Yellow Fever Emergence: Role of Heterologous Flavivirus Immunity in Preventing Urban Transmission.

During major, recent yellow fever (YF) epidemics in Brazil, human cases were attributed only to spillover infections from sylvatic transmission with no evidence of human amplification. Furthermore, the historic absence of YF in Asia, despite abundant peridomestic Aedes aegypti and naive human populations, represents a longstanding enigma. We tested the hypothesis that immunity from dengue (DENV) and Zika (ZIKV) flaviviruses limits YF virus (YFV) viremia and transmission by Ae. aegypti . Prior DENV and ZIKV immunity consistently suppressed YFV viremia in experimentally infected macaques, leading to reductions in Ae. aegypti infection when mosquitoes were fed on infected animals. These results indicate that, in DENV- and ZIKV-endemic regions such as South America and Asia, flavivirus immunity suppresses YFV human amplification potential, reducing the risk of urban outbreaks. One-Sentence Summary: Immunity from dengue and Zika viruses suppresses yellow fever viremia, preventing infection of mosquitoes and reducing the risk of epidemics.

CD8 + T cell response promotes viral clearance and reduces chances of severe testicular damage in mouse models of long-term Zika virus infection of the testes.

Zika virus (ZIKV) causes human testicular inflammation and alterations in sperm parameters and causes testicular damage in mouse models. The involvement of individual immune cells in testicular damage is not fully understood. We detected virus in the testes of the interferon (IFN) α/ß receptor -/- A129 mice three weeks post-infection and found elevated chemokines in the testes, suggesting chronic inflammation and long-term infection play a role in testicular damage. In the testes, myeloid cells and CD4 + T cells were absent at 7 dpi but were present at 23 days post-infection (dpi), and CD8 + T cell infiltration started at 7 dpi. CD8 -/- mice with an antibody-depleted IFN response had a significant reduction in spermatogenesis, indicating that CD8 + T cells are essential to prevent testicular damage during long-term ZIKV infections. Our findings on the dynamics of testicular immune cells and importance of CD8 + T cells functions as a framework to understand mechanisms underlying observed inflammation and sperm alterations in humans.

6-Nitrodopamine Is the Most Potent Endogenous Positive Inotropic Agent in the Isolated Rat Heart.

BACKGROUND: 6-nitrodopamine released from rat isolated atria exerts positive chronotropic action, being more potent than noradrenaline, adrenaline, and dopamine. Here, we determined whether 6-nitrodopamine is released from rat isolated ventricles (RIV) and modulates heart inotropism. METHODS: Catecholamines released from RIV were quantified by LC-MS/MS and their effects on heart inotropism were evaluated by measuring left ventricular developed pressure (LVDP) in Langendorff's preparation. RESULTS: 6-nitrodopamine was the major released catecholamine from RIV. Incubation with L-NAME (100 µM), but not with tetrodotoxin (1 µM), caused a significant reduction in 6-nitrodopamine basal release. 6-nitrodopamine release was significantly reduced in ventricles obtained from L-NAME chronically treated animals. 6-nitrodopamine (0.01 pmol) caused significant increases in LVDP and dP/dtmax, whereas dopamine and noradrenaline required 10 pmol, and adrenaline required 100 pmol, to induce similar increases in LVDP and dP/dtmax. The infusion of atenolol (10 nM) reduced basal LVDP and blocked the increases in LVDP induced by 6-ND (0.01 pmol), without affecting the increases in LVDP induced by 10 nmol of dopamine and noradrenaline and that induced by adrenaline (100 nmol). CONCLUSIONS: 6-nitrodopamine is the major catecholamine released from rat isolated ventricles. It is 1000 times more potent than dopamine and noradrenaline and is selectively blocked by atenolol, indicating that 6-ND is a main regulator of heart inotropism.

Zoonotic mosquito-borne arboviruses: Spillover, spillback, and realistic mitigation strategies.

Emerging zoonotic mosquito-borne viruses pose increasing health threats because of growing mosquito population, geographic expansions, and control challenges. We emphasize the need for global preparedness to effectively mitigate the health, societal, and economic impacts of spillover by these viruses through proactive measures of prediction, surveillance, prevention, and treatment.

Testicular pathological alterations associated with SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the etiologic agent of the coronavirus disease 2019 (COVID-19), which caused one of the pandemics with the highest mortalities with millions of deaths and hundreds of millions of cases to date. Due to its potential for airborne transmission, many studies have focused on SARS-CoV-2 primarily as a respiratory disease. However, the spread of SARS-CoV-2 to non-respiratory organs has been experimentally demonstrated and clinically observed. During autopsy studies, histopathological lesions, and disruption of the blood-testes barrier (BTB) have been observed in the male reproductive tract. Here, we review findings from both autopsy cases and animal models that demonstrate testicular disease due to COVID-19 and present an overview of the pathological alterations that occur in the testes resulting from SARS-CoV-2 infection and explore its potential mechanisms.

Measurement of 6-cyanodopamine, 6-nitrodopa, 6-nitrodopamine and 6-nitroadrenaline by LC-MS/MS in Krebs-Henseleit solution. Assessment of basal release from rabbit isolated right atrium and ventricles.

This study presents the validation of a sensitive method for the determination of 6-nitrodopa, 6-nitrodopamine, 6-nitroadrenaline and 6-cyanodopamine in Krebs-Henseleit solution by LC-MS/MS with ESI+ . HRMS was used to precisely characterize the structures of the fragment ions. The method was applied to investigate the catecholamine basal release from rabbit isolated atria and ventricles. The atria and ventricles were suspended separately in a 5 ml organ bath containing Krebs-Henseleit solution with ascorbic acid (3 mM), gassed (95%O2 /5%CO2 ) at 37°C for 30 min. Strata-X 33 µm SPE cartridges were employed for the extraction of the catecholamines and the internal standard 6-nitrodopamine-d4 . The catecholamines were separated employing a 150 × 3 mm Shim-pack GIST C18-AQ (3 mm particle size), placed in an oven at 40°C and perfused by 65% of mobile phase A (MeCN/H2 O; 90/10, v/v) + 0.4% CH3 COOH and 35% mobile phase B (deionized H2 O) + 0.2% CH2 O2 at 320 µl/min in isocratic mode. The method was linear at 0.1-20 ng/ml. The method was used to identify for the first-time basal release of the three nitrocatecholamines mentioned above and a member of a novel class of catecholamines, the cyanocatecholamines.

Rutin ameliorates nitrergic and endothelial dysfunction on vessels and corpora cavernosa of diabetic animals.

Endothelial dysfunction is an early complication of diabetes and it is related to both micro- and macroangiopathies. In addition, >70% of diabetic patients develop autonomic neuropathies. Increased oxidative stress has a major role in the development of both nitrergic and endothelial dysfunction. The aim of this work is to evaluate whether rutin, a potent antioxidant, could ameliorate nitrergic and/or endothelial dysfunction in diabetic animals. Primary and secondary treatment protocols with rutin were investigated on rat aortic rings and the mesenteric arteriolar bed, and on rabbit aortic rings and corpora cavernosa (RbCC) from both euglycemic and alloxan-diabetic animals. Acetylcholine endothelium-dependent and sodium nitroprusside endothelium-independent relaxations were compared in tissues from euglycemic or diabetic animals. Electrical field stimulation (EFS)-induced relaxation was performed only in the RbCC. Endothelial-dependent relaxations were blunted by 40% in vessels and neuronal relaxation was blunted by 50% in RbCC taken from diabetic animals when compared to euglycemic animals. Pre-treatment with rutin restored both neuronal and endothelial dependent relaxations in diabetic animals towards the values achieved in control euglycemic tissues. Rutin was able to ameliorate both endothelial dysfunction and nitrergic neuropathy in animal experimental models. Rutin could be a lead compound in the primary or secondary preventive ancillary treatment of endothelial and nitrergic dysfunction in the course of diabetes.

Dielectric Characterization of Fabric Aggregates around the 2.45 GHz ISM Band under Various Humidity, Density, and Temperature Conditions.

Fabric permittivity is critical for the manufacturing of wearable sensors and antennas as well as predicting how fabrics interact with electromagnetic fields. Engineers should also understand how permittivity changes under different temperatures, densities, and moisture content values, or when several fabrics are mixed in aggregates, when designing future applications such as microwave dryers. The permittivity of cotton, polyester, and polyamide fabric aggregates is investigated in this paper for a wide range of compositions, moisture content levels, density values, and temperature conditions around the 2.45 GHz ISM band using a bi-reentrant resonant cavity. The obtained results show extremely comparable responses for all characteristics investigated for single and binary fabric aggregates. Permittivity always increases as temperature, density, or moisture content levels rise. Moisture content is the most influential characteristic, causing enormous variations in the permittivity of aggregates. Fitting equations are supplied for all data, with exponential functions used to accurately model variation in temperature and polynomial functions employed to precisely model density and moisture content variations with low error levels. The temperature permittivity dependence of single fabrics without the influence of air gaps is also extracted from fabric and air aggregates by using complex refractive index equations for two-phase mixtures.

Permittivity measurements for raw and boiled quinoa seeds versus temperature, bulk density, and moisture content.

Quinoa is a nutrient-rich pseudocereal that is gaining popularity in European countries since it is gluten-free and an interesting source of fat, proteins, minerals, and amino acids. However, up to date, the electric permittivity of quinoa seeds has not been measured and, therefore, this hampers the possibility of designing optimized recipes for its microwave processing. In this work, the permittivity of both raw and boiled quinoa seeds is measured around 2.45 GHz at several conditions for temperature, moisture content, and bulk density. The grain kernel permittivity is also estimated from the Complex Refractive Index (CRI) mixture equation and different bulk density measurements. The obtained results show different temperature behaviours for raw and boiled seeds, whereas the permittivity of quinoa seeds versus moisture content and bulk density was as expected: the permittivity (both the dielectric constant and loss factor) levels increased as the observed variables did. From the measured data, it can be concluded that both raw and boiled quinoa can be processed with microwave technology, although care must be taken with raw quinoa grain kernels since the permittivity increases significantly with temperature, and a thermal runaway may occur.

6-Nitrodopamine potentiates contractions of rat isolated vas deferens induced by noradrenaline, adrenaline, dopamine and electric field stimulation.

6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that is released from the rat isolated vas deferens, and has been characterized as a major modulator of the contractility of rat isolated epididymal vas deferens (RIEVD). Drugs such as tricyclic antidepressants, α1 and ß1ß2 adrenoceptor blockers, act as selective antagonists of the 6-ND receptor in the RIEVD. In the rat isolated atria, 6-ND has a potent positive chronotropic action and causes remarkable potentiation of the positive chronotropic effects induced by dopamine, noradrenaline, and adrenaline. Here, whether 6-ND interacts with the classical catecholamines in the rat isolated vas deferens was investigated. Incubation with 6-ND (0.1 and 1 nM; 30min) caused no contractions in the RIEVD but provoked significant leftward shifts in the concentration-response curves to noradrenaline, adrenaline, and dopamine. Pre-incubation of the RIEVD with 6-ND (1 nM), potentiated the contractions induced by electric-field stimulation (EFS), whereas pre-incubation with 1 nM of dopamine, noradrenaline or adrenaline, did not affect EFS-induced contractions. In tetrodotoxin (1 µM) pre-treated (30 min) RIEVD, pre-incubation with 6-ND (0.1 nM) did not cause leftward shifts in the concentration-dependent contractions induced by noradrenaline, adrenaline, or dopamine. Pre-incubation of the RIEVD with the α2A-adrenoceptor antagonist idazoxan (30 min, 10 nM) did not affect dopamine, noradrenaline, adrenaline, and EFS-induced contractions. However, when idazoxan (10 nM) and 6-ND (0.1 nM) were simultaneously pre-incubated (30 min), a significant potentiation of the EFS-induced contractions of the RIEVD was observed. 6-nitrodopamine causes remarkable potentiation of dopamine, noradrenaline, and adrenaline contractions on the RIEVD, due to activation of adrenergic terminals, possibly via pre-synaptic adrenoceptors.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

Prevalence of anxiety and depression in parents of children with autism spectrum disorder during the first wave of the COVID-19 pandemic on Northeast Brazil / Prevalência de ansiedade e depressão em pais de crianças com transtorno do espectro autista durante a primeira onda da pandemia de COVID-19 no Nordeste do Brasil

ABSTRACT Objective: Estimate the prevalence of anxiety and depression symptoms in parents of children with autism spectrum disorder (ASD) during the first wave of the Coronavirus Disease (COVID-19) pandemic, comparing them with parents of neurotypical children and with other mental disorders. Methods: Responses from 211 participants were collected from an online form about familial behavior during the pandemic, and the Portuguese version of the HADS scale (Hospital Anxiety and Depression Scale). Results: Anxiety symptoms were present in 51% of the sample and depression was present in 35.1%. The prevalence of depression and anxiety symptoms was 58% and 44.4% respectively in the group of parents of children from the ASD; 50.3% e 32.2% for parents of neurotypical children; and 40% and 35% in the group of parents of children with other mental disorders. Conclusions: The prevalence of anxiety and depression in parents during the pandemic was greater than that of before the pandemic period. The prevalence of anxiety symptoms, as well as the mean scores of anxiety and depression symptoms, were significantly higher for the group of parents of children with the ASD. The results point to the need for additional care for parents and caregivers of children with ASD, since the social isolation adopted as a measure to contain the pandemic seemed to pose as a risk factor for negative psychological effects especially in this group.

RESUMO Objetivo: Estimar a prevalência de sintomas de ansiedade e depressão em pais de crianças com transtorno do espectro autista (TEA), durante a primeira onda da pandemia da doença causada pelo coronavírus SARS-CoV-2 (COVID-19), comparando com pais de crianças típicas e com outros transtornos mentais. Métodos: Foram coletadas respostas de 211 participantes a partir de um questionário on-line sobre comportamento familiar durante a pandemia e da versão em português da escala HADS (Hospital Anxiety and Depression Scale). Resultados: Sintomas de ansiedade estiveram presentes em 51% da amostra e de depressão, em 35,1%. A prevalência de sintomas de ansiedade e depressão foi de 58% e 44,4%, respectivamente, no grupo de pais de crianças com TEA, 50,3% e 32,2% no grupo de pais de crianças típicas e 40% e 35% no grupo de pais de crianças com outros transtornos mentais. Conclusões: As prevalências de ansiedade e depressão em pais durante a quarentena foram superiores àquelas fora do período pandêmico. As prevalências de sintomas de ansiedade, bem como as médias dos escores de sintomas de ansiedade e depressão, foram maiores no grupo de pais de crianças com TEA. Os resultados indicam a necessidade de atenção aos pais e cuidadores das crianças com TEA, já que o isolamento social adotado como medida de contenção da pandemia se constituiu como um fator de risco para impactos psicológicos negativos especialmente nesse grupo.

Quantification of cyclic AMP and cyclic GMP levels in Krebs-Henseleit solution by LC-MS/MS: Application in washed platelet aggregation samples.

Cyclic Nucleotides are important in regulating platelet function. Increases in 3'5'-cyclic adenosine monophosphate (cAMP) and 3'5'-cyclic guanosine monophosphate (cGMP) inhibit platelet aggregation and are pharmacological targets for antiplatelet therapy. Here we report an improved method for determining cAMP and cGMP concentrations and, for the first time, in washed platelet supernatants by combining high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). Characteristic peaks of the substrates, cGMP or cAMP and their internal standards were identified in negative-ion electrospray ionisation using multiple reaction monitoring. Compared with previously reported methods, the method presented here shows high precision with the lowest lower limit of quantification (LLoQ) to date (10 pg/mL). The effect of a novel catecholamine, 6-nitrodopamine, on cyclic nucleotide levels was quantified. Our results showed that this new method was fast, sensitive, and highly reproducible.

Release of 6-nitrodopamine modulates vascular reactivity of Pantherophis guttatus aortic rings.

6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels and Chelonoidis carbonaria aortic rings. The synthesis/release of 6-ND is inhibited by either pre-incubation of the vessels with the nitric oxide (NO) synthase inhibitor L-NAME or by mechanical removal of the endothelium. 6-ND causes powerful vasorelaxation, acting as a potent and selective dopamine D2-like receptor antagonist. Basal release of 6-ND from Panterophis guttatus endothelium intact and denuded aortic rings was quantified by LC-MS/MS. In order to evaluate the interaction of 6-ND with other catecholamines, aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. Endothelium intact aortic rings presented basal release of 6-ND, which was significantly reduced by previous incubation with L-NAME (100 µM). In endothelin-1 (3 nM) pre-contracted endothelium intact aortic rings, 6-ND (10pM-1 µM) and the dopamine D2-receptor antagonist L-761,626 (10 pM-1 µM) induced concentration-dependent relaxations, which were not affected by incubation with L-NAME but greatly reduced in endothelium-removed aortic rings. 6-ND (0.1-1 µM) produced significant rightward shifts of the concentration-response curves to dopamine in L-NAME pre-treated endothelium-intact (pA2 7.01) rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND (1 µM). The EFS-induced contractions of L-NAME pre-treated endothelium-intact aortic rings were significantly inhibited by incubation with 6-ND (1 µM). The results indicate that 6-ND released from Pantherophis guttatus aortic rings is coupled to NO release and represents a new mechanism by which NO can modulate vascular reactivity independently of cGMP production.

Vaccination Decreases the Infectious Viral Load of Delta Variant SARS-CoV-2 in Asymptomatic Patients.

The Delta variant of SARS-CoV-2 has caused many breakthrough infections in fully vaccinated individuals. While vaccine status did not generally impact the number of viral RNA genome copies in nasopharyngeal swabs of breakthrough patients, as measured by Ct values, it has been previously found to decrease the infectious viral load in symptomatic patients. We quantified the viral RNA, infectious virus, and anti-spike IgA in nasopharyngeal swabs collected from individuals asymptomatically infected with the Delta variant of SARS-CoV-2. Vaccination decreased the infectious viral load, but not the amount of viral RNA. Furthermore, vaccinees with asymptomatic infections had significantly higher levels of anti-spike IgA in their nasal secretions compared to unvaccinated individuals with asymptomatic infections. Thus, vaccination may decrease the transmission risk of Delta, and perhaps other variants, despite not affecting the amount of viral RNA measured in nasopharyngeal swabs.

Dynamic Permittivity Measurement of Ground-Tire Rubber (GTR) during Microwave-Assisted Devulcanization.

Many efforts are being made to find innovative ways of recycling rubber from end-of-life tires (ELTs), also called ground tire Rubber (GTR). Recycling through devulcanization allows the reintroduction of rubber back into the manufacturing industry. Such a process requires providing enough energy to break the sulfur links, while preventing damage to the polymeric chain. Microwave heating is controllable, efficient, and it does not rely on conventional heating mechanisms (conduction, convection) which may involve high heating losses, but rather on direct dielectric heating. However, to adequately control the microwave-assisted devulcanization performance, a thorough knowledge of the GTR permittivity versus temperature is required. In this work, GTR permittivity was monitored during its devulcanization. A resonant technique based on a dual-mode cylindrical cavity was used to simultaneously heat rubber and measure its permittivity at around 2 GHz. The results show sharp changes in the GTR permittivity at 160 and 190 °C. After the GTR cooled down, a shifted permittivity evidences a change in the GTR structure caused by the devulcanization process. Microwave-assisted devulcanization effectiveness is proven through time-domain nuclear magnetic resonance (NMR) measurements, by verifying the decrease in the cross-link density of processed GTR samples compared to the original sample.

Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.

6-nitrodopamine is a major endogenous modulator of human vas deferens contractility.

BACKGROUND: Rat isolated vas deferens releases 6-nitrodopamine (6-ND), and the spasmogenic activity of this novel catecholamine is significantly reduced by tricyclic compounds such as amitriptyline, desipramine, and carbamazepine and by antagonists of the α1 -adrenergic receptors such as doxazosin, tamsulosin, and prazosin. OBJECTIVES: To investigate the liberation of 6-ND by human epididymal vas deferens (HEVDs) and its pharmacological actions. METHODS: The in vitro liberation of 6-ND, dopamine, noradrenaline, and adrenaline from human vas deferens was evaluated by LC-MS/MS. The contractile effect of the catecholamines in HEVDs was investigated in vitro. The action of tricyclic antidepressants was evaluated on the spasmogenic activity ellicited by the catecholamines and by the electric-field stimulation (EFS). The tissue was also incubated with the inhibitor of nitric oxide (NO) synthase L-NAME and the release of catecholamines and the contractile response to EFS were assessed. RESULTS: 6-ND is the major catecholamine released from human vas deferens and its synthesis/release is inhibited by NO inhibition. The spasmogenic activity elicited by EFS in the human vas deferens was blocked by tricyclic antidepressants only at concentrations that selectively antagonize 6-ND induced contractions of the human vas deferens, without affecting the spasmogenic activity induced by dopamine, noradrenaline, and adrenaline in this tissue. Incubation of the vas deferens with L-NAME reduced both the 6-ND release and the contractions induced by EFS. DISCUSSION AND CONCLUSION: 6-ND should be considered a major endogenous modulator of human vas deferens contractility and possibly plays a pivotal role in the emission process of ejaculation. It offers a novel and shared mechanism of action for tricyclic antidepressants and α1 -adrenergic receptor antagonists.

6-NitroDopamine is an endogenous modulator of rat heart chronotropism.

6-Nitrodopamine (6-ND) is released by rat vas deferens and exerts a potent contractile response that is antagonized by tricyclic antidepressants and α1-, ß1- and ß1/ß2-adrenoceptor antagonists. The release of 6-ND, noradrenaline, adrenaline and dopamine from rat isolated right atria was assessed by tandem mass spectrometry. The effects of the catecholamines were evaluated in both rat isolated right atria and in anaesthetized rats. 6-ND was the major catecholamine released from the isolated atria and the release was significantly reduced in nitric oxide synthase inhibitor L-NAME pre-treated atria or in atria obtained from L-NAME chronically treated animals, but unaffected by tetrodotoxin. 6-ND (1 pM) significantly increased the atrial frequency, being 100 times more potent than noradrenaline and adrenaline. Selective ß1-blockers reduced the atrial frequency only at concentrations that prevented the increases in atrial frequency induced by 6-ND 1pM. Conversely, ß1-blockade did not affect dopamine (10 nM), noradrenaline (100 pM) or adrenaline (100 pM) effect. The reductions in atrial frequency induced by the ß1-adrenoceptor antagonists were absent in L-NAME pre-treated atria and in atria obtained from chronic L-NAME-treated animals. Tetrodotoxin did not prevent the reduction in atrial frequency induced by L-NAME or by ß1-blockers treated preparations. In anaesthetized rats, at 1 pmol/kg, only 6-ND caused a significant increase in heart rate. Inhibition of 6-ND synthesis by chronic L-NAME treatment reduced both atrial frequency and heart rate. The results indicate that 6-ND is a major modulator of rat heart chronotropism and the reduction in heart rate caused by ß1-blockers are due to selective blockade of 6-ND receptor.

6-Nitrodopamine is an endogenous selective dopamine receptor antagonist in Chelonoidis carbonaria aorta.

Chelonoidis carbonaria aortic rings present endothelium-derived release of dopamine, noradrenaline, adrenaline and 6-nitrodopamine (6-ND). Here it was investigated whether 6-ND release is coupled to nitric oxide (NO) synthesis and its action on the vascular smooth muscle reactivity. Basal release of 6-ND from aortic rings in the absence and presence of the NO synthesis inhibitor L-NAME was quantified by LC-MS-MS. Aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. The tissues were allowed to equilibrate for 1 h before starting the experiments. The release of 6-ND was significantly reduced by previous incubation with L-NAME. 6-ND (up to 300 µM) had no contractile activity in the aortic rings. 6-ND (1, 3 and 10 µM) produced significant rightward shifts of the concentration-response curves to dopamine in endothelium-intact (pA2 6.09) and L-NAME pre-treated endothelium-intact (pA2 7.06) aortic rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND. The EFS (16 Hz)-induced aortic contractions were significantly inhibited by incubation with 6-ND (10 µM). In the thromboxane A2 mimetic U-46619 (30 nM) pre-contracted endothelium intact aortic rings, 6-ND (1 nM-1 µM) and the dopamine D2-receptor antagonist haloperidol (1 nM-1 µM) induced concentration-dependent relaxations. The relaxations were not present in endothelium-removed aortic rings but they were not affected by incubation with L-NAME in endothelium-intact aortic rings. The results indicate that the synthesis of this novel catecholamine in Chelonoidis carbonaria aortic rings is coupled to NO release and that 6-ND acts as a highly selective dopamine D2-like receptor antagonist.