Cytosolic expression of aldehyde dehydrogenase-2 is essential for nitroglycerin bioactivation by cultured cells

Abstract

We have recently shown that aldehyde dehydrogenase-2 (ALDH2) is mainly cytosolic in murine vascular tissue. Relaxation of ALDH2 knockout aortas to nitroglycerin (GTN) was restored by cytosolic but not by mitochondrial overexpression of ALDH2. To find out whether GTN bioactivation is determined by the endogenous distribution of ALDH2, we studied two types of cultured cells with strictly different ALDH2 localization for GTN-induced formation of 1,2-glycerol dinitrate (1,2-GDN) and accumulation of intracellular cGMP. Chloral hydrate (1 mM) and daidzin (0.1 mM) were used as ALDH inhibitors. Quantitative immunoblotting revealed that porcine aortic endothelial cells (PAECs) and rat lung fibroblasts (RFL-6 cells) expressed the protein predominantly in mitochondrial and cytosolic fractions, respectively. Interestingly, ALDH2-catalyzed GTN metabolism required addition of dithiothreitol (DTT), indicating that the cultured cells lack an essential reductant that is present in blood vessels. In the presence of 10 μM GTN and 2 mM DTT, the rates of 1,2-GDN formation were 0.5 ± 0.2 and 1.3 ± 0.4 pmol min −1 mg −1 in PAECs and RFL-6 cells, respectively. The maximal cGMP levels observed with 10 μM GTN in the presence of 2 mM DTT and 1 kU SOD/ml were 26 ± 4 and 87 ± 16 pmol/10 6 cell in PAECs and RFL-6 cells, corresponding to about 2- and 5-fold increases of basal levels, respectively. These results show that cytosolic expression of ALDH2 is essential for GTN bioactivation, indicating limited access of GTN to the mitochondrial matrix. Further work is needed to identify the endogenous reductant that was replaced by DTT in the present study.