R, Murphy D. L, McGinniss M. Hum Genet 34 , — Biochem Biophys. H Evidence for a specific monoamme oxidase associated with sympathetic nerves Neuropharmacology 10 , — Graham R C. J The histochemlcal demonstration of monoamine oxidase activity by coupled peroxidatic oxidation.
Histochem Cytochem 13 , — Green A. M A colorimetric method for the estimation of monoamme oxidase. Biochem J 78 , — Guha S. R Purification and solubilization of monoamine oxidase of rat liver mitochondria Biochem Biophys Res.
Anal Chem. Substrate specificity in various mammalian species Biochem Pharmacol 18 , — Hanker J. S, Kusuk C J. E, and Pearse A G E. Histochemie 33 , — Harada M. New enzyme in liver. Biochem J. Harris E. Neurochem 38 , — Hidaka H. Tokyo , 62 , — Horita A. Phar acol. Houslay M. Biochem Pharmacol 29 , — F b The nature of the electrophoretitally separable multiple forms of rat liver monoamine oxidase. Huang R. Hussein L. Jarrott B. J, Neurochem. Kan J.
Kawai S. Kamilo K. Kim H. J Bzochem. Kishimoto S. Kline N. Psychopathol 19 , Suppl. Knoll J. Advances in Biocheical Psychopharmacology , Costa E. Kobes R. Methods 3 , — Kochli H. Kraml M A rapid microfluorometric determination of monoamine oxrdase Biochem. Res Commun. Kupfer D. Psychiatry , — La Motte R. H, Schmidt D. Littlewood J. Psychiatry 47 , — Loomer H.
C, and Kline N. Psychlaty Assn. Psychzatric Res. Ther , 7— Lowe M C, Reichenback D. D, and Horita A Extraneuronal monoamine oxidase in rat heart Biochemical characterization and electron microscopic locahzation. J Phurmacol Exp. Ther , — Mason W D and Olson C. L Differential amperometric measurement of monoamme oxidase activity at tubular carbon electrode, Anal Chem.
May M. E A caution in the use of tritlated substrates for monoamine oxldase assays J, Neurochem. McCaman R E. A substrate for aromatic L-amino acid decarboxylase liberating an enzyme-activated irreversible inhibitor of monoamine oxidase. J, Am. Soc , — McEwen C. D An amine oxldase in normal human serum.
J Lab Clin. Med 62 , — C Human liver mitochondnal monoamine oxldase. Determinants of substrate and inhibitor speaflaties. Blochemisfry 8 , — Mendlewlcz J. H L-Deprenil, a selective monoamine oxidase type B inhibitor in the treatment of depression: A double blind evaluation. Meyerson L. Anal Biochem. Murphy D. H, and Richelson E Substrate-and inhibitor-related characteristics of monoamine oxidase in C6 rat glial cells. Nagatzu T. Tokyo 59 , — Tokyo 60 , — Nimmo G.
Nissinen E. Nohta H. Obata F. Tokyo 69 , — Otsuka S. The constitution of vallesiachotamine. Therefore, vallesiachotamine-like alkaloids can be easily detected in alkaloid fractions by LC-DAD, based also on previous knowledge of the species or genus. This MIA had already been isolated from several different families, including Apocynaceae Evans et al. The alkaloids of Rhazya orientalis.
Phytochemistry 7, Cheng et al. Indole alkaloids from cultivated Vinca major. Tetrahedron 70, Cytotoxic 7S-oxindole alkaloids from Gardneria multiflora. Ethnobotany, phytochemistry and pharmacology of Uncaria Rubiaceae. Phytochemistry 66, Specifically in Rubiaceae family, this alkaloid had already been described for several species of Palicoureeae Robbr.
Indole alkaloids and semisynthetic indole derivatives as multifunctional scaffolds aiming the inhibition of enzymes related to neurodegenerative diseases - a focus on Psychotria L. Solis et al. Alkaloids from Cephaelis dichroa. Phytochemistry 33, Novel Bis monoterpenoid indole alkaloids from Psychotria bahiensis.
Borhidi Berger et al. In vitro antiproliferative effects of the indole alkaloid vallesiachotamine on human melanoma cells. Passos et al. Monoamine oxidase inhibition by monoterpene indole alkaloids and fractions from Psychotria suterella and Psychotria laciniata. Enzyme Inhib. The pharmacological potential of vallesiachotamine was also reported.
Shang et al. Pharmacological evaluation of Alstonia scholaris: anti-inflammatory and analgesic effects. The authors concluded that the cytotoxicity observed for this compound is mainly due to apoptosis and necrosis processes Soares et al. In addition to the reported activities, different central effects were also described for Z -vallesiachotamine, including butyrylcholinesterase BChE and monoamine oxidase-A MAO-A inhibitory activity Passos et al.
Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors. Phytochemistry 86, Alkaloids from Psychotria target sirtuins: in silico and in vitro interaction studies. Planta Med. COMT is a methyltransferase S-adenosylmethionine-dependent responsible for catalyzing the methylation of catecholamines, such as dopamine, leading to their inactivation. The MB-COMT has a high affinity for catecholamine neurotransmitters, being found in neurons and glial cells of rat brains Shirakawa et al.
Neuronal expression of the catechol-O-methyltransferase nRNA in neonatal rat suprachiasmatic nucleus. Neuroreport 15, However, little is known regarding the regional distribution and cellular localization of COMT in the human brain.
Kastner et al. Gene regulation by hypoxia and the neurodevelopmental schizophrenia. Neuropsychopharmacology 28, COMT is the major enzyme responsible for dopamine degradation in the prefrontal cortex. The role of COMT in the dopaminergic transmission makes this enzyme an interesting target for the discovery of new substances for the treatment of diseases such as schizophrenia, obsessive-compulsive disorder, bipolar disorder, anxiety, and panic disorder Brisch et al.
Dopamine-glutamate abnormalities in the frontal cortex associated with the catechol-O-methyltransferase COMT in schizophrenia. Brain Res. Entacapone, a catechol-O-methyltransferase inhibitor, improves the motor activity and dopamine content of basal ganglia in a rat model of Parkinson's disease induced by Japanese encephalitis virus. Taking into account the already described BChE, MAO-A, and sirtuin inhibitory effects described for vallesiachotamine alkaloids, the present study aimed at developing and implementing a protocol for the evaluation of COMT activity suitable for the screening of plant extracts and isolated natural products.
Further, we used the optimized protocol to evaluate the potential effects of vallesiachotamine on COMT activity, and an in silico approach to elucidate the molecular mechanisms involved in the interactions between vallesiachotamine and COMT. Z -Vallesiachotamine was isolated from Psychotria laciniata Vell. Briefly, the dried leaves were extracted with EtOH at room temperature. The solvent was removed under vacuum and the residual extract dissolved in 1 N HCl and exhaustively extracted with CH 2 Cl 2 in order to remove non-polar constituents.
The COMT inhibitory activity was assessed based on methods previously described in the literature Kurkela et al. Microplate screening assay to identify inhibitors of human catechol-O-methyltransferase. B: Enzym. The general reaction is presented below:. For setting up the experimental conditions, different concentrations of the porcine COMT and of the methyl donor substrate, SAM, were tested.
COMT was evaluated in concentrations of 0. The concentration of the catechol substrate aesculetin was kept constant at 0. An incision was made in the skin to expose the skull, and then three holes with diameters of 0. The reference electrode was positioned superficially in the cortical tissue contralateral to the hemisphere where the CFM and stimulating electrode were implanted.
Electrode coordinates were defined by the rat brain atlas 35 based on the skull position using the bregma as a reference point with coordinates anteroposterior AP , mediolateral ML , and dorsoventral DV. CFMs were fabricated as previously described Louis, MO at one end. The silica tubing was then attached to a nitinol wire Nitinol 1, an alloy of nickel and titanium; Fort Wayne Metals, IN with a commercial silver conductive paste Sigma-Aldrich, St.
The carbon fiber-attached nitinol wire was insulated with polyimide tubing 0. PEDOT:Nafion coating was applied to the exposed carbon fiber by electrical deposition with a mixture of poly 3,4-ethylenedioxythiophene and Nafion to increase their sensitivity and selectivity to DA and reduce in vivo biofouling Teflon-coated silver wire A-M Systems, Inc. FSCV was applied with a conventional triangular waveform — 0.
Before recording, the CFM and the electrical stimulating electrode were gradually adjusted until a robust phasic DA signal was detected. Before drug treatment, the carbon microelectrode was positioned and stabilized to the right recording site to optimize the DA signal.
We quantified the DA degradation kinetics with the exponential decay time rate constant: K , which was fitted to a one-phase exponential curve. M-CSWV was performed using the same equipment used in the FSCV experiment except for an in-house built current-to-voltage amplifier without an analog filter to preserve sharp current responses to the square pulse. Data processing included temporal averaging, filtering, and simulating background currents.
The basal DA response was extracted from the 2-dimensional voltammogram using the DA-kernel method. Five cyclic square waveforms, which consisted of a large-amplitude square wave modulation on top of a symmetric staircase waveform, were applied in sequence at a frequency of 0. The experimental protocol of basal-level monitoring was performed by the same protocol as the FSCV experiment.
In contrast to the FSCV experiment, however, rats were constantly measured without electrical stimulation. Pharmacological effects on tonic DA concentrations were calculated in a circular DA area extracted from the maximum amplitude of the pseudocolor plot. After incision, the skull was cleaned and leveled in the coronal and sagittal planes using the bregma and lambda as reference points. Using a dental drill, holes were made bilaterally in the skull targeting the striatum using the following coordinates 37 : AP, 0.
Virus injection was performed using a microinfusion pump Legato , KD Scientific at a rate of 0. After at least 2 weeks of recovery, the mice were used for experiments. The brain was quickly excised from the skull and submerged in ice-cold dissection buffer in mM : After the first electrical stimulation, we chose approximately ten different regions of interest ROIs that responded to stimulation. Before drug treatment, the baseline phasic DA release was measured by acquiring 3 successive similar levels of DA signals.
All experiments were performed in voltage-clamp mode. Whole-cell recordings were performed on striatal neurons located in the dorsal striatum.
Most of these recorded striatal neurons were medium spiny neurons by morphology. However, we did not further characterize them as D1-positive or D2-positive neurons.
The frequency and amplitude of spontaneous inhibitory postsynaptic currents sIPSCs before bicuculline administration were detected and analyzed by Template Search in Clampfit Data from multiple independent experiments were assumed to have normal variance. All data were tested to determine whether they were normally distributed. All raw data are presented as dot plots in each graph. The numbers of animals used are described in the corresponding figure legends or on each graph.
Experimental groups were balanced in terms of animal age, sex, and weight. The sample size was determined based on the expected difference from our pilot studies using similar protocols in animals without carrying out a formal power analysis. At least three independent repeats were performed for each experiment. No outliers were excluded. As previously defined 38 , the phasic DA level indicates the evoked synaptic dopamine concentration, while the basal DA level or tonic DA level indicates the steady-state level of extrasynaptic dopamine concentration, which is a combination of tonic DA neuronal activity and spontaneous phasic activity.
We first tested whether the pharmacological inhibition of the two isoenzymes differentially affected the phasic release of striatal DA by performing in vivo FSCV with electrical stimulation of the MFB of a rat We also confirmed that vehicle injection did not significantly alter the evoked DA current The time-series plot of each evoked DA response was extracted at the dopamine oxidation potential right. The black line and red line show the concentration change before and after drug injection, respectively.
Consistently, a previous report showed that DA clearance is mediated mostly by DA uptake through the DA transporter DAT , which is expressed mainly by neighboring DAergic neurons but minimally by astrocytes in vivo Another possibility is that the amount of DA released in clorgyline-treated animals may saturate DAT, thus widening the peak. These possibilities await future investigation. The amplitude of the electrical stimulation-evoked DA response was dependent on the intensity of the electrical stimulation Fig.
The dotted line indicates the level of phasic DA release before drug treatment. Due to the unstable nature of the background currents inherent in the FSCV technique, we should subtract the background signals, which limit the measurement of changes in basal DA levels.
The M-CSWV consisted of a square-wave oscillation superimposed on a symmetric staircase waveform that was applied consecutively multiple times Fig. The basal DA level, indicated by oxidation peaks seen in red in the region of interest marked with a circle in the range of 0.
Only the clorgiline-injected group showed a significant overall increase in basal DA concentration during the min recording compared with the saline-injected, KDSinjected, and even selegiline-injected groups Fig. The changes in tonic DA concentration changes were quantified right.
We have demonstrated that the inhibition of MAO-B does not affect either phasic or basal DA levels in rodents, which is inconsistent with the traditional belief. We recently demonstrated that striatal MAO-B is physiologically important for astrocytic GABA synthesis, which is the main cause of tonic inhibition of neighboring neurons Therefore, we performed a whole-cell patch-clamp recording of striatal neurons to record a tonic inhibition current in the presence of MAO-A and MAO-B inhibitors in rats Fig.
On the other hand, there was no significant difference in the amplitude and frequency of spontaneous inhibitory postsynaptic currents across the groups Fig. On the other hand, MAO-A inhibition by clorgiline significantly increased the levels of phasic and basal DA, which could be attributed to a blockade of DA metabolism.
Taken together, our findings provide conclusive in vivo evidence for resolving the controversy on the role of MAO-B in DA metabolism, which has continued over several decades. The development of specific inhibitors against MAO-A clorgiline and MAO-B selegiline in the s made it possible to investigate which is the major enzyme for DA metabolism between the two isoenzymes.
Through intense investigations on this issue in the s 13 , 14 , 15 , 16 , both MAO-A and MAO-B were reported to be responsible for DA metabolism in the human brain, but the contributions varied among brain regions However, direct evidence has been lacking. Human studies from postmortem brain homogenates have provided only indirect evidence. On the other hand, several in vivo microdialysis and high-performance liquid chromatography HPLC studies in live rodents cast doubt on the previous belief in the s that MAO-B was the key enzyme for DA degradation by demonstrating that the pharmacological inhibition of MAO-B minimally alters the basal DA concentration 5 , 18 , 19 , 20 , 21 , Although microdialysis and HPLC methods have advantages such as high chemical specificity 45 and sensitivity 46 , 47 , 48 for detecting DA, they have several critical drawbacks.
Second, it is difficult to study the mechanisms of fast synaptic release and the reuptake of DA with microdialysis, both of which critically affect the extracellular DA concentration 53 , These critical drawbacks have hindered widespread that MAO-B is less responsible for DA degradation, which is against the traditional belief. M-CSWV is a recently developed voltammetric technique to measure tonic DA concentration by using cyclic square wave voltammetric waveforms in conjunction with a delayed holding potential period to control DA adsorption to the CFME surface.
In addition to these electrochemical approaches, our ex vivo DA imaging with a newly developed DA sensor, GRAB DA2m , which yields a high spatiotemporal resolution, high specificity, and high sensitivity, demonstrated consistent results with the findings from FSCV experiments. Several previous studies have suggested that a possible mode of action of MAO-B inhibitors in PD patients involves the stabilization of mitochondria and the induction of the antiapoptotic Bcl-2 protein family and neurotrophic factors 58 , 59 , In addition to DA, other transmitters such as epinephrine, norepinephrine, and serotonin could be metabolized by MAO in the brain.
How should we resolve the discrepancy between the results from in vitro and in vivo studies regarding MAO-B? Because the level of DAT expression in striatal astrocytes is very low, the released DA is minimally taken up by astrocytes, in which MAO-B is mainly expressed under physiological conditions In addition to the striatum, DAT is expressed mainly in neurons but not in astrocytes in most brain regions, including the substantia nigra, nucleus accumbens, and cortical areas 63 , 64 , Shih, J.
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