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Effect of overexpression of human aldehyde dehydrogenase 2 in LLC-PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation.
Br J Pharmacol. 2013 Feb; 168(4):978-87.BJ

Abstract

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 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 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.

Authors+Show Affiliations

Department of Biomedical and Molecular Sciences, Faculty of Health Sciences, Queen's University, Kingston, Ontario, Canada.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22994391

Citation

D'Souza, Y, et al. "Effect of Overexpression of Human Aldehyde Dehydrogenase 2 in LLC-PK1 Cells On Glyceryl Trinitrate Biotransformation and cGMP Accumulation." British Journal of Pharmacology, vol. 168, no. 4, 2013, pp. 978-87.
D'Souza Y, Ji Y, Bennett BM. Effect of overexpression of human aldehyde dehydrogenase 2 in LLC-PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation. Br J Pharmacol. 2013;168(4):978-87.
D'Souza, Y., Ji, Y., & Bennett, B. M. (2013). Effect of overexpression of human aldehyde dehydrogenase 2 in LLC-PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation. British Journal of Pharmacology, 168(4), 978-87. https://doi.org/10.1111/j.1476-5381.2012.02220.x
D'Souza Y, Ji Y, Bennett BM. Effect of Overexpression of Human Aldehyde Dehydrogenase 2 in LLC-PK1 Cells On Glyceryl Trinitrate Biotransformation and cGMP Accumulation. Br J Pharmacol. 2013;168(4):978-87. PubMed PMID: 22994391.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effect of overexpression of human aldehyde dehydrogenase 2 in LLC-PK1 cells on glyceryl trinitrate biotransformation and cGMP accumulation. AU - D'Souza,Y, AU - Ji,Y, AU - Bennett,B M, PY - 2011/12/30/received PY - 2012/07/29/revised PY - 2012/09/11/accepted PY - 2012/9/22/entrez PY - 2012/9/22/pubmed PY - 2013/7/17/medline SP - 978 EP - 87 JF - British journal of pharmacology JO - Br J Pharmacol VL - 168 IS - 4 N2 - 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 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 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. SN - 1476-5381 UR - https://www.unboundmedicine.com/medline/citation/22994391/Effect_of_overexpression_of_human_aldehyde_dehydrogenase_2_in_LLC_PK1_cells_on_glyceryl_trinitrate_biotransformation_and_cGMP_accumulation_ L2 - https://doi.org/10.1111/j.1476-5381.2012.02220.x DB - PRIME DP - Unbound Medicine ER -