RESUMEN
A bundle of initiatives to integrate human immunodeficiency virus (HIV) and tuberculosis (TB) services was assessed for the impact on antiretroviral therapy (ART) initiation at a TB referral hospital in Irkutsk, Russian Federation, from February 2014 to December 2015. The ART initiation rates in 166 ART-naïve patients undergoing anti-tuberculosis treatment (34.1% with multidrug or extensively drug-resistant TB) increased significantly from 14 (17%) pre-intervention to 44 (54%) post-intervention (P < 0.001). A survey of TB hospital staff identified administrative prioritisation as the most important initiative for increasing ART initiation.
Un ensemble d'initiatives visant à intégrer les services relatifs au virus de l'immunodéficience humaine (VIH) et à la tuberculose (TB) a été évalué en termes d'impact sur la mise en route du traitement antirétroviral (TAR) dans un hôpital de référence de la TB à Irkoutsk, Fédération de Russie, entre février 2014 et décembre 2015. Les taux de mise en route du TAR chez 166 patients n'en ayant jamais reçu et traités pour TB (34,1% avec une TB multi-résistante ou ultra-résistante) ont significativement augmenté de seulement 14 (17%) avant l'intervention à 44 (54%) après l'intervention (P < 0,001). Une enquête auprès du personnel de la TB a identifié la priorisation administrative comme l'initiative la plus importante dans l'augmentation de l'initiation du TAR.
Se evaluó un conjunto de iniciativas encaminadas a integrar los servicios de atención de la infección por el virus de la inmunodeficiencia humana (VIH) y la tuberculosis (TB), con el objeto de determinar la repercusión de la integración sobre el comienzo del tratamiento antirretrovírico (TAR) en el hospital de referencia de la TB de Irkutsk, en la Federación de Rusia, de febrero del 2014 a diciembre del 2015. La tasa de iniciación del TAR en 166 pacientes que nunca lo habían recibido y que estaban en curso de tratamiento antituberculoso (34,1% con TB multirresistente o extremadamente multirresistente) aumentó de manera significativa de solo 14 pacientes antes de la intervención (17%) a 44 pacientes después de la misma (54%; P < 0,001). Al interrogar al personal encargado de la TB en este hospital de referencia, se puso en evidencia que la priorización administrativa del TAR constituía la iniciativa de más había influido en el incremento de su utilización.
RESUMEN
BACKGROUND AND PURPOSE: Expression of inducible NOS (iNOS) is important in certain inflammatory diseases. We determined if the hormone aldosterone, a mineralocorticoid receptor (MR) agonist, affects LPS activation of iNOS expression in rat aortic smooth muscle cells (RASMC). EXPERIMENTAL APPROACH: Cultured RASMC were treated with LPS, with or without agonists/antagonists of steroid receptors. iNOS expression was determined by nitrite assays on culture medium removed from treated cells and by immunoblotting of cell protein extracts. KEY RESULTS: LPS (1 µg·mL(-1) ) increased nitrite and iNOS protein above that in control (untreated) cells. These effects of LPS were reduced by aldosterone (0.1-10 µM). The MR antagonists, eplerenone (10 µM) and spironolactone (10 or 50 µM), did not inhibit these actions of 1 µM aldosterone, but the latter were prevented by 10 µM mifepristone, a glucocorticoid (GR) and progestogen receptor (PR) antagonist. Mifepristone also prevented the reduction of LPS-induced nitrite increase produced by 1 µM dexamethasone (GR agonist) and 10 µM progesterone (PR agonist). Spironolactone (10-50 µM) by itself decreased LPS-induced increases in nitrite and iNOS protein. Mifepristone (10 µM) partially reversed these effects of 10 µM spironolactone, but not those of 50 µM; the effects of 50 µM spironolactone were also unchanged when mifepristone was increased to 50 µM. CONCLUSIONS AND IMPLICATIONS: This pharmacological profile suggests that aldosterone, and possibly 10 µM spironolactone, use mechanisms that are dependent on PR and/or GR, but not MR, to inhibit iNOS induction in RASMC. With 50 µM spironolactone, other inhibitory mechanisms requiring further investigation may become predominant.
Asunto(s)
Aldosterona/farmacología , Aorta Torácica/efectos de los fármacos , Lipopolisacáridos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Esteroides/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/enzimología , Células Cultivadas , Eplerenona , Masculino , Mifepristona/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Ratas , Ratas Sprague-Dawley , Espironolactona/análogos & derivados , Espironolactona/farmacologíaRESUMEN
Treatment of rat aortic smooth muscle cells (RASMC) with 1 or 100 microg ml-1 lipopolysaccharide (LPS) for 20-24 h led to expression of the inducible form of nitric oxide synthase (iNOS) as detected by Western blotting for iNOS protein, and by determination of increased cellular nitrite formation. LPS-induced nitrite production was inhibited almost completely by concomitant treatment of cells with LPS and either (a) pyrrolidine dithiocarbamate (PDTC, 25 microm), an antioxidant inhibitor of NF-kappaB activation; (b) N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 20 and 40 microm), a proteasomal inhibitor which prevents NF-kappaB activation; (c) nordihydroguaiaretic acid (NDGA, 10 and 50 microm), a lipoxygenase inhibitor; or (d) apocynin (2, 3.5 and 5 m m), an inhibitor of NADPH oxidase. Gel-shift assays using nuclear protein extracts incubated with a 32P-labelled DNA binding probe for NF-kappaB detected two electrophoretically separable complexes containing NF-kappaB. A faster migrating complex obtained when using both LPS-treated and untreated cells appeared to represent a basal or constitutive NF-kappaB activity, whereas a slower band was found only after LPS-treatment. The latter band was abolished when using cells treated for 1 h with LPS in the presence of PDTC (25 microm) or TPCK (20 microm), but was not inhibited by NDGA (50 microm) or apocynin (3.5 m m). The basal band was unaffected by any of the cell signalling inhibitors. Densitometry of Western blots indicated that LPS-induced iNOS protein expression was inhibited to a similar extent (between 74 and 87%) by the latter concentrations of PDTC, TPCK, NDGA and apocynin. The ability of PDTC and TPCK to abolish LPS-specific NF-kappaB activation, while also producing considerable inhibition of iNOS protein expression and nitrite formation, suggests that induction of iNOS by LPS in RASMC involves NF-kappaB-dependent transcription. However, the failure of NDGA and apocynin to prevent NF-kappaB activation, at least during early stages (up to 1 h) of its nuclear accumulation, suggests that these agents may affect cell signalling pathways which regulate iNOS induction by another mechanism to be determined.
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Aorta Torácica/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Lipopolisacáridos/farmacología , Músculo Liso Vascular/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Acetofenonas/farmacología , Animales , Aorta Torácica/citología , Western Blotting , Técnicas In Vitro , Masculino , Masoprocol/farmacología , Músculo Liso Vascular/citología , NADPH Oxidasas/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Nitritos/antagonistas & inhibidores , Nitritos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Tiocarbamatos/farmacología , Clorometilcetona de Tosilfenilalanila/farmacologíaRESUMEN
Partial depolarisation of smooth muscle in endothelium-denuded rat aortic ring preparations, by increasing physiological buffer KC1 concentrations from 4.7 to 14.7 mM, produced a leftward shift of concentration response curves (CRCs) to the alpha 1-adrenoceptor agonist noradrenaline (NA), phenylephrine and methoxamine, without changing maximal responses, whereas maximal responses to clonidine (CLON), also an alpha 1-agonist in this tissue were considerably increased. Partial depolarisation did not alter responses to 10 nM NA or 100 nM CLON in Ca2+(-free) buffer, but significantly increased the contractions obtained on adding Ca2+ back in the presence of the agonists. The potentiation of NA (2.5 and 5 nm) contractions by partial depolarisation was prevented by the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine (NIF, 1 microM). NIF did not significantly affect NA CRCs in 4.7 mM KCl, whereas responses in 14.7 mM KCl were significantly decreased, indicating VOCC recruitment by NA only in the latter condition. Initial depletion of intracellular Ca2+ stores with 1 microM thapsigargin (THAP) in Ca2+(-free) buffer did not alter NA CRCs subsequently obtained in normal Ca2+. However, after THAP-pretreatment, these NA responses (in both 4.7 and 14.7 mM KC1) were attenuated by NIF, indicating that VOCCs were activated by NA in THAP-treated tissues. SKF 96365 (SKF, 30 microM), which can block VOCC and non-VOCC routes of extracellular Ca2+ influx, inhibited NA responses in 4.7 mM and 14.7 mM KCl, possibly implying a role for both types of Ca2+ entry in contractions. However, the greater inhibitory effects of SKF in THAP-pretreated tissues, probably reflected the mobilisation of VOCCs by NA following THAP exposure, because SKF was shown separately to block VOCC-mediated contractions in tissues depolarised with 100 mM KCl alone. 10 microM niflumic acid, an inhibitor of Ca2+(-activated) Cl- channels, did not affect responses to NA in 4.7 mM or 14.7 mM KC1, suggesting that VOCC opening induced by NA in 14.7 mM KCl was not due to depolarisation produced by alpha 1-adrenoceptor induced Cl- efflux. CRCs for NA were unaffected by pretreatment of rings with 100 ng ml-1 pertussis toxin (PT), suggesting a lack of involvement of PT-sensitive G proteins in the contractions obtained either in 4.7 or 14.7 mM KCl. BMY 7378 (100 microM), a selective antagonist for alpha 1D-adrenoceptors, competitively inhibited NA contractions with apparent pKB values of 8.7 +/- 0.2 and 8.4 +/- 0.1 in 4.7 mM and 14.7 mM KCl, respectively. Pretreatment of rings with chloroethylclonidine (100 microM), an irreversible antagonist of alpha 1B-and alpha 1D-adrenoceptors, produced similar rightward shifts in CRCs to NA by 3.2 +/- 0.2 and 3.7 +/- 0.3 log concentration units in 4.7 mM and 14.7 mM KCl, respectively, without changing maximal responses. Inositol phosphate (IP) turnover produced by NA in aortic rings was not significantly different in 4.7 mM compared with 14.7 mM KCl. As a whole, these results suggest that partial depolarisation of the rat aorta with KCl enhances alpha 1-adrenoceptor mediated contractions predominantly via the alpha 1D-subtype, and by a mechanism to be identified which allows greater recruitment of VOCCs by NA. In addition, the ability of THAP-pretreatment also to enhance VOCC activation by NA suggests that Ca2+ release from, or prevention of its reuptake into, intracellular stores may contribute to those processes leading to VOCC opening.
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Agonistas alfa-Adrenérgicos/farmacología , Aorta/efectos de los fármacos , Calcio/fisiología , Norepinefrina/farmacología , Cloruro de Potasio/farmacología , Receptores Adrenérgicos alfa 1/fisiología , Animales , Aorta/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Técnicas In Vitro , Fosfatos de Inositol/biosíntesis , Masculino , Nifedipino/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Vasoconstricción/fisiologíaRESUMEN
The demonstration of semicarbazide-sensitive amine oxidase (SSAO) activity in some freshly-dispersed cell preparations and in particular types of cells grown in culture, provides increasing opportunities for investigating the importance of SSAO in various aspects of cellular function. Assays of benzylamine and methylamine metabolism in homogenates of cultured cells have established clearly that SSAO is expressed in rat and pig vascular (aortic) smooth muscle cells, as well as in rat non-vascular (anococcygeus, trachea) smooth muscle, brown and white adipocytes. However, to date little or no SSAO activity has been detected in cultures of human vascular smooth muscle cells grown from blood vessels (e.g. umbilical artery) known to contain the enzyme, and the reason for this is not yet apparent. However, those cell cultures expressing SSAO are offering useful experimental models for studying biochemical and toxicological consequences upon cellular function which may result from the metabolism of various aromatic and aliphatic amines suggested to be possible physiological and xenobiotic substrates of the enzyme.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Músculo Liso Vascular/enzimología , Músculo Liso/enzimología , Adipocitos/enzimología , Tejido Adiposo Pardo/enzimología , Animales , Aorta , Células Cultivadas , Humanos , Cinética , Ratas , Especificidad por Sustrato , Porcinos , TráqueaRESUMEN
The possibility that increased levels of the activity of the semicarbazide-sensitive amine oxidase (SSAO) might, to some extent, compensate for the loss of monoamine oxidase (MAO) activity in the atypical form of Norrie Disease, was examined using the rat as a model. Long-term treatment with the MAO inhibitor tranylcypromine (1 mg/kg/day) resulted in sustained inhibition of MAO-A and MAO-B activities in liver and brain. After one week, the SSAO activity in heart had increased by 79% above the control levels. This increase was maintained for 3 weeks. Since such alterations might result from enzyme induction, the turnover of the enzyme was studied in cultured cells from rat aortic smooth muscle. The time-course of recovery of enzyme activity following irreversible inhibition by MDL 72145 corresponded to a half-life of approximately 6 days for this process.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Encéfalo/enzimología , Hígado/enzimología , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Músculo Liso Vascular/enzimología , Miocardio/enzimología , Tranilcipromina/farmacología , Alilamina/análogos & derivados , Alilamina/farmacología , Amina Oxidasa (conteniendo Cobre)/efectos de los fármacos , Animales , Aorta/enzimología , Células Cultivadas , Femenino , Isoenzimas/antagonistas & inhibidores , Masculino , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The ability of allylamine (AA) administration to produce vascular lesions resembling atherosclerotic disease in animals, has been linked to metabolism of AA to the toxic aldehyde acrolein (ACR) by a semicarbazide-sensitive amine oxidase (SSAO) found in plasma and in vascular smooth muscle. Here, we have assessed the cytotoxicity of AA and ACR towards cultured human umbilical vein endothelial cells (HUVEC). After 20h treatment, 50 microM AA alone had no effect and 100 microM AA produced a modest reduction in cell viability. However, both concentrations produced considerable cell death when incubated with HUVEC in the presence of human umbilical artery homogenate as a source of human vascular SSAO activity. The cytotoxic actions of 50 microM AA were not altered by coincubation with 100 microM pargyline (an inhibitor of monoamine oxidase, MAO) but were completely prevented by 100 microM semicarbazide (SSAO inhibitor) and propargylamine (MAO/SSAO inhibitor). ACR at 50 and 100 microM was considerably cytotoxic, but had little effect at 5 and 10 microM. Since SSAO can also metabolize the biogenic aliphatic amine methylamine to formaldehyde (FA), effects of this aldehyde upon HUVEC were also studied. 50 microM FA did not significantly alter HUVEC viability whereas 200 microM FA produced a small but significant reduction in viability. However, 200 microM FA (but not 50 microM FA) was highly cytotoxic in HUVEC pretreated for 24h with the glutathione (GSH) synthesis inhibitor, D.L-buthionine sulphoximine (200 microM). These results suggest that endothelial integrity or function in blood vessels could be affected by these aliphatic aldehydes as environmental pollutants, dietary contaminants, and possible products of metabolic pathways, including those involving the action of SSAO upon biogenic and xenobiotic aliphatic amines. The availability of GSH-dependent mechanisms for metabolizing these aldehydes may have an important protective influence.
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Acroleína/farmacología , Alilamina/farmacología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Endotelio Vascular/efectos de los fármacos , Formaldehído/farmacología , Glutatión/metabolismo , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Butionina Sulfoximina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Humanos , Inhibidores de la Monoaminooxidasa/farmacología , Pargilina/análogos & derivados , Pargilina/farmacología , Propilaminas/farmacologíaRESUMEN
Mammalian plasma and tissues contain various soluble and membrane-bound enzymes which metabolize the synthetic amine benzylamine particularly well. The sensitivity of these enzymes to inhibition by semicarbazide and related compounds suggests that they contain a cofactor with a reactive carbonyl group, which has been proposed to be either pyridoxal phosphate, pyrroloquinoline quinone or (more recently) 6-hydroxydopa. It is not yet clear if all of these semicarbazide-sensitive amine oxidases (SSAOs) are copper-dependent enzymes. A variety of compounds have now been identified as relatively selective inhibitors to distinguish the SSAOs from other amine oxidases, in order to investigate the properties of SSAOs and their potential role in biogenic and xenobiotic amine metabolism in vivo. While plasma SSAO is soluble, most tissue SSAOs appear to be membrane-bound, probably plasmalemmal enzymes, which may be capable of metabolizing extracellular amines. Vascular (and non-vascular) smooth muscle cells have particularly high SSAO activity, although recently the enzyme has been found in other cell types (e.g. adipocytes, chondrocytes, odontoblasts) implying a functional importance not restricted solely to smooth muscle. The substrate specificity of plasma and tissue SSAOs shows considerable species-related variations. For example, while some endogenously-occurring aromatic amines such as tyramine and tryptamine are metabolized well by SSAO in homogenates of rat blood vessels, and also in vitro inhibition of SSAO can potentiate vasoconstrictor actions of these amines in rat vascular preparations, these amines are poor substrates for human SSAO, thus complicating attempts to generalize possible physiological roles for these enzymes. Vascular SSAO can metabolize the xenobiotic aliphatic amine, allylamine, to the cytotoxic aldehyde acrolein and this has been linked to the ability of allylamine administration to produce cardiovascular lesions in experimental animals, sometimes mimicking features of atherosclerotic disease. Recent studies showing that the endogenously-occurring aliphatic amines methylamine and aminoacetone are metabolized in vitro to formaldehyde and methylglyoxal, respectively, by SSAO in some animal (including human) tissues, suggest the possibility that toxicological consequences upon cellular function could result if such conversions occur in vivo.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Mamíferos/metabolismo , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Amina Oxidasa (conteniendo Cobre)/química , Animales , Supervivencia Celular/fisiología , Humanos , Estructura Molecular , Ratas , Fracciones Subcelulares/enzimología , Especificidad por SustratoRESUMEN
High speed (105,000 g/60 min) membrane fractions from rat aorta homogenates metabolized the aliphatic amine aminoacetone (AA) to methylglyoxal (MG) with a Km of 19 +/- 3 microM, and Vmax of 510 +/- 169 nmol MG/hr/mg protein. This deaminating activity appears to be due to a semicarbazide-sensitive amine oxidase (SSAO), which is associated with smooth muscle cells in blood vessels of the rat and other species. AA was a competitive inhibitor (Ki of 28 +/- 6 microM) of the metabolism of benzylamine, a synthetic amine often used as an assay substrate for SSAO. AA is produced endogenously from mitochondrial metabolism of threonine and glycine, and thus could be a physiological substrate for SSAO, whereas the production of MG by SSAO could have cytotoxic implications for cellular function.
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Acetona/análogos & derivados , Amina Oxidasa (conteniendo Cobre) , Aorta/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Semicarbacidas/farmacología , Acetona/metabolismo , Animales , Bencilaminas/metabolismo , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Cinética , Masculino , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Pargilina/análogos & derivados , Pargilina/farmacología , Propilaminas/farmacología , Piruvaldehído/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/enzimologíaRESUMEN
Although the existence of a membrane-bound (probably plasmalemmal) semicarbazide-sensitive amine oxidase (SSAO) is well established in various mammalian tissues, and especially within vascular smooth muscle, its importance and the possible consequences of its metabolism of certain physiological and xenobiotic amines in vivo are under continuing investigation. In this respect, there are major species-related differences in substrate specificity determined in vitro, not only towards the synthetic amine benzylamine, but also towards some other aromatic amines (e.g. tyramine, tryptamine, 2-phenylethylamine, dopamine, histamine) which are possible endogenous substrates. Inhibition of SSAO can potentiate the pharmacological activity of some amines in isolated tissue (e.g. blood vessel) preparations from some species. Recent evidence has accumulated that SSAO may also be involved in metabolizing endogenous aliphatic amines such as methylamine and aminoacetone, focussing attention on the fact that the aldehyde products (formaldehyde and methylglyoxal, respectively) are potentially cytotoxic agents. Indeed, SSAO has been implicated in experimental models of cardiovascular toxicity involving conversion of the industrial aliphatic amine allylamine to acrolein. In summary, metabolism by SSAO may reduce the physiological/pharmacological effects of some amines, but the resulting metabolites (aldehydes, H2O2) may also have important actions.
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Amina Oxidasa (conteniendo Cobre) , Monoaminooxidasa/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Semicarbacidas/metabolismo , Animales , Bencilaminas/farmacocinética , Unión Competitiva , Humanos , Cinética , Especificidad por SustratoRESUMEN
Semicarbazide-sensitive amine oxidase (SSAO), occurs not only in vascular smooth muscle but also in other cell types (e.g. adipocytes, chondrocytes, odontoblasts), probably in the plasma membrane. Although certain aromatic biogenic amines (e.g. tryptamine, tyramine, beta-phenyl-ethylamine) may be endogenous substrates for SSAO in species such as the rat, the weak activity of SSAO in human tissues towards these amines makes this less likely in man. However SSAO in human and rat vascular homogenates readily converts the aliphatic biogenic amines methylamine and aminoacetone to formaldehyde and methylglyoxal, respectively. Also the xenobiotic aliphatic amine allylamine produces cardiovascular damage in experimental animals by a mechanism which involves its deamination by SSAO to acrolein. Further metabolism of these toxic aliphatic aldehydes may involve glutathione-dependent pathways. Thus, SSAO may be involved not only in the removal of physiologically-active endogenous/xenobiotic amines, but resulting metabolite (aldehyde/H2O2?) formation could also influence cellular function.
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Amina Oxidasa (conteniendo Cobre) , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/fisiología , Semicarbacidas/farmacología , Aminas/química , Aminas/metabolismo , Animales , Aminas Biogénicas/metabolismo , Humanos , Ratas , Especificidad por Sustrato , Distribución Tisular , Xenobióticos/metabolismoRESUMEN
The lung has been shown to be potentially important in the metabolism of amines. Since SSAO has been demonstrated to be active towards some volatile short-chain aliphatic amines in other tissues, the current study determined the specificity and kinetic constants for the metabolism by bovine lung SSAO, of several aliphatic and aromatic amines some of which have been suggested to be physiological substrates (e.g. methylamine, aminoacetone and beta-phenylethylamine), and others (e.g. benzylamine) which are non-physiological. In the case of benzylamine, an inhibition at high substrate concentration was observed. Kinetic assays ruled out the possibility that this inhibition was caused by products of the deamination of benzylamine, and consequently it is suggested that these results may indicate the presence of two binding sites for the interaction of benzylamine with SSAO.
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Amina Oxidasa (conteniendo Cobre) , Aminas/metabolismo , Pulmón/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Semicarbacidas/farmacología , Animales , Bencilaminas/metabolismo , Bovinos , Cinética , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidoresRESUMEN
The metabolism of [14C]methylamine (MA) by amine oxidase activity in rat white and brown adipose tissue homogenates, and in mature adipocytes from these tissues has been studied. Oxidation of MA was completely inhibited by 0.1-1 mM semicarbazide, without being affected by the monoamine oxidase (MAO) inhibitor, pargyline (1 mM), indicating that MA is metabolized by semicarbazide-sensitive amine oxidase (SSAO) and not by MAO. The mean Km for MA deamination in all of these sources was around 250-300 microM. SSAO activity towards MA was also demonstrated in white and brown pre-adipocytes, transformed to the adipose phenotype by treatment in culture for 7 days with lipogenic agents. These results are similar to previous findings that SSAO in vascular smooth muscle is able to metabolize aliphatic amines such as MA, and furthermore suggest that SSAO may play a role in adipose tissue function and/or maturation.
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Tejido Adiposo Pardo/enzimología , Tejido Adiposo/enzimología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Metilaminas/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Animales , Cinética , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Von Hippel Lindau disease (VHL) is a rare autosomal dominant disease associated with tumors and cysts in multiple organ systems. The VHL disease gene is tightly linked to the polymorphic DNA marker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomeric and RAF1 on its centromeric side. Two additional markers, D3S1038 and D3S601, have also been identified, and these markers, like D3S720, are very tightly linked to VHL. Previously 93 cosmid clones were mapped to the larger region, 3p24.2-pter, surrounding the VHL disease gene. Using a Southern-based screening strategy on pools of YAC clones we have isolated a contig of overlapping YAC clones that extends about 0.7 megabase centromeric, and about 1.3 megabases telomeric of D3S720 and contains all three tightly linked VHL markers. Individual YACs in this contig were hybridized to grids containing cosmids localized between 3p24.2-pter and to several cosmids localized by fluorescent in situ hybridization (FISH) to 3p25. A total of 28 cosmids were positioned on this contig of overlapping YAC clones. We have also identified homologous YAC clones to many additional cosmid clones localized between 3p24.2-p25, although these have not yet been precisely localized relative to the contig of YAC clones. This contig of YAC clones probably contains the VHL disease gene and should facilitate the isolation and characterization of this gene.
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Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 3 , Enfermedad de von Hippel-Lindau/genética , Secuencia de Bases , Southern Blotting , Centrómero , Mapeo Cromosómico , Cósmidos , Cartilla de ADN , Genes Dominantes , Marcadores Genéticos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo , TelómeroRESUMEN
The aliphatic amine aminoacetone has been described previously as a product of mitochondrial metabolism of threonine and glycine. Here, aminoacetone is shown to be deaminated to methylglyoxal by supernatants obtained by low speed centrifugation (600 g/10 min) of human umbilical artery homogenates, and also by membrane fractions isolated by high speed centrifugation (105,000 g/60 min) of these supernatants. Metabolism of 100 microM aminoacetone was completely inhibited by 1 mM propargylamine and MDL 72145, drugs which are capable of inhibiting the membrane-bound semicarbazide-sensitive amine oxidase (SSAO) activity found in vascular smooth muscle cells, whereas 1 mM pargyline and deprenyl which are inhibitors of monoamine oxidase, were without inhibitory effect. Estimated kinetic constants (at pH 7.8) for aminoacetone metabolism were Km = 92 microM; Vmax = 270 nmol/hr/mg protein. In addition, aminoacetone was a competitive inhibitor (Ki = 83 microM and 128 microM in low speed supernatants and high speed membrane fractions, respectively) of [14C]benzylamine metabolism by SSAO in this tissue. Aminoacetone would appear to be an endogenously occurring amine with a Km for metabolism by SSAO far lower than other aliphatic and aromatic biogenic amines examined previously as potential physiological substrates for the human vascular enzyme and possible implications of this are discussed.
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Acetona/análogos & derivados , Amina Oxidasa (conteniendo Cobre) , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Piruvaldehído/metabolismo , Semicarbacidas/farmacología , Arterias Umbilicales/enzimología , Acetona/metabolismo , Bencilaminas/metabolismo , Humanos , Cinética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Arterias Umbilicales/efectos de los fármacosRESUMEN
The capacity of the vascular enzyme, semicarbazide-sensitive amine oxidase (SSAO), to metabolize methylamine to the potentially toxic product, formaldehyde, was tested using rat aortic homogenates and purified porcine aortic SSAO. Formaldehyde production in incubations of enzyme source with methylamine (1 mM) was detected by high performance liquid chromatography and product was confirmed by desorption chemical ionization mass spectrometry (DCI-MS). Inhibitor studies using the specific SSAO inhibitor semicarbazide and the monoamine oxidase inhibitor pargyline indicate that SSAO is responsible for metabolism of methylamine to formaldehyde. These results suggest the possibility that elevated methylamine found in several pathologic states (such as uremia and diabetes mellitus), or generated from exogenous sources, could result in overproduction of formaldehyde in tissues with high SSAO activity, especially blood vessels.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/farmacología , Formaldehído/análisis , Metilaminas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Animales , Carcinógenos/farmacología , Cromatografía Líquida de Alta Presión , Masculino , Músculo Liso Vascular/metabolismo , Pargilina/farmacología , Ratas , Ratas Endogámicas , Semicarbacidas/farmacología , PorcinosRESUMEN
An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0.1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amino oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 microM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol (mg prot.)-1 h-1, and in plasma of 48 nmol (mL serum)-1 h-1. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 microM (plasma), 222 microM (umbilical artery), and Vmax values of 28 nmol (mL serum)-1 h-1 (plasma) and 377 nmol (mg prot.)-1 h-1 (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 microM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 microM) and [14C]MA (800 microM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Metilaminas/metabolismo , Músculo Liso Vascular/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Semicarbacidas/farmacología , Aminopropionitrilo/farmacología , Animales , Aorta Torácica/enzimología , Clorgilina/farmacología , Colorimetría , Diálisis , Humanos , Técnicas In Vitro , Cinética , Masculino , Músculo Liso Vascular/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/sangre , Proteínas/metabolismo , Ratas , Arterias Umbilicales/enzimologíaRESUMEN
Metabolism of 5 microM benzylamine (BZ) by rat aortic cultured smooth muscle cells (SMC) is inhibited almost completely by 10(-3) M semicarbazide and 10(-6) M propargylamine, but is little affected by 10(-4) M and 10(-3) M pargyline and clorgyline, indicating BZ metabolism predominantly by the semicarbazide-sensitive amine oxidase (SSAO) previously characterized in rat aortic homogenates. Km values of 7-9 microM for BZ metabolism by SSAO in SMC cultures, indicate similar, if not identical properties, to the enzyme in the parent blood vessel.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Músculo Liso Vascular/enzimología , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Animales , Aorta Torácica/citología , Aorta Torácica/enzimología , Bencilaminas/metabolismo , Células Cultivadas , Clorgilina/farmacología , Técnicas In Vitro , Cinética , Masculino , Inhibidores de la Monoaminooxidasa/farmacología , Músculo Liso Vascular/citología , Pargilina/farmacología , RatasAsunto(s)
Alilamina/farmacología , Amina Oxidasa (conteniendo Cobre) , Aminas/farmacología , Inhibidores de la Monoaminooxidasa/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Alilamina/análogos & derivados , Animales , Masculino , Especificidad de Órganos , Ratas , Ratas EndogámicasRESUMEN
The effects of amine oxidase inhibitors upon the daily urinary excretion of monomethylamine (MMA), dimethylamine (DMA), trimethylamine (TMA) and ammonia in the rat have been examined. Administration of hydralazine (5 mg kg-1) or semicarbazide (100 mg kg-1), drugs which irreversibly inhibit semicarbazide-sensitive amine oxidases (SSAO) but not monoamine oxidase (MAO), enhanced MMA excretion by around three- to six-fold above pretreatment levels, whereas no effect of pargyline (25 mg kg-1), a selective irreversible inhibitor of MAO was found. No apparent changes in DMA or TMA excretion in response to drug-treatment were observed. Ammonia excretion also was generally unchanged except for an apparent marked increase (approximately four-fold) over the 24 h following semicarbazide, a result which might be explained if ammonia is a degradation product of semicarbazide metabolism in the rat. With recent evidence that MMA is a substrate in-vitro for SSAO activities, results here may indicate that SSAO or related enzymes are involved in endogenous MMA turnover.