Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in V_max of both MAO isoenzymes in serum-free medium, but no change in K_m. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.     

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[³H]glutamate. The apparent binding constant K _d was 135 nM and 393 nM and the maximal binding capacity B_max 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC₅₀ in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [³H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.     

Effect of riboflavin on monoamine oxidase activity in cultured neuroblastoma cells

Monoamine oxidase (MAO) depends on a covalently attached FAD cofactor for activity. Activity is depressed in mouse neuroblastoma cells (NIE-115) grown in synthetic N2 medium lacking riboflavin. MAO activity in depleted cells is stimulated by added riboflavin, and this recovery is blocked by inhibitors of RNA and protein synthesis, and not by an inhibitor of protein glycosylation. Recovery from riboflavin depletion appears to depend upon new RNA and protein synthesis, and not on the addition of FAD cofactor to an inactive MAO precursor.     

随机突变提高单胺氧化酶活性

前期获得了一个对底物美西律具有一定活性的单胺氧化酶突变体A-1(F210V/L213C)。为进一步提高其酶活性,利用MegaWHOP PCR构建了库容约为104的随机突变库。筛选后获得了一个最优突变酶ep-1,比活力为A-1的189%。选择性测定结果表明,酶的对映体选择性有较大提高,E值由101提高到282;动力学常数测定揭示,酶催化效率有较大提高,kcat/Km由0.001 51 mmol/(L?s)提高到0.002 89 mmol/(L?s)。和A-1酶相比,在所测定的11种胺类底物中,ep-1对其他7种底物的比活力有较明显提高,对其他4种底物的比活力变化不大。序列分析表明,ep-1的突变为T162A。分子动力学模拟结果提示,该突变主要通过修正通道氨基酸的二级结构和扩大活性口袋来发挥作用。     

In vitro andin vivo effect of organophosphate pesticides on monoamine oxidase activity in rat brain

The effects of some organophosphate pesticides, e.g. lebaycid, metacid and metasystox on the monoamine oxidase (MAO) activity in rat brain mitochondria have been studied. These pesticides cause significant inhibition of MAO activityin vitro but have negligible effects on its activityin vivo.     

Properties of monoamine oxidase in control and Lesch-Nyhan fibroblasts

Monoamine oxidase activity of the A type was measured in homogenates of cultured human skin fibroblasts. Twenty-four control lines had activities ranging over fifty-fold with an apparent bimodal distribution. Activity in fibroblasts from 20 patients with the Lesch-Nyhan syndrome fell in the low portion of the normal distribution with a mean activity about 50% that of the control mean (p<0.05). In a subgroup of control and Lesch-Nyhan lines with levels of enzyme activity from 0.9 to 179 pmol/min/mg protein, monoamine oxidase was similar with respect to apparent K_m for tryptamine, thermal stability at 56 C, and sensitivity to clorgyline. Thus the lower mean levels of activity observed in the Lesch-Nyhan as compared to control fibroblasts were not associated with other altered properties of the enzyme. The bimodal distribution of enzyme activity suggests that a genetic polymorphism for monoamine oxidase may control levels of activity expressed in fibroblasts.M. R. C. C. was funded by the Dystonia Medical Research Foundation. This work was supported by grants from USPHS—NS12105 and GM20124—and from the National Foundation-March of Dimes.     

In vitro andin vivo effect of chloropromazine,imipramine and lithium chloride on monoamine oxidase activity in rat brain mitochondria

Chloropromazine (CPZ) and imipramine at a concentration of 1×10^–3 M inhibit rat brain mitochondrial monoamine oxidase activity in vitro by 70 and 55% respectively, while lithium, even at a concentration of 0.05 M, inhibits the activity of this enzyme very negligibly (4%). In vivo, these drugs at a dose level of 56 mg CPZ, 76 mg Jimipramine and 76 mg lithium chloride/Kg body wt., did not cause any observable variation from normal in brain mitochondrial monoamine oxidase activity.To whom correspondence should be addressed.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells

Abstract Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

3D-QSAR method on indole and pyrrole inhibitors of monoamine oxidase type A

Monoamine oxidase A (MAO-A) converts norepinephrine and serotonin to an oxidative form. These monoamine neurotransmitters have important roles in depression. The MAO-A inhibitors have been discovered for neurodegenerative disease therapy. In order to design novel MAO-A inhibitors, in this study, the quantitative structure-activity relationships for the combined series of indoles and pyrroles were elucidated and the structural conditions to show good inhibitory effects on MAO-A were derived. This result can help us design new inhibitors irrespective of their specific moiety.     

Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells

The effects of Mg²⁺ on the glutamate-, kainate-, N-methyl-d-aspartate- and quisqualate-induced influx of⁴⁵Ca²⁺ were studied in cultured cerebellar granule cells. The N-methyl-d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg²⁺. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg²⁺, whereas N-methyl-d-aspartate was most effective in totally Mg²⁺-free media.d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca²⁺ influx by 1 mM quisqualate in 0.1-mM Mg²⁺ medium. The effect of 10 M quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-d-aspartate, (RS)-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg²⁺ medium. The efficacy of quisqualate in inducing influx of Ca⁺ and the selectivity of antagonists for different receptors are also modified by extracellular Mg²⁺.     

Colorimetric assay for monoamine oxidase in tissues using peroxidase and 2,2'-azinodi(3-ethylbenzthiazoline-6-sulfonic acid) as chromogen

Monoamine oxidase is assayed in tissue by a colorimetric reaction using horse radish peroxidase and 2,2'-azinodi(ethylbenzthiazoline-6-sulfonic acid to measure H2O2 formed during oxidation of amines. The method has a coefficient of variation of approximately 2.5% and provides results comparable with those of radiometric assay. Monoamine oxidase activities in rat liver mitochondria and crude mitochondrial fraction from brain and with tyramine as a substrate were 18.9 +/- 0.4 and 4.61 +/- 0.15 nmol/min/mg of protein, respectively, using this method. Kinetic parameters of liver and brain monoamine oxidase with various substrates and inhibitors appeared to be the same when determined by either colorimetric or radiometric methods.     

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.     

Differential negative coupling of type 3 metabotropic glutamate receptor to cyclic GMP levels in neurons and astrocytes

Metabotropic receptors may couple to different G proteins in different cells or perhaps even in different regions of the same cell. To date, direct studies of group II and group III metabotropic glutamate receptors' (mGluRs) relationships to second messenger cascades have reported negative coupling of these receptors to cyclic AMP (cAMP) levels in neurons, astrocytes and transfected cells. In the present study, we found that the peptide neurotransmitter N-acetylaspartylglutamate (NAAG), an mGluR3-selective agonist, decreased sodium nitroprusside (SNP)-stimulated cyclic GMP (cGMP) levels in cerebellar granule cells and cerebellar astrocytes. The mGluR3 and group II agonists FN6 and LY354740 had similar effects on cGMP levels. The mGluR3 and group II antagonists beta-NAAG and LY341495 blocked these actions. Treatment with pertussis toxin inhibited the effects of NAAG on SNP-stimulated cGMP levels in rat cerebellar astrocytes but not in cerebellar neurons. These data support the conclusion that mGluR3 is also coupled to cGMP levels and that this mGluR3-induced reduction of cGMP levels is mediated by different G proteins in cerebellar astrocytes and neurons. We previously reported that this receptor is coupled to a cAMP cascade via a pertussis toxin-sensitive G protein in cerebellar neurons, astrocytes and transfected cells. Taken together with the present data, we propose that mGluR3 is coupled to two different G proteins in granule cell neurons. These data greatly expand knowledge of the range of second messenger cascades induced by mGluR3, and have implications for clinical conditions affected by NAAG and other group II mGluR agonists.     

Terfenadine induces toxicity in cultured cerebellar neurons: A role for glutamate receptors

Summary Exposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2h exposure, 20µM terfenadine reduced the number of surviving neurons by 75%, and as low as 10nM terfenadine induced significant neurotoxicity after 5 days of exposure. Neuronal sensitivity to terfenadine changed with age in culture, and at 25 days in culture neurons appeared to be much less sensitive than at 5 or 9–17 days in culture. Neurotoxicity by terfenadine could not be prevented by high concentrations of histamine (5 mM), but it was significantly delayed by blocking NMDA or non-NMDA glutamate receptors with MK-801 or CNQX respectively, suggesting the involvement of excitatory transmission mediated by glutamate in the neurotoxicity induced by terfenadine in these neurons. We also found that the presence of terfenadine (5,µM) unveiled the potential excitotoxicity of the non-NMDA receptor agonist AMPA (100µM), and reduced the concentration of glutamate necessary to induce excitotoxicity, compared to untreated cultures. These results suggest a role for terfenadine in the modulation of the excitotoxic response mediated in cerebellar neurons through ionotropic glutamate receptors.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.     

Effects of medium glutamine,glutamate, and ammonia on glutamine synthetase activity in cultured mouse astroglial cells

Mouse astroglial cells were grown during the last week of culture in either glutamine-free or glutamine-containing medium. The addition of cortisol to the glutamine-containing medium resulted in a doubling of astroglial glutamine synthetase (GS) activity. Withdrawal of glutamine from the medium resulted in a 50% elevation of GS and addition of cortisol to such a medium resulted in a further increase in GS which was not additive to glutamine withdrawal. Both in glutamine-free and glutamine-containing medium, the addition of glutamate resulted in a depression of both basal and cortisol induced GS activity. The simultaneous addition of ammonia plus glutamate to the culture medium ameliorated the glutamate mediated depressive effects on cortisol induced but not basal GS activity. Glutamine withdrawal from the culture medium resulted in an astroglial protein deficit. The addition of ammonia to the medium considerably reduced this deficit and the addition of glutamate completely eliminated this protein deficit.     

New catalytic properties of monoamine oxidase immobilized in Langmuir-blodgett films with amphiphilic polyelectrolytes

Langmuir-Blodgett (LB) films of monoamine oxidase (MAO) have been formed on the surface f a polypropylene membrane using amphiphilic polyelectrolytes. The enzyme activity of such protein-polyelectrolyte films was measured by a Clark electrodes. It was shown that in LB films thus formed the use of amphiphilc polyelectrolytes, MAO activity was higher than in polyelectrolyte-free LB films. Immobilization of MAO with branched polyethylenimine modified on 12% by laurylchain led to pronounced changes in its catalytic properties. The dependence of the enzyme's kinetic parameters on amphiphilic polyelectrolyte structures was discussed. (c) 1994 John Wiley & Sons, Inc.     

Structure of rat monoamine oxidase A and its specific recognitions for substrates and inhibitors

Monoamine oxidase (MAO), a mitochondrial outer membrane enzyme, catalyzes the degradation of neurotransmitters in the central nervous system and is the target for anti-depression drug design. Two subtypes of MAO, MAOA and MAOB, are similar in primary sequences but have unique substrate and inhibitor specificities. The structures of human MAOB complexed with various inhibitors were reported early. To understand the mechanisms of specific substrate and inhibitor recognitions of MAOA and MAOB, we have determined the crystal structure of rat MAOA complexed with the specific inhibitor, clorgyline, at 3.2A resolution. The comparison of the structures between MAOA and MAOB clearly explains the specificity of clorgyline for MAOA inhibition. The fitting of serotonin into the binding pockets of MAOs demonstrates that MAOB Tyr326 would block access of the 5-hydroxy group of serotonin into the enzyme. These results will lead to further understanding of the MAOA function and to new anti-depression drug design. This study also presents that MAOA has a transmembrane helix at the C-terminal region. This is the first crystal structure of membrane protein with an isolated transmembrane helix.