TITLE

Canalicular Retention as an in Vitro Assay of Tight Junctional Permeability in Isolated Hepatocyte Couplets: Effects of Protein Kinase Modulation and Cholestatic Agents

AUTHOR(S)
Roma, Marcelo G.; Orsler, Dominic J.; Coleman, Roger
PUB. DATE
May 1997
SOURCE
Fundamental & Applied Toxicology;1997, Vol. 37 Issue 1, p71
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
A simple, fast method to evaluate acute changes of tight junctional permeability in isolated hepatocyte couplets is proposed. The method consists of the recording of the number of canalicular vacuoles able to retain the previously accumulated fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF), as visualized by inverted fluorescent microscopy, following acute exposure to the compounds under study. The method was validated by (i) making a systematic documentation of the effect on CLF retention of a variety of hormonal modulators (vasopressin and phorbol esters), as well as several cholestatic (taurolithocholic acid, cyclosporin A, and estradiol 17β-glucuronide) and hepatotoxic agents (menadione, A23187, and t-butyl hydroperoxide), all known to affect biliary permeability in intact liver, and (ii) carrying out a comparative analysis of the results obtained with those recorded using rapid canalicular access of horseradish peroxidase (HRP) as an alternative procedure. The compounds tested all decreased canalicular vacuolar retention of CLF in a dose-dependent manner. Vasopressin- and phorbol ester-induced decline in CLF retention were prevented by pretreatment with the protein kinase C inhibitors H-7 and staurosporine, thus confirming a role for this enzyme in canalicular permeability regulation. A significant direct correlation (r = 0.934, p < 0.001) was obtained when the decrease in canalicular retention of CLF was compared with the increment in the canalicular access of HRP. Image analysis revealed that cellular fluorescence was not increased following exposure to these compounds, suggesting a paracellular rather than transcellular route for CLF egress. These results all support canalicular vacuolar retention of CLF as a suitable method to readily evaluate acute changes in tight junctional permeability in isolated hepatocyte couplets induced by physiological modulators or hepatotoxic agents.
ACCESSION #
82418342

 

Related Articles

  • Eupatilin attenuates bile acid-induced hepatocyte apoptosis. Park, Su Cheol; Yoon, Jung-Hwan; Kim, Won; Gwak, Geum-Youn; Kim, Kang Mo; Lee, Sung-Hee; Lee, Soo-Mi; Lee, Hyo-Suk // Journal of Gastroenterology;Aug2006, Vol. 41 Issue 8, p772 

    In cases of cholestasis, bile acids induce hepatocyte apoptosis by activating death receptor-mediated apoptotic signaling cascades. Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is a pharmacologically active ingredient found in Artemisia asiatica and exhibits cytoprotective effects against...

  • Bile Acids Affect Liver Mitochondrial Bioenergetics: Possible Relevance for Cholestasis Therapy. Rolo, Anabela P.; Oliveira, Paulo J.; Moreno, António J. M.; Palmeira, Carlos M. // Toxicological Sciences;Sep2000, Vol. 57 Issue 1, p177 

    It has been pointed out that intracellular accumulation of bile acids cause hepatocyte injury in cholestatic disease process. This study was aimed to test if cytotoxicity of these compounds is mediated through mitochondria dysfunction. Bile acids effects on isolated rat liver mitochondrial were...

  • Activation-dependent degradation of protein kinase Cη. Kang, Beom-Sik; French, Olivia G; Sando, Julianne J; Hahn, Chang S // Oncogene;8/31/2000, Vol. 19 Issue 37, p4263 

    Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKCη, we expressed the novel PKCs η and θ and...

  • Effects of phorbol ester on vesicle dynamics as revealed by total internal reflection fluorescence microscopy. Enming Zhang; Renhao Xue; Jianchow Soo; Peng Chen // Pflugers Archiv European Journal of Physiology;Oct2008, Vol. 457 Issue 1, p211 

    Exocytosis of neurotransmitter or hormone-filled vesicles is a highly dynamic process regulated by various proteins and lipids. As mainly revealed indirectly by the electrophysiological methods, exocytosis is believed to involve multiple kinetic steps in which vesicles transit from one state to...

  • Association of PKC δ and active Src in PMA-treated MCF-7 human breast cancer cells. Shanmugam, Malathy; Krett, Nancy L; Peters, Carl A; Maizels, Evelyn T; Murad, Faris M; Kawakatsu, Hisaaki; Rosen, Steven T; Hunzicker-Dunn, Mary // Oncogene;4/2/98, Vol. 16 Issue 13, p1649 

    Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC δ. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC δ and catalytically active PKC δ....

  • Regulation of protein kinase C-δ and -ε isoforms by phorbol ester treatment of LLC-PK1 renal epithelia. Clarke, Hilary; Ginanni, Nicole; Soler, Alejandro Peralta; Mullin, James M. // Kidney International;Sep2000, Vol. 58 Issue 3, p1004 

    Examines the subcellular distribution and down-regulation of protein kinase (PKC)-delta and PKC-epsilon isoforms in phorbol ester-treated LLC-PK epithelia. Use of the Western immunoblot in detecting the cellular localization of PKC delta and epsilon; Role of o-tetradecanoyl phorbol treatment in...

  • Modulation of Hepatocyte Apoptosis: Cross-talk Between Bile Acids and Nuclear Steroid Receptors. Solá, S.; Amaral, J. D.; Aranha, M. M.; Steer, C. J.; Rodrigues, C. M. P. // Current Medicinal Chemistry;2006, Vol. 13 Issue 25, p3039 

    The efficient removal of unwanted cells, such as senescent, damaged, mutated or infected cells is crucial for the maintenance of normal liver function. In fact, apoptosis has emerged as a potential contributor to the pathogenesis of a number of hepatic disorders, such as viral hepatitis,...

  • Interlobular Bile Duct Cholestatic Disease is Aberrant Cytokeratin by Hepatocytes Loss in Pediatric Associated with 7 Expression. Ernst, Linda M.; Spinner, Nancy B.; Piccoli, David A.; Mauger, Joanne; Russo, Pierre // Pediatric & Developmental Pathology;Sep/Oct2007, Vol. 10 Issue 5, p383 

    The objective of this study was to determine whether aberrant hepatic expression of cytokeratin 7 (CK7) and/or other putative stem cell markers is seen in pediatric cholestatic diseases. Eighteen liver biopsies and 14 liver explants from pediatric patients with extrahepatic biliary atresia...

  • RhoA signaling in phorbol ester-induced apoptosis. Zee-Fen Chang; Hsiao-Hui Lee // Journal of Biomedical Science;Mar2006, Vol. 13 Issue 2, p173 

    Exposure of cells to phorbol ester activates protein kinase C (PKC) to induce apoptosis or differentiation, depending on the cellular context. In erythroblastic cell lines, TF-1 and D2, upregulation of the RhoA signaling promotes phorbol ester-induced apoptosis through activating Rho-associated...

Share

Read the Article

Courtesy of THE LIBRARY OF VIRGINIA

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics