular Membrane Vesicles

European Journal of Biochemistry, 1983

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane ves... more Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.

Research paper thumbnail of Structural and Functional Hepatocyte Polarity and Liver Disease

Journal of Hepatology, 2015

Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canali... more Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canalicular bile. They have a uniquely organized polarity with a basal membrane facing liver sinusoidal endothelial cells, while one or more apical poles can contribute to several bile canaliculi jointly with the directly opposing hepatocytes. Establishment and maintenance of hepatocyte polarity is essential for many functions of hepatocytes and requires carefully orchestrated cooperation between cell adhesion molecules, cell junctions, cytoskeleton, extracellular matrix and intracellular trafficking machinery. The process of hepatocyte polarization requires energy and, if abnormal, may result in severe liver disease. A number of inherited disorders affecting tight junction and intracellular trafficking proteins have been described and demonstrate clinical and pathophysiological features overlapping those of the genetic cholestatic liver diseases caused by defects in canalicular ABC transporters. Thus both structural and functional components contribute to the final hepatocyte polarity phenotype. Many acquired liver diseases target factors that determine hepatocyte polarity, such as junctional proteins. Hepatocyte depolarization frequently occurs but is rarely recognized because hematoxylin-eosin staining does not identify the bile canaliculus. However, the molecular mechanisms underlying these defects are not well understood. Here we aim to provide an update on the key factors determining hepatocyte polarity and how it is affected in inherited and acquired diseases.

Research paper thumbnail of Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

American Journal of Physiology - Gastrointestinal and Liver Physiology, 2003

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several AT... more Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canali...

Research paper thumbnail of Bilirubin conjugation in the spiny dogfish, Squalus acanthias, the small skate, Raja erinacea and the winter flounder, Pseudopleuronectes americanas

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1980

Bile pigments from spiny dogfish (Squalus acanthias), small skate (Raja erinacea) and winter flou... more Bile pigments from spiny dogfish (Squalus acanthias), small skate (Raja erinacea) and winter flounder (Pseudopleuronectes americanas) were quantitated after chromatography of tetrapyrroles and azopigment analysis. 2. Bilirubin monoglucuronide, unconjugated bilirubin, bilirubin diglucuronide and unidentified pigments were present in decreasing order of abundance. 3. UDPglucuronate glucuronyl transferase and bilirubin glucuroniside glucuronosyl transferase activities were present in all three fish livers, and had temperature optima of 37°C. 4. The distribution of the two enzyme activities were different in liver fractions of the three fish.

Research paper thumbnail of Dismutation of Bilirubin Monoglucuronide

Enzymatic Basis of Detoxication, 1980

Research paper thumbnail of Disorders of Bilirubin Metabolism

The Liver, 2009

FIGURE 20.1. Mechanism of heme ring opening and subsequent reduction of biliverdin to bilirubin.

Research paper thumbnail of Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles

European Journal of Biochemistry, 1984

Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal pl... more Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal plasma-membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle-associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high-affinity and low-affinity GSH transport systems; the former has an apparent Km of 0.34 mM for GSH and V of 1.4 nmol X mg protein-1 X 20 s-1, and the latter has an apparent Km of 3.3 mM and V of 3.9 nmol X mg protein-1 X 20 s-1. Both Km values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier-mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S-benzylglutathione inhibited GSH transport by the vesicles; the low-affinity transport system was inhibited more markedly than the high-affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low-affinity transport system for GSH. S-Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S-benzylglutathione or GSH. The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter-organ metabolism of these compounds.

Research paper thumbnail of Biliary transport of glutathione disulfide studied with isolated rat-liver canalicular-membrane vesicles

European Journal of Biochemistry, 1984

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study ... more Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH-groups in membrane proteins. Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier-mediated transport for GSSG (Q10 X 1.6) and seems to exclude simple diffusion. Concentration dependence of GSSG uptake shows Michaelis-Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol X min-1 X mg protein-1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics. It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.

Research paper thumbnail of Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization

Proceedings of the National Academy of Sciences, 2013

Research paper thumbnail of Intracellular Trafficking of Bile Salt Export Pump (ABCB11) in Polarized Hepatic Cells: Constitutive Cycling between the Canalicular Membrane and rab11-positive Endosomes

Molecular Biology of the Cell, 2004

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into... more The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by...

Research paper thumbnail of Interaction of coding region mutations and the Gilbert-type promoter abnormality of the UGT1A1 gene causes moderate degrees of unconjugated hyperbilirubinaemia and may lead to neonatal kernicterus

Journal of Medical Genetics, 2001

Research paper thumbnail of Isolation of multiple normal and functionally defective forms of uridine diphosphate-glucuronosyltransferase from inbred Gunn rats

Journal of Clinical Investigation, 1987

Gunn rats are a mutant strain of Wistar rats that have unconjugated hyperbilirubinemia due to abs... more Gunn rats are a mutant strain of Wistar rats that have unconjugated hyperbilirubinemia due to absence of hepatic uridine diphosphate-glucuronosyltransferase (UDPGT; EC. 2.4.1.17) activity toward bilirubin. We isolated five UDPGT isoforms from solubilized microsomal fractions from liver of inbred Wistar (RHA) rats and congeneic Gunn rats. UDPGT isoform V (elution pH 7.5) from Wistar (RHA) rats is active toward bilirubin and 4'-hydroxydimethylaminoazobenzene. The corresponding isoform from Gunn rat liver was enzymically inactive but exhibited normal elution pH and mobility on NaDodSO4/polyacrylamide gel electrophoresis (Mr 53,000), and was recognized by a UDPGT-specific antiserum. UDPGT isoform I (elution pH 8.7) from Wistar (RHA) and Gunn rats was active toward 4-nitrophenol. The isoform from Gunn rat liver had only 10% of normal UDPGT activity, however UDPGT activity increased to normal upon addition of 15 mM diethylnitrosamine in vitro. Isoforms II (elution pH 8.4), III (elution pH 8.0), and IV (elution pH 7.8) from Gunn rats had normal UDPGT activities, except that Isoform IV was inactive toward bilirubin.

Research paper thumbnail of Taurocholate transport by rat liver canalicular membrane vesicles. Evidence for the presence of an Na+-independent transport system

Journal of Clinical Investigation, 1984

Research paper thumbnail of Submitted: 05/10/11

polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway. Proc Natl Acad Sci USA 2011; 108:1403–8; ... more polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway. Proc Natl Acad Sci USA 2011; 108:1403–8; PMID:21220320;

Research paper thumbnail of Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes

Journal of Biological Chemistry

Research paper thumbnail of Uptake of bilirubin glucuronides by isolated rat hepatocytes

Gastroenterologia Japonica

The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in ord... more The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in order to characterize the mechanism by which bile pigments are transported by the liver. The BDG uptake by hepatocytes was saturable. The uptake was inhibited by bilirubin, sulfobromophthalein, and bilirubin monoglucuronide, but not by taurocholate. The uptake was not affected by replacement of sodium with other cations except for choline. Only when sodium was replaced with choline, was significant decrease in uptake observed. When chloride was replaced with nitrate, BDG uptake decreased, but it was not changed by replacement with sulfate. Metabolic inhibitors did not affect BDG uptake significantly. Thus bile pigments share a common sodium-independent and electrogenic potential-dependent transporter in liver cell membranes. A high concentration of albumin interferes with BDG uptake.

Research paper thumbnail of The Role of Albumin in the Hepatic Transport of Bilirubin: Studies in Mutant Analbuminemic Rats1

The Journal of Biochemistry

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hep... more Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.

Research paper thumbnail of Hepatic Uptake of Bilirubin Diglucuronide: Analysis by Using Sinusoidal Plasma Membrane Vesicles1

The Journal of Biochemistry, 1990

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of biliru... more In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.

Research paper thumbnail of AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

PloS one, 2016

Mitochondrial damage is the major factor underlying drug-induced liver disease but whether condit... more Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitoc...

Research paper thumbnail of Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo

Hepatology (Baltimore, Md.), Oct 11, 2016

Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play criti... more Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), ...

Research paper thumbnail of The Mechanism of Biliary Secretion of Reduced Glutathione. Analysis of Transport Process in Isolated Rat-Liver Canalicular Membrane Vesicles

European Journal of Biochemistry, 1983

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane ves... more Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.

Research paper thumbnail of Structural and Functional Hepatocyte Polarity and Liver Disease

Journal of Hepatology, 2015

Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canali... more Hepatocytes form a crucially important cell layer that separates sinusoidal blood from the canalicular bile. They have a uniquely organized polarity with a basal membrane facing liver sinusoidal endothelial cells, while one or more apical poles can contribute to several bile canaliculi jointly with the directly opposing hepatocytes. Establishment and maintenance of hepatocyte polarity is essential for many functions of hepatocytes and requires carefully orchestrated cooperation between cell adhesion molecules, cell junctions, cytoskeleton, extracellular matrix and intracellular trafficking machinery. The process of hepatocyte polarization requires energy and, if abnormal, may result in severe liver disease. A number of inherited disorders affecting tight junction and intracellular trafficking proteins have been described and demonstrate clinical and pathophysiological features overlapping those of the genetic cholestatic liver diseases caused by defects in canalicular ABC transporters. Thus both structural and functional components contribute to the final hepatocyte polarity phenotype. Many acquired liver diseases target factors that determine hepatocyte polarity, such as junctional proteins. Hepatocyte depolarization frequently occurs but is rarely recognized because hematoxylin-eosin staining does not identify the bile canaliculus. However, the molecular mechanisms underlying these defects are not well understood. Here we aim to provide an update on the key factors determining hepatocyte polarity and how it is affected in inherited and acquired diseases.

Research paper thumbnail of Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

American Journal of Physiology - Gastrointestinal and Liver Physiology, 2003

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several AT... more Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canali...

Research paper thumbnail of Bilirubin conjugation in the spiny dogfish, Squalus acanthias, the small skate, Raja erinacea and the winter flounder, Pseudopleuronectes americanas

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1980

Bile pigments from spiny dogfish (Squalus acanthias), small skate (Raja erinacea) and winter flou... more Bile pigments from spiny dogfish (Squalus acanthias), small skate (Raja erinacea) and winter flounder (Pseudopleuronectes americanas) were quantitated after chromatography of tetrapyrroles and azopigment analysis. 2. Bilirubin monoglucuronide, unconjugated bilirubin, bilirubin diglucuronide and unidentified pigments were present in decreasing order of abundance. 3. UDPglucuronate glucuronyl transferase and bilirubin glucuroniside glucuronosyl transferase activities were present in all three fish livers, and had temperature optima of 37°C. 4. The distribution of the two enzyme activities were different in liver fractions of the three fish.

Research paper thumbnail of Dismutation of Bilirubin Monoglucuronide

Enzymatic Basis of Detoxication, 1980

Research paper thumbnail of Disorders of Bilirubin Metabolism

The Liver, 2009

FIGURE 20.1. Mechanism of heme ring opening and subsequent reduction of biliverdin to bilirubin.

Research paper thumbnail of Glutathione transport across hepatocyte plasma membranes. Analysis using isolated rat-liver sinusoidal-membrane vesicles

European Journal of Biochemistry, 1984

Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal pl... more Transport of reduced glutathione (GSH) and its derivatives was studied in rat-liver sinusoidal plasma-membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited transport of GSH into an osmotically active intravesicular space. Equilibrium uptake of vesicle-associated GSH was similar to that of free ligands which can be trapped by the intravesicular space of sinusoidal membrane samples. Kinetic analysis of the transport process revealed that the sinusoidal membrane vesicles have high-affinity and low-affinity GSH transport systems; the former has an apparent Km of 0.34 mM for GSH and V of 1.4 nmol X mg protein-1 X 20 s-1, and the latter has an apparent Km of 3.3 mM and V of 3.9 nmol X mg protein-1 X 20 s-1. Both Km values are lower than intrahepatic GSH levels, indicating that GSH transport across the sinusoidal membrane occurs via carrier-mediated mechanism and argues simple diffusion mechanism. The presence of oxidized glutathione (GSSG) or S-benzylglutathione inhibited GSH transport by the vesicles; the low-affinity transport system was inhibited more markedly than the high-affinity transport system. This suggests that these glutathione derivatives interacted preferentially with the low-affinity transport system for GSH. S-Dinitrophenylglutathione was also transported by the vesicles by a process which was inhibited by GSH and GSSG. The vesicles also transported GSSG, and this transport was markedly inhibited by S-benzylglutathione or GSH. The transport systems in sinusoidal plasma membranes may function in vivo in translocating GSH and its derivatives from hepatocytes into plasma and play an important role in inter-organ metabolism of these compounds.

Research paper thumbnail of Biliary transport of glutathione disulfide studied with isolated rat-liver canalicular-membrane vesicles

European Journal of Biochemistry, 1984

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study ... more Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH-groups in membrane proteins. Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier-mediated transport for GSSG (Q10 X 1.6) and seems to exclude simple diffusion. Concentration dependence of GSSG uptake shows Michaelis-Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol X min-1 X mg protein-1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics. It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.

Research paper thumbnail of Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization

Proceedings of the National Academy of Sciences, 2013

Research paper thumbnail of Intracellular Trafficking of Bile Salt Export Pump (ABCB11) in Polarized Hepatic Cells: Constitutive Cycling between the Canalicular Membrane and rab11-positive Endosomes

Molecular Biology of the Cell, 2004

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into... more The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by...

Research paper thumbnail of Interaction of coding region mutations and the Gilbert-type promoter abnormality of the UGT1A1 gene causes moderate degrees of unconjugated hyperbilirubinaemia and may lead to neonatal kernicterus

Journal of Medical Genetics, 2001

Research paper thumbnail of Isolation of multiple normal and functionally defective forms of uridine diphosphate-glucuronosyltransferase from inbred Gunn rats

Journal of Clinical Investigation, 1987

Gunn rats are a mutant strain of Wistar rats that have unconjugated hyperbilirubinemia due to abs... more Gunn rats are a mutant strain of Wistar rats that have unconjugated hyperbilirubinemia due to absence of hepatic uridine diphosphate-glucuronosyltransferase (UDPGT; EC. 2.4.1.17) activity toward bilirubin. We isolated five UDPGT isoforms from solubilized microsomal fractions from liver of inbred Wistar (RHA) rats and congeneic Gunn rats. UDPGT isoform V (elution pH 7.5) from Wistar (RHA) rats is active toward bilirubin and 4'-hydroxydimethylaminoazobenzene. The corresponding isoform from Gunn rat liver was enzymically inactive but exhibited normal elution pH and mobility on NaDodSO4/polyacrylamide gel electrophoresis (Mr 53,000), and was recognized by a UDPGT-specific antiserum. UDPGT isoform I (elution pH 8.7) from Wistar (RHA) and Gunn rats was active toward 4-nitrophenol. The isoform from Gunn rat liver had only 10% of normal UDPGT activity, however UDPGT activity increased to normal upon addition of 15 mM diethylnitrosamine in vitro. Isoforms II (elution pH 8.4), III (elution pH 8.0), and IV (elution pH 7.8) from Gunn rats had normal UDPGT activities, except that Isoform IV was inactive toward bilirubin.

Research paper thumbnail of Taurocholate transport by rat liver canalicular membrane vesicles. Evidence for the presence of an Na+-independent transport system

Journal of Clinical Investigation, 1984