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Biomedical subjects

A Parini

Publications and source records attributed to A Parini.

At least 19 recordsLinked to original sources
PubMed2005

[Cardiac cellular therapy: from cells to the first clinical uses].

Despite the improvement in revascularisation techniques, coronary artery disease remains the principal aetiology of cardiac failure in developed countries. The therapeutic management of cardiac failure has been improved over recent years, yet cardiac failure is still associated with significant morbidity and mortality. As cardiac transplantation lacks donors, techniques that allow myocardial regeneration represent an attractive alternative. To date, several types of cells are under study and are suitable for implantation into infarcted myocardium (myoblasts, medullary stem cells...). Following good preclinical study results, the first human cell therapy trials, using the intramyocardial route, have begun, in the course of aorto-coronary bypass surgery in patients with chronic ischaemic cardiopathy and little altered left ventricular function, and then in those with ventricular dysfunction. Different modes of administration of these cell therapy products are under study and could be envisaged in clinical situations such as just after infarction in order to improve ventricular remodelling with an intracoronary injection technique. As for every new treatment, there are numerous problems to resolve, from understanding the relevant mechanisms of cellular transplantation, to the secondary effects that it could entail. Nevertheless, cardiac cellular transplantation is expanding rapidly and with the evolution of techniques it allows a glimpse of a new field of treatment for cardiac failure.

Cell Transplantation↗
PubMed2003

Differential substrate specificity of monoamine oxidase in the rat heart and renal cortex.

Although it is known that substrate specificities differ with species and within each species with the tissues, in the rat heart no natural substrate was found for MAO-B. beta-phenylethylamine (beta-PEA) has always been considered the "endogenous" substrate of MAO B. We thought worthwide to evaluate the effect of Ro 41-1049 and lazabemide, both members of a class of highly selective, mechanism-based and reversible inhibitors for MAO-A and MAO B, respectively on the metabolization of beta-PEA by the rat heart. Also the lack of molecular data on rat heart MAOs, prompted us to better characterize rat heart MAOs, both kinetically and using molecular biology techniques. K(m) values for deamination of beta-PEA in the rat heart were 13-fold those in the kidney, by contrast, K(m) values for deamination of 5-HT were quite similar in both tissues. Unexpectedly, the selective MAO-A inhibitor Ro 41-1049 was by far the most potent inhibitor of beta-PEA (20 microM) deamination in the rat heart, while clorgyline, another MAO A inhibitor, and lazabemide, a MAO B inhibitor, had intermediate efficacy; selegiline was found unable to inhibit deamination of beta-PEA. In the rat renal cortex lazabemide and selegiline both inhibited beta-PEA deamination. The reduction of beta-PEA concentration to just 200 nM, the use of heart membranes instead of tissue homogenates or the use of heart membranes pre-treated with 1% digitonine failed to change this pattern of inhibition. Semicarbazide was found not to alter deamination of beta-PEA. Western blot showed the presence of both isoforms (55 kd and 61 kd) in the renal cortex. In the heart there was a predominance of the A form, the B form being undetected. The RT-PCR products for both MAO-A and MAO-B, were found to have the expected sizes. In conclusion, we found mRNA for MAO-B but were unable to detect the protein itself or its activity when using beta-PEA as the substrate.

Amine Oxidase (Copper-Containing)↗
PubMed2001

Monoamine oxidase in developing rat renal cortex: effect of dexamethasone treatment.

The expression of the biogenic amine degrading enzyme monoamine oxidases-A and -B depends on several factors including regional distribution, development and hormonal environment. In the present study, we investigated the expression of monoamine oxidases in developing kidney and their regulation by dexamethasone treatment. Immunoblots and enzyme assays, performed using [14C]5-hydroxytriptamine and [14C]beta-phenylethylamine as substrates for monoamine oxidases-A and -B, respectively, showed that monoamine oxidase-A is the isoenzyme largely predominant in 9-day-old rats renal cortex. Experiments performed in 5-week-old rats showed an increase in monoamine oxidase-B activity and a decrease in monoamine oxidase-A activity and substrate affinity. The changes of monoamine oxidase-A activity and affinity were mimicked by dexamethasone treatment (0.60 mg/kg body weight injected subcutaneously three times at intervals of 24 h) of 9-day-old rats. In contrast, dexamethasone administration induced a modification of monoamine oxidase-B activity opposite to that found between 9-day- and 5-week-old rats. Dexamethasone treatment did not modify immunoreactivity and mRNA corresponding to monoamine oxidases-A and -B indicating that changes of enzyme activities were unrelated to regulation of protein synthesis and mRNA turnover. These results show that monoamine oxidases-A and -B are differently expressed in developing renal cortex and are regulated by dexamethasone treatment.

Animals↗
PubMed2001

Dopamine induces ERK activation in renal epithelial cells through H2O2 produced by monoamine oxidase.

BACKGROUND: The rat renal proximal tubule cells contain a large amount of monoamine oxidase, which catalyzes the oxidative deamination of catecholamines such as dopamine (DA). The aim of this study is to investigate the potential role of hydrogen peroxide (H2O2) produced by monoamine oxidase (MAO) isoform on regulation of cell signaling and function. METHODS: Primary rat proximal tubular cells, which contain almost exclusively MAO-A, and human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA were treated with DA or tyramine in the presence or the absence of some inhibitors. Then, Shc protein tyrosine phosphorylation and extracellular-regulated kinase (ERK) activation were evaluated by immunoprecipitation/immunoblot analysis and cell proliferation by [3H]thymidine incorporation or cell counting. RESULTS: In rat proximal tubule cells, DA induced tyrosine phosphorylation of Shc, ERK activation, and a significant increase in DNA synthesis. The involvement of MAO-dependent H2O2 generation induced by DA (5 micromol/L) was supported by the demonstration that the DA effects were (1) fully prevented by cell pretreatment with the MAO inhibitor pargyline, the antioxydant N-acetylcysteine (NAC), and the DA uptake inhibitor GBR 12909; (2) not abrogated by the D1 and D2 receptor antagonists; (3) observed in HEK 293 MAO-B cells but not in HEK 293 wild-type cells, which do not express MAO; and (4) similar to those induced by another MAO substrate, tyramine. CONCLUSIONS: Taken together, these results show that in addition to the effects related to receptor stimulation, DA, and probably the other catecholamines, may induce some of its effects through the MAO-dependent H2O2 production.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Hydrogen peroxide generation by monoamine oxidases in rat white adipocytes: role on cAMP production.

In rat, white adipocytes monoamine oxidases (EC 1.4.3.4.) generate hydrogen peroxide (H(2)O(2)). Recent studies suggested that, in addition to its toxic features, H(2)O(2) may behave as a cell second messenger. In the present study, using fluorimetric and chemiluminescence (CL) assays, we showed that tyramine degradation by monoamine oxidases in intact adipocytes resulted in the concentration-dependent generation of H(2)O(2). In addition, we found that, in the presence of low tyramine concentrations, forskolin-dependent cAMP production was significantly increased as compared to that of the control and this increase was prevented by the monoamine oxidase inhibitor pargyline or by the H(2)O(2) trapping system homovanillic acid-peroxidase. Finally, we demonstrated that tyramine degradation by monoamine oxidases increased the ability of isoproterenol to induce cell lipolysis. Taken together, these data suggest that H(2)O(2) produced during substrate degradation by monoamine oxidases may participate in the regulation of adipocyte metabolism.

Adipocytes↗
PubMed2000

Monoamine oxidase B induces ERK-dependent cell mitogenesis by hydrogen peroxide generation.

The mitochondrial enzyme monoamine oxidase (MAO) A and B catalyze the oxidative deamination of various endogenous and exogenous biogenic amines. In the present study, we used human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA to investigate the potential role of hydrogen peroxide (H(2)O(2)) produced by MAO-B isoform as an intracellular messenger involved in regulation of cell signaling and function. The MAO substrate tyramine induced tyrosine phosphorylation of Shc, ERK activation, and an increase in DNA synthesis in HEK 293 expressing MAO-B, but not in wild type HEK 293 cells, which do not express MAO. Tyramine effects were fully prevented by cell pretreatment with the MAO inhibitor pargyline or the antioxidant N-acetylcysteine. These results show that MAO-B induces MAPK/ERK activation and cell mitogenesis through H(2)O(2) production.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Analysis of the pharmacological and molecular heterogeneity of I(2)-imidazoline-binding proteins using monoamine oxidase-deficient mouse models.

The I(2) subgroup of imidazoline-binding sites was identified as monoamine oxidases (MAOs), but it is unclear whether there are I(2)-binding sites located on proteins distinct from MAOs. To address this issue, we characterized I(2)-binding proteins in liver and brain of wild-type and MAO A- and MAO B-deficient mice. I(2)-binding sites were identified using [(3)H]idazoxan and the photoaffinity adduct 2-[3-azido-4-[(125)I]iodophenoxyl]methylimidazoline ([(125)I]AZIPI). [(3)H]Idazoxan labeled binding sites with ligand recognition properties typical of I(2) sites in both brain and liver of wild-type mice. High-affinity, specific [(3)H]idazoxan binding were not altered in MAO A knockout (KO) mice. In contrast, [(3)H]idazoxan binding was completely abolished in both liver and brain of MAO B KO mice. In wild-type mice, [(125)I]AZIPI photolabeled three proteins with apparent molecular masses of approximately 28 (liver), approximately 61 (brain), and approximately 55 kDa (liver and brain). The photolabeling of each protein was blocked by the imidazoline cirazoline (10 microM). Photolabeling of the approximately 61- and approximately 55-kDa proteins was not observed in MAO A and B KO mice, respectively. In contrast, photolabeling of the liver approximately 28-kDa protein was still observed in MAO-deficient mice, indicating that this protein is unrelated to MAOs. These data indicate that I(2) imidazoline-binding sites identified by [(3)H]idazoxan reside solely on MAO B. The binding sites on MAO A and the liver approximately 28-kDa protein may represent additional subtypes of the family of the imidazoline-binding sites.

Adrenergic alpha-Antagonists↗
PubMed2000

Imidazoline-binding domains on monoamine oxidase B and subpopulations of enzyme.

A series of phenoxy-substituted methylimidazoline derivatives were synthesized and used to define the ligand recognition properties of the imidazoline-binding domain (IBD) on monoamine oxidase (MAO)-B and its role in substrate processing. The rank order of potency for selected compounds in competitive binding studies with the imidazoline [(3)H]idazoxan was different from that in enzyme activity assays, suggesting that the IBD and the site involved in enzyme inhibition are distinct. IC(50) values for inhibition of MAO-B activity by imidazoline/guanidinium ligands were one to two orders of magnitude greater than ligand concentrations that probably saturate the IBD, but were equal to the K(d) values of these ligands in competitive binding assays with the reversible MAO-B inhibitor [(3)H]Ro 19-6327. In addition, the degree of enzyme inhibition by these ligands was similar in platelet and liver, tissues exhibiting 10-fold differences in the amount of the IBD-accessible enzyme subpopulation. These data suggested that the inhibitory effect of these compounds on MAO-B activity involved a secondary interaction with the enzyme domain recognizing the inhibitor Ro 19-6327 and does not involve interaction with the IBD. Subsequent radioligand-binding studies indicated that human liver MAO-B actually existed as two distinct populations that differed in the accessibility of their IBD. The relatively small amounts of MAO-B possessing an accessible IBD ( approximately 5% in human liver) precludes determination of the functional consequences of ligand binding to the IBD. This subpopulation of MAO-B may be selectively regulated or generated in different individuals or tissues and targeted by pharmacologically active compounds in a cell type-specific manner.

Binding Sites↗
PubMed1999

High expression of monoamine oxidases in human white adipose tissue: evidence for their involvement in noradrenaline clearance.

The clearance of plasma adrenaline and noradrenaline by human adipose tissue suggests the expression of the catecholamine-degrading enzyme monoamine oxidases and of catecholamine transport systems in adipocytes. In the present study, we identified and characterized the monoamine oxidases and an extraneuronal noradrenaline transporter expressed in human adipocytes. Enzyme assays using the monoamine oxidase A/B substrate [14C]tyramine showed that abdominal and mammary human adipocytes contain one of the highest monoamine oxidase activities in the body. Characterization of the enzyme isoforms by inhibition profiles of [14C]tyramine oxidation and Western and Northern blot analyses showed that mRNAs and proteins related to both monoamine oxidases A and B were expressed in adipocytes. Quantification of each enzyme isoform performed by enzyme assay and Western blot showed that monoamine oxidase A was predominant, representing 70-80% of the total enzyme activity. In uptake experiments, the monoamine oxidase substrate [3H]noradrenaline was transported into white adipocytes (Vmax 0.81+/-0.3 nmol/30 min/100 mg of lipid, Km 235+/-104 microM). The inhibition of [3H]noradrenaline uptake by specific inhibitors indicated that white human adipocytes contain an extraneuronal-type noradrenaline transporter. Competition studies of [14C]tyramine oxidation showed that noradrenaline is metabolized by monoamine oxidases in intact cells. In conclusion, the concomitant expression of monoamine oxidases and of a noradrenaline transporter in human white adipocytes supports the role of the adipose tissue in the clearance of peripheral catecholamines. These results suggest that adipocytes should be considered as a previously unknown potential target of drugs acting on monoamine oxidases and noradrenaline transporters.

Adipose Tissue↗
PubMed1999

Relationship between I2 imidazoline binding sites and monoamine oxidase B in liver.

Biochemical and pharmacologic studies suggest that I2 imidazoline binding sites (I2BS) represent a heterogeneous family of membrane proteins. Indeed, the imidazoline binding sites located on monoamine oxidases (MAO) A and B display different pharmacologic properties. Recent results suggest that in liver and brain, I2BS may be located on proteins distinct from MAOs. The following observations indicate that in liver and brain, [3H]idazoxan binds exclusively to I2BS located on MAO-B: (1) size exclusion chromatography of digitonin-solubilized preparations from rabbit and human liver showed that [3H]idazoxan-specific binding eluted only in two peaks (approximately 175,000 and approximately 100,000 Da, corresponding to 90% and 10% of the recovered [3H]idazoxan binding) which also contained MAOs as determined by [14C]tyramine oxidation and Western blot analysis; (2) according to previous results obtained in various human and rat tissues, experiments performed in mice liver and brain showed that idazoxan was a potent inhibitor of [125I]-AZIPI photoincorporation to MAO-B but not to MAO-A; (3) in MAO-deficient transgenic mice, [3H]idazoxan binding to liver and brain membranes was completely abolished in MAO-B knockout mice and was not affected in MAO-A knockout mice. Together, these results show that in both liver and brain, I2BS are located exclusively on MAO-B. The imidazoline binding site on MAO-A, which photoincorporates [125I]-AZIPI and displays a low affinity for idazoxan, may not belong to the family of the I2 imidazoline binding sites.

Animals↗
PubMed1999

Reactive oxygen species production by monoamine oxidases in intact cells.

Monoamine oxidase (MAO) A and B are mitochondrial enzymes involved in the oxidative deamination of endogenous and exogenous amines. At present, the production of H2O2 by MAO in intact cells and its functional consequences in cell function have not been extensively investigated. The aim of this study was to define whether, in intact cells, the metabolism of small amounts of MAO substrates was able to induce a detectable H2O2 production. Hydrogen peroxide production was measured using a luminol-amplified chemiluminescence assay in three cell types, rat mesangial cells, rabbit proximal tubule cells and Hep-G2 cells, containing different MAO A/MAO B ratios. Our results showed that cell incubation with tyramine (50 micromol/l) led to a time-dependent H2O2 generation which was fully inhibited by MAO A (clorgyline and RO 41-1049) and MAO B (selegiline and RO 19-6327) inhibitors. The extent of inhibition of H2O2 production by selective inhibitors was in agreement with the amount of MAO isoforms expressed in each cell type, as determined by Western blot analysis and enzyme assay. Altogether, these findings show that, in a normal cell environment, MAO can be a source of reactive oxygen species which could have a functional impact on cell functions. In addition, we propose the luminol-amplified chemiluminescence assay as a rapid and sensitive procedure to characterize the monoamine oxidase isoforms and their regulation in intact cells.

Animals↗
PubMed1999

Characterization of monoamine oxidase isoforms in human islets of Langerhans.

In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms A & B are present in human pancreas. Two complementary approaches indicated that both MAO A and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO A (approximately 61 kDa) and B (approximately 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO A and B in islets could be the first step toward the characterization of the functional properties of these enzymes in the endocrine pancreas.

Blotting, Western↗
PubMed1999

Serotonin metabolism in rat mesangial cells: involvement of a serotonin transporter and monoamine oxidase A.

BACKGROUND: Serotonin is one of the factors regulating mesangial cell proliferation, and convergent evidence supports its involvement in the development of glomerulonephritis. In this study, we identified a serotonin transporter and the amine-degrading enzyme monoamine oxidases (MAOs) in mesangial cells, and we studied their involvement in serotonin degradation. METHODS: MAOs were characterized in membrane preparations and intact mesangial cells by enzyme assay using [14C]5-hydroxytryptamine and [14C]beta-phenylethylamine as specific substrates for MAO-A and MAO-B, respectively, and by Western blot analysis. The expression of a serotonin transporter was determined by [14C]5-hydroxytryptamine uptake experiments and Western blot. Mesangial cell proliferation was measured by BrdU incorporation. RESULTS: Quantitation of the MAO isoforms by enzyme assay and Western blot analysis showed that MAO-A was largely predominant in mesangial cells, accounting for approximately 90% of the total enzyme population. The MAO substrate [14C]serotonin was transported into mesangial cells by a saturable uptake system (Vmax 310 +/- 36 pmol/30 min/mg protein; Km 5.9 +/- 1.4 microM) displaying the pharmacological properties of a serotonin transporter. The expression of a serotonin transporter was confirmed by Western blot analysis. MAO activity measured in intact cells showed that after accumulation into mesangial cells, [14C]serotonin was metabolized by MAO-A. Finally, serotonin-mediated mesangial cell proliferation was significantly increased after irreversible MAO inhibition. CONCLUSIONS: Our results suggest that serotonin concentration and function in glomeruli may be regulated in part by its transport into mesangial cells and degradation by MAO-A.

Amino Acid Sequence↗
PubMed1998

Characterization of [3H]idazoxan binding proteins in solubilized membranes from rabbit and human liver.

Several studies have shown that I2 imidazoline binding sites are localized on monoamine oxidases. Recent results obtained after solubilization of rat brain membranes and analysis by size-exclusion chromatography suggested the existence of additional I2 imidazoline binding sites located on proteins distinct from monoamine oxidases. In order to define whether such binding sites are expressed in human and rabbit liver, we solubilized I2 imidazoline binding sites and monoamine oxidases and compared their elution profile by size-exclusion chromatography. I2 binding sites were labeled using [3H]idazoxan. Monoamine oxidases were identified by the measure of [14C]tyramine oxidation and Western blot analysis using an anti-MAO-A/MAO-B polyclonal antiserum. After solubilization of rabbit or human liver using 1% digitonin, 90% of [3H]idazoxan binding eluted in a major peak corresponding to a Mr of approximately 175000 Da. A minor peak, (Mr approximately equal to 100000 Da) representing 10% of the recovered [3H]idazoxan binding, was also observed. [14C]tyramine oxidation as well as immunoreactive bands corresponding to MAOs were exclusively detected in fractions containing [3H]idazoxan binding. These results show that solubilized I2 imidazoline binding sites distinct from monoamine oxidases are not detectable in rabbit and human liver.

Adrenergic alpha-Antagonists↗
PubMed1998

The renal monoamine oxidases: pathophysiology and targets for therapeutic intervention.

The mitochondrial enzyme monoamine oxidases A and B are among the major metabolic agents for the degradation of the biogenic amines adrenaline, noradrenaline, dopamine and serotonin. The fact that the kidney contains a large amount of monoamine oxidase suggests that the renal effects of biogenic amines might depend in part on the activity of these enzymes.

Animals↗