Phenacetin N-deacetylase and its non-identity with the serotonin sensitive aryl acylamidase of brain

Phenacetin N-deacetylase was characterized in monkey brain. The enzyme needed Triton X-100 for maximal extraction and it had a high specific activity in cerebellum and in the nuclear fraction of whole monkey brain. It differed from the brain aryl acylamidase in both the regional and subcellular distributions. Brain aryl acylamidase purified by affinity chromatography was ineffective in deacetylating phenacetin. All the potent inhibitors of brain aryl acylamidase such as serotonin, tryptamine, acetylcholine and its analogues and neostigmine had no effect on phenacetin deacetylase. However, brain pehnacetin deacetylase was moderately inhibited by indole-3-acetic acid and 5-hydroxy indole-3-acetic acid properties similar to those of liver aryl acylamidase. Acetaminophen was not deacetylated by the brain phenacetin deacetylase.     

Hydrolysis of 3′,4′-dichloropropionanilide by an aryl acylamidase from Taraxacum officinale

An aryl acylamidase (aryl-acylamine amidohydrolase, E.C. 3.5. 1.a) which hydrolyses the herbicide propanil (3′,4′-dichloropropionanilide), was isolated from dandelion roots and partially purified and characterized. Specificity tests on the enzyme revealed that it could hydrolyse various chlorine ring-substituted propionanilides and 3,4-dichloroanilide alkyl compounds. The partially purified enzyme was inhibited by several sulfhydryl reagents and metal ions. The pH optimum was broad, between 7·4 and 7·8. The apparent activation energy, determined from an Arrhenius plot, was 9·0 kcal/mol (37 700 J/mol) for the hydrolysis of 3′,4′-dichloropropionanilide. The apparent K_m was 1·7 × 10⁻⁴ M with propanil as substrate.     

Rat brain aryl acylamidase: multiple forms and inhibition effects of LSD, serotonin and related compounds

At least two forms of aryl acylamidase (E.C.3.5.1.13, AAA) were separated from rat brain extracts by ammonium sulfate precipitation (33–60% saturation) and subsequent Bio-Gel column chromatography. Fraction AAA-1 showed pH optimum at 7.5 whereas AAA-2 showed a pH optimum at 5.5 AAA-1 activity was markedly inhibited at pH 7.5 by d-LSD and 2-Br-LSD, moderately inhibited by 5-HT and slightly inhibited by tryptamine but it was not affected by 1-LSD, at 0.1 mM concentration. AAA-2 was only moderately inhibited at pH 5.5 by d-LSD and 2-Br-LSD but not affected by 1-LSD, 5-HT or tryptamine at the same concentrations. Catecholamines and their structurally related drugs had no significant effects on either enzyme activity. Kinetic studies with AAA-1 indicated competitive inhibition by d-LSD with a K_i value of 4.90 ± 0.61 μM.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

Synthesis,affinity and specificity of 18F-setoperone,a potential ligand for in-vivo imaging of cortical serotonin receptors

Setoperone, a piperidine derivative known for its potent serotonin and moderate dopamine receptor blocking properties was labelled with the positron emitter ¹⁸F using a nucleophilic substitution on the nitro derivative. The general pattern of the in-vivo and in-vitro rat brain distribution of this new radioligand was consistent with the mapping of serotonin (5HT₂) and dopamine (D₂) receptors. The cortical binding of ¹⁸F-setoperone was selectively inhibited by ketanserin and not by sulpiride. The affinity of the radiofluorinated ligand for the serotonin receptors was in the nanomolar range (K_d = 0.7 nM).     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k_cat = 0.13 ± 0.02 min ? ¹ and K_s = 0.67 ± 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k_cat = k₂). Though the aryl acylamidase activity of albumin is low (k_cat/K_s = 195 M^{? 1}min^{? 1}), because of its high concentration in human plasma (0.6–1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

PAF antagonists: different effects on platelets, neutrophils, guinea pig ileum and PAF-induced vasolidation in isolated rat lung

The effects of structurally different PAF receptor blockers were investigated in platelets, neutrophils, guinea pig ileum, rat isolated lung and rat isolated pulmonary artery. PAF caused serotonin release from platelets and a characteristic shape change and adhesion of neutrophils. The antagonists (CV 3988, alprazolam, 48740 RP and Merck-Sharp and Dohme L-652, 731) inhibited platelet serotonin release but not neutrophil shape change adhesion or lysosomal enzyme release. The antagonists in high concentrations (10⁻⁵ −10⁻⁴M) inhibited nonspecifically the PAF-induced (10⁻⁸M) guinea pig ileum contraction, but were ineffective at concentrations which inhibited platelet responses. In the rat lung the compounds, in high concentrations, partially inhibited the low dose PAF-induced pulmonary vasodilation and the high dose PAF induced pulmonary vasoconstriction and edema. Our data indicate that some platelet PAF antagonists may be ineffective in blocking the action of PAF on neutrophils and smooth muscle preparations and suggest either PAF-receptor independent actions of PAF or different classes of PAF receptors.     

A new acylamidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> TA37 can hydrolyze N-substituted amides

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4′-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7–8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K _m) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.     

5HT2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition

Contractile responses to serotonin were examined $\text{in}$$\text{vitro}$ in the longitudinal portal vein to determine whether such responses were mediated by the interaction of serotonin with 5HT₁ receptors (those that preferentially bind [³H]serotonin) or 5HT₂ receptors (those that preferentially bind [³H]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the affinity for serotonin receptors in the rat portal vein and the ability to bind to 5HT₂, but no 5HT₁ receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED₅₀ value) relative to contractions produced by the non-cumulative addition of serotonin. Affinities of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentration-response curves compared to affinities obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT₂ receptor affinities of antagonists in the rat portal vein are best determined by the shift of non-cumulative responses to serotonin.     

Rat brain de-acetylating activity: stereospecific inhibition by LSD and serotonin-related compounds.

A de-acetylase (aryl acylamidase, E.C.3.5.1.13) has been isolated and partially characterized from rat brain. Previous studies have shown that this enzymatic activity is inhibited by low concentrations of serotonin. This report examines the effects of closely related tryptamine derivatives and demonstrates that enzymatic activity is stereospecifically inhibited by LSD. Similar enzymatic activity from liver was found to be insensitive to any of the compounds tested. The significance of these findings with regard to well known serotonin-LSD interactions is discussed.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the ? 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.     

Aryl acylamidase from Rhodococcus erythropolis NCIB 12273

Summary A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a K_m for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen. Offprint requests to: P. A. Vaughan     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Effect of Chronic Corn Consumption on Serotonin Content of Rat Brain

SEROTONIN, a putative neurotransmitter in the mammalian central nervous system, is synthesized in the brain by the 5-hydroxylation and decarboxylation of the essential amino-acid L-tryptophan^1,2. The control of serotonin biosynthesis seems to involve a different mechanism from that responsible for catecholamine biosynthesis^3,4 in its dependence on the availability of the amino-acid precursor^5,6. Thus, small doses of tryptophan that do not increase brain or plasma tryptophan concentrations beyond their normal daily ranges cause significant increases in the serotonin concentration of rat brain⁷. Conversely, the chronic ingestion of diets lacking in tryptophan (with casein hydrolysates or amino-acid mixtures substituted for natural proteins) depresses brain serotonin levels^8–10. The dependence of serotonin biosynthesis on tryptophan availability probably arises from the unusually high substrate K _M that characterizes tryptophan hydroxylase¹. It seems likely that this enzyme normally functions in an unsaturated state; hence physiological increases in intraneuronal tryptophan could drive the hydroxylation of the amino-acid and, ultimately, its conversion to serotonin.     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.     

Effects of N-substituted phenyltetrahydropyridines on cerebral high-affinity synaptosomal uptake of dopamine and other monoamines in several mammalian species

Effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (N-methyl-PTP) and its N-propyl congener (N-propyl-PTP) on the high-affinity uptake of tritiated dopamine (DA), norepinephrine, and serotonin by striatal or cerebral cortical synaptosomes were evaluated in several species (rat, guinea pig, rabbit, calf, and man). Both compounds inhibited uptake of 0.1 ωM labeled amines at IC₅₀s of 5–10 ωM. Effects of N-methyl-PTP were competitive, reversible, somewhat more potent, and more selective for serotonin than were actions of N-propyl-PTP. Similar effects were found in all species. Neither agent inhibited binding of ³H-labeled spiperone or ADTN to DA receptor sites. ³H-N-methyl-PTP did not appear to be taken up selectively into DA neurons. N-methyl-PTP was highly toxic to the rat in doses that did not alter the metabolism of DA or serotonin in brain. These results, overall, do not provide strong support for the hypothesis that reported neurotoxic actions of N-methyl-PTP are mediated by neuron-specific local transport and intracellular accumulation, or account for species differences in the actions of this toxin, but do suggest interactions with brain monoamine neurons. The actions of the neurotoxic effects of N-methyl-PTP remain unclear.