A LC-MS/MS method for the simultaneous determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma and its clinical application in patients with chronic kidney disease.

The aim of this study was to develop a high-performance liquid chromatography-tandem mass spectrometry method for the determination of 6-cyanodopamine, 6-nitrodopamine, 6-nitrodopa, 6-nitroadrenaline and 6-bromodopamine in human plasma samples. Strata-X 33 µm solid-phase extraction cartridges were used for the extraction of the catecholamines from human plasma samples. The catecholamines were separated in a 150 × 3 mm Shim-pack GIST C18-AQ column with 3 µm particle size, placed in an oven at 40°C and perfused with 82% mobile phase A (acetonitrile-H2O; 90:10, v/v) + 0.4% acetic acid and 18% mobile phase B (deionized H2O) + 0.2% formic acid at a flow rate of 340 µl/min in isocratic mode. The injected volume was 4 µl and the run lasted 4 min. The method was linear from 0.1 to 20 ng/ml and the lower limit of quantification was 0.1 ng/ml for all analytes. The method was applied to evaluate the plasma levels of catecholamines in plasma of patients with chronic kidney disease and allowed the detection for the first time of circulating levels of the novel catecholamines 6-bromodopamine and 6-cyanodopamine.

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.

5-S-cysteinyl-dopamine, a neurotoxic endogenous metabolite of dopamine: Implications for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and is characterized for being an idiopathic and multifactorial disease. Extensive research has been conducted to explain the origin of the disease, but it still remains elusive. It is well known that dopamine oxidation, through the endogenous formation of toxic metabolites, is a key process in the activation of a cascade of molecular events that leads to cellular death in the hallmark of PD. Thio-catecholamines, such as 5-S-cysteinyl-dopamine, 5-S-glutathionyl-dopamine and derived benzothiazines, are endogenous metabolites formed in the dopamine oxidative degradation pathway. Those metabolites have been shown to be highly toxic to neurons in the substantia nigra pars compacta, activating molecular mechanisms that ultimately lead to neuronal death. In this review we describe the origin, formation and the toxic effects of 5-S-cysteinyl-dopamine and its oxidative derivatives that cause death to dopaminergic neurons. Furthermore, we correlate the formation of those metabolites with the neurodegeneration progress in PD. In addition, we present the reported neuroprotective strategies of products that protect against the cellular damage of those thio-catecholamines. Finally, we discuss the advantages in the use of 5-S-cysteinyl-dopamine as a potential biomarker for PD.

Extracellular activity of NBAD-synthase is responsible for colouration of brown spots in Ceratitis capitata wings.

After the emergence of the Ceratitis capitata imago, the pale and folded wings are expanded and sclerotized to acquire the definitive form and to stabilize the cuticle. The wings of this fly show a specific pattern of brownish and black spots. Black spots are pigmented by melanin, whereas there was scarce information about the development of the brownish spots. N-beta-alanydopamine (NBAD) is the main tanning precursor in C. capitata body cuticle, and we hypothesized that it may be responsible for the colouration of the brownish spots. We determined the topology and timing of NBAD synthesis and deposition to attain the species-specific colouration pattern. We demonstrated that during the first hours the colour of the brownish spots was principally determined by the tanning of the hairs. Haemolymph circulation through the veins is required to tan the wings. We confirmed that soon after wing spreading, most of the wing epidermal cells disappeared. Thus, the tanning of the brown spots was accomplished when the wing lamina was devoid of cells. NBAD synthase (NBAD-S; Ebony protein in D. melanogaster) activity in wings was detected in pharate adults and lasted several days after the emergence, even after the end of the tanning process. This observation is in contrast to epidermal NBAD-S activity in the body, where it was nearly undetectable 48 h post emergence. Our results indicate that NBAD-S was exported and deposited into the extracellular matrix of the brown spot areas before cell death and that tanning occurs through gradual export of NBAD precursors (dopamine and b-alanine) from veins.

Antagonistic effect of dopamine structural analogues on human GABAρ1 receptor.

GABAergic and dopaminergic pathways are co-localized in several areas of the central nervous system and recently several reports have shown co-release of both neurotransmitters. The GABA-A receptor (ß and ρ1 subunits) is modulated by dopamine (DA) and, interestingly, GABAρ1 can be modulated by several biogenic amines. Here we explored the effects of the metabolites of the dopaminergic pathway and other structural analogues of DA on GABAρ1 and the DA gated ion channel (LGC-53) from Caenorhabditis elegans expressed in Xenopus laevis oocytes. Our findings show an antagonistic effect of the metabolite 3-Methoxytyramine (3-MT, IC50 = 285 ± 30 µM) with similar potency compared to DA on induced GABA currents; however, it was inactive on LGC-53. The structural DA analogues and metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 2-phenylethylamine (ß-PEA) and 4-amino-1-butanol (4-AM-1-OH), antagonized GABAρ1 currents, whereas ß-PEA acted as partial agonists on LGC-53, indicating that the putative binding sites of both receptors may share structural characteristics. These results suggest that the DA metabolites 3-MT, DOPAC and HVA modulate GABAρ1 and possibly affect the activity of the receptors that include this subunit in vivo.

Modulação imunológica da relação mãe e filhote / Immunological modulation of the mother-pup interaction

O comportamento maternal (CM) em mamíferos tem características específicas. O período logo após o parto é particularmente sensível a alterações fisiológicas que podem modular a expressão deste comportamento importante. Mudanças comportamentais observadas em animais doentes são consideradas comportamento doentio (CD). A exposição ao LPS, uma endotoxina derivada da parede de uma bactéria gran negativa, durante a gravidez pode causar doenças mentais. A fim de investigar, uma possível relação entre CM e CD, os animais foram tratados com LPS. Para o estudo do CM e agressivo, quarenta ratas foram divididos em quatro grupos, dois controles e dois grupos experimentais, com dez animais cada. O grupo experimental recebeu 100µg/kg de LPS por via i.p, e grupo controle o veículo de endotoxina, após quarenta e oito horas de administração de LPS, ou seja, no quinto dia de lactação, as observações começaram. Para escolha deste dia, ratas virgens e ratas lactantes foram divididas em quatro grupos, dois controles e dois experimentais, com dez fêmeas cada. O peso corporal, consumo de água, ração, e a temperatura corporal foram medidas para cento e vinte horas. As fêmeas do grupo controle foram observadas da mesma forma, mas foram tratados com o veículo do LPS. Observamos que: 1) Em ratas virgens e lactantes o tratamento com LPS modificou a temperatura e peso corporal, consumo de água e ração; 2) No período de lactação houve redução da latência para busca do primeiro filhote. Na prole verificou-se que: 3) Houve alteração no padrão de vocalização dos filhotes cujas mães foram expostas ao LPS no terceiro dia de lactação; 4) houve alteração no burst e fagocitose de enutrofilos no vigésimo primeiro dia de lactação após desafio com a endotoxina indicativo de maior resposta ao LPS. Concluiu-se que a exposição de ratos ao LPS facilita o comportamento maternal, mas promove alterações na sua prole relacionadas à interação entre mãe-filhote e aumento na resposta a um desafio imunológico

Maternal behavior (MB) in mammals has specific characteristics. The time period just after parturition is particularly sensitive to physiological changes that can modulate the expression of this important behavior. Behavioral changes observed in sick animals, are considered as sick behavior (SB). Exposure to LPS, an endotoxin derived from the wall of a gran negative bacteria, during pregnancy might cause mental diseases. In order to investigate, a possible relationship between MB and SB, animals were treated with LPS. For the study of MB and maternal aggressive behavior, 40 rats were divided in 4 groups, 2 control and 2 experimental groups. The experimental group received 100µg/kg LPS by ip, and control group the vehicle of endotoxin, after 48 hours of LPS administration the observations of SB began. For choice these days, 20 virgin and 20 lactating rats were divided in 4 groups, 2 control and 2 experimental. They received ip 100µg/kg. Body weight, water and feed consumption, and body temperature were measured for 120h. Control females were observed in the same way, but they were treated with vehicle of LPS. The results showed that: 1) In 48 hours after the LPS treatment, virgin and lactating rats showed increased body temperature, loss of body weight, increased water consumption and decreased food consumption, 2) In 48 hours after the treatment with LPS, lactating rats showed reduced latency to retrieve the first pup to the nest. In the offspring of mothers treated with LPS it was found that: 3) Pups form mothers treated with LPS on the 5th day of lactation showed changes in the vocalization pattern; 4) Those pups showed changes in oxidative burst and phagocytosis on 21th day of lactation. It is concluded that exposure of rats to LPS promoted changes in the in the interaction between mother and pups

The role of N-beta-alanyldopamine synthase in the innate immune response of two insects.

Insects trigger a multifaceted innate immune response to fight microbial infections. We show that in the yellow mealworm, Tenebrio molitor, septic injuries induce the synthesis of N-beta-alanyldopamine (NBAD), which is known as the main sclerotization precursor of insect brown cuticles. We demonstrate that NBAD synthase is induced in the epidermis of the mealworm and of the Medfly, Ceratitis capitata, by infection with Escherichia coli. Our results indicate that synthesis of NBAD seems to be a novel component of the overall innate immune response in insects.

Constitutive activity of N-beta-alanyl-catecholamine ligase in insect brain.

N-Beta-Alanyldopamine (NBAD) is the primary catechol tanning agent precursor in typical brown or yellow insect cuticle. The insect integument enzyme responsible for the synthesis of NBAD was reported to be expressed solely in the epidermis, and only at the time of cuticle sclerotization. However, in this study we demonstrate directly that the enzyme also is expressed in a constitutive manner in the neural system of insects. The requirements and kinetic parameters of the brain-associated enzyme appear similar to those of the epidermis-associated enzyme in Ceratitis capitata. The brain-associated enzyme also was able to catalyze the in vitro synthesis of N-beta-alanylnorepinephrine (NBANE) and beta-alanyl derivatives of other biogenic amines. A melanic mutant of C. capitata, niger, was unable to conjugate beta-alanine with dopamine or other amines in either the epidermis or the brain. This result strongly supports the idea that these enzymes actually are expressed from a single gene and that differences in regulation must exist that account for the constitutive expression in the neural system. Similar results were obtained in Drosophila melanogaster and other insects. From these data, a number of questions arise about the role of beta-alanyl derivatives of biogenic amines and other compounds in insect brain and similarly, in the mammalian CNS.

Catecholamine-beta-alanyl ligase in the medfly Ceratitis capitata.

Dopamine (DA) and norepinephrine (NE) derivatives play an important role in the sclerotization and pigmentation of insect cuticles by serving as precursors for cuticular cross-linking. Protein preparations from prepupae of the medfly, Ceratitis capitata, were able to conjugate beta-alanine with DA producing N-beta-alanyldopamine (NBAD) or with NE, synthesizing N-beta-alanylnorepinephrine (NBANE). The latter reaction has been demonstrated for the first time. Apparent kinetic parameters were obtained for both substrates, DA (V(max)=30.7+/-6.0 pmol min(-1) mg(-1); K(m)=29.5+/-3.5 microM) and NE (V(max)=16.1+/-6.6 pmol min(-1)mg(-1); K(m)=89.0+/-8.3 microM). The same protein seems to be responsible for both enzymatic activities, judging from several criteria like identical behavior under heat inactivation as well as identical Mg2+ and Mn2+ dependent stimulation and Co2+ inhibition. Furthermore, the melanic mutants niger of C. capitata and ebony(4) of D. melanogaster, known to be defective for NBAD synthase, were also unable to synthesize NBANE. The protein preparation acylated tyrosine with much less efficiency, to produce sarcophagine (beta-alanyltyrosine). Strikingly, extracts from the melanic mutants were unable to synthesize sarcophagine. Our results strongly suggest that the enzymatic activity previously known as NBAD synthase is in fact a novel catalytic protein showing broad substrate specificity. We propose to identify it as catecholamine-beta-alanyl ligase.

Characterization of the mechanism involved in the relaxant response of dopexamine in the guinea pig pulmonary artery in vitro.

Dopexamine is a synthetic catecholamine used for the management of low-cardiac-output states. The purpose of this study was to characterize some of the mechanisms underlying dopexamine-mediated relaxation in the guinea pig pulmonary artery (PA) in vitro. Dopexamine (EC50, 1.2 microM; Rmax, 100%), like dobutamine (EC50, 1.4 microM, Rmax, 93.3%), prostacyclin (PGI2; EC50, 37 nM; Rmax, 96.2%), sodium nitroprusside (EC50, 370 pM; Rmax, 96.9%), forskolin (EC50, 47 pM: Rmax, 98.6%), and SKF 38393 (EC50, 120 nM; Rmax, 100%), caused graded relaxation in rings of PA precontracted by phenylephrine. The dopexamine vasorelaxation was antagonized by propranolol (1 microM), SCH 23390 (100 nM, a D1-dopamine antagonist), sulpiride (1 microM), glibenclamide (30 microM), tetraethylammonium (3 mM), apamin (100 nM), charybdotoxin (100 nM), SQ 22536 (10 microM, an adenylyl cyclase inhibitor), KT 5720 (10 microM, a protein kinase A inhibitor) and by calcitonin gene-related peptide (CGRP) or vasoactive intestinal peptide (VIP)-receptor antagonists (both 100 nM), as well as by chymotrypsin (1 U/ml). Neither the prior incubation of N(G)-nitro-L-arginine (100 pM), indomethacin (1 microM), nor removal of the vascular endothelium interfered with dopexamine vasorelaxation response in PA. Thus dopexamine relaxation in PA is mediated by activation of beta-adrenoceptors and dopamine receptors, and by the opening of both low- and high-conductance Ca2+-activated K+ channels, partially through adenosine triphosphate (ATP)-sensitive K+ channels. In addition, dopexamine-induced relaxation in PA seems to involve the release of peptides such as VIP and CGRP, an effect mediated by a cyclic adenosine monophosphate (cAMP)-dependent mechanism.

Levels of biogenic amines in larvae and adults of the rat hookworm, Nippostrongylus brasiliensis (Nematoda).

Norepinephrine, 5-hydroxytryptophan, octopamine, dihydroxyphenylacetic acid, N-acetyldopamine, dopamine, 5-hydroxyindoleacetic acid, N-acetylserotonin, tyramine, tryptophan and serotonin in larvae (third free stage and parasitic stages) and adult males and females (at defined ages during the intestinal phase) of the parasitic nematode Nippostrongylus brasiliensis were quantified simultaneously by high-performance liquid chromatography with electrochemical detection. Biogenic amine levels depended on the stage, the age and the sex of parasites and on environmental conditions. Their physiological roles in reproductively competent adults of this nematode are discussed in relation to exuviation and egg laying. Parallel fluctuations in free ecdysteroids and norepinephrine were observed in females from the same worm populations.

Coronary vascular activity of the novel inotropic pro-drug ibopamine and the de-esterified active form epinine.

The effects of the novel inotropic pro-drug, ibopamine, and the de-esterified active form, epinine (N-methyldopamine), were investigated in isolated canine circumflex coronary arteries in vitro. Both ibopamine and epinine produced concentration-dependent contractions of isolated canine coronary arteries, with epinine being approximately 7-fold more potent than ibopamine. The coronary vasoconstrictor response produced by ibopamine was inhibited completely by the irreversible alpha-adrenoceptor antagonist, phenoxybenzamine, whereas the response produced by epinine was transformed into relaxation which was inhibited by the beta-adrenoceptor antagonist, propranolol. The results indicate that ibopamine has the capacity to produce coronary arterial vasoconstriction, but that this activity may be partially offset by the beta-adrenoceptor-mediated activity of the active form, epinine.