Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Mitochondrial Ca uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO) production. However, it is not entirely clear if the organelles support or counteract NO biosynthesis by taking up Ca. The objective of this study was to verify whether or not mitochondrial Ca uptake influences Ca-triggered NO generation by endothelial NO synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO probes, the geNOps, and Ca sensors to monitor single cell NO and Ca dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP)-generating agonist. Mitochondrial Ca uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca-triggered NO increase independently of global cytosolic Ca signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca sequestration and Ca-induced NO signals. The positive correlation between mitochondrial Ca elevation and NO production was independent of eNOS phosphorylation at serine. Our findings emphasize that manipulating mitochondrial Ca uptake may represent a novel strategy to control eNOS-mediated NO production.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC50 value of ~0.2 μM and maximal cGMP formation at 10 μM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one–oxidized (ferric) sGC was moderate, reaching ~10%–15% of maximal activity. Dilution experiments of cinaciguat/Tween 20–preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide–sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.

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