Uptake and Metabolization of Serotonin by Granulosa Cells Form a Functional Barrier in the Mouse Ovary.

Serotonin (5-HT) plays an essential role in regulating female reproductive function in many animals. 5-HT accumulates in the mammalian ovary with the involvement of membrane serotonin transporter SERT and is functionally active in the oocytes of growing follicles, but shows almost no activity in follicular cells. In this study, we clarified the interplay between 5-HT membrane transport and its degradation by monoamine oxidase (MAO) in the mammalian ovary. Using pharmacologic agents and immunohistochemical staining of the cryosections of ovaries after serotonin administration in vitro, we demonstrated the activity of transport and degradation systems in ovarian follicles. The MAO inhibitor pargyline increased serotonin accumulation in the granulosa cells of growing follicles, indicating the activity of both serotonin uptake and degradation by MAO in these cells. The activity of MAO and the specificity of the membrane transport of serotonin was confirmed in primary granulosa cell culture treated with pargyline and fluoxetine. Moreover, the accumulation of serotonin is more effective in the denuded oocytes and occurs at lower concentrations than in the oocytes within the follicles. This confirms that the activity of SERT and MAO in the granulosa cells surrounding the oocytes impedes the accumulation of serotonin in the oocytes and forms a functional barrier to serotonin.

New insights in the metabolism of oxybutynin: evidence of N-oxidation of propargylamine moiety and rearrangement to enaminoketone.

1. Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects. 2. We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography-tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration. 3. A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring. 4. The functional activity of Oxy-EK was investigated on the muscarinic receptors (M1-3) demonstrating its lack of antimuscarinic activity. 5. Despite the presence of the α,ß-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.

Synthesis and biological evaluation of novel propargyl amines as potential fluorine-18 labeled radioligands for detection of MAO-B activity.

The aim of this project was to synthesize and evaluate three novel fluorine-18 labeled derivatives of propargyl amine as potential PET radioligands to visualize monoamine oxidase B (MAO-B) activity. The three fluorinated derivatives of propargyl amine ((S)-1-fluoro-N,4-dimethyl-N-(prop-2-ynyl)-pent-4-en-2-amine (5), (S)-N-(1-fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (10) and (S)-1-fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (15)) were synthesized in multi-step organic syntheses. IC(50) values for inhibition were determined for compounds 5, 10 and 15 in order to determine their specificity for binding to MAO-B. Compound 5 inhibited MAO-B with an IC(50) of 664 ± 48.08 nM. No further investigation was carried out with this compound. Compound 10 inhibited MAO-B with an IC(50) of 208.5 ± 13.44 nM and compound 15 featured an IC(50) of 131.5 ± 0.71 nM for its MAO-B inhibitory activity. None of the compounds inhibited MAO-A activity (IC(50) > 2 µM). The fluorine-18 labeled analogues of the two higher binding affinity compounds (10 and 15) (S)-N-(1-[(18)F]fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (16) and (S)-1-[(18)F]fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (18) were both prepared from the corresponding precursors 9A, 9B and 14A, 14B by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography experiments on human postmortem brain tissue sections were performed with 16 and 18. Only compound 18 demonstrated a high selectivity for MAO-B over MAO-A and was, therefore, chosen for further examination by PET in a cynomolgus monkey. The initial uptake of 18 in the monkey brain was 250% SUV at 4 min post injection. The highest uptake of radioactivity was observed in the striatum and thalamus, regions with high MAO-B activity, whereas lower levels of radioactivity were detected in the cortex and cerebellum. The percentage of unchanged radioligand 18 was 30% in plasma at 90min post injection. In conclusion, compound 18 is a selective inhibitor of MAO-B in vitro and demonstrated a MAO-B specific binding pattern in vivo by PET in monkey. It can, therefore, be considered as a candidate for further investigation in human by PET.

15N magnetic resonance hyperpolarization via the reaction of parahydrogen with 15N-propargylcholine.

(15)N-Propargylcholine has been synthesized and hydrogenated with para-H(2). Through the application of a field cycling procedure, parahydrogen spin order is transferred to the (15)N resonance. Among the different isomers formed upon hydrogenation of (15)N-propargylcholine, only the nontransposed derivative contributes to the observed N-15 enhanced emission signal. The parahydrogen-induced polarization factor is about 3000. The precise identification of the isomer responsible for the observed (15)N enhancement has been attained through a retro-INEPT ((15)N-(1)H) experiment. T(1) of the hyperpolarized (15)N resonance has been estimated to be ca. 150 s, i.e., similar to that reported for the parent propargylcholine (144 s). Experimental results are accompanied by theoretical calculations that stress the role of scalar coupling constants (J(HN) and J(HH)) and of the field dependence in the formation of the observed (15)N polarized signal. Insights into the good cellular uptake of the compound have been gained.

Site-specific protein propargylation using tissue transglutaminase.

Transglutaminases (TGases) catalyse the transamidation of glutamine residues with primary amines. Herein we report the first FRET-based activity assay for the direct detection of the ligation (transamidation) reaction mediated by tissue TGase (TG2). This novel assay was then used in a microtiter plate-based screen of a library of 18 potential amine substrates. From this screen it was discovered that propargyl amine serves as an excellent substrate for TG2. Subsequently, propargyl amine and 2-azidoethyl amine were validated independently as TG2 substrates with K(M) values of 44 ± 4 µM, and 0.99 ± 0.06 mM, respectively. In a proof-of-principle protein labelling experiment, the protein casein was selectively functionalized with propargyl amine using TG2 and subsequently fluorescently labelled through a dipolar cycloaddition reaction with an azido-fluorescein conjugate. This application demonstrates the strong potential of using TG2 for site-specific protein modification through a combination of enzymatic and bioorthogonal chemistry.

Interplay between bacterial deubiquitinase and ubiquitin E3 ligase regulates ubiquitin dynamics on Legionella phagosomes.

Legionella pneumophila extensively modulates the host ubiquitin network to create the Legionella-containing vacuole (LCV) for its replication. Many of its virulence factors function as ubiquitin ligases or deubiquitinases (DUBs). Here, we identify Lem27 as a DUB that displays a preference for diubiquitin formed by K6, K11, or K48. Lem27 is associated with the LCV where it regulates Rab10 ubiquitination in concert with SidC and SdcA, two bacterial E3 ubiquitin ligases. Structural analysis of the complex formed by an active fragment of Lem27 and the substrate-based suicide inhibitor ubiquitin-propargylamide (PA) reveals that it harbors a fold resembling those in the OTU1 DUB subfamily with a Cys-His catalytic dyad and that it recognizes ubiquitin via extensive hydrogen bonding at six contact sites. Our results establish Lem27 as a DUB that functions to regulate protein ubiquitination on L. pneumophila phagosomes by counteracting the activity of bacterial ubiquitin E3 ligases.

Antioxidant properties of propargylamine derivatives: assessment of their ability to scavenge peroxynitrite.

A series of arylpropargylamines, variously substituted in the hydrogen in p-position and in the propargyl moiety, were studied as potential peroxynitrite scavengers. The scavenging activity of these compounds was evaluated through peroxynitrite (ONOO-)-mediated oxidation of dichlorofluorescin and linoleic acid by measuring the dichlorofluorescein formation and oxygen consumption, respectively. Among tested compounds, only 1-phenylpropargylamine (AP3) promoted concentration-dependent inhibition of ONOO(-)-induced dichlorofluorescin and linoleic acid oxidation with IC50 values of 637 and 63 microM, respectively. The AP3 spectral changes in UV-visible absorbance properties in the presence of peroxynitrite suggested the formation of a new compound. This was identified by gas-chromatograph-mass spectrometer analysis as phenylpropargyl alcohol. Structure-activity relationship analysis indicated that the scavenging activity of AP3 was due to the aminopropargyl moiety and availability of the nitrogen electron pair. This data suggested that AP3 could be considered a lead compound for the synthesis of new ONOO- scavenger derivatives.

[Molecular mechanisms of the neuroprotective effect of (-)-deprenyl]. / A (-)-deprenyl neuroprotektív hatásának molekuláris mechanizmusai.

(-)-Deprenyl, the irreversible inhibitor of monoamine oxidase B, has been used for decades in the therapy of Parkinson's disease. It improves parkinsonian symptoms due to its dopamine potentiating and antioxidant properties and presumedly delays disease progression. Its complex pharmacological action cannot be explained solely by its monoamine oxidase B inhibitory property. Recently, (-)-deprenyl has been demonstrated to exert antiapoptotic, neuroprotective effects on a number of in vitro and in vivo models in a dose significantly lower than required for monoamine oxidase B inhibition. (-)-Deprenyl and related propargylamines prevent apoptotic cell death by preserving the integrity of the mitochondrion that may be based on the activation of a complex transcriptional program. The changes in gene expression initiated by propargylamines incited to search for further possible target molecules that would explain more accurately the antiapoptotic effect of these compounds. The latest molecular targets include such classical metabolic enzymes, the homologues of which may participate in the regulation of gene expression as a part of transcriptional factor complexes. Some of the propargylamine targets--glyceraldehyde-3-phosphate dehydrogenase, poly(ADP-ribose) polymerase, nuclear amine oxidases--have already been demonstrated to be capable of transforming the metabolic changes in the cell to transcriptional responses. Data are accumulating about the relationship of these enzymes and propargyl compounds, but the real significance of this issue will only be established by future research.

Characterization of monoamine oxidase activity present in human granulocytes and lymphocytes.

The characterization of monoamine oxidase (MAO) activity in lymphocytes and granulocytes was studied by using cells prepared from human blood. The specific activities of the enzyme towards beta-phenylethylamine (PEA), benzylamine (Bz), tyramine (TYR) and 5-hydroxytryptamine (5-HT) were found to be 5-times higher in lymphocytes than in granulocytes. The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by the lack of effect of semicarbazide on the benzylamine oxidation. The presence of MAO-B was corroborated by the inhibition of PEA oxidation with nanomolar deprenyl concentrations and by inhibition of TYR oxidation with high clorgyline concentrations, as well as by the simple sigmoid curve obtained in both cases. These results, together with the substrate preferences, suggest that the MAO activity of human granulocytes and lymphocytes is predominantly of the B form. For each fraction the kinetic constants were determined towards PEA, TYR and Bz as substrates. The Km values were similar for both cellular samples, whereas the Vmax values were higher in lymphocytes than in granulocytes. MAO-B was titrated with [3H]pargyline in order to find out the number of active sites. The corresponding molecular concentration, Kcat values and turnover number showed the presence of related enzymes in human granulocytes and lymphocytes.

Mechanism-based inactivation of porcine kidney diamine oxidase by 1,4-diamino-2-butene.

Cis- and trans-1,4-diamino-2-butene are substrates and potent inactivators of porcine kidney diamine oxidase. Evidence from absorption and NMR spectra indicates that both are oxidized to pyrrole. Both substrates are irreversible mechanism-based inactivators of the enzyme, although the trans isomer is more potent and results in complete inactivation in a reaction which follows pseudo-first-order kinetics with an apparent Ki of 0.34 mM and a second-order inactivation constant of 500 M-1 s-1. Under the same conditions, 46% of the activity remains when the enzyme is reacted with cis-1,4-diamino-2-butene. Trans-4-amino-2-butenal, the product of oxidation of the trans diamine, has been synthesized and shown to undergo cyclization to pyrrole in a concentration-dependent manner, approaching second-order at low concentrations. Trans-4-amino-2-butenal is itself a potent irreversible inhibitor with IC50 of 2.5 microM. We propose that the irreversible inactivation by both cis- and trans-1,4-diamino-2-butene involves attack by a protein-based nucleophilic residue on the unsaturated aminoenal products of the enzymatic reactions, resulting in a covalent adduct. Cyclization of the cis-aminoenal to pyrrole is much more rapid than in the trans case, thus it is less available for inhibitory reaction with the protein.

Inhibition of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolic activity of porcine FAD-containing monooxygenase by selective monoamine oxidase-B inhibitors.

The MPTP metabolic activity of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was inhibited considerably by deprenyl and pargyline, selective MAO-B inhibitors, and they showed typical competitive inhibition. Deprenyl and pargyline, amine derivatives were also examined as to whether they are substrates for the FMO. It was found that deprenyl and pargyline are excellent substrates for the FMO. The Ki and Km values of deprenyl and pargyline for the FMO are 14 microM and 9 microM, and 14.3 microM and 11.6 microM, at pH 8.0 and 25 degrees C, respectively.

Similar distribution of monoamine oxidase (MAO) and parkinsonian toxin (MPTP) binding sites in human brain.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) causes parkinsonism in humans and other species. We found [3H] MPTP binding sites that were saturable, specific, and of high affinity. In autoradiographic studies, the highest binding densities of [3H] MPTP occurred in the hypothalamus, interpeduncular nucleus, and ependymal lining of the ventricles. High to moderate binding was seen in the dentate gyrus, caudate, putamen, substantia nigra, and cingulate cortex. The distribution of [3H] MPTP binding correlated with the distribution of [3H] pargyline binding to MAO. Human substantia nigra contains more MPTP binding sites than rat substantia nigra, and this may explain the sensitivity of humans to the neurotoxic effects of MPTP.

Biodistribution of a positron-emitting suicide inactivator of monoamine oxidase, carbon-11 pargyline, in mice and a rabbit.

Carbon-11 (11C) pargyline, which is a suicide inactivator of Type B monoamine oxidase (MAO), was synthesized by the reaction of N-demethylpargyline with 11CH3I. Biodistribution was investigated in mice, and positron tomographic images of the heart and lung in a rabbit were obtained. The distribution of 11C after administration of [11C]pargyline was measured in several organs and blood at various time intervals. After 30 min its concentrations in the organs were constant. Subcellular distribution studies in the brain, lung, liver, and kidney showed that 59-70% of the 11C became acid-insoluble and 9-33% was present in the crude mitochondrial fraction at 60 min after injection. However, a high loading dose influenced the subcellular distribution but had little effect on tissue distribution. The uptakes of the 11C in each organ except for the kidney and spleen seemed to correlate with the in vitro enzymatic activity of Type B MAO. At high loading dose a nonspecific uptake was observed.

Effect of antibiotics on the binding of pargyline to monoamine oxidase in cultured hepatocytes.

The laser dye rhodamine 123 has been used to establish that the binding of [3H]pargyline to monoamine oxidase is a more sensitive indicator of mitochondrial perturbation than measurements of protein synthesis, secretion, or degradation. The amount of monoamine oxidase labelled depends on the antibiotic used. The labelling was considerably lower in the presence of gentamycin than in the presence of either chloramphenicol or of penicillin and streptomycin. The accumulation of gentamycin within the cells was the cause of the reduced labelling of monoamine oxidase which was not accompanied by an alteration in the metabolism of pargyline. The gentamycin effect can be prevented by incubating the cells in medium supplemented with methylamine prior to adding the monoamine oxidase inhibitor. Long term culture of cells with gentamycin can result in the cell culture medium becoming dark brown. Under these conditions the monoamine oxidase labelling is increased due to an inhibition of pargyline metabolism. The results indicate that the choice of antibiotic is important in patients being treated with both antibiotics and monoamine oxidase inhibitors.

Determination of monoamine oxidase concentrations in rat liver by inhibitor binding.

The concentrations of monoamine oxidase-A and -B have been determined in mitochondria, mitochondrial outer membranes and microsomes from Sprague-Dawley and Wistar rats by determining the binding of tritium-labelled pargyline. Although the amounts of each form present depended on the source and the preparation method, this was paralleled by the specific activity such that the molecular turnover number was found to remain constant. The catalytic constants, kcat/Km, which represents the apparent second-order rate constant for the combination of enzyme and substrate, were about 0.13 and 2.1 sec-1 X microM-1 for 5-hydroxytryptamine and 2-phenethylamine, respectively, regardless of the source. Estimations of the amounts of the two forms by determining the concentrations of the inhibitors clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition were shown to give overestimates of the true values because of the non-specific binding of these inhibitors to sites other than the monoamine oxidase active site.

Fate of the monoamine oxidase inhibitor pargyline in cultured hepatocytes.

The interaction of the monoamine oxidase inhibitor pargyline with cultured hepatocytes has been studied. [Phenyl-3, benzyl-3H] pargyline (38 nM) rapidly enters the cells and a plateau of incorporation into a trichloroacetic acid insoluble form (monoamine oxidase) is reached after 2 hr. The level of labelling is lower in freshly isolated cells than in those in later culture. The maximum incorporation accounts for only 6% of the added radioactivity and produces a 9% inhibition of monoamine oxidase activity. The remaining [3H] pargyline is metabolized and quickly accumulates in the cell culture medium in a form which cannot label exogenous mitochondria. The metabolism of pargyline varies both qualitatively and quantitatively with culture age. In 0 hr and 20 hr-cultured cells one metabolite preferentially appears whilst in 140 hr cultured hepatocytes at least three metabolites are formed. The metabolism of [3H] pargyline in early culture is consistent with a cytochrome P-450 involvement. The use of [3H] pargyline to label monoamine oxidase in cultured hepatocytes offers several attractive features for studying the turnover of this enzyme. These include speed of interaction, non-reutilization, application to normal cells, controlled inhibition of monoamine oxidase and metabolism of non-specific label.

Differences in the structures of monoamine oxidases A and B in rat clonal cell lines.

[3H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in two rat cell lines were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [3H]Pargyline was bound to MAO A and B in a crude mitochondrial fraction from rat hepatoma cell line MH1C1 and to MAO A in a mitochondrial fraction from rat glioma line C6. Specific radiolabeling of proteins associated with type A or B activity in the hepatoma samples was achieved by incubation with selective B or A inhibitors, respectively, prior to [3H]pargyline binding. Following [3H]pargyline binding, the samples were solubilized by heating in sodium dodecyl sulfate (SDS) in the presence of a reducing agent. SDS-PAGE of [3H]pargyline bound samples revealed a radiolabeled protein band of apparent molecular weight (mol. wt) 63,000 daltons associated exclusively with MAO A activity and a band of apparent mol. wt 60,000 associated exclusively with MAO B activity. Furthermore, when SDS-solubilized, [3H]pargyline-labeled MAO A and B proteins from these cell lines were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [3H]pargyline-labeled peptides were obtained. These findings indicate that the A and B forms of MAO activity are associated with enzyme molecules of different primary covalent structures determined by different gene loci.

4-Fluoro-3-nitrophenyl azide, a selective photoaffinity label for type B monoamine oxidase.

The effects of 4-fluoro-3-nitrophenyl azide (FNPA) on types A and B monoamine oxidase in rat brain cortex were studied using serotonin and phenylethylamine as substrates respectively. FNPA competitively inhibited the oxidative deamination of both serotonin (Ki = 3 microM) and phenylethylamine (Ki = 0.78 microM) in the dark. Upon photoirradiation in the presence of FNPA, a photodependent inhibition of type B MAO activity resulted. This photodependent inhibition was apparently irreversible since there was no recovery of activity upon washing of the photolyzed FNPA-enzyme mixture. Additional evidence for the photoinduced covalent binding of FNPA to type B MAO is that non-competitive inhibition kinetics resulted after photolysis. The specificity of the photodependent incorporation of FNPA to type B MAO was shown by the protective effect of phenylethylamine and by decreased [3H]pargyline labeling after the enzyme was photolyzed with FNPA. Under the same experimental conditions, only minimal photodependent inhibition of type A MAO by FNPA was found. The observed difference in the efficiencies of the photodependent inactivation of the two types of MAO by FNPA suggests that there is a conformational or a structural difference in the active sites of the two types of MAO. The active site of type B MAO could be characterized by utilizing FNPA as a photoaffinity labeling probe.

The metabolic fate of pargyline in rat liver microsomes.

The availability of a sensitive analytical assay for the simultaneous quantitation of pargyline (PARG) and four of its major metabolites have made possible a detailed study on the metabolism of the drug in rat liver microsomes with emphasis put on comparisons between optional N-dealkylation reactions and N-oxide formation. Pargyline is a lipophilic amine with a low pKa-value of 6.6 and undergoes extensive metabolism. The conversion of the substrate is rapid and comprizes three N-dealkylation and one N-oxidation reactions, yielding N-benzylpropargylamine (BPA), N-methyl-propargylamine (MPA), N-benzylmethylamine (BMA) and pargyline N-oxide (PNO), respectively. Phenobarbital (PB) pretreatment of the rats causes a pronounced increase in the metabolism with about 90% of the substrate being consumed within the first minute of incubation at 100 microM substrate concentration. At this substrate concentration the most pronounced induction is seen in the formation of BPA and also in its further metabolism, while levels of BMA and MPA remain fairly constant. Pargyline N-oxide is the most abundant metabolite in microsomes from untreated rats and its formation is not increased by PB induction. Moreover, the inhibition of PNO formation by typical cytochrome P-450 inhibitors is marginal, while that of BPA, BMA and MPA formation is not. N-Debenzylation, yielding MPA, is the least important of the N-dealkylation reactions and the effect of PB induction on this reaction becomes noticeable only at high substrate concentrations. The studies suggest that various cytochrome P-450 enzymes are involved in the N-dealkylation reactions of PARG while N-oxidation appears to occur mainly by a cytochrome P-450-independent pathway. As propiolaldehyde, a potential hepatotoxin, is formed concomitant to BMA, and as PNO, under certain conditions, can decompose to acrolein, another well-known hepatotoxin, both these quantitatively important metabolic routes have to be considered in evaluating the toxicity of pargyline.

Photoaffinity labeling of beef liver monoamine oxidase-B by 4-fluoro-3-nitrophenyl azide.

4-Fluoro-3-nitrophenyl azide (FNPA) competitively inhibited beef liver monoamine oxidase-B (MAO-B) in the dark (Ki = 2.8 microM). Upon irradiation in the presence of FNPA, a concentration-dependent photoinactivation of MAO-B was observed. The kinetic analysis showed that the photoinactivation of MAO-B resulted in a decrease in Vmax but no change in Km. This result suggests that an irreversible linkage may be formed between the enzyme and the photolyzed FNPA. When [3H]FNPA was photoirradiated with the purified MAO-B, a single radioactive band associated with MAO-B was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The photo-dependent incorporation could be protected by phenylethylamine, the substrate for MAO-B, in a concentration-dependent manner. Complete tryptic-chymotryptic digestion of [3H]FNPA-labeled MAO-B resulted in three radioactive peaks on Sephadex G-25 column chromatography. With the same digestion and separation procedures, only one major radioactive peak was observed for the [3H]pargyline-labeled MAO-B, and its elution volume was different from that of [3H]FNPA-labeled peptides. These results suggest that, upon photolysis, FNPA may incorporate into a region in the active site of MAO-B which may be different from the pargyline binding site--the FAD prosthetic group of the enzyme.