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M Unzeta

Publications and source records attributed to M Unzeta.

At least 19 recordsLinked to original sources
PubMed2006

The decrease of NAD(P)H has a prominent role in dopamine toxicity.

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2006

Protective effect of N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF9601N) on mitochondrial permeability transition.

PF9601N, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine, an monoamine oxidase (MAO) B inhibitor, has shown neuroprotective properties against dopaminergic toxins. To elucidate the mechanisms involved in this protection, the effect of PF9601N on mitochondria was assessed. PF9601N prevents mitochondrial swelling, drop in the electrical potential and oxidation of sulfhydryl groups, glutathione and pyridine nucleotides induced by Ca(2+). These observations demonstrate the protective effect of PF9601N on the induction of mitochondrial permeability transition. This protection is due to the interaction of the secondary protonated amino group in the molecule with pore-forming structures and to its antioxidant property, rather than to inhibition of MAO B activity. PF9601N also prevents the release of cytochrome c from mitochondria, suggesting its potential inhibitory effect on mitochondria-mediated apoptosis. The low IC(50) value for this inhibition, in comparison with deprenyl, make it a more efficient compound than propargylamines and other amines in protecting the bioenergetic functions of mitochondria.

Animals↗
PubMed2003

Activation of human lung semicarbazide sensitive amine oxidase by a low molecular weight component present in human plasma.

Semicarbazide-sensitive amine oxidase (SSAO) encodes a wide family of enzymes named E.C.1.4.3.6 [amine:oxygen oxidoreductase (deaminating) (copper containing)] that metabolises primary aliphatic and aromatic amines. It is present in almost all vascularised and nonvascularised mammalian tissues, and it is also present in soluble form in plasma. SSAO appears to show different functions depending on the tissue where it is expressed. Here we describe, for the first time, the activation of the SSAO from human lung by human plasma. The extent of activation was greater when the human plasma came from diabetic and heart infarcted patients. A kinetic mechanism of such effect is proposed. The activation was lost after the plasma was dialysed, indicating a low molecular weight component (MW <3800 Da) to be responsible. The activator component is heat stable and resistant to proteolysis by chymotrypsin and trypsin and also resistant to perchloric acid treatment. However, treatment with 35% formic acid, completely abolished activation, suggesting involvement of lipid material. The possibility of that lysophosphatidylcholine (LPC), an amphiphilic phospholipid derived from the phosphatidylcholine, the major component in plasma accumulated in pathological conditions, was studied. LPC was shown to behave as a "competitive activator" of human lung SSAO at concentrations below its critical micellar concentration (CMC value=50 microM). Thus LPC may be a component of the SSAO activatory material present in human plasma.

Amine Oxidase (Copper-Containing)↗
PubMed2003

Localization of monoamine oxidase A and B and semicarbazide-sensitive amine oxidase in human peripheral tissues.

Monoamine oxidase (MAO) A and B and semicarbazide-sensitive amine oxidase (SSAO) localizations in peripheral human tissues were compared by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3) and a rabbit polyclonal anti-bovine SSAO antibody. Immunoreactivities of the samples, obtained from 6 routine autopsy cases, showed different distributions in the tissues studied (heart, lung, duodenum, liver, pancreas, spleen, thyroid gland, adrenal gland and kidney). The relative MAO-A, MAO-B and SSAO distributions indicated a widespread distribution of these enzymes in the human body that is characterized by a matching cellular pattern in only few tissues. These differences suggest that each amine oxidase may play a specific function in, at least some, peripheral tissues.

Journal Article↗
PubMed2003

Protective effect of N-(2-propynyl)-2-(5-benzyloxyindolyl) methylamine (PF 9601N), a novel MAO-B inhibitor, on dopamine-lesioned PC12 cultured cells.

Oxidative stress may play a role in the pathogenesis of Parkinson's disease. We have standardised a new model of dopaminergic-cell toxicity that uses dopamine, which is able to generate free radicals, as a toxin. The effect of this catecholamine on cell viability (MTT staining) was dose-dependent, reaching 65% of cell loss at a dopamine concentration of 300 microM. In this model, the protective effect of a novel MAO-B inhibitor, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF 9601N), was studied and compared with the effect of L-deprenyl assayed under the same experimental conditions. Whereas PF 9601N (50 microM and 100 microM) showed a significant protective effect, this was not the case with L-deprenyl. This different behaviour could be explained in terms of difference in antioxidant capacity. The toxicity induced in PC12 cells by 300 microM dopamine was partially reversed by incubating it in the presence of GBR-12909, a dopamine-transporter blocker. The results indicated that, besides the intracellular toxicity effect of dopamine, another non-specific extracellular mechanism could be involved.

Animals↗
PubMed2001

Tissue activity and cellular localization of human semicarbazide-sensitive amine oxidase.

Semicarbazide-sensitive amine oxidase (SSAO), widely distributed in highly vascularized mammalian tissues, metabolizes endogenous and xenobiotic aromatic and aliphatic monoamines. To assess whether its physiological role in humans is restricted to oxidation, we used an immunohistochemical approach to examine the cellular localization of SSAO in human peripheral tissues (adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and blood vessels) and also analyzed its subcellular localization. The results are in agreement with the specific activities also determined in the same samples and are discussed with reference to the tissue distribution of monoamine oxidase A and B. Together with the oxidative deamination of monoamines, SSAO cellular localization indicates that, in most human peripheral tissues, it might participate in the regulation of physiological processes via H(2)O(2) generation. (J Histochem Cytochem 49:209-217, 2001)

Amine Oxidase (Copper-Containing)↗
PubMed2001

Semicarbazide-sensitive amine oxidase substrates stimulate glucose transport and inhibit lipolysis in human adipocytes.

Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes scavenging biogenic or exogenous amines and generating hydrogen peroxide. We asked whether human adipose tissue could express SSAO. Since hydrogen peroxide exhibits pharmacological insulin-like effects, we also tested whether its endogenous production by SSAO could mimic several insulin effects on adipocytes, such as stimulation of glucose uptake and inhibition of lipolysis. The benzylamine oxidation by human adipose tissue was inhibited by semicarbazide or hydralazine and resistant to pargyline or selegiline. It was due to an SSAO activity localized in adipocyte membranes. A protein of 100-kDa and a 4-kb mRNA corresponding to SSAO were identified in either mammary or abdominal subcutaneous fat depots. In isolated adipocytes, SSAO oxidized similarly benzylamine and methylamine that dose dependently stimulated glucose transport in a semicarbazide-sensitive manner. Antioxidants also inhibited the benzylamine and methylamine effects. Moreover, the ability of diverse substrates to be oxidized by adipocytes was correlated to their effect on glucose transport. Benzylamine and methylamine exerted antilipolytic effects with a maximum attained at 1 mM. These results show that human adipocytes express a membrane-bound SSAO that not only readily oxidizes exogenous amines and generates H(2)O(2), but that also interplays with glucose and lipid metabolism by exerting insulin-like actions. Based on these results and the fact that variations in plasma levels of the soluble form of SSAO have been previously reported in diabetes, we propose that determination of adipocyte SSAO, feasible on subcutaneous microbiopsies, could bring relevant information in pathologies such as obesity or diabetes.

Adipocytes↗
PubMed2000

A spectrophotometric method for determining the oxidative deamination of methylamine by the amine oxidases.

Previously published studies on the oxidative deamination of methylamine by the amine oxidases have determined the formation of radioactively labeled formaldehyde from [(14)C]methylamine. The present work describes a coupled spectrophotometric assay, using formaldehyde dehydrogenase, for the continuous determination of the oxidative deamination of methylamine by semicarbazide-sensitive amine oxidase (SSAO) and its potential use for determining methylamine concentrations in plasma. In this assay, the formaldehyde produced by methylamine deamination is further oxidized to formate, with the reduction of NAD(+), by formaldehyde dehydrogenase. The NADH generated is monitored continuously at 340 nm. Interference from the presence of a rotenone-insensitive NADH oxidase activity in crude tissue homogenates and microsomal fractions can be minimized by pretreating samples with Triton X-100 or substituting NAD(+) by APAD(+) in the coupled assay. This relatively inexpensive and reproducible assay procedure avoids the use of radioactively labeled material.

Aldehyde Oxidoreductases↗
PubMed2000

Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes.

The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37 degrees C. After an initial lag-period, activation was first-order with time and complete after approx. 20 h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37 degrees C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6 M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37 degrees C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in V(max) for benzylamine oxidation, with no significant alterations in the K(m) or the K(si) for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others.

Amine Oxidase (Copper-Containing)↗
PubMed2000

Molecular determinants of MAO selectivity in a series of indolylmethylamine derivatives: biological activities, 3D-QSAR/CoMFA analysis, and computational simulation of ligand recognition.

A series of indolylmethylamine derivatives were assayed toward MAO-A and MAO-B inhibition. The K(i) values of these compounds are in the range from 0.8 to >10(6) nM for MAO-A or from 0.75 to 476000 nM for MAO-B. The most selective MAO-A or MAO-B inhibitors elicit a ratio of K(i) in the order of 1500 or 1000, respectively. Comparison of MAO-A and MAO-B CoMFA models showed that both the steric and electrostatic properties at the 5 position of the indole ring are determinant for MAO selectivity. Computational simulations of the complex between this part of the ligand and Phe-208 of MAO-A or Ile-199 of MAO-B, experimentally identified as responsible for substrate selectivity, allowed us to further characterize the nature of these enzyme-inhibitor interactions.

Amines↗
PubMed2000

The novel type B MAO inhibitor PF9601N enhances the duration of L-DOPA-induced contralateral turning in 6-hydroxydopamine lesioned rats.

The present study examined the effect of the highly potent and selective MAO B inhibitor PF9601N on L-DOPA-induced rotational behavior in unilateral nigrostriatal 6-hydroxydopamine lesioned rats. Three doses of PF9601N (20, 40 and 60mg/kg) were administered 30 min before an injection of L-DOPA (25mg/kg), and both contralateral and ipsilateral rotational behavior was measured. In addition, we also studied the effect produced by another MAO B inhibitor, deprenyl (20mg/kg), the MAO A inhibitor, clorgyline (20mg/kg), and the dopamine reuptake inhibitor, GBR2909 (7.5 mg/kg) on L-DOPA-induced rotational behavior. The results showed that PF9601N plus L-DOPA significantly enhanced the duration of contralateral rotational behavior with respect to L-DOPA plus vehicle in a dose-related manner. At the dose of 40 and 60mg/kg, PF9601N produced significantly more overall contralateral turning than L-DOPA plus vehicle, and at the dose of 60mg/kg, PF9601N produced significantly more turning behavior than L-DOPA plus deprenyl. These results suggest that PF9601N may be used as a novel tool in the treatment of Parkinson's disease.

Adrenergic Agents↗
PubMed2000

FA-70, a novel selective and irreversible monoamine oxidase-A inhibitor: effect on monoamine metabolism in mouse cerebral cortex.

A series of indolealkylamine derivatives has been previously designed and evaluated with the aim of finding the most potent and selective novel monoamine oxidase (MAO) inhibitors to be used in the therapy of neurological and affective disorders. Among them, FA70, a 5-hydroxy-indolealkylamine derivative, has been characterized in vitro as a potent, irreversible, and mechanism-based inhibitor of the MAO-A isoform. The comparison with clorgyline, analyzed under the same experimental conditions, confirmed FA70 as the most potent MAO-A inhibitor. The ex vivo effect of FA70 on MAO activity in mouse cerebral cortex was similar to that observed in vitro, showing more efficacy than in peripheral tissues. The ex vivo effect of FA70 on amine metabolism also was evaluated after acute and chronic treatment, and the results showed that between both MAO isoforms, MAO-A is the only one responsible for monoamine metabolism in this region of the brain. The ex vivo effect of FA70 on dopamine content was correlated with the activation effect on tyrosine hydroxylase activity, the enzyme responsible for the regulation of the limiting step in catecholamine synthesis.

Animals↗
PubMed2000

On the primary structure of membrane-bound semicarbazide-sensitive amine oxidase (SSAO).

Mammalian semicarbazide-sensitive amine oxidase (SSAO) activities are a diverse group of copper dependent enzymes within the classification EC 1.4.3.6. [amine:oxygen oxidoreductase (deaminating) (copper-containing)]. They include plasma amine oxidase and the membrane-bound enzyme. Although soluble plasma SSAO from several species have been successfully purified and cloned, relatively little work has been carried out on the molecular properties of the tissue-bound enzyme. At present there is not conclusive evidence that allows to conclude whether or not plasma SSAO is released from the tissue-bound enzyme. This review focuses upon recent progress made in determining the primary structure of the membrane-bound SSAO.

Amine Oxidase (Copper-Containing)↗
PubMed2000

Neuroprotective aspects of a novel MAO-B inhibitor PF9601N.

PF9601N is an acetylenic tryptamine derivative devoid of amphetamine-like properties, that behaves as suicide MAO-B inhibitor more potent than l-deprenyl. It is highly selective towards MAO-B and it neuroprotects from the neurotoxicity induced in C57Bl/6 adult mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PF9601N also shows in vitro antioxidant properties by inhibiting the dopamine autoxidation. A potential therapeutic use in Parkinson's disease treatment is proposed for this compound.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Involvement of Ca2+ in dopamine release in striatal rat slices by PF9601N and L-deprenyl.

Rat striatal slices were incubated 1 hour in Krebs buffer with carbogen by continuous perfusion. When both MAO-B inhibitors PF9601N and l-deprenyl were added in the incubation medium, at 200 microM concentration, a diminution in the dopamine content was observed. The decrease in dopamine content was partially calcium dependent and showed a different mechanism between both compounds. When 1 mM concentration EGTA was added at the Krebs incubation medium without calcium, dopamine content was partially recovered, being not affected in case of l-deprenyl. When 50 microM dantrolene (a calcium vesicular release inhibitor) was added in the Krebs incubation medium without calcium, dopamine was partially recovered for l-deprenyl, being not affected in case of PF9601N. These data indicate that dopamine release is extracellular calcium dependent in case of PF9601N, whereas l-deprenyl depends on calcium intracellular origin.

Animals↗
PubMed1999

Relevance of benzyloxy group in 2-indolyl methylamines in the selective MAO-B inhibition.

1. Previous studies with indolyl derivatives as monoamine oxidase (MAO) inhibitors have shown the relevance of the indole structure for recognition by the active site of this enzyme. We now report a new series of molecules with structural features which determine the selectivity of MAO inhibition. 2. A benzyloxy group attached at position 5 of the indole ring is critical for this selective behaviour. Amongst all of these benzyloxy-indolyl methylamines, N-(2-propynyl)-2-(5-benzyloxyindol)methylamine FA-73 was the most potent MAO-B 'suicide' inhibitor studied. 3. The Ki values for MAO-A and MAO-B were 800+/-60 and 0.75+/-0.15 nM, respectively. These data represent a selectivity value of 1066 for MAO-B, being 48 times more selective than L-deprenyl (Ki values of 376+/-0.032 and 16.8+/-0.1 nM for MAO A and MAO-B, respectively). The IC50 values for dopamine uptake in striatal synaptosomal fractions from rats were 150+/-8 microM for FA-73 and 68 +/- 10 microM for L-deprenyl whereas in human caudate tissue the IC50 values were 0.36+/-0.015 microM for FA-73 and 0.10+/-0.007 microM for L-deprenyl. Moreover, mouse brain MAO-B activity was 90% ex vivo inhibited by both compounds 1 h after 4 mg kg(-1) administration, MAO-A activity was not affected. 4. These novel molecules should provide a better understanding of the active site of monoamine oxidase and could be the starting point for the design of further selective, non-amphetamine-like MAO-B inhibitors with therapeutic potential for the treatment of neurological disorders.

Animals↗
PubMed1999

Presence of SSAO in human and bovine meninges and microvessels.

In spite that SSAO enzyme is widely distributed in almost all tissues, specially in vascularized ones, its presence in brain microvessels is still controversy. Our results resolve this question showing that both human and bovine cerebrovascular tissues do contain the SSAO enzyme. This was achieved biochemically, using benzylamine and methylamine as substrates, and by immunoblot analysis, using polyclonal antibodies anti-SSAO that recognized a 100 kDa single band in tissue homogenates.

Amine Oxidase (Copper-Containing)↗