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Effect of overexpression of human aldehyde dehydrogenase 2 in LLC‐PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation
Author(s) -
D'Souza Y,
Ji Y,
Bennett BM
Publication year - 2013
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.2012.02220.x
Subject(s) - aldh2 , transfection , biotransformation , soluble guanylyl cyclase , chemistry , aldehyde dehydrogenase , cell culture , activator (genetics) , biochemistry , small interfering rna , pharmacology , microbiology and biotechnology , biology , enzyme , receptor , gene , genetics , guanylate cyclase
Background And Purpose 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. Experimental Approach 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. Key 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 μ M GTN did not alter GTN ‐induced c GMP accumulation compared with wild‐type or PK 1 vector cells. Furthermore, siRNA ‐mediated depletion of ALDH2 had no effect on GTN ‐induced cGMP formation. Conclusions And Implications 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 .