title: Monoclonal antibody against α-actinin 4 from human umbilical vein endothelial cells inhibits endothelium-dependent vasorelaxation.
abstract: BACKGROUND
  This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity.
  
  
  METHODS
  Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity.
  
  
  RESULTS
  Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody.
  
  
  CONCLUSIONS
  These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.
authors.affiliation: Department of Physiology, School of Medicine, Konkuk University, Chungju, Korea.
authors.forename: Kyung-Jong
authors.initials: KJ
authors.lastname: Won
authors.forename: Kang Pa
authors.initials: KP
authors.lastname: Lee
authors.forename: Dong-Ku
authors.initials: DK
authors.lastname: Kim
authors.forename: Seung Hyo
authors.initials: SH
authors.lastname: Jung
authors.forename: Chang-Kwon
authors.initials: CK
authors.lastname: Lee
authors.forename: Dong Hyen
authors.initials: DH
authors.lastname: Lee
authors.forename: Su Yeol
authors.initials: SY
authors.lastname: Yu
authors.forename: Se Hyung
authors.initials: SH
authors.lastname: Park
authors.forename: Hwan Myung
authors.initials: HM
authors.lastname: Lee
authors.forename: Bokyung
authors.initials: B
authors.lastname: Kim
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000185:Actinin / Q000032:analysis / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D056404:Chaperonin Containing TCP-1 / Q000032:analysis / Q000276:immunology; D004730:Endothelium, Vascular / Q000502:physiology*; D061307:Human Umbilical Vein Endothelial Cells / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D043587:Receptor, Muscarinic M3 / Q000032:analysis; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*
chemical_list: C111453:ACTN4 protein, human; D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D043587:Receptor, Muscarinic M3; D000185:Actinin; D056404:Chaperonin Containing TCP-1; D000109:Acetylcholine; D009638:Norepinephrine