The first evidence of endogenous production of nitrocatecholamine was generated by the observation that noradrenaline levels detected by microdialysis of the rat hypothalamic paraventricular nucleus were decreased when the tissue was perfused with a solution containing nitric oxide (Shintani et al., 1996). Using 6-nitronoradrenaline synthetized by bubbling NO gas in a solution containing noradrenaline hydrochloride as a standard, extracts from the porcine brain were analyzed via electrochemical detection linked to high-pressure chromatography, and a peak with an identical retention time to the standard was identified and characterized as 6-nitronoradrenaline by UV spectrometry, mass spectrometry (MS), and Nuclear Magnetic Resonance (NMR) spectroscopy (Shintani et al., 1996). The basal release of dopamine (Britto-Júnior et al., 2020) and 6-nitrodopamine (6-ND; Britto-Júnior et al., 2021a) has been initially described from the human umbilical artery (HUA) but later identified also from other vascular tissues, such as the aortic rings of Chelonoidis carbonarius (Britto-Júnior et al., 2022a), Pantherophis guttatus (Lima et al., 2022), and Callithrix spp. (Britto-Júnior et al., 2023a) and in non-vascular tissues, such as rat-isolated atria (Britto-Júnior et al., 2022b) and rat- (Britto-Júnior et al., 2021b) and human-isolated vas deferens (Britto-Júnior et al., 2022c). In the cardiovascular system, 6-ND acts as a potent vasodilator and presents both positive chronotropic and inotropic effects (Zatz and De Nucci, 2023). In all the tissues mentioned above, pre-incubation of the tissues with the NO synthase (NOS) inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) caused a significant reduction in the synthesis/release of 6-ND, indicating a major role of nitric oxide synthases in the biosynthesis of 6-ND. Indeed, the basal release of 6-ND was significantly reduced in the isolated atria of endothelial nitric oxide synthase (eNOS)^−/− mice but not affected when the atria were obtained from either nNOS^−/− or iNOS^−/− mice (Britto-Júnior et al., 2023b).

Acute decompensated heart failure is a complex and life-threatening clinical syndrome associated with recurrent hospitalizations and a high mortality rate (Greenberg, 2012), and the positive inotropic catecholamines, such as noradrenaline and adrenaline, can be therapeutically used to overcome end-organ hypoperfusion (Monnet et al., 2023). Although there are known factors that can precipitate acute heart failure, such as myocardial ischemia, cardiac arrhythmias, infections, and non-compliance with medication (Follath, 2009), the molecular mechanism(s) involved are not known. Since 6-ND is the most potent endogenous positive chronotropic and inotropic agent described (Britto-Júnior et al., 2023c), understanding its biosynthetic pathway may provide insights into the physiopathology of acute heart failure.

This study evaluated the effects of the pharmacological modulation of the redox environment on the production/release of 6-ND and dopamine from human umbilical arteries, rat-isolated right atrium, and rat-isolated vas deferens in vitro.

The results obtained indicate a potential major role of endogenous hydrogen peroxide and peroxidases in the biosynthesis of 6-ND in human umbilical cord vessels. Hydrogen peroxide is an end product of several metabolic reactions by membrane-associated NADPH oxidases (NOX) and can also be generated as a by-product of mitochondrial respiration (Winterbourn, 2020). Hydrogen peroxide is produced by endothelial cells; in experiments performed with small mesenteric arteries from mice, we loaded the arteries with dichlorodihydrofluorescein diacetate (DCF), a peroxide-sensitive fluorescence dye (Ohba et al., 1994), and observed them using a laser confocal microscope. Stimulation with acetylcholine caused a significant increase in fluorescence in the endothelium, which was virtually abolished by pre-incubation of the endothelial cells with catalase (Matoba et al., 2000). Hydrogen peroxide can cross membranes, facilitated by aquaporins (Erudaitius et al., 2017; Bestetti et al., 2020). Hydrogen peroxide is rapidly metabolized by peroxiredoxins, catalase, and GSH peroxidase (Winterbourn, 2020) and can also react with CO₂, forming peroxymonocarbonate (HCO₄ ⁻). The significant reduction in the basal release of 6-ND obtained with catalase supports the importance of hydrogen peroxide in 6-ND biosynthesis. The peroxidase mimetic ebselen consumes hydrogen peroxide for the oxidation of reduced glutathione (Sies, 1993). Indeed, peroxiredoxins and glutathione peroxidases are abundant throughout the cells and are very efficient in metabolizing H₂O₂, with constant rates in the 10⁶–10⁸ M^-1 s^-1 range (Winterbourn, 2020). However, an alternative mechanism by which ebselen decreases the basal release of 6-ND could be due to its ability to inhibit eNOS (Zembowicz et al., 1993).

Rat skeletal muscle cell cultures produce hydrogen peroxide, and intense electrical stimulation increases H₂O₂ formation (Silveira et al., 2003). Exposure of rat cardiomyocytes to H₂O₂ causes gradual increases in intracellular calcium (Wang et al., 1999). In rat-isolated perfused beating atria, the average concentration of H₂O₂ in perfusate during the low stimulation period was 3.23 ± 0.55 nM and that during the high stimulation period was 7.00 ± 0.62 nM (Gao et al., 2011). In the isolated working heart preparation, an infusion of hydrogen peroxide (6 and 60 μM) caused a concentration-dependent increase in the heart rate (Shattock et al., 1982). Although H₂O₂ plays a role in heart chronotropism, further investigation is needed to determine whether the increase in the atrial rate induced by H₂O₂ reported here is due to either an increase in the basal release of 6-ND or increased [Ca⁺²]_i. Although the increase was sensitive to NO inhibition, the finding that it was transient is surprising since the positive chronotropic effect of 6-ND in rat isolated atria is prolonged even after the preparation is washed (Britto-Júnior et al., 2022b).

Superoxide anion (O₂ ⁻) is generated by the action of both NADH (Turrens and Boveris, 1980) and NADPH oxidases and xanthine oxidases. The superoxide anion reacts with nitric oxide, generating peroxynitrite (Blough and Zafiriou, 1985; Beckman and Koppenol, 1996). Although the main pathway for O₂ ^- elimination in biological systems is SOD-catalyzed dismutation to H₂O₂ and O₂, NO is the only biomolecule known to react fast enough and to be produced at sufficient concentrations to outcompete SOD for its reaction with O₂ ^-, yielding peroxynitrite. The nitration induced by peroxynitrite could be due to several different mechanisms: the formation of the nitronium cation (NO⁺) or the formation of the nitrogen dioxide radical ( ^• NO₂). The formation of the nitronium cation is favored over the formation of nitrogen dioxide radical in the presence of free metal ions. Another mechanism by which peroxynitrite can generate the nitrogen dioxide radical ( ^• NO₂) is through the reaction with CO₂, yielding nitrosoperoxocarboxylate (ONOOCO₂ ⁻) that rapidly decays into carbonate radical ( ^• CO₃) and nitrogen dioxide radical ( ^• NO₂), promoting one-electron oxidations and nitrations (Radi, 2022). However, our results indicate that peroxynitrite formation is not involved in the biosynthesis of 6-nitrodopamine since the synthesis/release of 6-ND is augmented with pre-incubation with SOD. Uric acid has been associated with decreased NO release by cultured endothelial cells, and it increases intracellular superoxide formation (Papežíkova et al., 2013). Interestingly, uric acid is also considered a radical scavenger and antioxidant (Becker et al., 1991). However, since uric acid did not affect the release of 6-ND from the umbilical artery, it indicates that these actions of uric acid may not occur in the human umbilical artery. It is interesting that in the presence of an NO donor, in contrast to wild-type NADPH oxidase 4 (NOX4), only superoxide-producing NOX4 mutants generated peroxynitrite (Takac et al., 2011). Peroxynitrite is known to be highly reactive with thiols (Radi, 2018), forming complexes with hemoglobin, methionine, and tyrosine (Bartesaghi and Radi, 2018). Whether nitration of tyrosine may result in the synthesis of nitrocatecholamines is yet to be demonstrated.

NADPH oxidase (NOX) enzymes were first identified in neutrophils and macrophages (Sylvester et al., 2022); during phagocytosis, NOX activation produces O₂ ⁻ that causes pathogen killing. In contrast to other oxidases, NADPH oxidases (NOX) produce superoxide anion as their primary and sole function (Altenhöfer et al., 2015). NOX1 is particularly abundant in the colonic epithelium and vascular smooth muscle cells (Szanto et al., 2005), whereas NOX2 is mainly found in phagocytes (Moghadam et al., 2021). NOX4 is expressed in the vascular wall and endothelial cells (Konior et al., 2014) and NOX5, although absent in rodents, is expressed in human vascular tissue (Touyz et al., 2019). GKT137831 belongs to the structural class of pyrazolopyridine-diones, acts as a preferential direct inhibitor of NOX1 and NOX4 (Aoyama et al., 2012), and does not inhibit NOX production by xanthine oxidase (Sedeek et al., 2010). The results presented herein indicate that the inhibition of NOX4 increases 6-ND biosynthesis, and this increase is dependent on the NO synthase pathway. In contrast, DPI presented a similar potency for all isoforms of NOX, and it also inhibited xanthine oxidase, acting as a general inhibitor of flavoproteins. GSK2795039 is a direct inhibitor of NOX2 and does not inhibit protein kinase C (PKC) or xanthine oxidase (Hirano et al., 2015). Our finding that 6-ND was increased with GKT137831 and the pan-inhibitor DPI presents another piece of evidence that the mechanism responsible for 6-ND synthesis does not require O₂ ⁻-induced peroxynitrite production. The failure of GSK2795039 to effect 6-ND synthesis/release was expected, as NOX2 is not expressed in vascular tissues.

The identity of the reactive oxygen products originated by NOX4 is controversial. Mouse vascular smooth muscle cells (Ellmark et al., 2005), rat aortic endothelial cells (Ago et al., 2004), and the membrane fraction of the human embryonic kidney-derived (HEK) 293 cells expressing NOX (Shiose et al., 2001) exhibit NADH- and NADPH-dependent superoxide-producing activities, whereas in cell lines expressing NOX4 upon tetracycline, the superoxide anion generation was almost undetectable, although hydrogen peroxide was produced (Serrander et al., 2007). This discrepancy could be attributed to the proposed perinuclear vesicle membrane location of NOX4 (von Löhneysen et al., 2008). Therefore, the superoxide production is confined to the intravesicular space, whereas hydrogen peroxide is freely diffusible and can be measured in the extracellular medium (Werner, 2003). In a membrane-free, partially purified preparation of NOX4, 90% of the electron flux through isolated NOX4 produced H₂O₂, and 10% formed superoxide (Nisimoto et al., 2014). Indeed, another source of endoplasmic reticulum (ER)-derived reactive oxygen species is NOX4, which releases O₂ ⁻ and H₂O₂ into the ER lumen to maintain its oxidizing environment (Lee et al., 2020). The kinetic mechanism of H₂O₂ formation is consistent with a mechanism involving the binding of one oxygen molecule, which is then sequentially reduced by the heme in two one-electron reduction steps, first to form a bound superoxide intermediate and then H₂O₂. Thus, it is possible that NO could react with the proposed bound superoxide intermediate. There are many sources for H₂O₂ production in endothelial cells (Cai, 2005). Indeed, more than 40 O₂ ⁻/H₂O₂-generating enzymes have been identified in humans (Sies and Jones, 2020). Thus, the mechanism by which NOX inhibition increases 6-ND biosynthesis/release should be related to the inhibition of superoxide anion production.

Since the O₂ ⁻/peroxynitrite pathway has been thoroughly excluded, what is the mechanism responsible for 6-ND biosynthesis? One interesting possibility is the reaction of nitrite with hydrogen peroxide, generating the nitrogen dioxide radical ( ^• NO₂). Hemoglobin oxidation in the erythrocyte lysate by submillimolar levels of NO₂ only occurs in the presence of high concentrations of H₂O₂ (Titov and Petrenko, 2005), and this process is slowed in the presence of catalase. The nitrite-dependent oxidation of oxyhemoglobin is not affected by incubation with superoxide dismutase, indicating that, similar to our results, superoxide anion does not play an important role (Keszler et al., 2008).

The increase in the basal release of 6-ND by hydrogen peroxide is dependent on NO production since it was inhibited by L-NAME. Hydrogen peroxide induces endothelium-dependent and endothelium-independent vasorelaxation (Qiao et al., 2014), and the exposure of large-sized arteries to hydrogen peroxide causes the activation of eNOS (Tejero et al., 2019). In bovine aortic endothelial cells, hydrogen peroxide caused a potent concentration-dependent increase in NO release, as detected by the NO-specific microelectrode (Cai et al., 2003). The release was due to eNOS phosphorylation induced by phosphoinositide 3-kinase (PI 3-kinase) since it was inhibited by pre-incubation of the endothelial cells with the PI-3 kinase inhibitors wortmannin (Arcaro and Wymann, 1993) and LY294002 (Vlahos et al., 1994) and by c-Src family tyrosine kinase (Zhao et al., 1992) since it was inhibited by Src inhibitor PP1 (Karni et al., 2003). Whether these biochemical pathways are involved in 6-ND biosynthesis/release by the human umbilical artery and rat atria is under current investigation. Although solid evidence for animal NOS-like enzymes in plants is lacking (Thomas et al., 2002), few alga species possess them (Santolini et al., 2016). It is interesting that hydrogen peroxide induction of NO production in the plant Vicia faba could be due to an NOS-like activity since it was inhibited by L-NAME (She et al., 2004). In plants, it is assumed that the increased NO production induced by hydrogen peroxide is via the activation of nitrate reductase (Lin et al., 2012). However, in Arabidopsis leaves, ultraviolet B triggered significant increases in hydrogen peroxide and NO levels were regulated by GPA1, the Gα-subunit of heterotrimeric G proteins (He et al., 2004). It is interesting that in co-cultures of calf pulmonary artery endothelial cells and rabbit pulmonary artery smooth muscle cells, incubation with H₂O₂ was associated with a decrease in cGMP accumulation (Marczin et al., 1992). Although these results indicated that H₂O₂ was reducing NO levels, one more plausible explanation is that H₂O₂ is increasing the synthesis of 6-ND. In human-washed platelets, 6-ND does not induce an increase in either cGMP or cAMP (Nash et al., 2022).

Molybdenum-containing enzymes of the xanthine oxidase family catalyze oxygen atom transfer reactions, and nitrite can be reduced to NO with xanthine oxidase (Maia, 2023). As mentioned before, the inhibition of NO synthase causes significant reductions in the basal release of 6-ND, but it does not abolish it. However, this pathway is unlikely to be involved in 6-ND biosynthesis since the xanthine oxidase inhibitor allopurinol (Pacher et al., 2006) failed to affect the basal release of 6-ND. However, the concept of “non-dedicated nitrite reductases” (Maia and Moura, 2018) as an alternative pathway for the NOS-independent synthesis of 6-ND deserves attention. It is interesting that the basal release of 6-ND and 6-nitroadrenaline from rabbit-isolated hearts is not affected by pre-treatment of the heart with L-NAME (Junior et al., 2023). One possible pathway for the nitrosation of dopamine, independent of NOS activity, would be the reduction of endogenous nitrite/nitrate since these ions are not only the products of the metabolism of NO, but they can also act as a reservoir (Shiva, 2013). Enzymes such as hemoglobin, myoglobin, xanthine oxidoreductase (Millar et al., 1998), and cytochrome P450 reductase (Li et al., 2006) can catalyze the reduction of nitrite or nitrate to generate NO. Peroxidases are iron-containing enzymes that can catalyze one- and two-electron oxidation reactions of small anionic molecules as electron donors, such as halides, thiocyanate, and nitrite, with hydrogen peroxide (García et al., 2023). Nitrite (NO₂ ⁻) is a symmetrical anion and, therefore, can undergo either oxidation or reduction, generating nitrogen dioxide ( ^• NO₂) or nitric oxide, respectively.

Diphenyleneiodonium is also considered a potent and irreversible inhibitor of nitric oxide synthase (Rand and Li, 1993; Szilagyi et al., 2016), yet it promoted a remarkable increase in the 6-ND synthesis/release from the umbilical artery, and this increase was sensitive to NOS inhibition since it was not observed in the L-NAME-treated umbilical artery. How should this apparent paradox be explained? The most likely explanation is that DPI either does not inhibit NOS in the human umbilical artery, or else it causes a transient inhibition of NOS. Indeed, DPI produces only a transient pressor response following systemic administration to animals, and the dose-dependent increases in the mean arterial pressure induced by L-NAME in conscious rats were shifted to the right by pre-treatment with DPI (Wang et al., 1995). Since 6-ND is a potent vasodilator (Zatz and De Nucci, 2023), it is possible that an increase in the basal release of 6-ND induced by DPI counteracts the vasoconstriction induced by the lack of NO in vivo.

However, how does DPI increase the release of 6-ND in the umbilical artery? Mammalian cells and tissues actively decompose NO (Gardner, 2005). NO dioxygenases (NODs) convert NO to nitrate; they bind O₂ to form a stable heme-Fe³⁺(O₂ ⁻) complex that reacts rapidly with NO, forming nitrate, and they are inhibited by DPI (Gardner et al., 2001). One possibility is that the inhibition of NOX by DPI could explain both the remarkable increase in 6-ND and the sensitivity to NOS inhibition. Another possibility would be that the inhibition of superoxide anion production by DPI plays a major role in 6-ND biosynthesis.

GKT137831 enhances the basal release of 6-ND from both rat-isolated atria and vas deferens. Since in the former tissue, eNOS is the main isoform responsible for 6-ND synthesis (Britto-Júnior et al., 2023b), whereas in the latter tissue, nNOS is the main isoform associated with 6-ND release (Britto-Júnior et al., 2024), one should consider that this mechanism of action could be relevant for the potential therapeutic effects of GKT137831. For instance, GKT137831 improves erectile function in diabetic rats (Zhao et al., 2015), and one possible explanation could be that GKT137831 induces increases in 6-ND in the corpus cavernosum. Although in vivo administration of GKT137831 induces dose-dependent reno- and atheroprotection in established micro- and macrovascular diseases (Gray et al., 2017), this is apparently the first report of a positive chronotropic effect of this NOX1/4 inhibitor. Since this effect was significantly reduced by pre-treatment of the atria with L-NAME, it is possible that the mechanism of action is related to the increase in 6-ND biosynthesis/release.

The increase in 6-ND levels induced by the NOX1/4 inhibitor in the rat-isolated vas deferens was accompanied by a concomitant decrease in dopamine levels. However, the increase in 6-ND levels (0.9 ng/mL) was significantly higher than the observed decrease in dopamine levels (0.3 ng/mL). In the rat atria, the increase in 6-ND levels was not accompanied by changes in dopamine levels. As proposed above, the main mechanism responsible for the increase in 6-ND levels by GKT137831 could be the inhibition of superoxide anion production, and this mechanism could also be responsible for an increase in dopamine availability. Dopamine is a very good antioxidant (Iuga et al., 2011), and although the superoxide anion is not a very reactive species, the hydroperoxyl radical (•OOH), which is the protonated form of the superoxide anion (DeGrey, 2002), presents higher reactivity and could contribute significantly to dopamine oxidation. The possible mechanisms involved in the synthesis/release of 6-ND are depicted in Figure 6.

The results indicate that hydrogen peroxide and peroxidases, rather than peroxynitrite, play a major role in the biosynthesis/release of 6-ND.