Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells

Biochem Pharmacol. 2009 Sep 15;78(6):648-55. doi: 10.1016/j.bcp.2009.05.012. Epub 2009 May 20.

Abstract

Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Caveolae / drug effects*
  • Caveolae / metabolism
  • Cell Line
  • Cell Polarity / drug effects
  • Cell Polarity / physiology
  • Cells, Cultured
  • Clathrin / metabolism
  • Clathrin / physiology*
  • Clathrin Heavy Chains / chemistry
  • Clathrin Heavy Chains / pharmacology
  • Drug Interactions
  • Endocytosis / drug effects*
  • Endocytosis / physiology
  • Ethanol / pharmacology*
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism
  • Humans
  • Liver / cytology
  • Membrane Microdomains / drug effects*
  • Membrane Microdomains / metabolism
  • Protein Transport
  • Receptors, Urokinase Plasminogen Activator / metabolism

Substances

  • Clathrin
  • Receptors, Urokinase Plasminogen Activator
  • Clathrin Heavy Chains
  • Ethanol