title: Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions.
abstract: Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4.
  
  
  CONCLUSIONS
  Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.
authors.affiliation: The Danish National Research Foundation Centre for Cardiac Arrhythmia and Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Sarah
authors.initials: S
authors.lastname: Mollerup
authors.forename: Johannes P
authors.initials: JP
authors.lastname: Hofgaard
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Braunstein
authors.forename: Ane
authors.initials: A
authors.lastname: Kjenseth
authors.forename: Edward
authors.initials: E
authors.lastname: Leithe
authors.forename: Edgar
authors.initials: E
authors.lastname: Rivedal
authors.forename: Niels-Henrik
authors.initials: NH
authors.lastname: Holstein-Rathlou
authors.forename: Morten Schak
authors.initials: MS
authors.lastname: Nielsen
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects*; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D009638:Norepinephrine / Q000494:pharmacology*; D017830:Octoxynol / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D000316:Adrenergic alpha-Agonists; D018031:Connexin 43; D056827:Endosomal Sorting Complexes Required for Transport; D061988:Proteasome Inhibitors; D011942:Receptors, Adrenergic, alpha; D017830:Octoxynol; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D009638:Norepinephrine