Catecholamines (CAs; norepinephrine (NE), dopamine and epinephrine) play important roles not only in the peripheral nervous system but in the central one. One of the inactivation pathways of CAs is the enzymatic metabolism by catechol-O-methyltransferase (COMT; EC2.1.1.6), which methylates their catechol moieties using S-adenosyl-L-methionine (SAMe) as a methyl donor.1) There are two COMT isoforms: in the cytoplasm as soluble COMT (S-COMT) and in association with membranes as membrane-bound COMT (MB-COMT).We have previously reported a rapid assay method for COMT activities using high-performance liquid chromatography (HPLC)-fluorescence detection.2) In this study, we applied the method to brain tissues, such as cerebral cortex, cerebellum, hypophysis and hypothalamus.In previous report using Sprague-Dawley (SD) rats, the cerebral cortex and the cerebellum were seemed to be important parts in brain for the metabolism of NE.3) Furthermore, the experiment with spontaneously hypertensive rats (SHR) suggested an association between MB-COMT in the cerebral cortex and blood pressure.4) Hence, in order to clarify the role of COMT in rat brain in relation to salt-sensitive hypertension, COMT activities in Dahl salt-sensitive (DS) rats, 5,6) which are model rats of salt-sensitive hypertension, were also examined.
MATERIALS AND METHODSReagent NE, normetanephrine (NMN), SAMe chloride salt and 4-hydroxy-3-methoxybenzylamine (HMBA) were purchased from Sigma (St. Louis, MO, U.S.A.). Ethylenediamine was from Sigma-Aldrich (Milwaukee, WI, U.S.A.). Imidazole and 1,4-dithiothreitol were obtained from Merck (Darmstadt, Germany). Acetonitrile and ethanol, both of HPLC grade, were purchased from Kanto Chemical Co. (Tokyo, Japan). All other reagents were of analytical grade.Animals Male DS and Dahl salt-resistant (DR) rats (6-7 weeks old) were purchased from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). The rats were randomly assigned to the normal-salt (NS, 0.3% NaCl) or high-salt (HS, 8% NaCl) group. The animals were housed under controlled environment (22-24°C and a 12-h light-dark cycle) with free access to tap water and diet. Systolic blood pressure was measured by the tail-cuff method every 2 weeks. All animals received human care in compliance with the National Institute of Health guideline.Preparation of Brain COMT Samples In anesthetized rats with pentobarbital, blood was removed from inferior vena cava, and cerebral cortex, cerebellum, hypophysis and hypothalamus were immediately removed and chilled on ice. All further procedures were the same with the previous reports.2,3) Each tissue was weighted and then homogenized. The homogenates were centrifuged at 100000ϫg for 30 min and the supernatants were stored for the determination of S-COMT. The pellet was washed and re-centrifuged at 100000ϫg for 30 min. Then, the pellet was suspended in buffer for the MB-COMT.COMT Assay Forty microliters of the S-COMT sample was incubated with 50 mM sodium phosphate buffer (pH 7.8) containing 1.5 mM NE, 2 mM MgCl 2 and 200 mM SAMe...