Spasmolytic Effect of Flopropione Does Not Involve Catechol-O- methyltransferase (COMT) Inhibition.

Flopropione (Flo) has been used for gallstone and urolithiasis as a spasmolytic agent almost exclusively in Japan. According to the package insert, its main mechanism is catechol-O-methyltransferase (COMT) inhibition and anti-serotonergic effect. This is obviously contrary to pharmacological common sense, but it is described that way in pharmacology textbooks and occurs in questions in the National Examination for Pharmacists in Japan. As this is a serious problem in education, we re-examined the action of Flo. The guinea pig ureter was hardly contracted by serotonin, but noradrenaline (NA) elicited repetitive twitch contraction, which was inhibited by Flo. The sphincter of Oddi (SO) exhibited a spontaneous repetitive twitch contraction, which was inhibited by NA and Flo. The inhibitory effect of NA was reversed by α- and ß-blockers, whereas that of Flo was not. Entacapone, a representative COMT inhibitor, did not affect the movement of the ureter and the SO. Nifedipine suppressed carbachol-induced contraction of the taenia coli, spontaneous movement of the SO, and NA-induced contraction of the ureter to almost the same extent, whereas Flo did not inhibit the taenia coli, but inhibited the contraction of the SO and the ureter. The inhibitory pattern of Flo resembled that of the ryanodine receptor agonist 4-chloro-m-cresol and the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate. It is concluded that COMT inhibition or serotonin inhibition is not involved in the spasmolytic action of Flo. Flo might act on ryanodine receptors and/or IP3 receptors, which are responsible for periodic Ca release from Ca stores, to disrupt coordinated Ca dynamics.

Cajanus cajan (L) Millsp seeds extract prevents rotenone-induced motor- and non-motor features of Parkinson disease in mice: Insight into mechanisms of neuroprotection.

ETHNOPHARMACOLOGICAL RELEVANCE: Cajanus cajan (L) Millsp (Fabaceae) seed decoction is used by traditional healers in Nigeria as nerve tonic, hence, could be beneficial in the treatment of Parkinson's disease (PD), a progressive and debilitating neurodegenerative disease that imposes great burden on the healthcare system globally. AIM OF THE STUDY: This study aimed at investigating the neuroprotective effect of ethanol seed extract of Cajanus cajan (CC) in the treatment of rotenone-induced motor symptoms and non-motor symptoms associated with PD. MATERIALS AND METHODS: To assess the protective action of CC on rotenone-induced motor- and non-motor symptoms of PD, mice were first pretreated with CC (50, 100 or 200 mg/kg, p.o.) an hour before oral administration of rotenone (1 mg/kg, p.o, 0.5% in carboxyl-methylcellulose) for 28 consecutive days and weekly behavioural tests including motor assessment (open field test (OFT), rotarod, pole and cylinder tests) and non-motor assessment (novel object recognition (NOR), Y-maze test (YM), forced swim and tail suspension, gastric emptying and intestinal fluid accumulation tests) were carried out. The animals were euthanized on day 28 followed by the collection of brain for assessment of oxidative stress, inflammatory markers and immunohistochemical analysis of the striatum (STR) and substantia nigra (SN). Phytochemicals earlier isolated from CC were docked with protein targets linked with PD pathology such as; catechol-O-methyltransferase (COMT), tyrosine hydroxylase (TH) and Leucine rich receptor kinase (LRRK). RESULTS: this study showed that CC significantly reduced rotenone-induced spontaneous motor impairment in OFT, pole, cylinder and rotarod tests in mice as well as significant improvement in non-motor features (significant reversal of rotenone-induced deficits discrimination index and spontaneous alternation behaviour in NORT and YM test, respectively, reduction in immobility time in forced swim/tail suspension test, gastrointestinal disturbance in intestinal transit time in mice. Moreso, rotenone-induced neurodegeneration, oxidative stress and neuroinflammation were significantly attenuated by CC administration. In addition, docking analysis showed significant binding affinity of CC phytochemicals with COMT, TH and LRRK2 receptors. CONCLUSION: Cajanus cajan seeds extract prevented both motor and non-motor features of Parkinson disease in mice through its antioxidant and anti-inflammatory effects. Hence, could be a potential phytotherapeutic adjunct in the management of Parkinson disease.

Acute effects of Δ9-tetrahydrocannabinol (THC) on resting state connectivity networks and impact of COMT genotype: A multi-site pharmacological fMRI study.

BACKGROUND: Cannabis produces various acute psychotropic effects, with marked individual differences. Cannabis use is a risk factor for developing psychotic disorders. The main component responsible for these effects is Δ9-tetrahydrocannabinol (THC). Here we investigated the neural basis of acute THC effects and its modulation by catechol-methyl-transferase (COMT) Val158Met genotype. METHODS: Resting state functional MRI data of healthy occasional cannabis users were combined and re-analyzed from three double-blind, placebo-controlled, within-subject pharmacological functional magnetic resonance imaging studies (total N=87). Functional connectivity after placebo and THC was compared in three functional networks (salience, executive and default mode network) and a network implicated in psychosis (the hippocampus-midbrain-striatum network). COMT genotype modulation of subjective effects and connectivity was examined. RESULTS: THC reduced connectivity in the salience network, specifically from the right insula to both the left insula and anterior cingulate cortex. We found a trend towards decreased connectivity in the hippocampus-midbrain-striatum network after THC. COMT genotype modulated subjective effects of THC, with strongest dysphoric reactions in Met/Met individuals. In addition, reduced connectivity after THC was demonstrated in the hippocampus-midbrain-striatum network of Met/Met individuals only. CONCLUSIONS: In this large multisite study we found that THC robustly decreases connectivity in the salience network, involved in processing awareness and salient information. Connectivity changes in the hippocampus-midbrain-striatum network may reflect the acute psychotic-like effects of THC. COMT genotype modulation of THC's impact on subjective effects and functional connectivity provides further evidence for involvement of prefrontal dopamine levels in the acute effects of cannabis.

Anti-cancer effect of entacaponeon esophageal cancer cells via apoptosis induction and cell cycle modulation.

BACKGROUND: Esophageal cancer (EC) is the sixth leading cause of cancer-related death, despite many advances in treatment, the survival of patients still remains poor. In recent years, the N6-methyladenosine (m6A) has been introduced as one of the most important modifications at the epitranscriptome level, with an important role in the mRNA regulation in various diseases, such as cancers. The m6A is regulated by different factors, including FTO as a demethylase. The m6A modification, especially through FTO overexpression has an oncogenic role in different cancer types such as EC. Recent studies showed that entacapone, a catechol-o-methyl transferase (COMT) inhibitor currently applied for Parkinson's disease, can inhibit FTO enzyme. AIMS: In this study, we aimed to investigate the effect of entacapone as an FTO inhibitor on the m6A level and also apoptosis and cell cycle response in KYSE-30 and YM-1 of esophageal squamous cancer cell (ESCC) lines. METHODS: Cell toxicity and IC50 of entacapone were evaluated using The MTT assay in YM-1 and KYSE-30 cells. Cells were treated into two groups: DMSO (control) and entacapone (mean IC50 ). Total RNA was extracted, and m6A levels were measured via the ELISA method. Subsequently, the apoptosis and cell cycle dys-regulation were detected by annexin-V-FITC/PI staining and PI staining via flow cytometry. RESULTS: Entacapone has the cytotoxicity effect on both esophageal cancer cell lines compared to normal PBMC cells. As well, entacapone treatment (140 µM) can induce apoptosis (KYSE-30: 50%. YM-1:22.6%) and has a modulatory effect on cell cycle progression in both YM-1 and KYSE-30 cells (p-value<.05). However, no significant difference in the m6A concentration was observed. CONCLUSION: Our findings suggested that entacapone has the inhibitory effect on ESCC cell lines through induction of the apoptosis and modulation of the cell cycle without toxicity on the normal PBMC.

Chronic coexposure to arsenic and estrogen potentiates genotoxic estrogen metabolic pathway and hypermethylation of DNA glycosylase MBD4 in human prostate epithelial cells.

BACKGROUND: Previously we reported that arsenic and estrogen cause synergistic effects in the neoplastic transformation of human prostate epithelial cells. In addition to receptor-mediated pathways, DNA-reactive estrogen metabolites have also been shown to play a critical role in mutagenicity and carcinogenicity. Both estrogen and arsenic are known prostate carcinogens, however, the effect of coexposure to these two chemicals on genes involved in estrogen metabolism is not known. Therefore, the objective of this study was to evaluate the role of arsenic and estrogen coexposure on the expression of estrogen receptors and estrogen metabolism-associated genes. Earlier, we also reported the synergistic effect of arsenic and estrogen on decreased expression of MBD4 genes that play an important role in DNA repair through its DNA glycosylase activity. To further understand the mechanism, the promoter methylation of this gene was also analyzed. METHODS: Total RNA and protein were isolated from RWPE-1 human prostate epithelial cells that were coexposed to arsenic and estrogen for a chronic duration (6 months). The expression of estrogen receptors, estrogen metabolism associated phase I genes (CYP 1A1, 1A2, 3A4, and 1B1) and phase II gene catechol-O-methyltransferase (COMT), as well as antioxidant MnSOD, were analyzed either at the RNA level by quantitative reverse transcriptase-polymerase chain reaction or at the protein level by western blot. Promoter methylation of MBD4 was analyzed by pyrosequencing. RESULTS: Expression of MnSOD and phase I genes that convert E2 into genotoxic metabolites 2-OH-E2 and 4-OH-E2 were significantly increased, whereas the expression of phase II gene COMT that detoxifies estrogen metabolites was significantly decreased in arsenic and estrogen coexposed cells. MBD4 promoter was hypermethylated in arsenic and estrogen coexposed cells. Coexposure to arsenic and estrogen has synergistic effects on the expression of these genes as well as in MBD4 promoter hypermethylation. CONCLUSIONS: These novel findings suggest that coexposure to arsenic and estrogen acts synergistically in the activation of not only the estrogen receptors but also the genes associated with genotoxic estrogen metabolism and epigenetic inactivation of DNA glycosylase MBD4. Together, these genetic and epigenetic aberrations provide the molecular basis for the potentiation of carcinogenicity of arsenic and estrogen coexposure in prostate epithelial cells.

Thermostability and catechol-O-methyltransferase inhibitory activity of acylated anthocyanins from purple yam.

The thermostability of purple yam was investigated to be used as natural colorants. In addition, the inhibitory properties of purple yam and its isolated anthocyanins toward human catechol-O-methyltransferase (COMT), a key neurotransmitter involved in Parkinson's disease and depression, were also investigated. The thermostability of purple yam was higher than that of the reference samples (purple sweet potato and purple potato). Quantitative HPLC analysis revealed that alatanin A (2) contributed to the thermostability of purple yam. Methanol extracts of purple yam exhibited the highest COMT inhibitory activity of the tested samples. Alatanin D (1) showed the highest inhibitory activity of the anthocyanins in purple yam (IC50 19 µM). This study revealed the thermostability and COMT inhibitory activity of purple yam and may lead to its use not only as a thermostable natural source of colorants, but also for the prevention and treatment of Parkinson's disease and depression.

Evaluation of Selected Natural Compounds as Dual Inhibitors of Catechol-O-Methyltransferase and Monoamine Oxidase.

BACKGROUND: The most effective symptomatic treatment of Parkinson's disease remains the metabolic precursor of dopamine, L-dopa. To enhance the efficacy of L-dopa, it is often combined with inhibitors of the enzymes, catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) B, key metabolic enzymes of L-dopa and dopamine. OBJECTIVE: This study attempted to discover compounds that exhibit dual inhibition of COMT and MAO-B among a library of 40 structurally diverse natural compounds. Such dual acting inhibitors may be effective as adjuncts to L-dopa and offer enhanced value in the management of Parkinson's disease. METHODS: Selected natural compounds were evaluated as in vitro inhibitors of rat liver COMT and recombinant human MAO. Reversibility of MAO inhibition was investigated by dialysis. RESULTS: Among the natural compounds morin (IC50 = 1.32 µM), chlorogenic acid (IC50 = 6.17 µM), (+)-catechin (IC50 = 0.86 µM), alizarin (IC50 = 0.88 µM), fisetin (IC50 = 5.78 µM) and rutin (IC50 = 25.3 µM) exhibited COMT inhibition. Among these active COMT inhibitors only morin (IC50 = 16.2 µM), alizarin (IC50 = 8.16 µM) and fisetin (IC50 = 7.33 µM) were noteworthy MAO inhibitors, with specificity for MAO-A. CONCLUSION: None of the natural products investigated here are dual COMT/MAO-B inhibitors. However, good potency COMT inhibitors have been identified, which may serve as leads for future development of COMT inhibitors.

Modification of COMT-dependent pain sensitivity by psychological stress and sex.

Catecholamine-O-methyltransferase (COMT) is a polymorphic gene whose variants affect enzymatic activity and pain sensitivity via adrenergic pathways. Although COMT represents one of the most studied genes in human pain genetics, findings regarding its association with pain phenotypes are not always replicated. Here, we investigated if interactions among functional COMT haplotypes, stress, and sex can modify the effect of COMT genetic variants on pain sensitivity. We tested these interactions in a cross-sectional study, including 2 cohorts, one of 2972 subjects tested for thermal pain sensitivity (Orofacial Pain: Prospective Evaluation and Risk Assessment) and one of 948 subjects with clinical acute pain after motor vehicle collision (post-motor vehicle collision). In both cohorts, the COMT high-pain sensitivity (HPS) haplotype showed robust interaction with stress and number of copies of the HPS haplotype was positively associated with pain sensitivity in nonstressed individuals, but not in stressed individuals. In the post-motor vehicle collision cohort, there was additional modification by sex: the HPS-stress interaction was apparent in males, but not in females. In summary, our findings indicate that stress and sex should be evaluated in association studies aiming to investigate the effect of COMT genetic variants on pain sensitivity.

Catechol-O-methyltransferase low activity genotype (COMTLL) is associated with low levels of COMT protein in human hepatocytes.

A single nucleotide polymorphism in the COMT (catechol-O-methyltransferase) gene that alters the amino acid sequence at codon 108 of S-COMT from val to met (val108met polymorphism) has been associated with a number of diseases and neuropsychiatric disorders. Several studies have shown that the met108 allele (COMTL) is associated with three to four-fold lower levels of COMT activity, compared to the val108 allele (COMTH), in extracts of human erythrocytes, liver and kidney tissue. We hypothesized that the differences in COMT activity observed in these studies were due to differing levels of COMT protein in cells and tissues with varying COMT genotypes. In order to address this, we obtained hepatocytes from 31 Caucasian female donors and determined their COMT genotype, COMT activity and COMT protein levels. We found that both cytosolic COMT activity and cytosolic COMT protein levels are lower in hepatocytes from COMTLL individuals, and that COMT activity levels correlate with COMT protein levels. Therefore, lower COMT activity seen in tissues and cells with the COMTLL genotype is likely due to lower COMT protein levels compared with tissues and cells from COMTHH individuals.

The association between panic disorder and the L/L genotype of catechol-O-methyltransferase.

To clarify the role of catechol-O-methyltransferase (COMT) polymorphism in panic disorder (PD), we investigated a large group of Korean PD patients (N = 178) and controls (N = 182) using a case-control study. We also assessed the response to paroxetine treatment and other clinical variables in the PD patients. The increase in the COMT(L) allele was not statistically significant in PD (p = 0.104). However, compared with the sum of the other genotypes, the frequency of the L/L genotype was significantly higher in PD (p = 0.042). The odd ratios (ORs) also indicated a significant effect of the homozygosity for the COMT(L) allele on an increased risk for PD (OR=2.38; 95% CI 1.03-5.51). In addition, patients with L/L genotype had higher trait-anxiety levels (p = 0.030) and poorer treatment response to paroxetine than those with other genotypes (p = 0.002). Our results suggest that the COMT L/L genotype is associated with PD and the genetic variant of the COMT enzyme may be related to the clinical severity and treatment response to paroxetine in PD.

Equine catechol estrogen 4-hydroxyequilenin is a more potent inhibitor of the variant form of catechol-O-methyltransferase.

Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.

Catechol estrogen metabolites and conjugates in different regions of the prostate of Noble rats treated with 4-hydroxyestradiol: implications for estrogen-induced initiation of prostate cancer.

Prostate carcinomas arise in 100% of Noble rats treated with estradiol and testosterone. We hypothesize that estrogens initiate prostate cancer mainly by formation of 4-catechol estrogens (CE), followed by their oxidation to catechol estrogen-3,4-quinones (CE-3,4-Q), which can react with DNA. To avoid cancer initiation, CE can be detoxified by catechol-O-methyltransferase (COMT), and CE-3,4-Q by conjugation with glutathione (GSH) or by reduction to CE, catalyzed by quinone reductase and/or cytochrome P450 reductase. To investigate the prostatic metabolism of estrogens, Noble rats were treated with the CE 4-hydroxyestradiol (4-OHE2) or estradiol-3,4-quinone (E2-3,4-Q), and CE metabolites and conjugates were analyzed in the four regions of the prostate, which differ in susceptibility to carcinoma formation. Following treatment of rats with 4-OHE2 (6 micromol/100 g body weight in 200 microl of trioctanoin/dimethylsulfoxide (4:1) by intraperitoneal injection) for 90 min, the non-susceptible ventral (VP) and anterior (AP) prostate had higher levels of 4-methoxyCE and GSH conjugates than the susceptible dorsolateral prostate (DLP) and periurethral prostate (PUP). After treatment with the same molar amount of E2-3,4-Q, the VP and AP contained more GSH conjugates, 4-CE and 4-methoxyCE than the susceptible DLP and PUP. These results suggest that prostate areas susceptible to carcinoma induction have less protection by COMT, GSH, and quinone reductase and/or cytochrome P450 reductase, favoring reaction of CE-3,4-Q with DNA, presumably to initiate cancer.

Catechol-O-methyltransferase decreases levodopa toxicity in vitro.

The purpose of this study was to examine the effects of 3-O-methylation by catechol-O-methyltransferase (COMT) on the toxicity of levodopa in neuronal cultures. High concentrations of levodopa are toxic in vitro. Therefore, there is concern that long-term treatment with levodopa in patients with Parkinson's disease might accelerate the rate of degeneration of nigrostriatal neurons. However, recent studies have suggested that, while levodopa is harmful in vitro, it may not be toxic in vivo. A possible defense mechanism is by means of metabolic shunting of levodopa excess to 3-O-methyldopa by COMT in peripheral and central nervous system tissues. In this study we examine whether the use of COMT inhibitor, which reduced the levels of 3-O-methyldopa, affect levodopa toxicity. Mice cerebellar granule neurons, PC12, and neuroblastoma cells were used, and their viability following exposure to levodopa and COMT with and without tolcapone, a COMT inhibitor, was measured by neutral red staining. Auto-oxidation of levodopa was evaluated using a spectrophotometer (690 nm). We found that 3-O-methyldopa, unlike levodopa, was not toxic to all cells examined. Addition of purified COMT to levodopa prevented its auto-oxidation and markedly attenuated its cytotoxicity in vitro. Additional tolcapone reversed the protective effect of COMT. The agent 3-O-methyldopa is not toxic to cell cultures. Catechol-O-methyltransferase attenuates toxicity of levodopa in vitro by its metabolism to nontoxic 3-O-methyldopa.

Catechol-O-methyltransferase inhibition increases the uptake of 11C-3-(3,4-dihydroxyphenyl)-L-alanine in the rat pancreas.

BACKGROUND: The objective of the present investigation was to evaluate the uptake and metabolism of 3-(3,4-dihydroxyphenyl-L-alanine) (L-DOPA) in the rat pancreas. METHODS: The procedure included intravenous injection of the positron-emitting radiotracer L-[beta-11C] DOPA (DOP) into unanaesthetized male Sprague-Dawley rats and evaluation of uptake of radioactivity in organs in animals only given the tracer and in animals given therapeutic doses of three different catechol-O-methyltransferase (COMT) inhibitors, OR-486, OR-611, or Ro 41-0960. Selected pancreati were homogenized, and the chemical form bearing the radioactivity was analysed with high-performance liquid chromatography (HPLC). RESULTS: The main finding was that the tracer uptake in the pancreas increased fourfold when the rats were pretreated with COMT inhibitors. Half maximum effect of OR-486 was found at a dose of 0.2 mg/kg. HPLC analysis showed that with COMT inhibitor, the radioactivity in the pancreas consisted of 90% DOPAC. When administering MAO-A and COMT inhibitor together, the pancreas radioactivity corresponded to dopamine. Also in the pig pancreas a significant increase of DOP was observed after COMT inhibition. CONCLUSIONS: This study has shown a high turnover of L-DOPA in the rat pancreas, which can be modulated to give enhanced levels of DOPAC or dopamine by COMT and MAO inhibition.

The role of extraneuronal amine transport systems for the removal of extracellular catecholamines in the rabbit.

As selective inhibitors of the extraneuronal monoamine uptake system (uptake2) suitable for in-vivo studies were not available, the question of whether uptake2 plays a definite role in vivo is largely unresolved. We attempted to resolve the question by using 1,1'-diisopropyl-2,4'-cyanine iodide (disprocynium24), a novel agent that blocks uptake2 in vitro with high potency. Anaesthetized rabbits were infused with 3H-labelled noradrenaline, adrenaline and dopamine, and catecholamine plasma clearances as well as rates of spillover of endogenous catecholamines into plasma were measured before and during treatment with either disprocynium24 or vehicle. Four groups of animals were studied: group I, no further treatment: group II, monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited; group III, neuronal uptake (uptake1) inhibited; group IV, uptake1 as well as MAO and COMT inhibited. Disprocynium24 (270 nmol kg-1 i.v. followed by an i.v. infusion of 80 nmol kg-1 min-1) did not alter heart rate and mean arterial blood pressure, but increased cardiac output by 22% and decreased the total peripheral vascular resistance by 16% with no difference between groups. When compared with vehicle controls, catecholamine clearances (normalized for the cardiac output of plasma) were decreased and spillover rates increased in response to disprocynium24. Although there were statistically significant between-group differences in baseline clearances (which decreased in the order: group I > group II > group III > group IV), the drug-induced clearance reductions relative to vehicle controls were similar in groups I to IV and amounted to 29-38% for noradrenaline, 22-31% for adrenaline and 16-22% for dopamine. Hence, there was still a significant % reduction in catecholamine clearances even after the combined inhibition of MAO and COMT, and there was no increase in the % reduction of clearances after inhibition of uptake1. Noradrenaline spillover increased in response to disprocynium24 in all four groups by 1.6- to 1.9-fold, whereas a 1.5- to 2.0-fold increase in adrenaline and dopamine spillover was observed in groups II and IV only. The results indicate that disprocynium24 interferes with the removal of circulating catecholamines not only by inhibiting uptake2, but also by inhibiting related organic cation transporters. As disprocynium24 increased the spillover of endogenous catecholamines into plasma even after inhibition of MAO and COMT, organic cation transporters may also be involved in the removal of endogenous catecholamines before they enter the circulation.

Evidence for saturation of catechol-O-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats.

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/l) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min-1 and 0.186 min-1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline. In isolated lungs perfused with 3H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l 3H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nitecapone (OR-462) on the pharmacokinetics of levodopa and 3-O-methyldopa formation in cynomolgus monkeys.

3-O-Methyldopa (OMD) is the principal circulating metabolite formed from exogenously administered levodopa. We studied the effect of nitecapone (OR-462), a novel inhibitor of catechol-O-methyltransferase (COMT), on OMD formation in cynomolgus monkeys following intravenous levodopa administration. The drug does not cross the blood-brain barrier, and therefore inhibits only peripheral OMD formation. At a dose of 5 mg/kg, nitecapone reduced the area under the OMD concentration-time curve by 50%. Inhibition of OMD production was maximal at 65% following a dose of 10 mg/kg. A dose of 15 mg/kg produced no further inhibition. The plasma pharmacokinetics of carbidopa, levodopa, and OMD in the monkeys were similar to those in humans. No adverse physiological effects of nitecapone were observed. In single-dose studies, OR-462 is an effective peripheral COMT inhibitor.

The O-methylation of 4-hydroxyestradiol is inhibited by 2-hydroxyestradiol: implications for estrogen-induced carcinogenesis.

O-Methylation of catecholestrogens catalyzed by catechol-O-methyltransferase provides a major route for the rapid metabolic clearance of these steroids. However, the metabolic clearance rate of 4-hydroxyestradiol (4-OH-E2) is considerably lower than that of 2-hydroxyestradiol, although 2- and 4-hydroxycatecholestrogens (2- and 4-OH-CE) have similar apparent affinities for the enzyme. To determine the reason for this apparent paradox we have examined whether the efficiency of O-methylation of 4-OH-E2 could be affected by other catecholestrogens or their O-methyl ethers. The ratio of 4-methoxyestradiol:4-hydroxyestradiol 3-methyl ether was 2.6 at pH 8.5, the pH optimum for the reaction. The O-methylation of 4-OH-E2 (apparent Km 10 microM) was inhibited by 2-hydroxyestradiol (2-OH-E2) but not by 2- or 4-methyoxyestrogens. The values for Km, Vmax as well as the slope for the methylation of 4-OH-E2 were altered by 2-OH-E2 indicating a mixed inhibition. The inhibition constant for the intercept 1/V'max versus 2-OH-E2 concentrations and the inhibition constant for the slope versus 2-OH-E2 concentrations were 35 and 5.7 microM, respectively. The inhibition of O-methylation of 4-OH-E2 by 2-OH-E2 increased with the pH. In target tissues of the carcinogenic action of estrogens such as the rat pituitary, hamster kidney, or mouse uterus in which 2- and 4-OH-CE are both generated in almost equal amounts, the inactivation of 4-OH-CE by O-methylation may be impeded. Consequently, 4-OH-E2 would remain available as substrate for redox cycling, generation of active radicals and DNA damage.

Enzymatic methylation of microsomal metabolites of benzo(a)pyrene.

These studies suggest that the microsomal metabolism of benzo(a)pyrene (BP) produces metabolites which can be methylated by the catechol-o-methyltransferase (COMT)/S-adenosylmethionine (SAM) enzyme/donor combination. Induced microsomes converted 12 to 15% of substrate BP to polar products. Approximately 0.06% of substrate BP was recovered as COMT/SAM-reactive substances. In tests for specificity, COMT/SAM was found to react with catechols, but not with dihydrodiols, quinones, a phenol, an epoxide, or 1,4-hydroquinone. Organic extracts of COMT/[14C]SAM incubations with BP were fractionated by high-performance liquid chromatography. The appearance of radiolabeled chromatographic bands required the presence of substrate BP, microsomes, and COMT/[14C]SAM. When the Ames mutagenesis assay was supplemented with COMT/SAM, a 36% reduction was observed in the number of revertant colonies induced by the microsomal oxidation of BP. In contrast, the mutagenic properties of 2-aminofluorene were not affected by COMT/SAM. These observations indicate that COMT/SAM does not generally inhibit mixed-function oxidase activity but rather reacts with substances which are activated by ring oxygenations.

Catechol-O-methyltransferase activity in cultured human skin fibroblasts from controls and patients with dystonia musculorum deformans.

Fibroblasts provide a source of living cells that can be obtained easily from humans and used to evaluate inherited differences in the activities of enzymes important in neurotransmitter and drug metabolism. Here, we describe biochemical characteristics of catechol-O-methyltransferase (COMT, EC 2.1.1.6) activity in homogenates of cultured human skin fibroblasts. Many properties of the enzyme, including apparent affinity for dihydroxybenzoic acid and S-adenosyl methionine, optimal pH and (Mg++), and inhibition by Ca++, are similar to those reported in lysates of human erythrocytes. Culture and assay conditions have been established for optimal and reproducible measurement of COMT activity in individual fibroblast lines. In 16 control lines, COMT activity ranged from 115 to 263 pmol/min/mg protein with a mean of 181 pmol/min/mg protein. Enzyme activity did not vary with the age or sex of the donor. The COMT activities in fibroblasts from eight patients with dystonia musculorum deformans, an inherited movement disorder of unknown etiology, were not significantly different from controls. Monoamine oxidase (MAO, EC 1.4.3.4) type A activity was measured in 12 lines from patients with dystonia, and values did not differ significantly from age- and sex-matched controls. We conclude that inherited variation in activity of these two catabolic enzymes is not sufficient to explain alterations in monoamine metabolism described in this disorder.