Catechol-O-methyltransferase Protein Research Articles

Insight into the Therapeutic Potential of a Bicyclic Hydroxypyridone Compound 2-[(2,4-Dichlorophenyl)methyl]-7-hydroxy-1,2,3,4-tetrahydro-8H-pyrido[1,2-a]pyrazin-8-one as COMT Inhibitor in the Treatment of Parkinson's Disease: A Molecular Dynamic Simulation Approach.

Parkinson's disease (PD) is one of the most targeted neurodegenerative diseases in clinical research. Awareness of research is due to its increasing number of affected people worldwide. The pathology of PD has been linked to several key proteins upregulation such as the catechol O-Methyltransferase (COMT). Hence, the synthesis of compounds possessing inhibitory capacity has been the frontline of research in recent years. Several compounds have been synthesized among which is the nitrocatechol. However, major limitations associated with the nitrocatechol scaffold include the inability to possess adequate CNS penetration properties and hepatic toxicity associated with the compounds. However, a series of bicyclic hydroxypyridones compounds were synthesized to evaluate their inhibitory potentials on COMT protein with compound 38 (c38) 2-[(2,4-dichlorophenyl)methyl]-7-hydroxy-1,2,3,4-tetrahydro-8H-pyrido[1,2-a]pyrazin-8-one shown to have a 40 fold increase level coverage in its IC50 over brain exposure when compared to the other synthesized compound. The molecular dynamics method was employed to understand the nature of interaction exhibited by c38. Molecular mechanics of c38 revealed a disruptive effect on the secondary structure of COMT protein. Per residue decomposition analysis revealed similar crucial residues involved in the favorable binding of c38 and tolcapone implicated its increased inhibitory capacity on COMT in preventing PD. Free binding energy (ΔGbind ) of c38 further revealed the inhibitory capacity towards COMT protein in comparison to the FDA approved tolcapone. Ligand mobility analysis of both compounds showed a timewise different mobility pattern across the simulation time frame at the active site pocket of the protein connoting the different inhibitory potency exhibited by c38 and tolcapone. Findings from this study revealed optimization of c38 could facilitate the discovery of new compounds with enhanced inhibitory properties towards COMT in treating PD.

Computational analysis of deleterious single nucleotide polymorphisms in catechol O-Methyltransferase conferring risk to post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is one of the prevalent neurological disorder which is drawing increased attention over the past few decades. Major risk factors for PTSD can be categorized into environmental and genetic factors. Among the genetic risk factors, polymorphisms in the catechol-O-methyltransferase (COMT) gene is known to be associated with the risk for PTSD. In the present study, we analysed the impact of deleterious single nucleotide polymorphisms (SNPs) in the COMT gene conferring risk to PTSD using computational based approaches followed by molecular dynamic simulations. The data on COMT gene associated with PTSD were collected from several databases including Online Mendelian Inheritance in Man (OMIM) search. Datasets related to SNP were downloaded from the dbSNP database. To study the structural and dynamic effects of COMT wild type and mutant forms, we performed molecular dynamics simulations (MD simulations) at a time scale of 300 ns. Results from screening the SNPs using the computational tools SIFT and Polyphen-2 demonstrated that the SNP rs4680 (V158M) in COMT has a deleterious effect with phenotype in PTSD. Results from the MD simulations showed that there is some major fluctuations in the structural features including root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuation (RMSF) and secondary structural elements including α-helices, sheets and turns between wild-type (WT) and mutant forms of COMT protein. In conclusion, our study provides novel insights into the deleterious effects and impact of V158M mutation on COMT protein structure which plays a key role in PTSD.

S176. A PRELIMINARY INVESTIGATION OF COMT GENE INVOLVEMENT IN COGNITIVE FLEXIBILITY AND ATTENTION IN SCHIZOPHRENIA SPECTRUM DISORDERS

BackgroundSchizophrenia spectrum disorders (SSD) are often characterised by a plateau or decline in cognitive abilities early in the prodrome. The cause of developmental alteration remains unknown, and investigation of genetic involvement in cognitive function in these disorders may assist the understanding of the underlying neurobiological mechanisms involved. Variation at two single nucleotide polymorphisms (SNPs) of the catechol-O-methyltransferase (COMT) gene have previously shown an influence on COMT protein levels and cognition; rs4680 and rs4818. Here we investigate the influence of the nonsynonymous “Val/Met” SNP rs4680 and a second functional SNP, rs4818, on tasks of cognitive flexibility and attention.MethodsThe sample comprised 48 healthy controls (HC; age = 31.95 ± 12.80; 25 males, 23 females), and 43 with a diagnosis of SSD (age = 41.64 ± 10.36; 26 males, 17 females). Measures of cognitive flexibility and attention included the Wisconsin Card Sorting Test (WCST), Continuous Performance Test-Identical Pairs version (CPT-IP), Trail Making Test (TMT), and the D-KEFS Colour Word Interference Test (CWIT). Due to small cohort sizes, in our preliminary analyses we chose to compare people who should be most severely affected because of inheriting COMT haplotypes associated with poor cognitive functioning (GG rs4818 / GG rs4680: G-G haplotype) to those with haplotypes associated with better cognitive functioning (CC rs4818 / AA rs4680: C-A haplotype). Multivariate analysis of variance factors included COMT haplotype, diagnosis (HC and SSD), and gender, with Bonferroni correction for multiple comparisons; age was included as a covariate. Analyses were also conducted based on a non-functional SNP of the COMT gene; rs165599, as a negative control.ResultsSSD exhibited reduced cognitive performance compared to HC; F(4, 75) = 8.810, p < .001. Investigation of C-A haplotype revealed an interaction with diagnosis on cognitive performance; F(8, 154) = 2.075, p = .041; SSD had reduced performance compared to HC for the WCST, CPT-IP, and TMT in C-A haplotypes (all p < .05). COMT haplotype also interacted with gender on cognitive performance (C-A haplotype; F(8, 154) = 2.315, p = .023, G-G haplotype; F(8, 154) = 2.706, p = .008). Males who were C-A non-carriers and /or G-G haplotype (high COMT activity groups) performed better on CPT-IP (both p < .05) and worse on CWIT (both p < .05) compared to females. Control SNP rs165599 revealed no main effects or significant interactions (all p > .05).DiscussionThe role of the COMT gene in the cognitive abilities of SSD remains contentious as gene expression does not differ from a healthy population. This preliminary analysis revealed an interaction between diagnosis and COMT haplotype, however, this only reached statistical significance for the C-A haplotype, where SSD with C-A haplotype and C-A non-carriers had reduced performance compared to HC on most tasks except TMT. The different effects found across the tasks, which probed various elements of cognitive flexibility and attention, supports a nuanced role of COMT in cognitive function. Further, high COMT activity was beneficial for males on CPT-IP but not CWIT compared to females. Gender interaction remains a significant consideration in studies of the COMT gene, likely involving the catechol-estrogens which are substrates of COMT. As expected there was no significant results with control SNP rs165599, indicating that findings were due to the influence of SNPs rs4680 and rs4818 on COMT activity.

In Silico Prediction of the Effects of Nonsynonymous Single Nucleotide Polymorphisms in the Human Catechol-O-Methyltransferase (COMT) Gene.

Catechol-O-methyltransferase (COMT) enzyme performs transfer of methyl group to endogenous and exogenous catechol substrates. The COMT enzyme draws interest because of its association with psychiatric, neurological and cardiovascular diseases, and several cancers. Moreover, many prescribed drugs, supplements, and their metabolites are used as substrates of COMT enzyme. The human COMT gene has 226 nonsynonymous single nucleotide polymorphisms (nsSNPs) according to public databases. Uncovering of the molecular impacts of nsSNPs on COMT enzyme function and structure may provide standpoint on how COMT nsSNPs affect enzyme activity and contribute to disease development. Therefore, we aimed in this study to predict possible structural and functional damaging effects of all knowns nsSNPs in COMT gene by applying various bioinformatics tools. Two hundred and twenty-six nsSNPs were obtained from Ensembl, HGMD, ClinVar, and dbSNP databases. Twenty-eight nsSNPs were found to be high-risk changes for protein structure. Some of them were detected in extremely conserved sequences have functional and structural properties. Besides, high-risk nsSNPs were also uncovered to change properties of native COMT protein. Our findings demonstrated the significance of COMT high-risk nsSNPs on protein structure and function. We expect that our results will be helpful in future studies concerning experimental evaluation of the COMT gene polymorphisms and/or the association between COMT polymorphisms and disease development.

COMT-Catalyzed Palmitic Acid Methyl Ester Biosynthesis in Perivascular Adipose Tissue and its Potential Role Against Hypertension.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [13C16]-PA was methylated to form [13C16]-PAME in incubation medium or the Krebs-Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [13C16]-PA methylation to form [13C16]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [13C16]-PA methylation to form [13C16]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis. SIGNIFICANCE STATEMENT: PAME is a key PVAT-derived relaxing factor. We for the first time demonstrate that PAME is synthesized through PA methylation via the S-5'-adenosyl-L-methionine-dependent COMT catalyzation pathway. Moreover, we confirmed PVAT dysfunction in the hypertensive state. COMT-dependent PAME biosynthesis is involved in Ang II receptor type 1-mediated blood pressure regulation, as evidenced by the reversal of decreased PAME biosynthesis in PVAT by losartan in hypertensive rats. This finding might help in developing novel therapeutic or preventive strategies against hypertension.

Analysis on the correlation between rs4680 polymorphic site of catechol-O-methyl transferase and postoperative pain of mastectomy

Objective To explore the correlation between rs4680 polymorphic site of catechol-O-methyl transferase (COMT) gene and postoperative pain so as to provide theoretical evidence for individualized medicine. Methods A total of eighty -three patients [aging 29-58, American Society of Anesthesiologists (ASA) Ⅰ or Ⅱ] scheduled for mastectomy under general anesthesia was selected. Polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) was adopted to identify their genotype of rs4680 polymorphic site of COMT. The patients were divided as a wild-type group(Val/Val, VV, n=51), a hybrid subtype group (Val/Met, VM, n=23) and a mutation-type group(Met/Met, MM, n=9). Then the tertiary structures of COMT protein before and after mutation were predicted through homology modeling and the physicochemical property of the protein was analyzed. Preoperative anesthesia was conducted by intravenous-inhalation combined anesthesia and postoperative anesthesia by patient controlled intravenous analgesia (PCIA). The number of effective and ineffective compressions of the analgesic pump within 24 h was recorded. Patients′ pain was assessed 2, 4, 8 h and 24 h after operation by the Numerical Rating Scale (NRS) and then its correlation with different genotypes was analyzed. Results The mutation of rs4680 polymorphic site of COMT has no significant effects on tertiary structures of COMT protein but it affected the physicochemical property of COMT protein. MM patients showed remarkably more PCIA press time than VV and VM patients 24 h after surgery (P<0.05). MM patients presented markedly more PCIA effective press time than VV patients (P<0.05). There was no significant difference in NRS score between the three genotypes at each postoperative point. Conclusions Single polymorphic site rs4680 may have little influence on individual difference of postoperative pain for patient with breast cancer. Key words: Catechol-O-methyl transferase; Gene polymorphism; Homology modeling; Postoperative pain; Breast cancer

Overexpression of catechol-O-methyltransferase occurs early in the progression of pancreatic cancer

Abstract Aims: Catechol-O-methyltransferase (COMT) plays a role in many kinds of cancer, and its high expression in pancreatic cancer has been found to be related to better prognosis. However, the expression and biological significance of COMT in multistep pancreatic cancerogenesis is not clear. Main methods: Six LSL-KrasG12D-positive and Pdx1-Cre-positive mice (KrasG12D mice), 6 wild-type mice and 1 human tissue microarray were used in this study. COMT protein expression was measured by immunohistochemistry, and DNA chips were made for use in COMT mRNA assays. Key findings: The expression of COMT in pancreatic intraepithelial neoplasias (PanINs) from KrasG12D mice was significantly higher than that in normal pancreatic tissue from wild-type mice, both in terms of protein level and mRNA expression. The median COMT immunohistochemistry expression scores in human PanIN-1 and PanIN-2 were 70 and 160, respectively, which were significantly lower than the score of 170 observed in pancreatic ductal adenocarcinoma (PDAC) (P = .024 and .037, respectively) and significantly higher than the score of 25 observed in pancreatitis (P = .01 and .008, respectively). Significance: In the development of pancreatic cancer from chronic pancreatitis and in the transition of PanIN to PDAC, we observed significantly increased expression of COMT. These data predict that the overexpression of COMT is an early event in the multistep process of pancreatic cancerogenesis.

Activities of the Dopaminergic System and Glutathione Antioxidant System in the Hippocampus of Stressed rats

The effects of chronic restraint stress (CRS, 2 h during 14 days) on gene expression of tyrosine hydroxylase (TH), catechol-O-methyltransferase (COMT), and glutathione peroxidase (GPx) were studied in the rat hippocampus. Changes in the dopamine (DA) concentration and activities of monoamine oxidases (MAO A and MAO B) and GPx in this cerebral structure of chronically stressed rats were also examined. The investigated parameters were quantified using real-time RT-PCR, Western blot analyses, and assay of enzymatic activity. We found that CRS decreased the TH protein level and DA concentration, which probably confirms the statement that de novo synthesis of DA is suppressed under stress conditions. The increased activities of MAO B, as well as the increased level of COMT protein, are believed to be related to intensified DA catabolism conditions. Also, a decreased activity of GPx in the hippocampus of chronically stressed animals was found. The increased enzymatic activity of MAO B negatively correlated with the reduced activity of GPx under the above-mentioned stress conditions. These events in the hippocampus of chronically stressed animals could synergistically cause oxidative damage to the mitochondria.

Catechol-O-methyltransferase (COMT) genotypes are associated with varying soluble, but not membrane-bound COMT protein in the human prefrontal cortex.

Catechol-O-methyltransferase (COMT) is an enzyme that catalyses the O-methylation, and thereby the inactivation, of catechol-containing molecules. In humans, it has been suggested that COMT modulates cognitive ability, possibly by regulating degradation of dopamine in the prefrontal cortex. Hence, it is significant that two COMT SNPs, rs4680 (c.472 G > A, p.Val158Met) and rs4818 (c.408 C > G), have been associated with cognitive ability in humans. We have shown these SNPs to be associated with levels of muscarinic M1 receptor mRNA in human cortex, which is significant as that receptor also regulates cognitive ability. We decided to determine if COMT genotype was associated with varying levels of COMT protein, as this could be a mechanism by which COMT genotype could be associated with changes in muscarinic M1 receptor mRNA levels. Hence, we measured COMT levels in prefrontal cortex obtained postmortem from 199 subjects, some of whom had a history of schizophrenia, major depressive disorders or bipolar disorders. Our data show, independent of diagnostic status, that genotype at rs4680 and rs4818, but not at rs737865 and rs165599, is associated with differing levels of soluble COMT (S-COMT), but not membrane-bound COMT (MB-COMT). These findings suggest that the association between COMT polymorphisms and cognitive functioning could be, at least in part, due to their association with varying levels of S-COMT. This is important as, unlike MB-COMT, the substrates targeted by S-COMT are likely to be intra-cellular rather than, like dopamine, located mainly in the synaptic vesicles or the extra-cellular space.

Abstract 1404: Role of catechol-O-methyltransferase gene in prostate cancer

Abstract Prostate cancer is one of the most common malignancies and ranks the second most common cause of cancer-related deaths in men in the United States. Catechol-O-methyltransferase (COMT) is a phase II enzyme that detoxifies various catechol compounds that are reactive toward DNA and damaging to the cell. Studies have shown COMT to play a protective role against cancers such as renal and breast, but their effect on prostate is not well understood. In this study, the biological properties and function of COMT in prostate cancer were studied. Expression of COMT was initially measured in normal/benign and cancerous prostate tissues by immunohistochemistry, and cell lines by real-time PCR and western blotting. Cancerous cells displaying the lowest levels was then transfected with COMT. Gene effect on various cellular properties such as cell proliferation, migration, invasion and apoptosis, as well as growth in athymic nude mice were determined. COMT protein expression was lower in cancer regions compared to benign and normal regions of prostate tissues. Cancerous DU145 and DuPro cells also had reduced mRNA levels of COMT but with undetectable protein levels. Interestingly, re-expressing COMT in DU145 and DuPro cells led to decreased cell proliferation, migration, wound healing ability and invasion, and increased apoptosis compared to vector control. COMT also inhibited cell tumor formation in animal models. As a possible target, the TNFRSF11B gene was upregulated due to COMT. These results demonstrate COMT to protect against prostate cancer progression and to have a functional role by affecting apoptosis. COMT may thus be a potential biomarker or therapeutic target for prostate cancer. Citation Format: Shigekatsu Maekawa, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Ryan K. Wong, Varahram Shahryari, Soichiro Yamamura, Sahana Majid, Sharanjot Saini, Laura Z. Tabatabai, Yukio Homma, Rajvir Dahiya, Yuichiro Tanaka. Role of catechol-O-methyltransferase gene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1404.

Deficiency in catechol-o-methyltransferase is linked to a disruption of glucose homeostasis in mice

2-methoxyestradiol (2-ME), an estrogen metabolite generated via catechol-o-methyltransferase (COMT), is multifunctional methoxy-catechol. Here, we report that COMT deficiency leads to glucose intolerance and 2-ME rescues COMT-deficient-associated metabolic defects. Liver COMT protein was suppressed in high fat diet (HFD)-fed or in pregnant mice. COMT suppression, by Ro41-0960 or siRNA, in HFD fed mice or in pregnant mice exacerbated glucose intolerance; 2-ME intervention ameliorated these defects. 2-ME effects on glucose tolerance were associated with AMPK phosphorylation in the liver and in islet cells. Metformin restored liver COMT protein levels, and metformin-induced liver AMPK phosphorylation was abolished by COMT inhibition. The amelioration in glucose tolerance by 2-ME was associated with biphasic insulin secretion in an environment-dependent manner. 2-ME-induced insulin secretion was associated with the AMPK phosphorylation, PDX-1 phosphorylation, and MST-1 suppression in MIN-6 cells. Furthermore 2-ME displayed PPARγ agonist-like activity. These results suggest that COMT is an enzyme to maintain glucose homeostasis and 2-ME is a potential endogenous multi-target anti-diabetic candidate.

Abstract 14360: Gene Transfer of Sarco/Endoplasmic reticulum Ca2+-ATPase 2a Restores Pulmonary Artery Endothelial Catechol-O-Methyltransferase Activity to Decrease Norepinephrine Levels in Pulmonary Hypertension

Elevated levels of norepinephrine (NE) may occur in pulmonary hypertension (PH), in part, when activity of catechol- O -methyltransferase (COMT) is decreased. COMT is expressed by pulmonary artery endothelial cells (PAEC), degrades NE, and is negatively regulated by aldosterone (ALDO) and cytosolic Ca 2+ . Expression of the cardiac isoform of the sarco/endoplasmic Ca 2+ -ATPase (SERCA2a) modulates ALDO and cytosolic Ca 2+ and is downregulated in pulmonary arteries in PH. We hypothesized that gene transfer of SERCA2a to pulmonary arteries would prevent the decrease in COMT activity and lower NE levels. Accordingly, human PAEC were infected with an adenovirus encoding SERCA2a to increase expression 4.2-fold (p&lt;0.01) or control virus and exposed to ALDO (10 -7 mol/L), which decreases COMT activity by 59.2 ± 6.2% (p&lt;0.01). Compared to controls, SERCA2a overexpression in PAEC had no effect on COMT mRNA or protein levels but increased COMT activity by 148.8 ± 14.5% (p&lt;0.01) by decreasing cytosolic Ca 2+ levels by 38.2 ± 6.7% (p&lt;0.05). This resulted in a decrease in NE levels in the medium (229.4 ± 20.6 vs. 139.9 ± 14.8 vs. pg/mg protein, p&lt;0.01) and collagen I expression. To examine the in vivo relevance of SERCA2a gene transfer on COMT activity, the rat monocrotaline model of PH was treated with inhaled adeno-associated virus 1 carrying SERCA2a (AAV1.SERCA2a). There was no difference in COMT expression in AAVI.SERCA2a-treated or saline control PH-rats; however, lung COMT activity was increased 139.8 ± 8.2% (p&lt;0.01) while lung levels of NE (366.7 ± 38.6 vs. 504 ± 30.2 pg/ml/mg, p&lt;0.01), serum ALDO, and pulmonary artery collagen 1 were decreased by AAV1.SERCA2a. In the pig pulmonary vein banding model of PH, inhaled AAV1.SERCA2a gene therapy decreased mean pulmonary artery pressure (54 ± 20 vs. 29 ± 5 mmHg, p&lt;0.03); increased COMT activity by 121.1 ± 6.2% (p&lt;0.05); and decreased levels of ALDO by 43.3 ± 4.6% (p&lt;0.03), NE by 43.3 ± 4.6% (p&lt;0.03), and collagen I in remodeled vessels. Taken together, these data indicate that gene therapy with inhaled AAV1.SERCA2a increases COMT activity by decreasing intracellular Ca 2+ , thereby decreasing NE levels. This extends the actions of SERCA2a beyond pulmonary vascular remodeling in PH to modulating levels of circulating catecholamines.

Nuclear factor-kappa B regulates pain and COMT expression in a rodent model of inflammation.

Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed protein complex regulating the transcription of genes involved in inflammation and pain. Increased NF-κB activity in immune and nervous system cells is linked to several chronic pain conditions in humans as well as inflammation and nerve injury-evoked pain in animals. A recent in vitro study further demonstrates that increased NF-κB activity in astrocytes decreases transcription of catechol-o-methyltransferase (COMT), an enzyme that inactivates catecholamines that cause pain. The purpose of the present study was to examine the relationship between systemic and astrocytic NF-κB activity, pain, and COMT expression in an animal model of inflammation. Results demonstrated that administration of the inflammatory stimulant complete Freund's adjuvant (CFA) led to increased pain and decreased COMT protein expression in an NF-κB-dependent manner. Specifically, we found that rats and mice receiving intraplantar CFA exhibited increased behavioral responses to mechanical and thermal heat stimuli. CFA-evoked pain was blocked in rats receiving a pre-emptive systemic dose of the NF-κB inhibitor MG132 and exacerbated in IKKca mice with constitutive NF-κB activity in astrocytes. Furthermore, we observed NF-κB-linked reductions in COMT expression in midbrain at 6h and 1d following CFA in rats and at 1h and 1d in forebrain and midbrain following CFA in IKKca mice. Collectively, these results demonstrate that systemic and astrocytic NF-κB activity drive inflammatory pain and regulate the expression of COMT in forebrain and midbrain structures.

O13. Catechol-O-methyltransferase deficiency leads to hypersensitivity on the pressor response against angiotensin II

Introduction Women with preeclampsia (PE) exhibit hypersensitivity on the pressor response against angiotensin II (AngII). Catechol-O-methyltransferase (COMT) metabolizes 2-hydroxyestradiol (2-OHE2) into 2-methoxyestradiol (2-ME) and COMT deficiency has shown to be associated with PE. Objectives We have hypothesized that COMT deficiency is reasonable to explain the molecular mechanisms of the hypersensitivity on the pressor response against AngII. Methods We utilized C57BL/6 male mice for all experiments. Mice were subjected to COMT inhibitor (COMTi: 25 mg/kg/day) or olive oil (Control) for 4 weeks, with or without low-dose AngII infusion (ANGII: 70 ng/kg/min) for last 3 weeks. The AngII-infused mice were treated with 2-ME (10 ng/day), 2-OHE2 (10 ng/day), or vehicle for last 1 week. At the end of the protocol, we obtained following 6 experimental groups; Control, ANGII, COMTi, COMTi+ANGII, COMTi+ANGII+2-ME, and COMTi+ANGII+2-OHE2. Also we performed similar experiments utilizing in vivo administration of siRNA of COMT (20 nmol/week) instead of COMTi. Results Neither ANGII nor COMTi exhibited significant alteration in blood pressure through the experimental protocol compared with Control. When compared to ANGII or COMTi, COMTi+ANGII displayed significantly higher blood pressure; COMTi+ANGII+2-ME decreased in blood pressure significantly when compared to COMTi+ANGII. 2-OHE2 treatments did not suppress the blood pressure of COMTi+ANGII. When COMT protein was analyzed in several organs of siRNA-administered mice (COMTsi); COMT protein level was significantly suppressed in liver when compared to control siRNA treated mice (Csi): either heart, kidney, or aorta was not affected. There was no difference in blood pressure between COMTsi and Csi; AngII infusion increased blood pressure in COMTsi but not in Csi: 2-ME administration significantly suppressed blood pressure of COMTsi+ANGII. The levels of soluble fms-related tyrosine kinase 1and placental growth factor were not significantly associated with blood pressure of any groups of mice; plasminogen activator inhibitor(PAI)-1 level was significantly higher in both AngII-infused COMT deficient mice (COMTi+ANGII and COMTsi+ANGII) compared to control mice of each experiment. Such PAI-1 induction was suppressed by 2-ME. Conclusion Deficiency in COMT and 2-ME are associated with the hypersensitivity on the pressor response against AngII infusion. Similar mechanisms could be relevant to pregnant status and PE.

Abstract 18587: Downregulation of Pulmonary Artery Endothelial Catechol-o-methyltransferase Activity by Aldosterone Increases Norepinephrine Levels in Pulmonary Arterial Hypertension

Elevated levels of norepinephrine (NE) occur in pulmonary arterial hypertension (PAH) and are determined, in part, by the activity of catechol- O -methyltransferase (COMT). COMT degrades catecholamines, is negatively regulated by calcium, and is expressed by pulmonary artery endothelial cells (PAEC). As hyperaldosteronism occurs in PAH and aldosterone (ALDO) influences calcium levels, we hypothesized that ALDO decreases COMT activity to increase NE levels in PAH. Accordingly, human PAEC were treated with ALDO (10 -7 mol/L), a level that is achieved clinically in PAH, for up to 72 h. Compared to vehicle-treated PAEC, ALDO decreased COMT activity by 59.2 ± 6.2% (p&lt;0.01) to increase NE levels in the medium (122.4 ± 11.8 vs. 210.7 ± 15.5 pg/mL/mg protein, p&lt;0.01). This occurred as a result of an ALDO-mediated decrease in COMT protein expression by 52.6 ± 9.3% (p&lt;0.01) as well as an increase in intracellular calcium levels (102.9 ± 21.0 vs. 167.7 ± 17.8 nmol/L, p&lt;0.05) to inhibit activity. These effects were abrogated by coincubation with spironolactone. To determine the in vivo relevance of these findings, COMT was examined in the rat monocrotaline model of PAH with confirmed hyperALDO. COMT was decreased (47.6 ± 10.2 %control, p&lt;0.05) in remodeled pulmonary arterioles with a concomitant increase in lung NE levels (432.8 ± 44.5 vs. 899.7 ± 34.2 pg/mL, p&lt;0.01) compared to control rats. In the porcine pulmonary vein banding model of pulmonary hypertension (PH-pigs) with elevated mean pulmonary artery pressure (15[13-15] vs. 35[27-43], p&lt;0.01) and pulmonary vascular resistance (PVR) index (1.97[1.74-2.28] vs. 5.78[2.61-8.75], p &lt;0.05), ALDO levels were also increased (27.1 ± 5.1 vs. 60.8 ± 10.6 pg/mL, p&lt;0.03) in advance of right heart failure as compared to sham controls. PH-pigs demonstrated a 48.3 ± 9.9% (p&lt;0.02) decrease in pulmonary vascular COMT expression and an increase in NE levels (114.6 ± 20.2 vs. 1,622.6 ± 489.2 pg/mL, p&lt;0.02) that correlated positively with ALDO levels (R 2 =0.58, p&lt;0.02). These findings were confirmed in patients with PAH. Together, these data indicate that there is crosstalk in the pulmonary vasculature between ALDO and the sympathetic nervous system to regulate NE levels in PAH, and thus, have implications for therapeutic interventions.

Green Tea Extract Inhibition of Human Leiomyoma Cell Proliferation Is Mediated via Catechol-O-Methyltransferase

Background/Aims: To investigate the inhibitory effect of green tea extract, epigallocatechin gallate (EGCG), on wild-type human leiomyoma (WT-HuLM) cells and its potential action via catechol-o-methyltransferase (COMT) activity. Methods: Cell proliferation of WT-HuLM and COMT gene-silenced HuLM (COMT-shRNA-HuLM) cells treated with 0 or 100 µ<smlcap>M</smlcap> EGCG for 7 days was measured using the MTT method. Total RNA and protein were extracted from cells treated with 0 or 100 µ<smlcap>M</smlcap> of EGCG for 48 h. Gene expression profiling was performed using Human Signal Transduction PathwayFinder. Proliferation cell nuclear antigen (PCNA), cyclin-dependent kinase 4 (Cdk4) and COMT protein levels were detected by Western blot analyses. COMT enzyme activity was evaluated by HPLC. Results: EGCG-treated WT-HuLM cells showed significantly decreased COMT expression (p < 0.001) and enzyme activity (p < 0.05) compared to untreated WT-HuLM cells, while COMT-shRNA-HuLM cells showed no significant change. At 100 μ<smlcap>M</smlcap> of EGCG, survival of WT-HuLM cells was significantly lower (p < 0.05) compared to COMT-shRNA-HuLM cells. EGCG treatment modulated multiple signaling pathways in WT-HuLM compared to untreated control, while changes were minimal or reversed in COMT-shRNA-HuLM cells. EGCG significantly decreased PCNA, Cdk4 and soluble COMT protein levels (p < 0.001) in WT-HuLM, but not in COMT-shRNA-HuLM cells. Conclusions: The antiproliferative and gene-modulating effects of EGCG on HuLM cells are mediated, at least partially, via its effect on COMT expression and enzyme activity.

Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity.

Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined.

Decreased Maternal Serum 2-Methoxyestradiol Levels are Associated with the Development of Preeclampsia

Background: 2-methoxyestradiol (2-ME), a natural metabolite of 17β-estradiol, is synthesized by catechol-O-methyltransferase (COMT). The aim of this study was to explore the maternal 2-ME concentration and placental COMT expression in the different trimesters of normal pregnancy and preeclamptic pregnancies, as well as the effects of 2-ME on cell proliferation and migration of HTR-8/SVneo under normoxic (20% O₂) and hypoxic (2.5% O₂) conditions. Methods: 2-ME levels were examined by ELISA. COMT protein expression was analyzed by Western blot and immunohistochemistry. Cell proliferation and migration were measured by crystal violet assay and transwell system under either normoxia or hypoxia. Results: Maternal 2-ME concentration was elevated with the progression of pregnancy, in contrast, 2-ME was lower in women diagnosed with mild preeclampsia (mPE; 23%) and severe preeclampsia (sPE; 32%) as compared with normotensive full term pregnancies. Meanwhile, preterm controls had lower levels of 2-ME than full term controls. Soluble cytoplasmic COMT (S-COMT), but not membrane-bound COMT (MB-COMT) levels in placentas were increased by 2.5 fold in the full term vs. the first trimester placentas. Furthermore, 2-ME suppressed cell proliferation under 20% O₂ but not 2.5% O₂, while 2-ME promoted cell migration under 2.5% but not 20% O₂in vitro. Conclusion: Considering 2.5% O₂ is a state more closely mimicking in vivo condition, these data suggest a decrease in 2-ME levels may inhibit trophoblast cell migration, possibly leading to PE.

Biological Effects of COMT Haplotypes and Psychosis Risk in 22q11.2 Deletion Syndrome

22q11.2 deletion syndrome (22q11.2DS) is the most common genetic syndrome associated with schizophrenia. The catechol-O-methyltransferase (COMT) gene is located in the obligatory deletion region, and possible associations between COMT variants and neuropsychiatric manifestations in 22q11.2DS have been reported. The purpose of the current study was to evaluate the effect of COMT hemizygosity and molecular haplotypes on gene expression and enzyme activity and its association with psychotic symptoms in 22q11.2DS. Lymphoblast samples were drawn from 53 individuals with 22q11.2DS and 16 typically developing control subjects. We measured COMT messenger (m)RNA and protein expression and enzyme activity using standard procedures. The presence of a psychotic disorder and cognitive deficits were also evaluated using structured testing. There was an approximately 50% reduction in COMT mRNA, protein, and enzyme activity levels in 22q11.2DS samples. Haplotype analysis revealed clear phenotypic differences between various Val-containing haplotypes on COMT-3' untranslated region extended mRNA, soluble COMT and membrane-bound proteins, and enzyme activity. The G variant of rs165599, a 3' untranslated region single nucleotide polymorphism, was associated with low levels of COMT expression and with the presence of psychosis and lower performance IQ scores in our 22q11.2DS sample. Finally, we demonstrate that the COMT rs74745580 "T" mutation is associated with absent soluble COMT expression and very low COMT activity in two 22q11.2DS individuals. Our findings confirm a robust effect of COMT hemizygosity on COMT activity and show complex interactions of variants within the COMT gene that influence COMT biology and confound conclusions based on associations with the Val158Met genotype alone.

Subcutaneous injection of orally tolerated proteins in the mother disturbs embryo implantation in mice

Subcutaneous injection of orally tolerated proteins in the mother disturbs embryo implantation in mice