Determination of differential activities of soluble and membrane-bound catechol-O-methyltransferase in tissues and erythrocytes

doi:10.1016/S0378-4347(99)00125-5

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 729, Issues 1–2, 11 June 1999, Pages 347-353

https://doi.org/10.1016/S0378-4347(99)00125-5 Get rights and content

Abstract

Catechol-O-methyltransferase (COMT) exists as two isoenzymes, a membrane-bound form (MB–COMT) and a soluble form (S–COMT), with different roles in the metabolism of catecholamines and other catechol compounds. This report documents an HPLC assay for separate estimation of S–COMT and MB–COMT activity and examines activities of the two isoezymes among different rat tissues and in human and rat erythrocytes. Activities of MB–COMT and S–COMT varied widely among tissues. There were higher activities of S–COMT than MB–COMT in all tissues except the adrenal medulla where MB–COMT was the predominant isoenzyme, consistent with the importance of this tissue and MB–COMT for the O-methylation of catecholamines. MB–COMT and S–COMT in rat and human erythrocytes showed divergent levels and patterns of activity. The assay represents a rapid and accurate method for quantifying MB–COMT and S–COMT in various tissues and examining the relative roles of COMT isoenzymes in the metabolism of catechol compounds in health and disease.

Introduction

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the conversion of the catecholamines to their O-methylated metabolites using S-adenosyl-l-methionine (SAM) as a methyl donor. There are two COMT isoenzymes: a membrane bound form (MB–COMT) and a soluble form (S–COMT). Investigations have mainly focused on the soluble form, present in most tissues at higher levels of activity than the membrane-bound enzyme [1]. Roth, however, noted that COMT activities are commonly determined as V_max values under saturating concentrations of substrate that favor S–COMT over MB–COMT [2]. Adjusting for the much higher affinity (i.e., lower K_m) for catecholamine substrate of MB–COMT than S–COMT, by dividing V_max values by respective K_m values, suggests that MB–COMT may be the more important isoenzyme involved in the metabolism of the low cytoplasmic concentrations of catecholamines at the extraneuronal sites where the enzyme is located.

The importance of MB–COMT for metabolism of catecholamines in vivo is supported by recent findings that more than 90% of the high levels of circulating metanephrines found in patients with pheochromocytoma are derived from O-methylation of catecholamines within tumors and that these tissues have a greater abundance of MB–COMT over S–COMT [3]. These observations are consistent with other reports that the adrenals, not the liver, are the most important source of circulating metanephrines, responsible for over 90% of circulating metanephrine and about 25% of normetanephrine [4].

The above considerations highlight the need for reliable measurements of both MB–COMT and S–COMT. In this paper a rapid and accurate method, using HPLC with electrochemical detection, for measurement of COMT activity is described. The method was applied to examine the differential tissue distribution of S–COMT and MB–COMT in relation to known sites of metanephrine production. Since clinical studies of COMT typically rely on measurements in readily accessible red cells [5], [6], [7], the utility of the assay for measurements of erythrocyte S–COMT and MB–COMT activity was also examined.

Section snippets

Reagents

S-adenosyl-l-methionine (SAM), pargyline, dihydroxybenzylamine (DHBA), and Triton X-100 were obtained from Sigma (St. Louis, MO, USA). Clorgyline and l-deprenyl were obtained from Research Biochemicals (Natick, MA, USA). All other reagents were analytical grade.

Preparation of tissue samples and erythrocytes

Cerebellum, cerebral cortex, salivary gland, lung, heart, spleen, liver, adrenals (cortex and medulla separated), kidney, vas deferens, and testes were harvested from male Sprague-Dawley rats (300–400 g) sacrificed by lethal injection of

Assay validation

The assay was highly linear (r=0.999) with time of incubation and remained linear for up to 90 min incubation times (Fig. 1). The assay was also highly linear (r=0.999) with respect to a broad range of concentrations of COMT enzyme present in the incubation mixture. The intra-assay coefficient of variation determined from ten repeated determinations of the same sample in the same assay run was 4.7%. The inter-assay coefficient of variation determined from sixteen determinations of the same

Discussion

This report describes and validates an assay for separate measurements of S–COMT and MB–COMT and documents considerable variations in absolute and relative activities of the two COMT isoezymes among different tissues. Importantly, this study establishes that compared to other tissues, the adrenal medulla shows a reverse of the normal pattern of higher activity of S–COMT compared with MB–COMT. Previous findings in humans [4] and dogs [11] indicated that the adrenals represent an important source

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Cited by (38)

Analytical methodologies for sensing catechol-O-methyltransferase activity and their applications
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Mammalian catechol-O-methyltransferases (COMT) are an important class of conjugative enzymes, which play a key role in the metabolism and inactivation of catechol neurotransmitters, catechol estrogens and a wide range of endobiotics and xenobiotics that bear the catechol group. Currently, COMT inhibitors are used in combination with levodopa for the treatment of Parkinson’s disease in clinical practice. The crucial role of COMT in human health has raised great interest in the development of more practical assays for highly selective and sensitive detection of COMT activity in real samples, as well as for rapid screening and characterization of COMT inhibitors as drug candidates. This review summarizes recent advances in analytical methodologies for sensing COMT activity and their applications. Several lists of biochemical assays for measuring COMT activity, including the probe substrates, along with their analytical conditions and kinetic parameters, are presented. Finally, the challenges and future perspectives in the field, such as visualization of COMT activity in vivo and in situ, are highlighted. Collectively, this review article overviews the practical assays for measuring COMT activities in complex biological samples, which will strongly facilitate the investigations on the relevance of COMT to human diseases and promote the discovery of COMT inhibitors via high-throughput screening.
2-Methoxyestradiol ameliorates metabolic syndrome-induced hypertension and catechol-O-methyltransferase inhibited expression and activity in rats
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Metabolic syndrome (MetS) is a common disorder that is associated with hypertension and poses a significant cardiovascular risk. Deactivation of extra-neuronal norepinephrine is mediated by catechol-O-methyltransferase (COMT). Endogenous 2-methoxyestradiol (2ME) is a product of COMT activity. The current study investigated the impact of 2ME on MetS-induced hypertension and the possible alterations in COMT expression and activity in rats. Animals were randomly divided into 4 groups. Group 1 received drinking water and standard food pellets. Groups 2, 3 and 4 were subjected to experimental induction of MetS. Animals in groups 3 and 4 were given daily IP injection of 2ME (25 and 50 mg/kg, respectively). MetS animals showed significant increases in body weight gain and visceral fat, fasting blood glucose and serum insulin and insulin resistance. Meanwhile, MetS was associated with a significant hypertriglyceridemia. Further, MetS significantly increased systolic, diastolic and mean arterial blood pressure. These effects were associated with significant reduction in COMT expression in the liver, kidneys and aorta as well as reduced hepatic activity. 2ME inhibited the alterations in body weight gain, visceral fat accumulation, fasting blood glucose and serum insulin, insulin resistance and serum triglycerides. Elevations in blood pressure were significantly inhibited by 2ME. Also, it attenuated the decrease in liver, kidney and aorta COMT expression and hepatic COMT activity. MetS was associated with elevated epinephrine and norepinephrine levels. Only the higher dose of 2ME significantly mitigated the rise in epinephrine level. In conclusion, 2ME protects against MetS-induced hypertension and averts COMT inhibited expression and activity.
A highly selective fluorescent probe for sensing activities of catechol-O-methyltransferase in complex biological samples
2016, Sensors and Actuators, B: Chemical
Citation Excerpt :
COMT is widely distributed throughout the organs of the body. The highest COMT activity in humans is in the liver, and s-COMT activity is prominent [18]. COMT protein in vertebrates appears mostly in a soluble form (as s-COMT), and only a minor fraction is in the particular form (as MB-COMT) [3,17].
Catechol-O-methyltransferase (COMT), one of the most important phase II drug metabolizing enzymes, plays important roles in the metabolism of endogenous and xenobiotic catechols. In this study, a highly selective fluorescent probe for sensing activities of catechol-O-methyltransferase in complex biological samples was discovered and well characterized. Under physiological conditions, COMT selectively catalyzes the conversion of the probe (7,8-dihydroxy-4-methylcoumarin, DHMC) to 7-hydroxy-8-methoxy-4-methylcoumarin (HMMC), which brings a strong turn-on fluorescence signal at 520 nm. The probe substrate has been used for monitoring the real activities of COMT in complex biological samples, as well as for rapid screening of potential COMT inhibitors which are useful in the treatment of Parkinson's diseases Furthermore, the probe has been successfully used to monitor endogenous COMT in living cells for the first time, and the results demonstrate that DHMC is cell membrane permeable and low toxic to the cells. All these features of DHMC suggested that this probe holds great promise for COMT-related regulation and inhibition assays in drug discovery, as well as for further investigation on the biological functions of COMT in living cells.
Liquid chromatographic methods for the quantification of catecholamines and their metabolites in several biological samples - A review
2013, Analytica Chimica Acta
The measurement of catecholamines and their metabolites in biological samples remains a current analytical challenge, in spite of the great diversity of methodologies that have been developed throughout the years. High-performance liquid chromatography is the standard method for their separation and quantification in biological samples, either coupled with electrochemical, fluorescence, chemiluminescence or mass spectrometry detection. This review summarizes the most important physicochemical properties of catecholamines, the wide panoply of sample preparation techniques and the main issues to consider during the development of chromatographic methods. The major difficulties encountered during the optimization of these procedures are related with the high tendency of catecholamines to oxidize and the very low quantities at which they exist in biological matrices. Herein, the most important aspects that ought to be considered during collection, treatment and storage of fluid and tissue samples intended for catecholamine analysis are underlined, the chromatographic conditions are compared and the technical advantages and limitations of each detection system are discussed.
Differential expression and activity of catechol-O-methyl transferase (COMT) in a generalist (Neotoma albigula) and juniper specialist (Neotoma stephensi) woodrat
2011, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
Citation Excerpt :
At the end of the trials, the animals were euthanized with CO2 and blood, kidney and liver tissues were harvested. While the liver tends to have the highest levels of COMT in the body, the kidney also shows high expression and activity of COMT (Ellingson et al., 1999; Männistö and Kaakkola, 1999; Tsunoda et al., 2002; Zhu, 2002). The expression and activity of COMT in the blood may mirror that occurring elsewhere in the body (Masuda et al., 2002).
Mammalian herbivores, particularly dietary specialists must have an efficient means to metabolize the high doses of plant secondary compounds they consume. We found previously that Neotoma stephensi, a juniper specialist, upregulated catechol-O-methyl transferase (COMT) mRNA almost seven fold in response to an ecologically relevant diet (70% juniper). To further investigate the relevance of this enzyme with respect to juniper metabolism, we compared the protein expression, activity and kinetics of the two forms of COMT, soluble (S-COMT) and membrane bound (MB-COMT), in the blood, kidneys and liver of N. stephensi on its natural juniper diet to that of N. stephensi fed an experimental diet of 70% juniper as well as a non-toxic control diet under laboratory conditions. In addition, we compared these results to that of Neotoma albigula, a generalist species, which consumes a diet of 25% juniper in the wild. The specialist consuming juniper under both field and laboratory conditions had increased S-COMT expression and activity in their livers and kidneys, and increased S-COMT activity in their blood compared to the specialist and generalist fed the control diet. The specialist showed expression and activity of S-COMT in their kidneys that was as high as or higher than that in their livers. The generalist had an elevated V_max for MB-COMT compared to the specialist that resulted in higher activity for MB-COMT than the specialist despite lower expression of MB-COMT in the generalist's livers and kidneys. This high activity MB-COMT may be in part responsible for differences in the behaviors of the generalist compared to the specialist. We conclude that S-COMT is important in the specialist's ability to consume high levels of juniper.
Distribution and functions of catechol-O-methyltransferase proteins. Do recent findings change the picture?
2010, International Review of Neurobiology
Citation Excerpt :
In the rat brain, S-COMT is the dominant form (Lundström et al., 1995), and also the actual S-COMT enzyme activity was higher than MB-COMT activity (Ellingson et al., 1999). Moreover, the enzyme activity of S-COMT and MB-COMT seems to be rather equal in most of the peripheral tissues studied (Table 1B, Ellingson et al., 1999). As stated above, at the subcellular level, MB-COMT has been detected from the RER of the COMT-overexpressing cells while S-COMT was observed from the cytosol and nucleus (Ulmanen et al., 1997).
Old and new results show that both catechol-O-methyltransferase (COMT) forms are found in all mouse tissues, demonstrating that COMT is a ubiquitous enzyme. Some novel findings are obvious when considering differences between old and new distribution data. In addition to the brain, membrane-bound form of COMT (MB-COMT) is found also in most peripheral mouse tissues at about equal amounts as soluble form of COMT (S-COMT), suggesting that their functions do not need to be very different. There are large differences between the species in the relative distribution of S-COMT and MB-COMT. According to the new data, it is evident that even in the animal tissues MB-COMT is not associated with the plasma membranes but with intracellular membranes, and that S-COMT resides not only in the cytoplasm but even in the nucleus.

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Short communicationDetermination of differential activities of soluble and membrane-bound catechol-O-methyltransferase in tissues and erythrocytes

Abstract

Introduction

Section snippets

Reagents

Preparation of tissue samples and erythrocytes

Assay validation

Discussion

Rev. Physiol. Biochem. Pharmacol

J. Pharm. Biomed. Anal.

J. Biol. Chem.

Lancet

Psychiatry Res.

Biol. Psychiatry

Eur. J. Pharmacol.

Clin. Chim. Acta

J. Chromatogr.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr. B.

Pharmacol. Rev

J. Clin. Endocrinol. Metab.

J. Clin. Endocrinol. Metab.

Science

Short communication
Determination of differential activities of soluble and membrane-bound catechol-O-methyltransferase in tissues and erythrocytes