title: Effect of overexpression of human aldehyde dehydrogenase 2 in LLC-PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation.
abstract: OBJECTIVE
  Recent studies suggest a primary role for aldehyde dehydrogenase 2 (ALDH2) in mediating the biotransformation of organic nitrates, such as glyceryl trinitrate (GTN), to the proximal activator of soluble guanylyl cyclase (sGC), resulting in increased cGMP accumulation and vasodilation. Our objective was to assess the role of ALDH2 in organic nitrate action using a cell culture model.
  
  
  METHODS
  Porcine renal epithelial (LLC-PK1) cells possess an intact NO-sGC-cGMP signaling system, and can be used as a biochemical model of organic nitrate action. We used a pcDNA3.1-human ALDH2 expression vector to establish a stably transfected cell line (PK1(ALDH2)) that overexpressed ALDH2, or small interfering RNA (siRNA) to deplete endogenous ALDH2, and assessed GTN biotransformation and GTN-induced cGMP formation.
  
  
  RESULTS
  ALDH2 activity in the stably transfected cells was approximately sevenfold higher than wild-type cells or cells stably transfected with empty vector (PK1(vector)); and protein expression, as assessed by immunoblot analysis, was markedly increased. In PK1(ALDH2), GTN biotransformation was significantly increased as a result of increased glyceryl-1,2-dinitrate formation compared to wild-type or PK1(vector). However, the incubation of PK1(ALDH2) with 1 or 10 μM GTN did not alter GTN-induced cGMP accumulation compared with wild-type or PK1(vector) cells. Furthermore, siRNA-mediated depletion of ALDH2 had no effect on GTN-induced cGMP formation.
  
  
  CONCLUSIONS
  In an intact cell system, neither overexpression nor depletion of ALDH2 affects GTN-induced cGMP formation, indicating that ALDH2 does not mediate the mechanism-based biotransformation of GTN to an activator of sGC.
authors.affiliation: Department of Biomedical and Molecular Sciences, Faculty of Health Sciences, Queen's University, Kingston, Ontario, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: D'Souza
authors.forename: Y
authors.initials: Y
authors.lastname: Ji
authors.forename: B M
authors.initials: BM
authors.lastname: Bennett
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000818:Animals; D018929:Cell Culture Techniques; D006152:Cyclic GMP / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D018374:LLC-PK1 Cells; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D005996:Nitroglycerin / Q000378:metabolism* / Q000493:pharmacokinetics; D034741:RNA, Small Interfering / Q000235:genetics; D013552:Swine; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D005996:Nitroglycerin; D006152:Cyclic GMP