Effect of overexpression of human aldehyde dehydrogenase 2 in<scp>LLC‐PK1</scp>cells on glyceryl trinitrate biotransformation and<scp>cGMP</scp>accumulation

<jats:sec><jats:title>Background And Purpose</jats:title><jats:p>Recent studies suggest a primary role for aldehyde dehydrogenase 2 (<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>) in mediating the biotransformation of organic nitrates, such as glyceryl trinitrate (<jats:styled-content style="fixed-case">GTN</jats:styled-content>), to the proximal activator of soluble guanylyl cyclase (<jats:styled-content style="fixed-case">sGC</jats:styled-content>), resulting in increased<jats:styled-content style="fixed-case">cGMP</jats:styled-content>accumulation and vasodilation. Our objective was to assess the role of<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>in organic nitrate action using a cell culture model.</jats:p></jats:sec><jats:sec><jats:title>Experimental Approach</jats:title><jats:p>Porcine renal epithelial (<jats:styled-content style="fixed-case">LLC‐PK1</jats:styled-content>) cells possess an intact<jats:styled-content style="fixed-case">NO‐sGC‐cGMP</jats:styled-content>signaling system, and can be used as a biochemical model of organic nitrate action. We used a<jats:styled-content style="fixed-case">pcDNA3</jats:styled-content>.1‐human<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>expression vector to establish a stably transfected cell line (<jats:styled-content style="fixed-case">PK1<jats:sub>ALDH2</jats:sub></jats:styled-content>) that overexpressed<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>, or small interfering RNA (<jats:styled-content style="fixed-case">siRNA)</jats:styled-content>to deplete endogenous<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>, and assessed<jats:styled-content style="fixed-case">GTN</jats:styled-content>biotransformation and<jats:styled-content style="fixed-case">GTN</jats:styled-content>‐induced<jats:styled-content style="fixed-case">cGMP</jats:styled-content>formation.</jats:p></jats:sec><jats:sec><jats:title>Key Results</jats:title><jats:p><jats:styled-content style="fixed-case">ALDH2</jats:styled-content>activity in the stably transfected cells was approximately sevenfold higher than wild‐type cells or cells stably transfected with empty vector (<jats:styled-content style="fixed-case">PK1<jats:sub>vector</jats:sub></jats:styled-content>); and protein expression, as assessed by immunoblot analysis, was markedly increased. In<jats:styled-content style="fixed-case">PK1<jats:sub>ALDH2</jats:sub></jats:styled-content>,<jats:styled-content style="fixed-case">GTN</jats:styled-content>biotransformation was significantly increased as a result of increased glyceryl‐1,2‐dinitrate formation compared to wild‐type or<jats:styled-content style="fixed-case">PK1<jats:sub>vector</jats:sub></jats:styled-content>. However, the incubation of<jats:styled-content style="fixed-case">PK1<jats:sub>ALDH2</jats:sub></jats:styled-content>with 1 or 10 μ<jats:styled-content style="fixed-case">M GTN</jats:styled-content>did not alter<jats:styled-content style="fixed-case">GTN</jats:styled-content>‐induced c<jats:styled-content style="fixed-case">GMP</jats:styled-content>accumulation compared with wild‐type or<jats:styled-content style="fixed-case">PK</jats:styled-content>1<jats:sub>vector</jats:sub>cells. Furthermore,<jats:styled-content style="fixed-case">siRNA</jats:styled-content>‐mediated depletion of<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>had no effect on<jats:styled-content style="fixed-case">GTN</jats:styled-content>‐induced<jats:styled-content style="fixed-case">cGMP</jats:styled-content>formation.</jats:p></jats:sec><jats:sec><jats:title>Conclusions And Implications</jats:title><jats:p>In an intact cell system, neither overexpression nor depletion of<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>affects<jats:styled-content style="fixed-case">GTN</jats:styled-content>‐induced<jats:styled-content style="fixed-case">cGMP</jats:styled-content>formation, indicating that<jats:styled-content style="fixed-case">ALDH2</jats:styled-content>does not mediate the mechanism‐based biotransformation of<jats:styled-content style="fixed-case">GTN</jats:styled-content>to an activator of<jats:styled-content style="fixed-case">sGC</jats:styled-content>.</jats:p></jats:sec>