The negative chronotropic effects of (±)-propranolol and (±)-4-NO2-propranolol in the rat isolated right atrium are due to blockade of the 6-nitrodopamine receptor.

The positive chronotropic action induced by 6-nitrodopamine (6-ND) is selectively blocked by ß1-adrenoceptor antagonists at concentrations that do not affect the positive chronotropic effect induced by dopamine, noradrenaline, and adrenaline. Here, the effects of ( ±)-propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranolol were investigated in the rat isolated right atrium. The atrium was mounted in glass chambers containing gassed (95%O2:5%CO2) and warmed (37 °C) Krebs-Henseleit's solution, and the isometric tension registered (PowerLab system). ( ±)-Propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranolol caused concentration-dependent falls in the spontaneous atrial frequency (pIC50: 4.80 ± 0.10, 4.64 ± 0.10, and 4.95 ± 0.10, respectively). The calculated pA2 values for ( ±)-propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranol on noradrenaline-induced positive chronotropism were 8.44 ± 0.08, 6.41 ± 0.07, and 9.21 ± 0.29, respectively. The positive chronotropism induced by 6-ND (10 pM) was blocked by ( ±)-propranolol (1 µM) and ( ±)-4-NO2-propranolol (30 nM), whereas ( ±)-7-NO2-propranolol (1 µM) had no effect on 6-ND-induced responses. The pIC50 of ( ±)-propranolol, ( ±)-4-NO2-propranolol, and ( ±)-7-NO2-propranolol were significantly shifted to the right in L-NAME-treated atria. The discrepancy between pA2 values of ( ±)-propranolol and its respective pIC50 indicates that the falls in atrial rate induced by ( ±)-propranolol should not be attributed to b-adrenergic antagonism. The reduced chronotropism by ( ±)-propranolol was unaffected by the sodium channel inhibitors tetrodotoxin and lidocaine but that was abolished in atria pre-treated with ( ±)-4-NO2-propranolol. The finding that ( ±)-propranolol reduces spontaneous atrial rate only in concentrations that affect 6-ND-induced positive chronotropism confirms the role of this catecholamine as an endogenous modulator of heart chronotropism. ( ±)-4-NO2-Propranolol behaves as a selective antagonist of 6-ND in the rat isolated atrium.

Basal release and relaxation responses to 6-nitrodopamine in swine carotid, coronary, femoral, and renal arteries.

Mammalian and reptilian vascular tissues present basal release of 6-nitrodopamine, which is reduced when the tissues are pre-incubated with the NO synthase inhibitor L-NG-Nitro arginine methyl ester (L-NAME), or when the endothelium is mechanically removed. 6-Nitrodopamine induces vasorelaxation in pre-contracted vascular rings by antagonizing the dopaminergic D2-like receptor. Here it was investigated whether male swine vessels (including carotid, left descendent coronary, renal, and femoral arteries) release 6-nitrodopamine, dopamine, noradrenaline, and adrenaline, as measured by liquid chromatography coupled to tandem mass spectrometry. The in vitro vasorelaxant action of 6-nitrodopamine was evaluated in carotid, coronary, renal, and femoral arteries precontracted by U-46619 (3 nM), and compared to that induced by the dopamine D2-receptor antagonist L-741,626. Expression of tyrosine hydroxylase and the neuromaker calretinin was investigated by immunohistochemistry. All vascular tissues presented basal release of endothelium-derived catecholamines. The relaxation induced by 6-nitrodopamine was not affected by preincubation of the tissues with either L-NAME (100 µM, 30-min preincubation) or the heme-site inhibitor of soluble guanylyl cyclase ODQ (100 µM, 30-min preincubation). Electrical field stimulation (EFS)-induced contractions were significantly potentiated by previous incubation with L-NAME, but unaffected by ODQ preincubation. The contractions induced by EFS were reduced by preincubation with either 6-nitrodopamine or L-741,626. Immunohistochemistry in all arteries revealed the presence of tyrosine hydroxylase in the endothelium, whereas immunoreactivity for calretinin was negative. Swine vessels present basal release of endothelium-derived catecholamines and expression of tyrosine hydroxylase in the endothelium. The vasodilation induced by 6-nitrodopamine is due to blockade of dopaminergic D2-like receptors.

Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility.

6-Cyanodopamine is a novel catecholamine released from rabbit isolated heart. However, it is not known whether this catecholamine presents any biological activity. Here, it was evaluated whether 6-cyanodopamine (6-CYD) is released from rat vas deferens and its effect on this tissue contractility. Basal release of 6-CYD, 6-nitrodopamine (6-ND), 6-bromodopamine, 6-nitrodopa, and 6-nitroadrenaline from vas deferens were quantified by LC-MS/MS. Electric-field stimulation (EFS) and concentration-response curves to noradrenaline, adrenaline, and dopamine of the rat isolated epididymal vas deferens (RIEVD) were performed in the absence and presence of 6-CYD and /or 6-ND. Expression of tyrosine hydroxylase was assessed by immunohistochemistry. The rat isolated vas deferens released significant amounts of both 6-CYD and 6-ND. The voltage-gated sodium channel blocker tetrodotoxin had no effect on the release of 6-CYD, but it virtually abolished 6-ND release. 6-CYD alone exhibited a negligible RIEVD contractile activity; however, at 10 nM, 6-CYD significantly potentiated the noradrenaline- and EFS-induced RIEVD contractions, whereas at 10 and 100 nM, it also significantly potentiated the adrenaline- and dopamine-induced contractions. The potentiation of noradrenaline- and adrenaline-induced contractions by 6-CYD was unaffected by tetrodotoxin. Co-incubation of 6-CYD (100 pM) with 6-ND (10 pM) caused a significant leftward shift and increased the maximal contractile responses to noradrenaline, even in the presence of tetrodotoxin. Immunohistochemistry revealed the presence of tyrosine hydroxylase in both epithelial cell cytoplasm of the mucosae and nerve fibers of RIEVD. The identification of epithelium-derived 6-CYD and its remarkable synergism with catecholamines indicate that epithelial cells may regulate vas deferens smooth muscle contractility.

GKT137831 and hydrogen peroxide increase the release of 6-nitrodopamine from the human umbilical artery, rat-isolated right atrium, and rat-isolated vas deferens.

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 (30 min) 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 pre-incubation with catalase (1,000 U/mL) significantly decreased it. Pre-incubation of the HUA with superoxide dismutase (SOD) (250 U/mL; 30 min) also significantly increased the basal release of 6-ND. Preincubation of the HUA with either allopurinol (100 µM) or uric acid (1 mM) had no effect on the basal release of 6-ND. Pre-treatment of the HUA with L-NAME (100 µM) prevented the increase in the basal release of 6-ND induced by GKT137831, diphenyleneiodonium, and H2O2. The results obtained indicate a major role of endogenous H2O2 and peroxidases as modulators of 6- ND biosynthesis/release and a lack of peroxynitrite contribution.

6-Nitrodopamine is an endogenous mediator of the rabbit corpus cavernosum relaxation.

BACKGROUND: 6-Nitrodopamine (6-ND) is a novel endogenous catecholamine that has a potent relaxant action on vascular smooth muscle in vitro. OBJECTIVES: To evaluate the basal release of 6-ND and noradrenaline from rabbit-isolated corpus cavernosum (RbCC) and its relaxing action on this tissue. METHODS: Rabbit corpus cavernosa were dissected and suspended in a 5-mL organ bath containing oxygenated Krebs-Henseleit's solution. 6-ND and noradrenaline release was quantified by liquid chromatography coupled to tandem mass spectrometry. The relaxant activity of 6-ND was assessed in RbCC strips pre-contracted with endothelin-1 (10 nM). RESULTS: Rabbit corpus cavernosum presented basal release of both 6-ND (2.9 ± 0.8 ng/mL, n = 12) and noradrenaline (1.7 ± 1.3 ng/mL, n = 12). The 6-ND release was reduced by pre-treatment with Nω-nitro-l-arginine methyl ester (l-NAME) (100 µM), whereas that of noradrenaline was unaffected. Tetrodotoxin (TTX, 1 µM) abolished the noradrenaline release but had no effect on 6-ND release, indicating a non-neurogenic origin for 6-ND. 6-ND and the selective dopamine D2-agonist L-741,626 caused concentration-dependent RbCC relaxations (pEC50 of 11 ± 0.15 and 11.15 ± 0.28, respectively). Pre-treatment with either l-NAME or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-on (ODQ) (100 µM) caused a rightward shift of the concentration-response curve to 6-ND, without affecting the L-741,626 responses. In TTX (100 nM)-pre-treated preparations, neither l-NAME nor ODQ shifted the 6-ND concentration-response curve. Dopamine, noradrenaline, and adrenaline caused concentration-dependent RbCC contractions. Pre-incubation with 6-ND concentration-dependently inhibited the dopamine-induced contractions, without affecting those induced by either noradrenaline or adrenaline. DISCUSSION AND CONCLUSION: 6-Nitrodopamine is the most potent endogenous relaxant agent in RbCC ever described and represents a novel mechanism by which NO causes corpus cavernosum smooth muscle relaxation. The finding that 6-ND acts as a truly selective dopamine D2-receptor antagonist indicates that the balance of dopamine and 6-ND release/synthesis may be the main mechanism that modulates corpus cavernosum smooth muscle tonus in vivo.

Alpha1-adrenergic blockers selectively antagonize the contractions induced by 6-nitrodopamine in the human vas deferens.

6-Nitrodopamine (6-ND) is released from human vas deferens and plays a modulatory role in the male ejaculation. Therapeutical use of α1-adrenoceptor antagonists is associated with ejaculatory abnormalities. To evaluate the effect of α1-adrenoceptor antagonists on the contractions induced by 6-ND, dopamine, noradrenaline, and adrenaline in the human epididymal vas deferens (HEVD). HEVD strips were suspended in glass chambers containing heated and oxygenated Krebs-Henseleit's solution. Cumulative concentration-response curves to catecholamines (10 nM-300 µM) were constructed in HEVD strips pre-incubated (30 min) with doxazosin (0.1-1 nM), tamsulosin (1-10 nM), prazosin (10-100 nM) and/or silodosin (0.1-10 nM). The effects of these α1-adrenoceptor antagonists were also evaluated in the electric-field stimulation (EFS, 2-32 Hz)-induced contractions. Doxazosin (0.1 nM) caused significant reductions in 6-ND-induced HEVD contractions without affecting the contractions induced by dopamine, noradrenaline, and adrenaline. Similar results were observed with tamsulosin (1 nM) and prazosin (10 nM). At these concentrations, these α1-adrenoceptor antagonists largely reduced the EFS-induced contractions. Silodosin (1 nM) caused concentration-dependent rightward shifts of the concentration-response curves to 6-ND but had no effect on the contractions induced by dopamine and adrenaline. Silodosin (0.1 nM) only inhibited the contractions induced by noradrenaline. Silodosin at 1 nM, but not at 0.1 nM, caused significant reductions in the EFS-induced contractions. The results reinforce the concept that 6-ND plays a major role in the human vas deferens contractility and indicate that the ejaculation disorders caused by doxazosin, tamsulosin, prazosin and silodosin cause in man, may be due to inhibition of the contractions induced by 6-ND rather than by the classical catecholamines dopamine, noradrenaline, and adrenaline.

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.

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.

Release of 6-nitrodopamine from human popliteal artery and vein.

6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels, and it causes vascular relaxation by acting as a dopamine D2-receptor antagonist. Here it was investigated whether human peripheral vessels obtained from patients who have undergone surgery for leg amputation release 6-ND, and its action in these tissues. Popliteal artery and vein strips present basal release of 6-ND, as measured by liquid chromatography coupled to tandem mass spectrometry. The release was significantly reduced when the tissues were pre-treated with the nitric oxide synthase inhibitor L-NAME (100 µM), or when the endothelium was mechanically removed. In U-46619 (3 nM) pre-contracted rings, 6-ND induced concentration-dependent relaxations (pEC50 8.18 ± 0.05 and 8.40 ± 0.08, in artery and vein rings, respectively). The concentration-dependent relaxations induced by 6-ND were unaffected in tissues pre-treated with L-NAME, but significantly reduced in tissues where the endothelium has been mechanically removed. In U-46619 (3 nM) pre-contracted rings, the selective dopamine D2 receptor antagonist L-741,626 also caused concentration-dependent relaxations (pEC50 8.92 ± 0.22 and 8.79 ± 0.19, in artery and vein rings, respectively). The concentration-dependent relaxations induced by L-741,626 were unaffected in tissues pre-treated with L-NAME, but significantly reduced in tissues where the endothelium has been mechanically removed. This is the first demonstration that 6-nitrodopamine is released from human peripheral artery and vein rings. The results also indicate that endothelium-derived dopamine is a major contractile agent in the popliteal artery and vein, and that selective dopamine D2-receptor antagonists such as 6-ND, may have therapeutic potential in the treatment of human peripheral vascular diseases.

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.

Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria.

6-Nitrodopamine (6-ND) is released from rat isolated atria being 100 times more potent than noradrenaline and adrenaline, and 10,000 times more potent than dopamine as a positive chronotropic agent. The present study aimed to investigate the interactions of 6-ND with the classical catecholamines, phosphodiesterase (PDE)-3 and PDE4, and the protein kinase A in rat isolated atria. Atrial incubation with 1 pM of dopamine, noradrenaline, or adrenaline had no effect on atrial frequency. Similar results were observed when the atria were incubated with 0.01 pM of 6-ND. However, co-incubation of 6-ND (0.01 pM) with dopamine, noradrenaline, or adrenaline (1 pM each) resulted in significant increases in atrial rate, which persisted over 30 min after washout of the agonists. The increased atrial frequency induced by co-incubation of 6-ND with the catecholamines was significantly reduced by the voltage-gated sodium channel blocker tetrodotoxin (1 µM, 30 min), indicating that the positive chronotropic effect of 6-ND is due in part to activation of nerve terminals. Pre-treatment of the animals with reserpine had no effect on the positive chronotropic effect induced by dopamine, noradrenaline, or adrenaline; however, reserpine markedly reduced the 6-ND (1 pM)-induced positive chronotropic effect. Incubation of the rat isolated atria with the protein kinase A inhibitor H-89 (1 µM, 30 min) abolished the increased atrial frequency induced by dopamine, noradrenaline, and adrenaline, but only attenuated the increases induced by 6-ND. 6-ND induces catecholamine release from adrenergic terminals and increases atrial frequency independently of PKA activation.

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.

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.

ß1- and ß1/ß2-adrenergic receptor antagonists block 6-nitrodopamine-induced contractions of the rat isolated epididymal vas deferens.

6-Nitrodopamine (6-ND) is an endogenous modulator of the contractility in the rat isolated epididymal vas deferens (RIEVD) and considered to be the main peripheral mediator of the emission process. Use of selective and unselective ß-adrenergic receptor antagonists has been associated with ejaculatory failure. Here, the effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists on RIEVD contractions induced by 6-ND, dopamine, noradrenaline, adrenaline, and electric-field stimulation (EFS) were investigated. The selective ß1-adrenergic receptor antagonists atenolol (0.1 and 1 µï»¿M), betaxolol (1 µï»¿M), and metoprolol (1 µï»¿M) and the unselective ß1/ß2-adrenergic receptor antagonists propranolol (1 and 10 µï»¿M) and pindolol (10 µï»¿M) caused significant rightward shifts of the concentration-response curve to 6-ND (pA2 6.41, 6.91, 6.75, 6.47, and 5.74; for atenolol, betaxolol, metoprolol, propranolol, and pindolol), but had no effect on dopamine-, noradrenaline-, and adrenaline-induced contractions. The effects of selective ß1- and ß1/ß2-adrenergic receptor antagonists at a higher concentration (atenolol 1 µï»¿M, betaxolol 1 µï»¿M, metoprolol 1 µï»¿M, propranolol 10 µï»¿M, and pindolol 10 µï»¿M) also reduced the EFS-induced RIEVD contractions in control, but not in RIEVD obtained from L-NAME-treated animals. The selective ß1-adrenoceptor agonist RO-363, the selective ß2-adrenoceptor agonist salbutamol, and the selective ß3-adrenoceptor agonist mirabegron, up to 300 µï»¿M, had no effect on the RIEVD tone. The results demonstrate that ß1- and ß1-/ß2-adrenoceptor receptor antagonists act as 6-ND receptor antagonists in RIEVD, further confirming the main role of 6-ND in the RIEVD contractility.

Quantification of 6-nitrodopamine in Krebs-Henseleit's solution by LC-MS/MS for the assessment of its basal release from Chelonoidis carbonaria aortae in vitro.

In this study, the development and validation of a method for quantification of 6-nitrodopamine in Krebs-Henseleit's solution by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with positive ion electrospray ionization is described. Aortic rings taken from tortoise were either denuded or left with endothelium intact (15 mm, N = 6) and were incubated for 30 min in 5 mL Krebs-Henseleit's solution in an organ bath. Solid phase extraction (SPE) was performed for aliquots of 1 mL of the supernatant. The separation of 6-nitrodopamine was obtained on a 150 mm × 3 mm Shim-pack GIST-HP C18 column, using 75% of mobile phase A consisted of deionized water with 0.1% formic acid (v/v) and 25% of mobile phase B consisted of acetonitrile/deionized water (50/50, v/v) + 0.1% formic acid at a flow rate of 350 µL/min in an isocratic mode. The method was linear over the concentration range of 0.1-20 ng/mL. The method was sensitive, precise and accurate for the assessment of the basal release of 6-nitrodopamine from Chelonoidis carbonaria aortae in vitro. The mean ± SEM concentrations of 6-nitrodopamine released from endothelium-intact and endothelium-denuded aortae were 0.44 ± 0.06 ng/mL and 0.18 ± 0.05 ng/mL, respectively. These results indicate that tortoise's aortae display a basal endothelium-derived 6-nitrodopamine release.

Determination of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by liquid chromatography coupled with tandem mass spectrometry and measurement of their basal release from Chelonoidis carbonaria aortae in vitro.

This study presented for the first time the development and validation of a sensitive method for quantification of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by LC-tandem mass spectrometry. Aliquots of 2.0 mL calibrators, quality controls, and samples of Krebs-Henseleit solution incubated with tortoise's aortic ring for 30 min were extracted by solid-phase extraction. Catecholamine separation was achieved on a 100 × 4.6 mm LiChrospher RP-8 column and the quantification was performed by a mass spectrometer equipped with an electrospray interface operating in positive ion mode. The run time was 4 min and the calibration curve was linear over the range of 0.1-20.0 ng/mL. The method was applied to the measurement of basal release of dopamine, noradrenaline, and adrenaline from the tortoise Chelonoidis carbonaria aortae in vitro. One aortic ring (30 mm) per tortoise (n = 5) was incubated for 30 min in a 5 mL organ bath filled with Krebs-Henseleit solution. The method demonstrated sensitivity, precision, and accuracy enough for its application in the measurement of basal release of these catecholamines from C. carbonaria aortic rings in vitro. The mean (standard deviation) concentrations of dopamine, noradrenaline, and adrenaline were 3.48 (2.55) ng/mL, 1.40 (0.57) ng/mL, and 1.87 (1.09) ng/mL, respectively.

The basal release of endothelium-derived catecholamines regulates the contractions of Chelonoidis carbonaria aorta caused by electrical-field stimulation.

The contractions of Chelonoidis carbonaria aortic rings induced by electrical field stimulation (EFS) are not inhibited by blockade of the voltage-gated sodium channels by tetrodotoxin but almost abolished by the α1/α2-adrenoceptor antagonist phentolamine. The objective of this study was to identify the mediator(s) responsible for the EFS-induced contractions of Chelonoidis carbonaria aortic rings. Each ring was suspended between two wire hooks and mounted in isolated 10 ml organ chambers filled with oxygenated and heated Krebs-Henseleit's solution. Dopamine, noradrenaline and adrenaline concentrations were analysed by liquid chromatography coupled to tandem mass spectrometry. The contractions caused by dopamine and EFS were done in absence and presence of the nitric oxide (NO) synthesis inhibitor L-NAME, the NO-sensitive guanylyl cyclase inhibitor ODQ, the D1-like receptor antagonist SCH-23390, the D2-like receptor antagonists risperidone, quetiapine, haloperidol, and the tyrosine hydroxylase inhibitors salsolinol and 3-iodo-L-tyrosine. Basal concentrations of dopamine, noradrenaline and adrenaline were detected in Krebs-Henseleit solution containing the aortic rings. The catecholamine concentrations were significantly reduced in endothelium-denuded aortic rings. L-NAME and ODQ significantly potentiated the dopamine-induced contractions. The D2-like receptor antagonists inhibited the EFS-induced contractions of the aortic rings treated with L-NAME, whereas SCH 23390 had no effect. Similar results were observed in the contractions induced by dopamine in L-NAME treated aortic rings. These results indicate that catecholamines released by endothelium regulate the EFS-induced contractions. This may constitute a suitable mechanism by which reptilia modulate specific organ blood flow distribution.This paper has an associated First Person interview with the first author of the article.

Endothelium-derived dopamine modulates EFS-induced contractions of human umbilical vessels.

Electrical field stimulation (EFS) induces contractions of both snake aorta and human umbilical cord vessels (HUCV) which were dependent on the presence of the endothelium. This study aimed to establish the nature of the mediator(s) responsible for EFS-induced contractions in HUCV. Rings with or without endothelium from human umbilical artery (HUA) or vein (HUV) were mounted in organ bath chambers containing oxygenated, heated Krebs-Henseleit's solution. Basal release of dopamine (DA), noradrenaline, and adrenaline was measured by LC-MS-MS. Cumulative concentration-response curves were performed with dopamine in the absence and in the presence of L-NAME or of dopamine antagonists. EFS studies were performed in the presence and absence of L-NAME, the α-adrenergic blockers prazosin and idazoxan, and the dopamine antagonists SCH-23390 and haloperidol. Tyrosine hydroxylase (TH) and dopa-decarboxylase (DDC) were studied by immunohistochemistry and fluorescence in situ hybridizations. Basal release of dopamine requires an intact endothelium in both HUA and HUV. TH and DDC are present only in the endothelium of both HUA and HUV as determined by immunohistochemistry. Dopamine induced contractions in HUA only in the presence of L-NAME. Dopamine-induced contractions in HUV were strongly potentiated by L-NAME. The EFS-induced contractions in both HUA and HUV were potentiated by L-NAME and inhibited by the D2-like receptor antagonist haloperidol. The α-adrenergic antagonists prazosin and idazoxan and the D1-like receptor antagonist SCH-23390 had no effect on the EFS-induced contractions of HUA and HUV. Endothelium-derived dopamine is a major modulator of HUCV reactivity in vitro.

Antioxidant and Vasodilatory Action of Grape Juices Produced in Different Regions of Brazil

Grapes and its derivatives (wines and juices) are rich in polyphenols that have high antioxidant and vasodilator capacity. These biological activities may vary in the juices marketed and produced in different regions of Brazil. Objectives: To determine the antioxidant and vasorelaxant effects of grape juice samples produced in different regions of Brazil. Methods: The content of phenolic compounds and antioxidant capacity were evaluated by the methods of Folin-Ciocalteau, DPPH, ABTS and a new electroanalytical approach (differential pulse voltammetry - DPV). Vasodilator effects were analyzed in isolated aorta from rats in an organ bath. Results: The samples from RJ and SP presented respectively the higher and lower phenolic content and also antioxidant capacity by the methods used (ABTS and DPPH). The results of the electrochemical index corroborate to the other tests, with the best results to RJ (21.69 ± 3.15 µA/V) and worse to the SP sample (11.30 ± 0.52 µA/V). In the vascular reactivity studies, the relaxation induced by each sample presented more distinct differences, following the order: RJ (87.9 ± 4.8%) > RS1 (71.6 ± 8.6%) > GO (56.2 ± 7.2%) > SP (39.9 ± 7.8%) > PR (39.4 ± 9.5%) > RS2 (19.5 ± 6.2%). Inhibition of endothelial NO practically abolished (p < 0.001) the relaxation for all samples, except one. Conclusion: The phenolic content and antioxidant capacity vary greatly among samples. The results obtained for the order of antioxidant activity were: RJ > RS1 > GO > RS2 > PR > SP. The juices were able to induce vascular relaxation at quite varied levels, and the RJ sample the most effective. The L-NAME practically blocked all samples except one (RS2)