Endogenous Monoamine Oxidase Research Articles

An (Immuno) Fluorescence Protocol for Monitoring Monoamine Oxidase A/B Protein Distribution Within the Cell.

The influence of a protein is not determined exclusively by its level of expression, but also by its localization within the cell. The literature often refers to the enzyme monoamine oxidase (MAO) as a mitochondrial enzyme, yet there is evidence that mitochondria-independent pools of MAO exist. These pools of MAO could exert distinct influences across physiological as well as pathological phenotypes. Fluorescence microscopy is a powerful tool for spatially resolving target proteins in cell and tissue preparations. This can rely on an antibody-based probe that targets the endogenous protein, e.g., immunofluorescence. In the event that antibodies might not be readily available or if one is interested in characterizing a variant of the wild-type protein, then a recombinant protein with a fluorescent fusion "tag" is preferred. We now describe a protocol for the detection of endogenous MAO using indirect immunofluorescence and a version of the protocol with minor modification for detecting (green) fluorescent protein-tagged MAOs. One observation we can highlight using these easily adaptable approaches is that MAO A and MAO B do not follow similar patterns of distribution throughout the cell, suggesting potential expression of MAO A and MAO B on distinct pools of mitochondria. Furthermore, distinct subcellular compartmentalization is suggested by the fact that a pool of MAO A, but not MAO B, is associated with certain lysosomal compartments. However, directed and quantitative studies will be required before any definitive statement can be made on these intriguing possibilities.

Resorufin Enters the Photodynamic Therapy Arena: A Monoamine Oxidase Activatable Agent for Selective Cytotoxicity.

A red-absorbing, water-soluble, and iodinated resorufin derivative (R1) that can be selectively activated with a monoamine oxidase (MAO) enzyme was synthesized, and its potential as a photodynamic therapy (PDT) agent was evaluated. R1 showed high 1O2 generation yields in aqueous solutions upon addition of MAO isoforms, and it was further tested in cell culture studies. R1 induced photocytotoxicity after being triggered by endogenous MAO enzyme in cancer cells with a much higher efficiency in SH-SY5Y neuroblastoma cells with high MAO-A expression. Additionally, R1 displayed differential cytotoxicity between cancer and normal cells, without any considerable dark toxicity. To the best of our knowledge, R1 marks the first example of a resorufin-based photosensitizer (PS) as well as the first anticancer drug that is activated by a MAO enzyme. Remarkably, the target PDT agent was obtained only in three steps as a result of versatile resorufin chemistry.

Detection of Lipase Activity in Cells by a Fluorescent Probe Based on Formation of Self-Assembled Micelles.

SummaryReliable and sensitive detection of lipase activity is essential for the early diagnosis and monitoring of acute pancreatitis or progression of digestive diseases. However, the available fluorescent probes for detection of lipase activity are only implemented in a hexane-water two-phase system due to the nature of heterogeneous catalysis of lipase, thus limiting their applications in direct imaging of lipase activity in living cells and tissues. Here we designed and synthesized a “turn on” fluorescent probe CPP based on self-assembled micelles for hydrolysis of lipase. The CPP probe exhibits high selectivity and excellent sensitivity for the detection of lipase in such a homogeneous system and is successfully applied for monitoring lipase activity in pancreatic AR42J cells, tissues, and serums. Taken together, the fluorescent CPP probe not only provides a tool for diagnostic potential in pancreatic disease but also demonstrates an application potential for micelle self-assembly-based development of biological probes.

Possible synergies between isatin, an endogenous MAO inhibitor, and antiparkinsonian agents on the dopamine release from striatum of freely moving rats.

Isatin is an endogenous indole that inhibits monoamine oxidase (MAO). When exogenously administered, it increases the striatal dopamine and acetylcholine levels and presents neuroprotective effects in the brain. Previous studies show that intrastriatal administration of isatin increased the in vivo dopamine release from striatum in a concentration-dependent form. In the present work, we investigated the effects of combined administration of isatin together with other substances actually used in antiparkinsonian pharmacotherapy on in vivo dopamine overflow. For this, we co-administered isatin with the MAO inhibitors selegiline and clorgyline, l-DOPA, the catechol-o-methyl-transferase (COMT) inhibitors tropolone and dinitrocatechol, with the dopaminergic agonist ropinirole, and with the psychostimulant caffeine, in order to evaluate possible synergies between these substances to increase the dopamine extracellular levels in freely moving rats. Intrastriatal administration of isatin (10 mM, 60 min) significantly increased dopamine release to 1164 ± 152%, compared to the baseline. Co-administration of isatin together with selegiline (1 mM) or clorgyline (1 mM) alone or in combinations showed a similar profile to increase in vivo dopamine release. Intrastriatal infusion of isatin together with antiparkinsonian drugs l-DOPA (25 μM), tropolone (1 mM), dinitrocatechol (100 μM), amantadine (5 mM) and caffeine (5 mM) significantly elevated extracellular dopamine levels more than any single drug, showing a good neurochemical synergy by improving the effect of isatin on the extracellular dopamine levels in the striatum. Infusion of isatin + ropinirole (5 mM) did not change the isatin-induced increase in dopamine overflow. These results could be useful to carry out further investigations with a possible clinical application.

Effect of monoamine oxidase inhibitors on ischaemia/reperfusion-induced acute kidney injury in rats

Increases in renal sympathetic nerve activity during ischaemia and renal venous norepinephrine levels after reperfusion play important roles in the development of ischaemia/reperfusion-induced acute kidney injury. In the present study, we examined the effect of isatin, an endogenous monoamine oxidase inhibitor, on renal venous norepinephrine levels, superoxide production after reperfusion, and ischaemia/reperfusion-induced acute kidney injury. Ischaemia/reperfusion-induced acute kidney injury was accomplished by clamping the left renal artery and vein for 45min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal superoxide production and norepinephrine overflow were elevated and significant renal tissue damage was observed following ischaemia/reperfusion injury. Intravenous injection of isatin (10mg/kg) at 5min before ischaemia increased the renal venous plasma norepinephrine level after reperfusion and aggravated ischaemia/reperfusion-induced renal dysfunction and histological damage. The excessive superoxide production after reperfusion was significantly suppressed by isatin administration, indicating that the inhibition of oxidative deamination effectively suppressed superoxide production. These data suggest that the exacerbation effect of isatin is associated, at least in part, with increased norepinephrine levels but not with superoxide production. To the best of our knowledge, this is the first report of isatin involvement in the pathogenesis and/or development of acute kidney injury.

Analysis of the serum isatin, an endogenous monoamine oxidase inhibitor, in aging and Alzheimer's disease

Background: Isatin, an endogenous monoamine oxidase (MAO)-A and -B inhibitor, distributes in all organs. Isatin makes monoamine in rat brain increased. MAO plays an important role in the metabolic regulation as metabolic enzyme of monoamine which is neurotransmitter substance. The MAO activity in both brain and platelet increase according to age. Several reports indicate that both A and B forms of MAO are altered in Alzheimer’s disease and Parkinson's disease.

The relation between isatin and selegiline by the alteration of serum isatin, an endogenous monoamine oxidase inhibitor, induced by selegiline hydrochloride in Parkinson's disease

Background: Monoamine oxidase (MAO) is an enzyme that metabolizes a wide range of monoamines and play an important role in the control of neurotransmitter concentrations. However, the detailed CNS functions of an endogenous MAO inhibitor, isatin, were not determined. Isatin was found to be a potent MAO inhibitor that is more active against MAO-B than MAO-A. Exogenously administered isatin increased dopamine (DA) levels in the rat striatum. Furthermore isatin improves motor dysfunction and the DA levels in the striatum in rat model of Parkinson’s disease(Ogata et al., 2003). We showed the low concentration in the serum isatin was found in Parkinson's disease patients as compared with controls.

Sensitive and Selective Ratiometric Fluorescence Probes for Detection of Intracellular Endogenous Monoamine Oxidase A.

Monoamine oxidase A (MAO-A) is known to widely exist in most cell lines in the body, and its dysfunction (unusually high or low levels of MAO-A) is thought to be responsible for several psychiatric and neurological disorders. Thus, a sensitive and selective method for evaluating the relative MAO-A levels in different live cells is urgently needed to better understand the function of MAO-A, but to our knowledge such a method is still lacking. Herein, we rationally design two new ratiometric fluorescence probes (1 and 2) that can sensitively and selectively detect MAO-A. The probes are constructed by incorporating a recognition group of propylamine into the fluorescent skeleton of 1,8-naphthalimide, and the detection mechanism is based on amine oxidation and β-elimination to release the fluorophore (4-hydroxy-N-butyl-1,8-naphthalimide), which is verified by HPLC analysis. Reaction of the probes with MAO-A produces a remarkable fluorescence change from blue to green, and the ratio of fluorescence intensity at 550 and 454 nm is directly proportional to the concentration of MAO-A in the ranges of 0.5-1.5 and 0.5-2.5 μg/mL with detection limits of 1.1 and 10 ng/mL (k = 3) for probes 1 and 2, respectively. Surprisingly, these probes show strong fluorescence responses to MAO-A but almost none to MAO-B (one of two isoforms of MAO), indicating superior ability to distinguish MAO-A from MAO-B. The high specificity of the probes for MAO-A over MAO-B is further supported by different inhibitor experiments. Moreover, probe 1 displays higher sensitivity than probe 2 and is thus investigated to image the relative MAO-A levels in different live cells, such as HeLa and NIH-3T3 cells. It is found that the concentration of endogenous MAO-A in HeLa cells is approximately 1.8 times higher than that in NIH-3T3 cells, which is validated by the result from an ELISA kit. Additionally, the proposed probes may find more uses in the specific detection of MAO-A between the two isoforms of MAO, thereby promoting our understanding of the behavior and function of MAO-A in living biosystems.

Therapeutic Potential of the Folkloric Medicinal Plant Rhazya stricta

Medicinal plants assumed principle role in folkloric medicine throughout history. They have been the subject of many recent studies for the evaluation of what have been ascribed to them of medicinal properties by means of modern techniques. For example Rhazya stricta decne of the Apocynaceae family is a widely distributed plant in Saudi Arabia. Extract of its leaves is prescribed in folkloric medicine for the treatment of various disorders such as diabetes, sore throat, helminthiasis, inflammatory conditions and rheumatism. The extract contains mainly alkaloids, glycosides, flavonoids, tannins and triterpenes. Several studies on rats and mice reported that the leaves extract causes sedation, analgesia, decreases motor activity and has anti-depressant, anti-oxidant activity, complex effect on brain endogenous monoamine oxidase activity and central–mediated hypotension. Moreover, some studies ascribed anticancer activities to indole alkaloids of Rhazya stricta. The genotoxicity of Rhazya stricta leaves was demonstrated by Baeshen and colleagues in a battery of tests. We also demonstrated various therapeutic properties of Rhazya stricta for the treatment of cancer, insulin insensitivity, MDRs (multi-drug resistant organisms), cardiovascular diseases, obesity and some other ailments. We are currently having collaboration with some international institutes in the United States and Europe for extensive studies on the whole genome of Rhazya stricta which may lead to future Natural Products Genomics and PDT (Phytodynamic Therapy).

1-Methyl-1,2,3,4-tetrahydroisoquinoline, an Endogenous Neuroprotectant and MAO Inhibitor with Antidepressant-Like Properties in the Rat

Oxidative stress is a major contributing factor in a range of brain pathologies and in the etiology of depression. 1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous substance which is present in the mammalian brain and exhibits neuroprotective, and monoamine oxidase (MAO)-inhibiting properties. In the present study, in order to investigate the potential role of 1MeTIQ as an antidepressant, we tested antidepressant-like effects of 1MeTIQ in comparison with desipramine (a classic antidepressant) in the forced swimming test (FST), and using HPLC methodology, we measured the concentrations of monoamines (dopamine, noradrenaline, serotonin) and the rate of their metabolism. 1MeTIQ given alone as well as in combination with desipramine produced an antidepressant-like effect and decreased the immobility time in the FST. Neurochemical data have shown that 1MeTIQ like desipramine, activated the noradrenergic system. However, the mechanism of action of 1MeTIQ is broader than the actions of desipramine, and 1MeTIQ inhibits the MAO-dependent oxidation of dopamine and serotonin in all investigated structures. We can conclude that 1MeTIQ exhibits antidepressant-like activity in the FST in the rat. The mechanism of its antidepressant action differs from desipramine and seems to be mostly associated with the inhibition of the catabolism of monoamines and their increased concentrations in the brain. 1MeTIQ seems to be very beneficial from the clinical point of view as a reversible MAO inhibitor with a significant antidepressant effects.

A review of phytochemistry, bioactivities and ethno medicinal uses of Rhazya stricta Decsne (Apocynaceae)

Rhazya stricta Decne is an important medicinal plant belonging to family Apocynaceae. Its chemical constituents, biological and pharmacological activities, and ethno medicinal uses have been reviewed in the present study. The reported phytochemical studies conducted on various parts of R. stricta plant revealed presence of over 100 alkaloids and few non-alkaloidal compounds. Most of the activities of the plant reside in its alkaloidal fractions. The compounds have been reported to exhibit ambulatory, antifungal, antimicrobial, antioxidant, endogenous monoamine oxidase (MAO) A and B inhibitory and herbicidal activities. They also showed effects on arachidonic acid (AA) metabolism, blood pressure, central nervous system (CNS), immunity, smooth muscles and glucose homeostasis. Different parts of the plant have been reported to be used in traditional medicine against various ailments such as diabetes, foot burning, skin diseases, stomach pain etc. But more work needs to be done regarding its medicinal importance (recording and exploring recipes) for the benei¬t of improving human health. As the plant of R. strictahas immense potential as an anti-microbial due to its richness of phytochemicals it possesses, therefore, such studies are recommended in various parts of the countries of Arabian Peninsula and the Indian subcontinent where it grows. Key words: Photochemistry, bioactivities, Rhazya stricta, apocynaceae.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for "genome editing" in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

Immunohistochemical detection of l-DOPA-derived dopamine within serotonergic fibers in the striatum and the substantia nigra pars reticulata in Parkinsonian model rats

On the basis of our previous studies in the normal rat [Arai, R., Karasawa, N., Geffard, M., Nagatsu, I., 1995. l-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study. Neurosci. Lett. 195, 195–198; Arai, R., Karasawa, N., Nagatsu, I., 1996a. Aromatic l-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study. Brain Res. 706, 177–179; Arai, R., Karasawa, N., Nagatsu, I., 1996b. Dopamine produced from l-DOPA is degraded by endogenous monoamine oxidase in neurons of the dorsal raphe nucleus of the rat: an immunohistochemical study. Brain Res. 722, 181–184] we have assumed that exogenously administered l-dihydroxyphenylalanine ( l-DOPA) is converted into dopamine (DA) in serotonergic (5-HT) fibers within the striatum (ST) and the substantia nigra pars reticulata (SNR). In the present study, an attempt was made to confirm the assumptions in Parkinsonian rats, which were produced by unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNC). The rats exhibiting more than 150 total controversial circles were regarded as satisfactory models of Parkinson disease (PD). Using a dual immunofluorescence histochemistry, we examined DA-immunoreactivity in the 5-HT fibers within the ST and the SNR of the PD model rats after l-DOPA was injected intraperitoneally. In experimental cases with the l-DOPA administration, DA-immunoreactivity was detected in 5-HT fibers in both the ST and the SNR on the 6-OHDA injection side; no DA-immunoreactivity was found in 5-HT fibers in the ST or the SNR in control cases without the l-DOPA administration. The results support the assumption that exogenously administered l-DOPA may be converted into DA within the 5-HT fibers in the ST and SNR of the PD model rats.

Immobilization stress increases endogenous monoamine oxidase (MAO) inhibitor in rat liver

Although the physiological role of endogenous monoamine oxidase (MAO) inhibitor still remains unclear, the present study examined whether or not immobilization stress (IMMO) induce MAO inhibitor. An endogenous inhibitor of MAO was separated by gel filtration from 105,000 g supernate in rat liver cytosol following IMMO. The molecular weight of this inhibitor was estimated to be 500–600 by gel filtration. This inhibitor was proved to be heat-stable resistant to protease treatment. IMMO for 2 h significantly decreased MAO. These results suggest that this inhibitor is induced by IMMO. MAO activity in rat liver might be regulated by the level of this inhibitor.

Isatin, an endogenous monoamine oxidase inhibitor, triggers a dose- and time-dependent switch from apoptosis to necrosis in human neuroblastoma cells

Isatin is an endogenous indole that is increased in stress, inhibits monoamine oxidase (MAO) B and improves bradykinesia and striatal dopamine levels in rat models of Parkinson's disease. Consequently, it has been suggested that isatin might be a possible treatment for Parkinson's disease although little is known about its effects on neural cell growth and survival. The aim of this study was to investigate the survival of dopaminergic human neuroblastoma (SH-SY5Y) cells following treatment with increasing concentrations of isatin. SH-SY5Y cells were exposed to isatin for defined time points, after which cell survival was determined by MTT assay. A combination of Annexin V binding and propidium iodide (PI) exclusion was used to distinguish apoptosis from necrosis in flow cytometry experiments and FACS profiles of permeabilised PI-labelled cells were employed for the assessment of cell cycle distribution. Isatin treatment (1–400 μM) for 24 h induced a significant dose-dependent increase in MTT metabolism by SH-SY5Y cells in culture, but this was not due to an increase in cell division. At the higher concentrations (200–400 μm) isatin triggered cell death, although MTT metabolism was still increased in the culture, suggesting that surviving cells were hypermetabolic. Following a longer (48 h) exposure, isatin was found to cause cell death in a dose-dependent manner; at lower concentrations (50 μM), the predominant mode of cell death was apoptosis while at the highest concentration (400 μm) increasing numbers of necrotic cells were also evident. Thus, in dopaminergic SH-SY5Y cells isatin induces cell death in dose- and time-dependent manner. This death occurred as a continuum of survival, apoptosis and necrosis. Our results re-emphasise that caution should be exercised when considering high doses of isatin as a putative anti-Parkinson's disease therapeutic.

Comparative Study of the Effects of Isatin, an Endogenous MAO-Inhibitor, and Selegiline on Bradykinesia and Dopamine Levels in a Rat Model of Parkinson’s Disease Induced by the Japanese Encephalitis Virus

We previously reported that exogenously administered isatin, an endogenous monoamine oxidase (MAO) inhibitor, significantly increased acetylcholine (ACh) and dopamine (DA) levels in the rat striatum. Selegiline [(−)-deprenil] was developed as a MAO-B inhibitor more than 30 years ago and widely used in the treatment of Parkinson’s disease. Effects of isatin or selegiline were investigated in Japanese encephalitis virus (JEV)-induced post-encephalitic parkinsonism rats by a pole test for detecting motor activity and by the determination of biogenic amine levels. Motor activity of JEV-induced rats receiving isatin (100 mg/kg per day for 1 week, i.p.) or selegiline (0.2 mg/kg per day for 1 week, i.p.) was significantly improved compared with that of untreated JEV-infected rats. Both isatin and selegiline prevented the decrease in striatal DA levels in JEV-rats. The increased turnover of DA (DOPAC/DA) induced by JEV was significantly inhibited by isatin, but not by selegiline. These results suggested that exogenously administered isatin and selegiline can improve JEV-induced parkinsonism by increasing DA concentrations in the striatum.

Dopamine-Derived Salsolinol Derivatives as Endogenous Monoamine Oxidase Inhibitors: Occurrence, Metabolism and Function in Human Brains

Salsolinol, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, is an endogenous catechol isoquinoline detected in humans by M. Sandler. In human brain, a series of catechol isoquinolines were identified as the condensation products of dopamine or other monoamines with aldehydes or keto-acids. Recently selective occurrence of the ( R)enantiomers of salsolinol derivatives was confirmed in human brain, and they are synthesized by enzymes in situ, but not by the non-enzymatic Pictet–Spengler reaction. A ( R)salsolinol synthase catalyzes the enantio-specific synthesis of ( R)salsolinol from dopamine and acetaldehyde, and ( R)salsolinol N-methyltransferase synthesizes N-methyl( R)salsolinol, which is further oxidized into 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion by non-enzymatic and enzymatic oxidation. The step-wise reactions, N-methylation and oxidation, induce the specified distribution of the N-methylated and oxidized derivatives in the human nigro-striatum, suggesting that these derivatives may be involved in the function of dopamine neurons under physiological and pathological conditions. As shown by in vivo and in vitro experiments, salsolinol derivatives affect the levels of monoamine neurotransmitters though the inhibition of enzymes related in the metabolism of catechol- and indoleamines. In addition, the selective neurotoxicity of N-methyl( R)salsolinol to dopamine neurons was confirmed by preparation of an animal model of Parkinson’s disease in rats. The involvement of N-methyl( R)salsolinol in the pathogenesis of Parkinson’s disease was further indicated by the increase in the N-methyl( R)salsolinol levels in the cerebrospinal fluid and that in the activity of its synthesizing enzyme, a neural ( R)salsolinol N-methyltransferase, in the lymphocytes prepared from parkinsonian patients. N-Methyl( R)salsolinol induces apoptosis in dopamine neurons, which is mediated by death signal transduction in mitochondria. In addition, salsolinol was found to function as a signal transmitter for the prolactin release in the neuro-intermediate lobe of the brain. These results are discussed in relation to role of dopamine-derived endogenous salsolinol derivatives as the regulators of neurotransmission, dopaminergic neurotoxins and neuro-hormonal transmitters in the human brain.

Isatin, an endogenous MAO inhibitor, improves bradykinesia and dopamine levels in a rat model of Parkinson's disease induced by Japanese encephalitis virus

Isatin, an endogenous monoamine oxidase (MAO) inhibitor, has an important role in the control of neurotransmitter concentration. We previously reported that exogenously administered isatin significantly increased acetylcholine (ACh) and dopamine (DA) levels in the rat striatum. In order to test the possibility of treating Parkinson's disease by isatin, we evaluated DA levels in the striatum and bradykinesia using a rat model of Parkinson's disease induced by the Japanese encephalitis virus (JEV). We have already reported that in adult Fischer rats infected with JEV at day 13, there was a marked decrease of tyrosine hydroxylase-positive neurons in the bilateral substantia nigra after 12 weeks. Effects of isatin were investigated in JEV-induced post-encephalitic parkinsonism rats by a pole test and high performance liquid chromatograph (HPLC) with an electrochemical detector (ECD). Isatin (100 mg/kg per day for 1 week, intraperitoneal injection) improved the bradykinesia observed in the JEV-induced parkinsonism rats. Dopamine (DA) concentrations in the JEV-infected rats were profoundly reduced in the striatum as compared with controls. Isatin also increased DA in the striatum of parkinsonism rats. These results suggest that isatin could be a possible treatment for Parkinson's disease as well as for post-encephalitic parkinsonism.

Quantitative liquid chromatography-mass spectrometry determination of isatin in urine using automated on-line extraction

Here we describe a simple, fast and sensitive liquid chromatography/mass spectrometry method with automated on-line extraction to quantify isatin, an endogenous monoamine oxidase, and atrial natriuretic peptide inhibitor, in urine. After derivatisation of isatin to isatinoxime with hydroxylamine hydrochloride and zinc sulfate precipitation, samples were loaded on the extraction column, washed and, after activation of the column-switching valve, backflushed onto the analytical column. Using electrospray ionisation, [M+H] + ions could be detected in the selected ion monitoring mode. The assay was linear from 5 to 5000 ng/ml ( r 2>0.99) and analytical recovery was >80%. Inter-assay precision for the quality control samples was less than 3% and inter-assay accuracy was within ±5%.

Effects of isatin on rotational behavior and DA levels in caudate putamen in Parkinsonian rats

Isatin was a potent endogenous monoamine oxidase (MAO) inhibitor that is more active against MAO-B than MAO-A. The acute effects of isatin on apomorphine (APO)-induced rotations were evaluated in Parkinsonian rats induced by 6-hydroxydopamine (6-OHDA) lesion. Furthermore, the effects of isatin on DA release in caudate putamen (CPu) of model and normal rats were monitored using fast cyclic voltammetry (FCV). The contents of monoamine transmitters and their metabolites in CPu of model and normal rats were also analyzed by high performance liquid chromatography with electrochemical detection after administration of isatin. Here we show that isatin (100 mg/kg, i.p.) apparently inhibited APO-induced rotations of Parkinsonian rats to 39.1±3.7% of the control ( n=12), while it had no apparent effects on electrical stimuli-induced DA release either in normal rats or in model rats. In addition, the content of 5-hydroxytryptamine but not DA was increased in both normal rats and model rats after isatin (100 mg/kg, i.p.) was administered ( P<0.01, n=6). The content of 5-hydroxyindole acetic acid was not changed. These results suggest that isatin cannot increase DA levels in rat CPu. Therefore, the effects of isatin on APO-induced rotations of our Parkinsonian rats could not attribute to its inhibition of DA catabolism as a MAO inhibitor.