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Bile acid composition of amniotic fluid and maternal serum in cholestasis of pregnancy and effect of ursodeoxycholic acid
125
Transport, biliary discuses, gallstones
LPS INDUCES CHOLESTASIS BY VESICULAR ACCUMULATION AND DOWNREGULATION OF THE MULTIDRUG RESISTANCE PROTEINZ. BXublh’. U. Warskulat’.
A. K Kurz’. D.
‘Medizinische Universit%sklinik, Klinik fur Gastroenterologie, Hepalologie Moorenstr. 5, D40225 Diisseldorf; ‘Abteilung Wr Tumorbmchemie, forschungszenrmm, Im Neuenhelmer Feld 280, D-69120 Heidelberg
und Infektiologie, Deutsches Krebs-
The Multidrug Resistance Protem2 (MRF’2)belongs to the ABC-transporters. In the liver it is located to the canalicular membrane of hepatocytes. Its absence is
associated with the Dubin-Johnson syndrome in humans. MRP2 transports endogenous compounds such as conjugated hilimbin, Leukotriene C4 and many xenohiotics into bile. We have demonstrated an osmodependent dynamic exchange of MRP2
between
the canalicular
membrane
and a vesicular
compartment
(GASTROENTEROLOGY 113,l I/97). This study was undertaken to characterise the possible involvement of such a vesicular accumulation of MRP2 under the influence of LPS which is known to rapidly induce cholestasis. Wistar rats were injected intraperitoneally with LPS (4mg/kg BW), LPS plus dexamethasone (ImgnCg BW), NaCl or dexamethasone alone. 16 hours later liverprobes were taken for immunohistochemistry and Northern blot analysis. Cryosections of liver samples were double stained for MRP2 and the tight junction associated protein ZO-1. In control (NaCl) or in dexamethasone treated rats MRP2 was confined to the canalicular membrane as evidenced by its localisation in relation to the tight junctions using laser scanning microscopy. In contrast, in LPS-treated rats a punctate staining of MRP2 occured aside of the tight junctions representing vesicular deposition of MRPZ. This dislocation of MRP2 was largely inhibited when dexamethasone was simultaneously injected to LPS. MRP2-mRNA in N&I-injected rats showed an abundant signal, while it was completely abolished in LPS-treated animals. Addition of dexamethasone diminished the effect of LPS on mRNA-level. In 24h cultured, primary rat hepatocytes, MRF’2-mRNA was only detectable in the presence of dexamethasone, its amount was further increased by hypoosmotic incubation in a genistein sensitive manner. In line with this finding, in cultured hepatocytes the MRP2-staining at the pseudocanalicular membrane was low in control, while it was clearly visible when dexamethasone was added to the medium. In conclusion LPS-induced cholestasis is mediated by a shift of MRP2 from a canalicular to an intracellular, suhcanalicular locatlon and by the reduction of MRP2mRNA. Dexamethasone reverses both effects, probably not only due to
immunomodulation, but also to a direct action on the hepatocellular level, since mRNA levels and MRP2-expression in cultured hepatocytes were only observed in the presence of Dexamethasone.
BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF THE COENZYME A (CoA) POOLS IN RATS WITH LONG-TERM CHOLESTASIS S. Kr’dhenbtihl. M. Ledermann. L.Kr&enbtihl. Depts. of Clinical Pharmacology and Visceral Surgery, Univ. of Beme, Switzerland Background: Rats with long-term bile duct ligation (BDL) have a reduced hepatic CoA content and imapired B-oxidation, compatible with a decrease in mitochondrial CoA. Aims: To quantify the hepatic mitochondrial and cytosolic CoA pools and to test the pools functionally in BDL and control rats (CON). Methods: BDL for 4 w, rats studied in the fed state. Isolation of liver mitochondria by differential centrifkgation, assessment of the CoA pools by a radioenzymatic method (J Clin Invest 1994;94: 1490). Functional assessment of the mitochondrial pool by monitoring (24 h) renal hippurate excretion after i.p. administration of benzoate (100 pmoYlO0 g bw) and of the cytosolic pool by monitoring acetylsulfamethoxazole (AcSMX) excretion after i.p. administration of SMX (10 mg/lOO g bw). Results: The mitochondrial CoA content was maintained in BDL, whereas the cytosolic and total hepatic CoA content were decreased (nmol/g liver wet weight, mean&d, *p
~!
Renal excretions ofhippurate (35&23% in BDL vs 48f9% of dose in CON) and AcSMX (37+9% in BDL vs 35+6% of dose in CON) were not different. Conclusions: The mitochondrial CoA pool is maintained in BDL and functions normally, whereas the cytosolic CoA pool is significantly decreased but has also a normal function.
1 P/CO7/666 EVIDENCE CARRIER-MEDIATED FOR BILIRUBIN TRANSPORT ACROSS THE BASAL PLASMA MEMBRANE OF HUMAN PLACENTA TROPHOBLAST AT TERM L. Pascolo, J.E. Bay&r’, M.A. Serrano$. D. Ostrow’. C. Tiribelli, J. Gonzalez-Gallego’, J.G. Marin”. BBCM Dept., Univ. Trieste, Italy; Dept. Physiology, Univ. Leon, Spain; ‘AMC, Univ. Amsterdam, The Netherlands; “School of Pharmacy, Univ. Salamanca, Spain. Though the main fate of bilirubin synthetised by the fetal liver is to be transferred to the mother for elimination, the mechanism(s) governing this important process is still unknown. Aim: To investigate unconjugated bilirubin (UCB) transfer across the basal (i.e., fetal facing) plasma membrane of the trophoblast (fTPM). Methods: Using a rapid-filtration technique, uptake of radiolabeled UCB by fTPM vesicles, obtained from normal human term placentas, was measured. UCB was solubilized in IO pM human serum albumin (fatty acid free) at pH 7.0 and free UCB concentration (Bf) varied from 10 to 100 nM. The possible effect of electric membrane potential was investigated by measuring UCB uptake upon addition of valinomycin (50 utgimg prot) in the presence of inwardly-directed K’ gradient. Na’-dependence and ATP-sensitivity of UCB transport were assessed by replacement of 50 mM NaCl for 50 mM choline chloride and by addition of 2mM MgATP or ATPyS, respectively. Results: UCB uptake by ffPM vesicles was a rapid process that reached steady-state at 60 s. The process was temperature-sensitive and was not modified by changes in plasma membrane potential. The rate of uptake vs (Bf) followed saturation kinetics with apparent K,=45.4*16.5 nM and V,,,= 49.5*8.8 pmol/mg proti20 s at 25°C. Either replacement of Nat by choline or addition of Mg’+-ATP or ATPyS to the medium had no effect on UCB uptake. Conclusions: These findings provide evidence for the presence of an electroneutral system for UCB transport across ffPM, which occurs independently of the presence of sodium gradients or ATP hydrolisis.
]
BILE ACID COMPOSITION OF AMNIOTIC FLUID AND MATERNAL SERUM IN CHOLESTASIS OF PREGNANCY AND EFFECT OF URSODEOXYCHOLIC ACID Cl Brites’, MY El-Mir”. CMP Rcdriques”. H van-Zeller’. JJG Marin”. *Molecular Pathcgenesis Center, Faculty of Pharmacy, University of Lisbon, Portugal and “Department of Physiology and Pharmacology, University of Salamanca, Spain, EackgrountiAlms: Ursodeoxycholic acid (UDCA) has been indicated as a useful drug for the treatment of intrahepatic cholestasis of pregnancy (ICP). The aim of this study was to investigate the effect of the disease on the bile acid profile in amniotic fluid (AF) from non-treated and UDCA-treated patients. hfeulods: Bile acids were assessed by gas chromatography-mass spectrometry and highperformance liquid chromatography in serum and AF from ICP patients non-treated (n=9) and treated (n=6) with UDCA (14 mglkg bw/day, during 14tI days) until delivery, as well as in AF from control pregnant women (n=Q Resulfa: In nontreated ICP patients, total bile acid (TBA) concentration in serum was 28.4kI2.3 pmol/L and cholic acid (CA) proportion was 55.9+6.6%. In the group where UDCA was administered, TBA decreased from 56.6k28.5 prnol/L at baseline to 25.7kI2.2 prnol/L after therapy. Individually, the most significant alteration was the reduction in serum CA proportion (horn 63.7*6.2% to 36.&6.1%, PcO.01) with UDCA administration. This reduction was accompanied by an increase (P~0.05) in the percentage of UDCA (20.5*5.3% Vs. 3.3*1.4%). TBA in AF from non-treated and treated ICP patients showad an elevation (18.3k5.8 prnol/L, RO.10, and 16.2k3.8 pmol/L, P
Transport, biliary discuses, gallstones
LPS INDUCES CHOLESTASIS BY VESICULAR ACCUMULATION AND DOWNREGULATION OF THE MULTIDRUG RESISTANCE PROTEINZ. BXublh’. U. Warskulat’.
A. K Kurz’. D.
‘Medizinische Universit%sklinik, Klinik fur Gastroenterologie, Hepalologie Moorenstr. 5, D40225 Diisseldorf; ‘Abteilung Wr Tumorbmchemie, forschungszenrmm, Im Neuenhelmer Feld 280, D-69120 Heidelberg
und Infektiologie, Deutsches Krebs-
The Multidrug Resistance Protem2 (MRF’2)belongs to the ABC-transporters. In the liver it is located to the canalicular membrane of hepatocytes. Its absence is
associated with the Dubin-Johnson syndrome in humans. MRP2 transports endogenous compounds such as conjugated hilimbin, Leukotriene C4 and many xenohiotics into bile. We have demonstrated an osmodependent dynamic exchange of MRP2
between
the canalicular
membrane
and a vesicular
compartment
(GASTROENTEROLOGY 113,l I/97). This study was undertaken to characterise the possible involvement of such a vesicular accumulation of MRP2 under the influence of LPS which is known to rapidly induce cholestasis. Wistar rats were injected intraperitoneally with LPS (4mg/kg BW), LPS plus dexamethasone (ImgnCg BW), NaCl or dexamethasone alone. 16 hours later liverprobes were taken for immunohistochemistry and Northern blot analysis. Cryosections of liver samples were double stained for MRP2 and the tight junction associated protein ZO-1. In control (NaCl) or in dexamethasone treated rats MRP2 was confined to the canalicular membrane as evidenced by its localisation in relation to the tight junctions using laser scanning microscopy. In contrast, in LPS-treated rats a punctate staining of MRP2 occured aside of the tight junctions representing vesicular deposition of MRPZ. This dislocation of MRP2 was largely inhibited when dexamethasone was simultaneously injected to LPS. MRP2-mRNA in N&I-injected rats showed an abundant signal, while it was completely abolished in LPS-treated animals. Addition of dexamethasone diminished the effect of LPS on mRNA-level. In 24h cultured, primary rat hepatocytes, MRF’2-mRNA was only detectable in the presence of dexamethasone, its amount was further increased by hypoosmotic incubation in a genistein sensitive manner. In line with this finding, in cultured hepatocytes the MRP2-staining at the pseudocanalicular membrane was low in control, while it was clearly visible when dexamethasone was added to the medium. In conclusion LPS-induced cholestasis is mediated by a shift of MRP2 from a canalicular to an intracellular, suhcanalicular locatlon and by the reduction of MRP2mRNA. Dexamethasone reverses both effects, probably not only due to
immunomodulation, but also to a direct action on the hepatocellular level, since mRNA levels and MRP2-expression in cultured hepatocytes were only observed in the presence of Dexamethasone.
BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF THE COENZYME A (CoA) POOLS IN RATS WITH LONG-TERM CHOLESTASIS S. Kr’dhenbtihl. M. Ledermann. L.Kr&enbtihl. Depts. of Clinical Pharmacology and Visceral Surgery, Univ. of Beme, Switzerland Background: Rats with long-term bile duct ligation (BDL) have a reduced hepatic CoA content and imapired B-oxidation, compatible with a decrease in mitochondrial CoA. Aims: To quantify the hepatic mitochondrial and cytosolic CoA pools and to test the pools functionally in BDL and control rats (CON). Methods: BDL for 4 w, rats studied in the fed state. Isolation of liver mitochondria by differential centrifkgation, assessment of the CoA pools by a radioenzymatic method (J Clin Invest 1994;94: 1490). Functional assessment of the mitochondrial pool by monitoring (24 h) renal hippurate excretion after i.p. administration of benzoate (100 pmoYlO0 g bw) and of the cytosolic pool by monitoring acetylsulfamethoxazole (AcSMX) excretion after i.p. administration of SMX (10 mg/lOO g bw). Results: The mitochondrial CoA content was maintained in BDL, whereas the cytosolic and total hepatic CoA content were decreased (nmol/g liver wet weight, mean&d, *p
~!
Renal excretions ofhippurate (35&23% in BDL vs 48f9% of dose in CON) and AcSMX (37+9% in BDL vs 35+6% of dose in CON) were not different. Conclusions: The mitochondrial CoA pool is maintained in BDL and functions normally, whereas the cytosolic CoA pool is significantly decreased but has also a normal function.
1 P/CO7/666 EVIDENCE CARRIER-MEDIATED FOR BILIRUBIN TRANSPORT ACROSS THE BASAL PLASMA MEMBRANE OF HUMAN PLACENTA TROPHOBLAST AT TERM L. Pascolo, J.E. Bay&r’, M.A. Serrano$. D. Ostrow’. C. Tiribelli, J. Gonzalez-Gallego’, J.G. Marin”. BBCM Dept., Univ. Trieste, Italy; Dept. Physiology, Univ. Leon, Spain; ‘AMC, Univ. Amsterdam, The Netherlands; “School of Pharmacy, Univ. Salamanca, Spain. Though the main fate of bilirubin synthetised by the fetal liver is to be transferred to the mother for elimination, the mechanism(s) governing this important process is still unknown. Aim: To investigate unconjugated bilirubin (UCB) transfer across the basal (i.e., fetal facing) plasma membrane of the trophoblast (fTPM). Methods: Using a rapid-filtration technique, uptake of radiolabeled UCB by fTPM vesicles, obtained from normal human term placentas, was measured. UCB was solubilized in IO pM human serum albumin (fatty acid free) at pH 7.0 and free UCB concentration (Bf) varied from 10 to 100 nM. The possible effect of electric membrane potential was investigated by measuring UCB uptake upon addition of valinomycin (50 utgimg prot) in the presence of inwardly-directed K’ gradient. Na’-dependence and ATP-sensitivity of UCB transport were assessed by replacement of 50 mM NaCl for 50 mM choline chloride and by addition of 2mM MgATP or ATPyS, respectively. Results: UCB uptake by ffPM vesicles was a rapid process that reached steady-state at 60 s. The process was temperature-sensitive and was not modified by changes in plasma membrane potential. The rate of uptake vs (Bf) followed saturation kinetics with apparent K,=45.4*16.5 nM and V,,,= 49.5*8.8 pmol/mg proti20 s at 25°C. Either replacement of Nat by choline or addition of Mg’+-ATP or ATPyS to the medium had no effect on UCB uptake. Conclusions: These findings provide evidence for the presence of an electroneutral system for UCB transport across ffPM, which occurs independently of the presence of sodium gradients or ATP hydrolisis.
]
BILE ACID COMPOSITION OF AMNIOTIC FLUID AND MATERNAL SERUM IN CHOLESTASIS OF PREGNANCY AND EFFECT OF URSODEOXYCHOLIC ACID Cl Brites’, MY El-Mir”. CMP Rcdriques”. H van-Zeller’. JJG Marin”. *Molecular Pathcgenesis Center, Faculty of Pharmacy, University of Lisbon, Portugal and “Department of Physiology and Pharmacology, University of Salamanca, Spain, EackgrountiAlms: Ursodeoxycholic acid (UDCA) has been indicated as a useful drug for the treatment of intrahepatic cholestasis of pregnancy (ICP). The aim of this study was to investigate the effect of the disease on the bile acid profile in amniotic fluid (AF) from non-treated and UDCA-treated patients. hfeulods: Bile acids were assessed by gas chromatography-mass spectrometry and highperformance liquid chromatography in serum and AF from ICP patients non-treated (n=9) and treated (n=6) with UDCA (14 mglkg bw/day, during 14tI days) until delivery, as well as in AF from control pregnant women (n=Q Resulfa: In nontreated ICP patients, total bile acid (TBA) concentration in serum was 28.4kI2.3 pmol/L and cholic acid (CA) proportion was 55.9+6.6%. In the group where UDCA was administered, TBA decreased from 56.6k28.5 prnol/L at baseline to 25.7kI2.2 prnol/L after therapy. Individually, the most significant alteration was the reduction in serum CA proportion (horn 63.7*6.2% to 36.&6.1%, PcO.01) with UDCA administration. This reduction was accompanied by an increase (P~0.05) in the percentage of UDCA (20.5*5.3% Vs. 3.3*1.4%). TBA in AF from non-treated and treated ICP patients showad an elevation (18.3k5.8 prnol/L, RO.10, and 16.2k3.8 pmol/L, P