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

Abstract

BACKGROUND AND PURPOSERecent 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 APPROACHPorcine 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 (PK1ALDH2) that overexpressed ALDH2, or small interfering RNA (siRNA) to deplete endogenous ALDH2, and assessed GTN biotransformation and GTN-induced cGMP formation. KEY RESULTSALDH2 activity in the stably transfected cells was approximately sevenfold higher than wild-type cells or cells stably transfected with empty vector (PK1vector); and protein expression, as assessed by immunoblot analysis, was markedly increased. In PK1ALDH2, GTN biotransformation was significantly increased as a result of increased glyceryl-1,2-dinitrate formation compared to wild-type or PK1vector. However, the incubation of PK1ALDH2 with 1 or 10 mM GTN did not alter GTN-induced cGMP accumulation compared with wild-type or PK1vector cells. Furthermore, siRNA-mediated depletion of ALDH2 had no effect on GTN-induced cGMP formation. CONCLUSIONS AND IMPLICATIONSIn 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. Abbreviations1,2-GDN, glyceryl-1,2-dinitrate; 1,3-GDN, glyceryl-1,3-dinitrate; ALDH, aldehyde dehydrogenase; DEA/NO, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine; GTN, glyceryl trinitrate; LLC-PK1, porcine renal proximal tubular epithelial cell; sGC, soluble guanylyl cyclase BJP British Journal of Pharmacology

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

“…ALDH activity in AS-30D mitochondria was similar to that in RLM when using acetaldehyde (Table 1) or malondialdehyde as substrate, but it was significantly lower with propionaldehyde (2.7 ± 0.7 vs. 6 ± 0.4 mU/mg mitochondrial protein; n = 3) and 4-hydroxy-2-nonenal (0.6 ± 0.2 vs. 1.4 ± 0.07 mU/mg mitochondrial protein; n = 3). In addition, the ALDH activity with acetaldehyde or propionaldehyde was similar to that reported for RLM (Svanas and Weiner, 1985) and mitochondria isolated from H4IIE-C3 hepatoma (Moon et al, 2005) and LLC-PK1 tumor cells (D'Souza et al, 2013). These proteomic and kinetic results revealed that tumor mitochondria have the enzymatic set (a) to drive OxPhos from different oxidizable sources including FFAs and (b) to inactivate several toxic aldehydes that protect OxPhos from impairment.…”

Comparison of rheumatoid arthritis (RA) and osteoarthritis (OA) based on microarray profiles of human joint fibroblast‐like synoviocytes

“…And they can eventually lead to destruction and loss of joint function, seriously affecting the life quality of the elderly. [1][2][3] The basic pathological characters of OA are cartilage degeneration, proliferation of subchondral bone, and synovitis, while persistent chronic synovitis, dysplasia of synovial membranes and pannus formation are the main changes in the RA pathology. [4][5][6] Because there are no blood vessels on the joint surface, fibroblast-like synoviocytes (FLSs) in the inner layer of the joint capsule are the only cells that can secrete lubricant to help joint movement and transport nutrients to maintain the physiology of chondrocytes.…”

Metabolomics-Driven Elucidation of Cellular Nitrate Tolerance Reveals Ascorbic Acid Prevents Nitroglycerin-Induced Inactivation of Xanthine Oxidase

“…Most textbooks and publications claim that ALDH2 is the key GTN-bioactivating enzyme, but our results, and findings published by others, suggest otherwise. The role of ALDH2 in GTN bioactivation has been recently questioned because siRNA-mediated knockouts and overexpression of ALDH2 had no significant effect on cGMP production in porcine epithelial cells ( D’souza et al, 2013 ). It has been proposed that ALDH2 acts as a scavenger of HNE and other reactive species, but in fact does not directly metabolize GTN ( D’souza et al, 2014 ).…”

“…We selected this ascorbic acid treatment protocol because it effectively attenuated nitrate tolerance, and it is a physiologically relevant dose. Similarly, the 1 μM GTN dose was chosen because it was previously shown to be effective at inducing nitrate tolerance with no effect on cell viability or proliferation in LLC-PK1 cells ( Hinz and Schröder, 1998b ; D’souza et al, 2013 ), while being a sufficient dose for the nitrite method detection limits ( Axton et al, 2016 ). A vehicle control of 2.3 μL acetonitrile was chosen to control for the 2.3 μL of acetonitrile added with the GTN treatment.…”