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1.
Pediatr Res ; 64(5): 498-504, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18596569

ABSTRACT

Retention of bile acids within the liver is a primary factor in the pathogenesis of cholestatic liver disorders, which are more common in human infants. The objective of this study was to evaluate developmental changes in mitochondrial factors involved in bile acid-induced hepatocyte injury. Hepatic mitochondria from adult rats (aged 9 wk) underwent a mitochondrial permeability transition (MPT) and release of cytochrome c upon exposure to glycochenodeoxycholic acid. In contrast, mitochondria from young rats (age 6-36 d) were resistant to MPT induction and cytochrome c release. Neither mitochondrial levels of MPT-associated proteins (voltage-dependent anion channel, cyclophilin D, or adenine nucleotide translocase), Bcl-2 family proteins, nor antioxidant enzymes explained this resistance. Mitochondria from young rats contained 2- to 3-fold higher alpha-tocopherol (alpha-TH). In vivo alpha-TH enrichment of adult hepatic mitochondria increased their MPT resistance. Tetra-linoleoyl cardiolipin (TL-CL), the primary molecular species of CL, was reduced in mitochondria of the young rat; however, enrichment with CL and TL-CL only modestly increased their MPT susceptibility. In conclusion, we observed an unexpected resistance in young rats to bile acid induction of mitochondrial cell death pathways, which may be related to developmental differences in membrane composition.


Subject(s)
Glycochenodeoxycholic Acid/metabolism , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , alpha-Tocopherol/metabolism , Age Factors , Animals , Cardiolipins/metabolism , Cell Death , Cytochromes c/metabolism , Mitochondria, Liver/enzymology , Mitochondria, Liver/pathology , Mitochondrial Permeability Transition Pore , Oxidation-Reduction , Rats
2.
Hepatology ; 46(2): 485-95, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17659596

ABSTRACT

UNLABELLED: Several genetic metabolic liver diseases share the pathological features of combined steatosis and cholestasis, or steatocholestasis. The aims of this study were to develop and characterize an in vivo model for steatocholestasis and to evaluate the effects of an antioxidant treatment on liver injury, oxidative stress, and mitochondrial perturbations in this model. Obese and lean Zucker rats received intravenous (IV) injections of glycochenodeoxycholic acid (GCDC) and were killed 4 hours later. Liver enzymes were measured; the liver histology was assessed, and hepatic mitochondria were analyzed for mitochondrial lipid peroxidation. In separate experiments, rats received daily injections of subcutaneous (SQ) vitamin E before GCDC infusion. Bile acid-induced injury (serum AST and ALT and liver histology) was more severe in the obese rats than in the lean rats, characterized predominantly by extensive cell necrosis with minimal evidence of apoptosis. SQ vitamin E provided significant protection against IV GCDC-induced hepatic injury, in vitro GCDC-induced permeability transition, and cytochrome C and apoptosis-inducing factor release from isolated mitochondria. CONCLUSION: Steatosis sensitizes the liver to bile acid-induced necrotic hepatocyte injury, which is responsive to vitamin E therapy.


Subject(s)
Fatty Liver/drug therapy , Vitamin D/administration & dosage , Adenosine Triphosphate/metabolism , Alanine Transaminase/blood , Animals , Apoptosis , Aspartate Aminotransferases/blood , Fatty Liver/pathology , Glycochenodeoxycholic Acid/toxicity , Injections, Subcutaneous , Liver/pathology , Male , Rats , Rats, Zucker
3.
J Pediatr Gastroenterol Nutr ; 43 Suppl 1: S4-9, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16819400

ABSTRACT

Cholestatic liver disorders account for a large proportion of chronic liver ailments in adults, children and infants, and are among the leading indications for liver transplantation in all age groups. Recent studies have begun to characterize the cellular and molecular mechanisms of hepatocyte injury caused by the retention of hydrophobic bile acids in cholestasis. Steatocholestasis is the combined presence of hepatic steatosis and cholestasis, common in genetic causes of metabolic liver disease in childhood. Retention of hydrophobic bile acids promotes hepatocellular injury and subsequent portal fibrosis in these conditions. Investigations at the mechanistic level have revealed that activation of hepatocyte death receptors, induction of oxidative stress, mitochondrial perturbations and activation of caspases are intracellular pathways that mediate hepatocyte injury. Several compounds in licorice root have been shown to modulate bile acid-induced apoptosis and necrosis of hepatocytes. Further investigations will be needed to identify novel molecular and cellular targets for which pharmaceuticals might be developed, to reduce liver injury and fibrosis in cholestasis and steatocholestasis.


Subject(s)
Cholestasis/physiopathology , Fatty Liver/physiopathology , Hepatocytes/metabolism , Liver/metabolism , Anti-Inflammatory Agents/therapeutic use , Cell Death/drug effects , Cholestasis/drug therapy , Cholestasis/metabolism , Cholestasis/pathology , Enterohepatic Circulation/drug effects , Fatty Liver/drug therapy , Fatty Liver/metabolism , Fatty Liver/pathology , Glycyrrhetinic Acid/therapeutic use , Hepatocytes/drug effects , Hepatocytes/pathology , Humans , Liver/drug effects , Liver/pathology , Liver/physiopathology , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Oxidative Stress/drug effects
4.
Am J Physiol Gastrointest Liver Physiol ; 290(4): G595-608, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16537972

ABSTRACT

Biliary excretion is the rate-limiting step in transfer of bilirubin, other organic anions, and xenobiotics across the liver. Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is the major transporter for conjugated endo- and xenobiotic-conjugated compounds into bile. Hormones regulate bilirubin and xenobiotic secretion into bile, which have dimorphic differences. Therefore, we examined the possible role of sex steroids and growth hormone in the regulation of Mrp2. In approximately 8-wk-old rats, mRNA, transcriptional activity, and hepatic content of Mrp2 were selectively increased fourfold (P < 0.001) in females compared with males. In males, estrogens increased and testosterone decreased Mrp2 mRNA and protein, whereas no significant effect was measured in females, suggesting either a direct effect on the liver or an alteration in growth hormone secretory pattern. After hypophysectomy, Mrp2 mRNA was markedly reduced and the effects of estrogens and testosterone on Mrp2 were prevented, supporting the role of pituitary hormones in controlling Mrp2 expression. Mrp2 increased following growth hormone infusion in males. Mrp2 mRNA was decreased in growth hormone-deficient "Little" mice. Growth hormone infusions in hypophysectomized rats partially restored Mrp2 levels, whereas thyroxine addition returned Mrp2 mRNA and protein to basal levels. Morphology as well as biochemical measurements demonstrated that Mrp2 was localized to the bile canaliculus in equal density in both genders, whereas hormone replacements increased Mrp2 in hypophysectomized animals. In cultured hepatocytes, thyroxine did not have an effect, but growth hormone alone and combined with thyroxine increased Mrp2 mRNA levels. In conclusion, Mrp2 levels are regulated by the combination of thyroxine and different growth hormone secretory patterns.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Gonadal Steroid Hormones/metabolism , Growth Hormone/metabolism , Liver/metabolism , Animals , Female , Gene Expression Regulation/physiology , Male , Rats , Rats, Sprague-Dawley , Sex Factors
5.
J Pediatr Gastroenterol Nutr ; 41(2): 235-43, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16056106

ABSTRACT

OBJECTIVES: Hydrophobic bile acids accumulate in the liver during cholestasis and are believed to cause hepatocellular necrosis and apoptosis in part through induction of the mitochondrial permeability transition (MPT) and the mitochondrial generation of oxidative stress. The purpose of this study was to determine if human hepatic mitochondria respond to bile acids in this manner. METHODS: The MPT was measured spectrophotometrically and morphologically in normal human liver mitochondria exposed to glycochenodeoxycholic acid (GCDC) with and without cyclosporin A, an inhibitor of the MPT, antioxidants, and tauroursodeoxycholic acid (TUDC). Hydroperoxide generation was measured by dichlorofluorescein fluorescence. Cytochrome c and apoptosis-inducing factor were assessed by immunoblotting. RESULTS: GCDC induced the MPT in a dose-dependent manner, which was inhibited by cyclosporin A, alpha-tocopherol, beta-carotene, idebenone, and TUDC. GCDC stimulated reactive oxygen species generation and release of cytochrome c and apoptosis-inducing factor, which were significantly inhibited by the antioxidants, cyclosporin A, and TUDC. CONCLUSIONS: Mitochondrial pathways of cell death are stimulated in human hepatic mitochondria exposed to GCDC consistent with the role of mitochondrial dysfunction in the pathogenesis of cholestatic liver injury. These results parallel those reported in rodents, supporting the extrapolation of mechanistic studies of bile acid toxicity from rodent to humans.


Subject(s)
Bile Acids and Salts/pharmacology , Hepatocytes/drug effects , Mitochondria, Liver/metabolism , Reactive Oxygen Species/metabolism , Antioxidants/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Cyclosporine/pharmacology , Cytochromes c , Dose-Response Relationship, Drug , Glycochenodeoxycholic Acid/pharmacology , Hepatocytes/enzymology , Hepatocytes/physiology , Humans , Hydrogen Peroxide , Ion Channels , Mitochondria, Liver/drug effects , Mitochondria, Liver/ultrastructure , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Spectrophotometry , Taurochenodeoxycholic Acid/pharmacology
6.
J Lab Clin Med ; 145(5): 247-62, 2005 May.
Article in English | MEDLINE | ID: mdl-15902097

ABSTRACT

UNLABELLED: Metabolic liver disorders cause chronic liver disease and liver failure in childhood. Many of these disorders share the histologic features of steatosis and cholestasis, or steatocholestasis. In this study we sought to (1) develop an in vitro model of steatocholestasis, (2) determine the mechanisms of cell death in this model, and (3) determine the role of mitochondrial disturbances in this model. METHODS: Hepatocytes were isolated from 8-week-old obese (fa/fa) and lean Zucker rats. Cell suspensions were treated with glycochenodeoxycholic acid (GCDC), after which reactive oxygen species (ROS) generation, oncotic necrosis, apoptosis, and ATP content were assessed. Isolated liver mitochondria were exposed to GCDC and analyzed for ROS generation, mitochondrial membrane-permeability transition (MPT), and cytochrome c release. Oncotic necrosis was significantly increased and apoptosis reduced in fa/fa hepatocytes exposed to GCDC compared with that in lean hepatocytes. Necrosis occurred by way of an ROS- and MPT-dependent pathway. Basal and dynamic ATP content did not differ between fa/fa and lean hepatocytes. GCDC stimulated ROS generation, MPT, and cytochrome c release to a similar extent in purified mitochondria from both fa/fa and lean rats. These findings suggest that fat-laden hepatocytes favor a necrotic rather than an apoptotic cell death when exposed to low concentrations of bile acids. The protective effects of antioxidants and MPT blockers suggest novel therapeutic strategies for the treatment of steatocholestatic metabolic liver diseases.


Subject(s)
Bile Acids and Salts/pharmacology , Fatty Liver , Hepatocytes/drug effects , Hepatocytes/pathology , Adenosine Triphosphate/analysis , Animals , Antioxidants/pharmacology , Apoptosis , Caspase Inhibitors , Cell Membrane Permeability/drug effects , Cytochromes c/metabolism , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Glycochenodeoxycholic Acid/pharmacology , Hepatocytes/metabolism , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Mitochondria, Liver/ultrastructure , Necrosis , Nutritional Status , Obesity , Rats , Rats, Zucker , Reactive Oxygen Species/metabolism
7.
J Biol Chem ; 280(11): 10556-63, 2005 Mar 18.
Article in English | MEDLINE | ID: mdl-15642733

ABSTRACT

The accumulation of hydrophobic bile acids results in cholestatic liver injury by increasing oxidative stress, mitochondrial dysfunction, and activation of cell signaling pathways. Licorice root and its constituents have been utilized as antihepatotoxic agents. The purpose of this study was to evaluate the potential modulation by a primary component of licorice root, glycyrrhizin (GL), and its metabolite, 18beta-glycyrrhetinic acid (GA), in a hepatocyte model of cholestatic liver injury. Preincubation of fresh rat hepatocyte suspensions with GL or GA reduced glycochenodeoxycholic acid (GCDC)-dependent reactive oxygen species generation, with GA more potent than GL. Interestingly, GL and GA had opposing effects toward GCDC-induced cytotoxicity; GA prevented both necrosis and apoptosis, whereas GL enhanced apoptosis. GCDC promoted activation of caspase 10, caspase 3, and PARP; all were inhibited by GA but not GL. Induction of apoptosis by GCDC was also associated with activation of JNK, which was prevented by GA. Activation of caspase 9 and dissipation of mitochondrial membrane potential were prevented by GA but not GL. In liver mitochondrial studies, GL and GA were both potent inhibitors of the mitochondrial permeability transition, reactive oxygen species generation, and cytochrome c release at submicromolar concentrations. Results from this study suggest that GL exhibits pro-apoptotic properties, whereas GA is a potent inhibitor of bile acid-induced apoptosis and necrosis in a manner consistent with its antioxidative effect.


Subject(s)
Bile Acids and Salts/metabolism , Glycyrrhetinic Acid/analogs & derivatives , Glycyrrhetinic Acid/pharmacology , Glycyrrhiza/chemistry , Glycyrrhizic Acid/pharmacology , Hepatocytes/metabolism , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Apoptosis , Caspase 10 , Caspase 3 , Caspase 9 , Caspases/metabolism , Cell Nucleus/metabolism , Cytochromes c/metabolism , Dose-Response Relationship, Drug , Flow Cytometry , Immunoblotting , JNK Mitogen-Activated Protein Kinases/metabolism , Liver/injuries , Liver/metabolism , MAP Kinase Kinase 4 , Male , Membrane Potentials , Microscopy, Fluorescence , Mitochondria/metabolism , Mitochondria, Liver/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Necrosis , Poly(ADP-ribose) Polymerases/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species , Time Factors
8.
Hepatology ; 40(4): 836-46, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15382115

ABSTRACT

The principal extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) involves formation of liver cysts derived from intrahepatic bile ducts. Autocrine and paracrine factors secreted into the cyst would be positioned to modulate the rate of hepatic cyst growth. The aim of this study was to identify potential growth factors present in human ADPKD liver cyst fluid. Cytokine array and enzyme-linked immunosorbent assay analysis of human ADPKD liver cyst fluid detected epithelial neutrophil attractant 78, interleukin (IL)-6 (503 +/- 121 pg/mL); and IL-8 (4,488 +/- 355 pg/mL); and elevated levels of vascular endothelial growth factor compared with non-ADPKD bile (849 +/- 144 pg/mL vs. 270 pg/mL maximum concentration). ADPKD liver cyst cell cultures also released IL-8 and vascular endothelial growth factor, suggesting that cystic epithelial cells themselves are capable of secreting these factors. Western blotting of cultured cyst cells and immunostaining of intact cysts demonstrate that cysteine-X-cysteine receptor 2, an epithelial neutrophil attractant 78 and IL-8 receptor, is expressed at the apical domain of cyst lining epithelial cells. Suggesting the cystic epithelial cells may exist in hypoxic conditions, electron microscopy of the ADPKD liver cyst epithelium revealed morphological features similar to those observed in ischemic bile ducts. These features include elongation, altered structure, and diminished abundance of apical microvilli. In conclusion, IL-8, epithelial neutrophil attractant 78, IL-6, and vascular endothelial growth factor may serve as autocrine and paracrine factors to direct errant growth of ADPKD liver cyst epithelia. Interruption of these signaling pathways may provide therapeutic targets for inhibiting liver cyst expansion.


Subject(s)
Cyst Fluid/metabolism , Cytokines/metabolism , Growth Substances/metabolism , Liver/metabolism , Polycystic Kidney, Autosomal Dominant/metabolism , Adult , Aged , Bile/metabolism , Cells, Cultured , Epithelial Cells/metabolism , Female , Humans , Ischemia/metabolism , Liver/pathology , Male , Middle Aged , Polycystic Kidney, Autosomal Dominant/pathology , Receptors, Interleukin-8B/metabolism
9.
Pediatr Res ; 55(5): 814-21, 2004 May.
Article in English | MEDLINE | ID: mdl-14764912

ABSTRACT

Hydrophobic bile acids are implicated in the pathogenesis of cholestatic liver disorders through mechanisms involving oxidative stress and mitochondrial dysfunction. Antioxidants ameliorate bile acid-induced cytotoxicity in rat hepatocyte suspensions. The purpose of the current study was to evaluate the potential protective role of beta-carotene (betaC), a putative fat-soluble antioxidant that is reduced in patients with cholestasis, against bile acid-induced hepatotoxicity. In freshly isolated rat hepatocyte suspensions that were exposed to the toxic hydrophobic bile acid glycochenodeoxycholic acid (100 or 500 microM), betaC (100 microM) decreased generation of reactive oxygen species by >50%, similar to the inhibition afforded by alpha-tocopherol. Commensurate with this antioxidant effect, 100 microM betaC also protected hepatocytes against both glycochenodeoxycholic acid-induced cellular necrosis and apoptosis, which was associated with reduction in caspase 3 activation, inhibition of mitochondrial cytochrome c release in rat hepatocytes, and prevention of the mitochondrial permeability transition in both liver mitochondria and rat hepatocytes. A lower concentration of betaC (50 microM) produced similar antioxidant and anti-apoptotic protection but with less inhibition against cell necrosis, suggesting that the higher concentration of betaC may have conferred additional cytoprotection not directly related to its antioxidant function. These results demonstrate that the antioxidant effects of betaC may provide hepatoprotection against cholestatic liver injury by preventing bile acid-induced oxidative stress and mitochondrial perturbations.


Subject(s)
Antioxidants/pharmacology , Apoptosis , Bile Acids and Salts/metabolism , Hepatocytes/metabolism , Hepatocytes/pathology , beta Carotene/metabolism , beta Carotene/physiology , Animals , Antioxidants/metabolism , Bile Acids and Salts/toxicity , Caspase 3 , Caspases/metabolism , Cholestasis , Cytochromes c/metabolism , Cytosol/metabolism , Enzyme Activation , Flow Cytometry , Glycochenodeoxycholic Acid/metabolism , Hepatocytes/drug effects , Immunoblotting , Liver/metabolism , Membrane Potentials , Mitochondria/metabolism , Mitochondria/pathology , Mitochondria, Liver/metabolism , Necrosis , Oxidative Stress , Rats , Reactive Oxygen Species , Time Factors
10.
Life Sci ; 71(3): 311-23, 2002 Jun 07.
Article in English | MEDLINE | ID: mdl-12034349

ABSTRACT

The synthetic steroid, pregnenolone-16-alpha-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental "stress". PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)beta, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)beta mRNA and also of its companion gene, RT1.B(I)alpha mRNA, became readily detectable within 3-6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)beta immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.


Subject(s)
Dexamethasone/pharmacology , Hepatocytes/metabolism , Histocompatibility Antigens Class II/biosynthesis , Pregnenolone Carbonitrile/pharmacology , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Steroid/agonists , Animals , Base Sequence , Cells, Cultured , Female , Glucocorticoids/pharmacology , Histocompatibility Antigens Class II/genetics , Molecular Sequence Data , Pregnane X Receptor , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley
11.
J Biol Chem ; 277(28): 25823-30, 2002 Jul 12.
Article in English | MEDLINE | ID: mdl-12006578

ABSTRACT

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.


Subject(s)
Apoptosis/drug effects , Glycochenodeoxycholic Acid/pharmacology , Hepatocytes/drug effects , Mitochondria, Liver/metabolism , Nitric Oxide/physiology , Animals , Enzyme Inhibitors/pharmacology , Flow Cytometry , Male , Nitric Oxide Synthase/antagonists & inhibitors , Rats , Rats, Sprague-Dawley
12.
Am J Physiol Cell Physiol ; 282(5): C1042-52, 2002 May.
Article in English | MEDLINE | ID: mdl-11940520

ABSTRACT

The present studies of cholangiocytes used complementary histological, biochemical, and electrophysiological methods to identify a dense population of subapical vesicles, quantify the rates of vesicular trafficking, and assess the contribution of the actin cytoskeleton to membrane trafficking. FM 1-43 fluorescence measured significant basal rates of total exocytosis (1.33 +/- 0.16% plasma membrane/min) in isolated cholangiocytes and apical exocytosis in cholangiocyte monolayers. Cell surface area remained unchanged, indicating that there was a concurrent, equivalent rate of endocytosis. FM 1-43 washout studies showed that 36% of the endocytosed membrane was recycled to the plasma membrane. 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; cAMP analog) increased exocytosis by 71 +/- 31%, whereas the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; protein kinase A inhibitor) diminished basal exocytosis by 53 +/- 11%. A dense population of 140-nm subapical vesicles arose, in part, from apical membrane endocytosis. Phalloidin staining showed that a subpopulation of the endocytosed vesicles was encapsulated by F-actin. Furthermore, membrane trafficking was inhibited by disrupting the actin cytoskeleton with cytochalasin D (51 +/- 13% of control) or jasplakinolide (58 +/- 9% of control). These studies indicate that there is a high rate of vesicular trafficking at the apical membrane of cholangiocytes and suggest that both cAMP and the actin cytoskeleton contribute importantly to these events.


Subject(s)
Actins/metabolism , Bile Ducts/metabolism , Biological Transport/physiology , Cell Membrane/metabolism , Depsipeptides , Transport Vesicles/metabolism , Animals , Antineoplastic Agents/pharmacology , Bile Ducts/drug effects , Bile Ducts/ultrastructure , Cell Line , Cell Polarity , Cyclic AMP/metabolism , Cytochalasin D/pharmacology , Cytoskeleton/metabolism , Endocytosis/physiology , Exocytosis/physiology , Fluorescent Dyes/metabolism , Nucleic Acid Synthesis Inhibitors/pharmacology , Peptides, Cyclic/pharmacology , Rats , Transport Vesicles/ultrastructure
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