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

ABSTRACT: 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.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.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 ?M 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.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.

SUBMITTER: D'Souza Y

PROVIDER: S-EPMC3631385 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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

D'Souza Y Y Ji Y Y Bennett B M BM

British journal of pharmacology 20130201 4

<h4>Background and purpose</h4>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.<h4>Experimental approach</h4>Porcine renal epithelial (LLC-PK1) cells possess an in ...[more]

PMID: 22994391

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Project description:A Phase I, double-blind, randomized, crossover study in healthy males (N=106) was conducted between March 21, 2004, and May 17, 2004, to determine the magnitude and duration of the hemodynamic interaction of avanafil (a phosphodiesterase type-5 inhibitor for treating males with erectile dysfunction) when coadministered with glyceryl trinitrate (NTG) compared with sildenafil and placebo. Subjects received avanafil (200 mg), sildenafil (100 mg), and placebo (on separate days) via the oral route followed by NTG (0.4 mg) 12, 8, 4, 1, or 0.5 hours post-dose via the sublingual route. Blood pressure (BP) and heart rate (HR) were assessed at defined intervals. Throughout the study (after administration of the study drug, and including the period after NTG administration), the effects of avanafil and sildenafil on BP and HR were significantly greatest overall, at the shortest (0.5-hour) time interval compared with placebo. By the 8- and 12-hour time intervals, no significant difference in BP or HR was observed for avanafil (8 and 12 hours) or sildenafil (12 hours) (p>0.05, compared with placebo). Compared with avanafil, sildenafil had a significantly greater effect when dosed 0.5 hours before NTG on standing HR (p=0.05); 1 hour before NTG on standing systolic blood pressure (SBP) (p<0.05), sitting SBP (p=0.01) and standing HR (p<0.01); and 12 hours before NTG on standing SBP (p=0.05). Throughout the study, symptomatic hypotension adverse events occurred in 27%, 29%, and 12%, and clinically significant reductions in standing SBP (?30 mmHg) occurred in 15%, 29%, and 12% of subjects dosed with avanafil, sildenafil, and placebo, respectively (overall treatment differences: p<0.01 and p<0.05, respectively). These data show that avanafil and sildenafil have no significant effect on BP and HR if administered to healthy males ?8 hours (avanafil) or ?12 hours (sildenafil) before a sublingual dose of NTG. However, results may differ in populations with known vascular disease, especially those using other concurrent pharmacotherapy. These findings may be of interest to clinicians who treat patients with erectile dysfunction and who also have a cardiovascular condition. Of note, the applicability of these results in such patients may be limited because the enrollment comprised healthy, normal subjects.

Dataset Information

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

Publications

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

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