Catechol-O-methyltransferase Protein Research Articles

Catechol-o-Methyltransferase Enzyme Activity and Protein Expression in Human Prefrontal Cortex across the Postnatal Lifespan

The prefrontal cortex (PFC) dopamine system, which is critical for modulating PFC function, undergoes remodeling until at least young adulthood in primates. Catechol-o-methyltransferase (COMT) alters extracellular dopamine levels in PFC, and its gene contains a functional polymorphism (Val(158)Met) that has been associated with variation in PFC function. We examined COMT enzyme activity and protein immunoreactivity in the PFC during human postnatal development. Protein was extracted from PFC of normal individuals from 6 age groups: neonates (1-4 months), infants (5-11 months), teens (14-18 years), young adults (20-24 years), adults (31-43 years), and aged individuals (68-86 years; n = 5-8 per group). There was a significant 2-fold increase in COMT enzyme activity from neonate to adulthood, paralleled by increases in COMT protein immunoreactivity. Furthermore, COMT protein immunoreactivity was related to Val(158)Met genotype, as has been previously demonstrated. The significant increase in COMT activity from neonate to adulthood complements previous findings of protracted postnatal changes in the PFC dopamine system and may reflect an increasing importance of COMT for PFC dopamine regulation during maturation.

Catechol- O-methyltransferase gene polymorphisms are associated with multiple pain-evoking stimuli

Variations in the gene encoding catechol- O-methyltransferase ( COMT) are linked to individual differences in pain sensitivity. A single nucleotide polymorphism (SNP) in codon 158 ( val 158 met), which affects COMT protein stability, has been associated with the human experience of pain. We recently demonstrated that three common COMT haplotypes, which affect the efficiency of COMT translation, are strongly associated with a global measure of pain sensitivity derived from individuals’ responses to noxious thermal, ischemic, and pressure stimuli. Specific haplotypes were associated with low (LPS), average (APS), or high (HPS) pain sensitivity. Although these haplotypes included the val 158 met SNP, a significant association with val 158 met variants was not observed. In the present study, we examined the association between COMT genotype and specific pain-evoking stimuli. Threshold and tolerance to thermal, ischemic, and mechanical stimuli, as well as temporal summation to heat pain, were determined. LPS/LPS homozygotes had the least, APS/APS homozygotes had average, and APS/HPS heterozygotes had the greatest pain responsiveness. Associations were strongest for measures of thermal pain. However, the rate of temporal summation of heat pain did not differ between haplotype combinations. In contrast, the val 158 met genotype was associated with the rate of temporal summation of heat pain, but not with the other pain measures. This suggests that the val 158 met SNP plays a primary role in variation in temporal summation of pain, but that other SNPs of the COMT haplotype exert a greater influence on resting nociceptive sensitivity. Here, we propose a mechanism whereby these two genetic polymorphisms differentially affect pain perception.

Estrogen Deficient Male Mice Develop Compulsive Behavior

Aromatase converts androgen to estrogen. Thus, the aromatase knockout (ArKO) mouse is estrogen deficient. We investigated the compulsive behaviors of these animals and the protein levels of catechol-O-methyltransferase (COMT) in frontal cortex, hypothalamus and liver. Grooming was analyzed during the 20-min period immediately following a water-mist spray. Running wheel activity over two consecutive nights and barbering were analyzed. COMT protein levels were measured by Western analysis. Six-month old male but not female ArKO mice develop compulsive behaviors such as excessive barbering, grooming and wheel-running. Excessive activities were reversed by 3 weeks of 17beta-estradiol replacement. Interestingly, the presentation of compulsive behaviors is accompanied by concomitant decreases (p < .05) in hypothalamic COMT protein levels in male ArKO mice. These values returned to normal upon 17beta-estradiol treatment. In contrast, hepatic and frontal cortex COMT levels were not affected by the estrogen status, indicating region- and tissue-specific regulation of COMT levels by estrogen. No differences in COMT levels were detectable between female animals of both genotypes. This study describes the novel observation of a possible link between estrogen, COMT and development of compulsive behaviors in male animals which may have therapeutic implications in obsessive compulsive disorder (OCD) patients.

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 ( COMT L ) is associated with three to four-fold lower levels of COMT activity, compared to the val108 allele ( COMT H ), 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 COMT LL individuals, and that COMT activity levels correlate with COMT protein levels. Therefore, lower COMT activity seen in tissues and cells with the COMT LL genotype is likely due to lower COMT protein levels compared with tissues and cells from COMT HH individuals.

Association between Catechol-O-Methyltransferase Gene Polymorphisms and Wearing-Off and Dyskinesia in Parkinson’s Disease

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates catecholamines, including levodopa. An amino acid change (Val-108-Met) in the COMT protein has been found to result in a change from high to low enzyme activity. In the present study, we genotyped 121 Japanese patients with Parkinson’s disease (PD) and 100 controls. Comparison of the allele frequencies revealed that homozygosity for the low-activity allele was significantly more common among PD patients than the controls (p = 0.047, odds ratio = 3.23). In addition, homozygosity for the low-activity allele was overrepresented in PD patients that exhibited the ‘wearing-off’ phenomenon (p = 0.045, odds ratio = 3.82) or dyskinesia (p = 0.030, odds ratio = 4.80) compared with controls, although these differences were not significant after Bonferroni’s correction. Our results may help understand the mechanism that cause complications of levodopa therapy in PD patients.

Human catechol- O-methyltransferase down-regulation by estradiol

Catechol- O-methyltransferase (COMT) is a crucial enzyme in dopamine and levodopa metabolism. Previously we reported that physiological concentrations of 17β-estradiol (E2) down-regulated steady-state 1.3-kb COMT mRNA levels in MCF-7 cells. In this study, we investigated whether similar reductions occurred in a glial cell line (U138MG) and whether COMT protein and activity levels paralleled the reduction in COMT mRNA levels in MCF-7 cells. In addition, we explored the mechanism of E2 action. E2 had no effect on COMT mRNA levels in U138MG cells, but significantly reduced COMT protein and activity in MCF-7 cells (activity by 53% at 10 −7 M of E2, by 45% at 10 −8 M, and by 28% at 10 −9 M relative to non-E2-treated cells). A specific estrogen receptor antagonist (ICI 182780) blocked these estrogenic effects. Estrogen receptor in nuclear extracts of MCF-7 cells, which were pretreated with E2 (10 −9 M) for 48 h, bound to the whole proximal and distal promoter regions, as determined by electrophoretic mobility shift analysis (EMSA). We propose that E2 decreased COMT activity through down-regulation of its gene and protein expression mediated via ER interaction with response elements in the promoter region of the gene. Our findings may explain the lower of COMT activity in women compared to that in men, and, in part, the beneficial effects of E2 therapy in post-menopausal Parkinson’s disease patients.

Reduced membrane-bound catechol-O-methyltransferase in the liver of spontaneously hypertensive rats.

We have previously reported that methylation of catecholamines by catechol-O-methyltransferase (COMT) was attenuated in spontaneously hypertensive rats (SHR) with acute hypotension as compared with that of Wistar-Kyoto (WKY) rats. Here we examined the soluble (S-) and membrane-bound (MB-) COMT activities and COMT protein in the liver, kidney, and erythrocytes in both strains. Both the activities and the amounts of MB-COMT in the liver were lower in SHR than in WKY rats, but no such trend was found in the kidney or erythrocytes. Nor was such a trend observed in any of these three tissues for S-COMT. These results indicate that liver MB-COMT may be a relevant factor in blood pressure regulation in rats.

Catechol-O-methyltransferase and Parkinson's disease.

Parkinson's disease (PD) is one of the main causes of neurological disability in the elderly. Levodopa is the gold standard for treating this disease, but chronic levodopa therapy is complicated by motor fluctuation and dyskinesia. The catechol-O-methyltransferase (COMT) inhibitors represent a new class of antiparkinsonian drugs. When coadministered with levodopa/decarboxylase inhibitor, 2 COMT inhibitors, tolcapone and entacapone have been shown to improve the clinical benefit of levodopa. COMT activity is genetically polymorphic, and individuals with the low activity (COMT(L/L)) genotype have a thermolabile COMT protein; studies suggest that this genotype is less common in Asians than in Caucasians. Differences in COMT activity may determine the individual response to levodopa and result in ethnic differences in PD susceptibility. Our recent study suggests that the COMTL allele can interact with the MAOB gene to increase the occurrence of PD in Taiwanese. In order to understand this new class of antiparkinsonian drugs, we review their basic properties, pharmacology, and clinical efficacy. The frequency distribution of COMT genetic polymorphisms among different populations and its implications in the etiology and drug response is also discussed.

Brain catecholamine metabolism in catechol‐O‐methyltransferase (COMT)‐deficient mice

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT-knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase-dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT-deficient mice are challenged with l-dihydroxyphenylalanine, they show an extensive accumulation of 3,4-dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage-dependent, brain-region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinson's disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.

Catecholamine metabolism in the brain by membrane-bound and soluble catechol-o-methyltransferase (COMT) estimated by enzyme kinetic values

A kinetic model was constructed to reevaluate the catecholamine metabolism in hypothetical brain homogenates. Earlier published kinetic values of recombinant membrane-bound (MB-) COMT and soluble (S-) COMT were combined with data suggesting that MB-COMT represents 70% and 30% of total COMT protein in human and rat brain, respectively. In the rat brain model L-DOPA and 3,4-dihydroxybenzoic acid were O-methylated mainly via S-COMT, while dopamine and noradrenaline, at low concentrations, were O-methylated slightly more by MB-COMT. In the human brain model dopamine and noradrenaline were metabolized primarily by MB-COMT. The ratio of meta (3-methoxy) over para (4-methoxy) product formation from 3,4-dihydroxybenzoic acid was higher for MB-COMT than S-COMT. It is suggested that MB-COMT clearly predominates the O-methylation of dopamine and noradrenaline also in vivo. Additionally, meta/para ratios could support the enrichment of either isoform of COMT in a homogenate sample.

Soluble and membrane-bound catechol- O-methyltransferase in normal and malignant mammary gland

The levels of 26 kDa-soluble (S) and 30 kDa-membrane-bound (MB) catechol- O-methyltransferase (COMT) polypeptides were determined in paired samples from normal and neoplastic breast tissue of 32 patients with breast cancer. Immunohistochemical staining showed that the COMT reaction in normal mammary tissue was restricted to the epithelial cells in the ducti and lobuli, whereas in the tumors a strong reaction was also seen in the malignant cells. The amounts of COMT proteins in tumors could not be correlated with various clinical or pathological parameters. Quantitative immunoblotting analysis revealed that the total amount of COMT proteins in tumors was more than 50% higher than in respective normal samples in 26 out of 32 patients. Five cases showed less than a 50% difference and in one case less COMT was detected in the tumor. In most cases the amount of both S- and MB-COMT forms was increased. The average amount of total COMT was 178±57 pg/μg total protein in normal tissue and 566±94 pg/μg total protein in tumor. Respective values for S-COMT were 137±52 pg/μg total protein in normal tissue and 369±62 pg/μg total protein in tumor and for MB-COMT 41±10 and 197±41 pg/μg total protein, respectively. Analysis of COMT-specific transcripts suggested that the COMT enzyme level in tumors is determined in some cases by transcriptional and in some cases by post-transcriptional mechanisms.

Global variation in the frequencies of functionally different catechol-O-methyltransferase alleles

Background: Catechol-O-methyltransferase (COMT) has been investigated as a candidate gene in many neurologic disorders involving catecholaminergic systems. The NlaIII restriction site polymorphism (RSP) at COMT is a G↔A (site absent↔site present) single nucleotide polymorphism (SNP) at nucleotide 322/472 (in the short or long mRNA) that results in a Val↔Met polymorphism at amino acid 108/158 (in soluble or membrane-bound) COMT protein and different enzyme activity levels, high for Val, low for Met. COMT enzyme activity is known to vary among ethnic groups, presumably as a result of different population frequencies of these COMT alleles. We have undertaken a direct survey of allele frequencies of this polymorphism in a global sample of populations.Methods: We typed 1314 individuals from 30 different populations using PCR of the relevant region followed by digestion with NlaIII and electrophoresis.Results: The frequencies of the low activity allele (COMT∗L, NlaIII site-present) vary significantly from 0.01 to 0.62. Europeans have nearly equal frequencies of the two alleles while the COMT∗H allele is much more common in populations in all other parts of the world. Sequencing in nonhuman primates indicates that COMT∗H is the ancestral allele in humans.Conclusions: This is the first global survey of the COMT∗L and COMT∗H allele frequencies, confirming and extending earlier studies to show significant world-wide variation. This is also the first study establishing the COMT∗L allele as the derived allele unique to humans. Henceforth, in any population-based association studies of this polymorphism, the control allele frequencies should be in agreement with these published values for corresponding ethnic groups.

Ethnic differences in catechol O-methyltransferase pharmacogenetics

Catechol O-methyltransferase (COMT) inactivates neurotransmitters, hormones and drugs such as levodopa. COMT activity is inherited in an autosomal recessive manner and individuals with low activity have thermolabile COMT protein. A low activity allele has been demonstrated at codon 108/158 of the soluble and membrane bound COMT protein, respectively, whereby a G to A transition results in a valine to methionine substitution, rendering the protein more thermolabile. As ethnic differences in erythrocyte COMT activity have been previously demonstrated, the frequency of low activity alleles were investigated in 265 British Caucasian, 99 British South-west Asian and 102 Kenyan individuals. Genotyping of COMT codon 108/158 was performed using a minisequencing method. Erythrocyte COMT activity was measured in 60 British Caucasian individuals by radiochemical assay. The frequency of low activity alleles was 0.54 in Caucasians, 0.49 in South-west Asians, and 0.32 in Kenyans. There was a much lower frequency of individuals with homozygous low activity allele in the Kenyan population (9%) than in Caucasians (31%) or South-west Asians (27%). Erythrocyte COMT activity was lower and less thermostable in individuals with homozygous low activity alleles. The data provide molecular evidence that low COMT is less common in African individuals than the Caucasian population.

No evidence for an association of affective disorders with high- or low-activity allele of catechol- o-methyltransferase gene

Catechol-o-methyltransferase (COMT) is an enzyme that inactivates biologically active or toxic catechols. Previous studies have yielded inconsistent results on the relationship between erythrocyte COMT activity and affective disorders. Recently an amino acid change (Val-108-Met) of the COMT protein was shown to determine high- and low-activity alleles of the enzyme. Using polymerase chain reaction and the restriction enzyme NLaIII, we genotyped 107 patients with bipolar disorder, 62 with unipolar depression, and 121 controls. Neither bipolar nor unipolar patients differ significantly in the genotypic or allelic frequency from the control group. Even when the bipolar and unipolar patients were pooled into a single group, the distributions of both the genotypes and the alleles for the patient group were similar to those for the controls. We conclude that genetic variation that determines high and low activities of COMT does not have a major effect on the vulnerability to affective disorders in our sample.

Expression and intracellular localization of catechol O-methyltransferase in transfected mammalian cells.

The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.

High and low activity alleles of catechol- O-methyltransferase gene: ethnic difference and possible association with Parkinson's disease

Catechol- O-methyltransferase (COMT) is an enzyme that inactivates catecholamines such as adrenaline, noradrenaline, dopamine, and levodopa. Recently an amino acid change (Val-108-Met) of the COMT protein was found to determine high and low activity alleles of the COMT gene. We genotyped 109 Japanese patients with Parkinson's disease (PD) and 153 controls by using polymerase chain reaction (PCR) amplification and digestion by the restriction enzyme NlaIII. The frequency of low activity allele in the controls was 0.29, which was significantly different from that reported in Caucasians (0.50). When comparison was made between patients with PD and controls, homozygosity for the low activity allele was significantly more common among the patients than among the controls ( P=0.017; odds ratio, 2.8, 95% CI 1.2–6.5), suggesting that homozygosity for the low activity allele may increase susceptibility to PD.

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.

Catechol- O-methyltransferase (COMT) in rat brain: immunoelectron microscopic study with an antiserum against rat recombinant COMT protein

Localization of catechol- O-methyltransferase (COMT) in rat cerebral cortex, neostriatum and cerebellar cortex was studied with pre-embedding immunoelectron microscopy using a specific antiserum raised against rat recombinant COMT protein. In all areas, immunoreactivity was found both in astrocytes and in neuronal processes. Reaction product was seen in the cytoplasm and in association with tubular structures of dendritic processes. Immunoreactivity was also located postsynaptically in dendritic spines and associated with the postsynaptic membrane. Strong immunoreaction was also seen in the cytoplasm of ependymal cells lining the ventricles, and in tanycytes in median eminence. The results suggest that postsynaptic dendritic spines and astrocytic processes may be the sites of catecholamine inactivation by COMT in rat brain.

Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection.

We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day.

Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line.

Human catechol-O-methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane-bound catechol-O-methyltransferase (MB-COMT; amino acids 1-271) and the soluble catechol-O-methyltransferase COMT (S-COMT; delta membrane anchor hCOMT; amino acids 27-271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high-level synthesis of catalytically active enzymes was obtained. Insertion of the human MB-COMT-coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV-hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol-O-methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37 degrees C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 microM for MB-COMT and 108 microM for S-COMT, indicating that recombinant MB-COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of approximately 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)