Serotonin concentrations in platelets, plasma, mitral valve leaflet, and left ventricular myocardial tissue in dogs with myxomatous mitral valve disease.

HYPOTHESIS/OBJECTIVES: Altered serotonin (5-hydroxytryptamine, 5HT) signaling is postulated in development and progression of canine myxomatous mitral valve disease (MMVD). Little is known regarding platelet, plasma, valvular, or myocardial 5HT concentration ([5HT]) in affected dogs. We quantified [5HT] in platelet-rich plasma (PRP), platelet-poor plasma (PPP), mitral valve leaflets (MV), and left ventricular myocardium (LV). ANIMALS: Forty-five dogs comprised 4 plasma groups of Cavalier King Charles Spaniels (CKCS) or non-CKCS, either healthy (CON) or MMVD affected: CKCS CON (n = 12); non-CKCS CON (n = 8); CKCS MMVD (n = 14); non-CKCS MMVD (n = 11). Twenty-four dogs comprised 3 tissue groups: MMVD (n = 8); other-HD (heart disease) (n = 7); non-HD, extracardiac disease (n = 9). METHODS: High-performance liquid chromatography measured PRP, PPP, MV, and LV [5HT]. RESULTS: Platelet-rich plasma platelet [5HT] was greater in CKCS CON (1.83 femtograms/platelet [fg/plt]; range, 0.20-4.76; P = .002), CKCS MMVD (1.58 fg/plt; range, 0.70-4.03; P = .005), and non-CKCS MMVD (1.72 fg/plt; range, 0.85-4.44; P = .003) versus non-CKCS CON (0.92 fg/plt; range, 0.63-1.30). There was no group difference in PPP [5HT]. MV [5HT] was significantly higher in MMVD (32.4 ng/mg; range, 8.4-106.7) versus non-HD (3.6 ng/mg; range, 0-28.3; P = .01) and LV [5HT] was significantly higher in MMVD (11.9 ng/mg; range, 4.0-104.8) versus other-HD (0.9 ng/mg; range, 0-10.1; P = .011) and non-HD (2.5 ng/mg; range, 0-6.9; P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Platelet [5HT] was highest in healthy CKCS and both MMVD groups, but plasma [5HT] showed no group differences. Tissue [5HT] was highest in MV and LV of MMVD-affected dogs, suggesting altered 5HT signaling as a potential feature of MMVD. Interactions of platelet, valvular, and myocardial 5HT signaling warrant further investigation.

First evidence of increased plasma serotonin levels in Tako-Tsubo cardiomyopathy.

BACKGROUND: There is no data about the serotonergic activity during the acute phase of Tako-Tsubo Cardiomyopathy (TTC). The objective of our study was to investigate evidence of serotonin release from patients with TTC in comparison with patients with ST elevation myocardial infarction (STEMI) and healthy control subjects (HCS). METHODS AND RESULTS: Plasma serotonin levels in 14 consecutive patients with TTC were compared with those in 14 patients with STEMI and 14 HCS. Plasma serotonin levels at admission were markedly higher in patients with TTC and STEMI as compared to HCS (3.9 ± 4.6, P = 0.02 versus control; 5.7 ± 5.6, P = 0.001 versus control; and 1 ± 0.4 ng/mL, resp.). There was no difference in serotonin levels between patients with TTC and those with STEMI (P = 0.33). CONCLUSION: This finding suggests that serotonin could participate to the pathophysiology of TTC.

Dose-dependent activation of distinct hypertrophic pathways by serotonin in cardiac cells.

There is substantial evidence supporting a hypertrophic action of serotonin [5-hydroxytryptamine (5-HT)] in cardiomyocytes. However, little is known about the mechanisms involved. We previously demonstrated that 5-HT-induced hypertrophy depends, in part, on the generation of reactive oxygen species by monoamine oxidase-A (MAO-A) (see Ref. 3). Cardiomyocytes express 5-HT(2) receptors, which may also participate in hypertrophy. Here, we analyzed the respective contribution of 5-HT(2) receptors and MAO-A in H9C2 cardiomyoblast hypertrophy. 5-HT induced a dose-dependent increase in [(3)H]leucine incorporation and stimulation of two markers of cardiac hypertrophy, ANF-luc and alphaSK-actin-luc reporter genes. Experiments using 1 microM 5-HT showed that hypertrophic response occurred independently from MAO-A. Using pharmacological inhibitors (M100907 and ketanserin), we identified a novel mechanism of action involving 5-HT(2A) receptors and requiring Ca(2+)/calcineurin/nuclear factor of activated T-cell activation. The activation of this hypertrophic pathway was fully prevented by 5-HT(2A) inhibitors and was unaffected by MAO inhibition. When 10 microM 5-HT was used, an additional hypertrophic response, prevented by the MAO inhibitors pargyline and RO 41-1049, was observed. Unlike the 5-HT(2A)-receptor-mediated H9C2 cell hypertrophy, MAO-A-dependent hypertrophic response required activation of extracellular-regulated kinases. In conclusion, our results show the existence of a dose-dependent shift of activation of distinct intracellular pathways involved in 5-HT-mediated hypertrophy of cardiac cells.

Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains.

AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.

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.

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.

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.

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.

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.

Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 imidazoline ligands in intact cells.

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.

I2-imidazoline binding sites and monoamine oxidase activity in human postmortem brain from patients with Parkinson's disease.

I2-imidazoline binding site (I2BS) has been identified with a regulatory site located on a sub-population of monoamine oxidase (MAO)-A and -B. Previous studies showed a modification of MAO and I2BS in the elderly and in neurodegenerative processes such as Alzheimer's disease. In the present study, we studied the potential modification of I2 binding sites and monoamine oxidases in Parkinson's disease. Putamen and cerebral cortex were collected from 17 normal subjects (79 +/- 12 yr) and 16 patients (76 +/- 9 yr) affected by Parkinson's disease. In mitochondrial preparations, radioligand binding studies with [3H]idazoxan showed that putamen and frontal cortex express equivalent amount of I2BS. The density and affinity of I2BS were similar in normal subjects (putamen: Bmax = 207 +/- 58 fmol/mg of protein, Kd = 10.1 +/- 3.4 nM; cerebral cortex: Bmax = 193 +/- 54 fmol/mg of protein, Kd = 12.8 +/- 6.8 nM) and Parkinson's disease patients (putamen: Bmax = 193 +/- 60 fmol/mg of protein, Kd = 9.8 +/- 4.6 nM; cerebral cortex: Bmax = 199 +/- 49 fmol/mg of protein, Kd = 15.9 +/- 8.1 nM). The activity of total monoamine oxidase and monoamine oxidase B, measured by [14C]tyramine and [14C]phenylethylamine oxidation, respectively, were higher in putamen than in cerebral cortex. No differences have been detected in the enzyme activity between normal and pathological subjects. These data suggest that, although MAO and I2BS may play a role in the development of Parkinson's disease, they are not altered in the chronic phase of this disease.

The elusive family of imidazoline binding sites.

Various imidazoline and guanidinium derivatives elicit diverse cellular responses in peripheral and nervous tissues that are often difficult to attribute to known receptor signalling systems. Biochemical, functional and clinical evidence suggests that some activities of these compounds may be related to their action on defined imidazoline binding sites, which have been recently characterized. Unexpectedly, and of particular significance, recent data indicate that two members of the family of imidazoline binding sites are identical to the A and B isoforms of monoamine oxidase. In this article, Angelo Parini and colleagues summarize the evidence for the characterization and location of imidazoline binding sites, and speculate on the clinical implications of compounds acting on these sites.

[Differential effect of estrogens in target tissues]. / Action différentielle des oestrogènes selon les tissus cibles.

Using ER 1D5 new antibody it is shown that the estrogen receptor has an extra-nuclear localisation in vascular cells. This work exhibits, moreover, that the polymorphism of the estrogen receptor is in part regulated at the translation step.