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6-Nitrodopamine (6-ND) is the predominant catecholamine released from isolated vascular tissues in both mammals and reptiles, with its release being significantly reduced by the NO synthesis inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME). The vasorelaxation induced by 6-ND is unaffected by either L-NAME or the soluble guanylate cyclase (sGC) inhibitor, ODQ, indicating an alternative mechanism of action. The vasorelaxant effect appears to be mediated through selective antagonism of dopamine D2 receptors rather than traditional nitric oxide (NO)-mediated pathways. This study examined the basal release of 6-ND, dopamine, noradrenaline, and adrenaline from the rabbit thoracic aorta by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Additionally, the effects of 6-ND and the dopamine receptor antagonist L741,626 on relaxation responses and electric-field stimulation (EFS)-induced contractions in aortic rings were assessed. Nitric oxide pathway inhibitors, including L-NAME, ODQ, and methylene blue, were utilized to assess the involvement of this pathway in 6-ND-induced vasorelaxation. Concentration–response curves for norepinephrine, epinephrine, and dopamine were generated in the presence and absence of 6-ND and L-741,626. The rabbit isolated aorta presented the basal release of endothelium-derived dopamine and 6-ND. Furthermore, 6-nitrodopamine and L-741,626 induced concentration-dependent relaxations in endothelin-1 pre-contracted aortic rings. The relaxations were reduced by the mechanical removal of the endothelium but unaffected by pre-treatment with L-NAME, ODQ, or methylene blue. Pre-incubation with 6-ND significantly reduced dopamine-induced contractions, while noradrenaline- and adrenaline-induced contractions remained unchanged. The findings demonstrated that endothelium-derived 6-ND is the most potent endogenous relaxant of the rabbit isolated aorta, and the mechanism is independent of the NO pathway and involved the blockade of dopamine D2 receptors. [ABSTRACT FROM AUTHOR]

Introduction: The human umbilical artery (HUA), rat-isolated right atrium, and rat-isolated vas deferens present a basal release of 6-nitrodopamine (6-ND). The basal release of 6-ND from these tissues was significantly decreased (but not abolished) when the tissues were pre-incubated with Nω-nitro-L-arginine methyl ester (L-NAME). Methods: In this study, the effect of the pharmacological modulation of the redox environment on the basal release of 6-ND was investigated. The basal release of 6-ND was measured using Liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results and Discussion: Pre-incubation (30min) of the tissues with GKT137831 (1 µM) caused a significant increase in the basal release of 6-ND from all tissues. In the HUA, pre-incubation with diphenyleneiodonium (DPI) (100 µM) also caused significant increases in the basal release of 6-ND. Preincubation of the HUA with hydrogen peroxide (H2O2) (100 µM) increased 6-ND basal release, whereas preincubation with catalase (1,000 U/mL) significantly decreased it. Pre-incubation of the HUA with superoxide dismutase (SOD) (250 U/mL; 30min) also significantly increased the basal release of 6-ND. Preincubation of the HUA with either allopurinol (100 µM) or uric acid (1mM) had no effect on the basal release of 6-ND. Pre-treatment of the HUAwith L-NAME (100 µM) prevented the increase in the basal release of 6-ND induced by GKT137831, diphenyleneiodonium, and H2O2. The results obtained indicate amajor role of endogenous H2O2 and peroxidases as modulators of 6-ND biosynthesis/release and a lack of peroxynitrite contribution. [ABSTRACT FROM AUTHOR]

We recently identified 6-nitrodopamine and other nitro-catecholamines (6-nitrodopa, 6-nitroadrenaline), indicating that the endothelium has the ability to nitrate the classical catecholamines (dopamine, noradrenaline, and adrenaline). In order to investigate whether drugs could be subject to the same nitration process, we synthesized 4-nitro- and 7-nitropropranolol as probes to evaluate the possible nitration of the propranolol by the endothelium. The separation of the enantiomers in very high yields and excellent enantiopurity was achieved by chiral HPLC. Finally, we used Riguera's method to determine the absolute configuration of the enantiomers, through double derivatization with MPA and NMR studies. [ABSTRACT FROM AUTHOR]

This study predicted dapaconazole clinical drug–drug interactions (DDIs) over the main Cytochrome P450 (CYP) isoenzymes using static (in vitro to in vivo extrapolation equation, IVIVE) and dynamic (PBPK model) approaches. The in vitro inhibition of main CYP450 isoenzymes by dapaconazole in a human liver microsome incubation medium was evaluated. A dapaconazole PBPK model (Simcyp version 20) in dogs was developed and qualified using observed data and was scaled up for humans. Static and dynamic models to predict DDIs following current FDA guidelines were applied. The in vitro dapaconazole inhibition was observed for all isoforms investigated, including CYP1A2 (IC50 of 3.68 µM), CYP2A6 (20.7 µM), 2C8 (104.1 µM), 2C9 (0.22 µM), 2C19 (0.05 µM), 2D6 (0.87 µM), and 3A4 (0.008–0.03 µM). The dynamic (PBPK) and static DDI mechanistic model-based analyses suggest that dapaconazole is a weak inhibitor (AUCR > 1.25 and <2) of CYP1A2 and CYP2C9, a moderate inhibitor (AUCR > 2 and <5) of CYP2C8 and CYP2D6, and a strong inhibitor (AUCR ≥ 5) of CYP2C19 and CYP3A, considering a clinical scenario. The results presented may be a useful guide for future in vivo and clinical dapaconazole studies. [ABSTRACT FROM AUTHOR]

The nitric oxide-mediated actions are mostly due to cyclic GMP (cGMP) formation, but cGMP-independent mechanisms, such as tyrosine nitration, have been suggested as potential signaling pathways modulating the NO-induced responses. However, the mechanisms that lead to tyrosine nitration in platelets are poorly studied, and the protein targets of nitration have not been identified in these cells. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the cGMP-independent mechanisms of the NO-donor sodium nitroprusside (SNP) that leads to inhibition of platelet adhesion. SNP concentration-dependently inhibited platelet adhesion, as observed at 15-min and 60-min adhesion. Additionally, SNP markedly increased the cGMP levels, and the soluble guanylate inhibitor ODQ nearly abolished the SNP-mediated cGMP elevations in all experimental conditions used. Nevertheless, ODQ failed to affect the adhesion inhibition obtained with 1.0 mM SNP at 15 rain. On the other hand, superoxide dismutase or peroxynitrite (ONOO-) scavenger epigal-locatechin gallate significantly reversed the inhibition of platelet adhesion by SNP (1 mM, 15 min). Western blot analysis in SNP (1 mM, 15 min)-treated platelets showed a single tyrosine-nitrated protein with an apparent mass of [approximately or equal to] 105 kDa. Nanospray LC-MS/MS identified the human [alpha]-actinin 1 cytoskeletal isoform (P12814) as the protein contained in the nitrated SDS gel band. Thus, tyrosine nitration of [alpha]-actinin, through ONOO--formation, may be a key modulatory mechanism to control platelet adhesion.

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor [N.sup.[Omega]]-nitro-L-arginine methyl ester (L-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 [+ or -] 11.6% of counted cells) and type III (24.7 [+ or -] 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x [10.sup.-8] M) and leukotriene [B.sub.4] ([LTB.sub.4], [10.sup.-8] M). L-NAME (but not D-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and [LTB.sub.4] (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and [LTB.sub.4]- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by L-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.

Aims: Inhaled nebulised unfractionated heparin (UFH) has a strong scientific and biological rationale that warrants urgent investigation of its therapeutic potential in patients with COVID‐19. UFH has antiviral effects and prevents the SARS‐CoV‐2 virus' entry into mammalian cells. In addition, UFH has significant anti‐inflammatory and anticoagulant properties, which limit progression of lung injury and vascular pulmonary thrombosis. Methods: The INHALEd nebulised unfractionated HEParin for the treatment of hospitalised patients with COVID‐19 (INHALE‐HEP) metatrial is a prospective individual patient data analysis of on‐going randomised controlled trials and early phase studies. Individual studies are being conducted in multiple countries. Participating studies randomise adult patients admitted to the hospital with confirmed SARS‐CoV‐2 infection, who do not require immediate mechanical ventilation, to inhaled nebulised UFH or standard care. All studies collect a minimum core dataset. The primary outcome for the metatrial is intubation (or death, for patients who died before intubation) at day 28. The secondary outcomes are oxygenation, clinical worsening and mortality, assessed in time‐to‐event analyses. Individual studies may have additional outcomes. Analysis We use a Bayesian approach to monitoring, followed by analysing individual patient data, outcomes and adverse events. All analyses will follow the intention‐to‐treat principle, considering all participants in the treatment group to which they were assigned, except for cases lost to follow‐up or withdrawn. Trial registration, ethics and dissemination: The metatrial is registered at ClinicalTrials.gov ID NCT04635241. Each contributing study is individually registered and has received approval of the relevant ethics committee or institutional review board. Results of this study will be shared with the World Health Organisation, published in scientific journals and presented at scientific meetings. [ABSTRACT FROM AUTHOR]

Abstract Background Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods Eosinophils were purified using a percoll gradient followed by immunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion.

Background and Purpose: ATB-346 is a hydrogen sulfide (H2 S)-releasing anti-inflammatory and analgesic drug. Animal studies demonstrated negligible gastrointestinal (GI) damage despite marked inhibition of COX activity and significant analgesic and anti-inflammatory effects. In humans, ATB-346 (250 mg once daily) was found to inhibit COX to the same extent as naproxen (550 mg twice daily).Experimental Approach: Two hundred forty-four healthy volunteers completed a 2-week, double-blind study, taking either ATB-346 (250 mg once daily) or naproxen (550 mg twice daily), with upper GI ulceration being examined endoscopically.Key Results: Forty-two per cent of the subjects taking naproxen developed at least one ulcer (≥3-mm diameter), while only 3% of the subjects taking ATB-346 developed at least one ulcer. The two drugs produced comparable and substantial (>94%) suppression of COX activity. Subjects in the naproxen group developed more ulcers per subject than ATB-346-treated subjects and a greater incidence of larger ulcers (≥5-mm diameter). The incidence of dyspepsia, abdominal pain, gastro-oesophageal reflux, and nausea was lower with ATB-346 than with naproxen. Subjects treated with ATB-346 had significantly higher plasma levels of H2 S than those treated with naproxen.Conclusions and Implications: This Phase 2B study provides unequivocal evidence for a marked reduction of GI toxicity of the H2 S-releasing analgesic/anti-inflammatory drug, ATB-346, as compared to the conventional dose of naproxen that produced equivalent suppression of COX.Linked Articles: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc. [ABSTRACT FROM AUTHOR]

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