Non-specific N-methyltransferase Research Articles

A SENSITIVE ASSAY FOR NON-SPECIFIC N-METHYLTRANSFERASE ACTIVITY IN RAT TISSUES BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ELECTROCHEMICAL DETECTION

Phenylethanolamine N-methyltransferase (PNMT) and non-specific N -methyltransferase (EC 2.1.1.28) catalyze the N-methylation of aromatic amines. PNMT is specific for phenylethanolamines such as noradrenaline (NA). and catalyzes the step in catecholamine biosynthesis, forming adrenaline (AD) from NA. PNMT activity is high in adrenal gland, whereas non-specific N-methyltransferase is distributed in various tissues such as the lungs. Borchardt et al. first reported a method to detect PNMT activity by high-performance liquid chromatography electrochemical detection (HPLC-EICD), which could demonstrate the activity only in the adrenal medulla and hypothalamus. Recently, Troeewicz et al. reported a highly sensitive assay method for PNMT using HPLC-EICD by which the activity in all regions of rat brains could be measured. The activity of non-specific N-methyltransferase in brain regions and peripheral tissues of the rat could be detected by a radioassay. However, there has been no repot on an assay method for non-specific N-methyltransferase using HPLC-EICD. In this paper, we describe a highly sensitive assay procedure for the activity of non-specific N-methyltransferase by high-performance reversed-phase ion pair chromatography with electrochemical detection. By this method, the non-specific N-methyltransferase activity could be determined in various rat brain regions and peripheral tissues.

Nonadrenal epinephrine-forming enzymes in humans. Characteristics, distribution, regulation, and relationship to epinephrine levels.

Animal studies indicate that nonadrenal tissues may synthesize epinephrine (E). Here we demonstrate phenylethanolamine N-methyltransferase (PNMT) and/or nonspecific N-methyltransferase (NMT) enzymatic activity in human lung, kidney, heart, liver, spleen, and pancreas. There was a significant overall correlation (r = 0.34) between tissue PNMT and E. PNMT and NMT in human tissues differed in substrate and inhibitor specificity, thermal stability, and antigenicity. By these criteria, PNMT in human lung and in human bronchial epithelial cells were indistinguishable from adrenal PNMT. PNMT and/or NMT activity were present in red blood cells (RBCs), and cancer cell lines. Human kidney, lung, and pancreas showed immunohistochemical staining with an antibody to adrenal PNMT. RBC PNMT activity was lower in males than females and was increased in hyperthyroidism and decreased in hypothyroidism. PNMT activity in a human bronchial epithelial cell line was dramatically increased by incubation with dexamethasone. E and 3H-E levels in plasma and urine during an intravenous infusion of 3H-E into humans indicated that kidney may synthesize half of urinary E. We conclude that PNMT and NMT are widely distributed in human tissues, that they may synthesize E in vivo and are influenced by glucocorticoid and thyroid hormones.

Novel toxins and Parkinson's disease: N-methylation and oxidation as metabolic bioactivation of neurotoxin.

In human brains, a series of monoamine-derived 1,2,3,4-tetrahydroisoquinolines and the 6,7-dihydroxy derivatives has been identified. A tetrahydroisoquinoline was found to cause parkinsonism in monkey, but its toxicity was not so potent as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Two metabolic steps were found to increase cytotoxicity of isoquinolines. N-Methylation by a non-specific N-methyltransferase was proved by in vivo and in vitro experiments. The N-methylated compound was oxidized into N-methylisoquinolinium ion by monoamine oxidase from human brain mitochondria. The oxidation was proved by microdialysis in the rat brain. The isoquinolinium ion was more cytotoxic than the two metabolic precursors. N-Methylation of dopamine-derived 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines was detected by in vivo microdialysis in the rat striatum, and their presence in the human brain was confirmed by GC-MS. The metabolic bioactivation may be a general pathway to produce neurotoxins as the pathogenic agents of Parkinson's disease.

Glucocorticoid induction of epinephrine synthesizing enzyme in rat skeletal muscle and insulin resistance.

Rat skeletal muscle contains two enzymes which can make epinephrine: phenylethanolamine N-methyltransferase (PNMT) and nonspecific N-methyltransferase. We studied the time-course and mechanism by which the glucocorticoid dexamethasone increases muscle PNMT activity. We also examined the hypothesis that increased muscle E synthesis may contribute to glucocorticoid-induced insulin resistance. Dexamethasone (1 mg/kg s.c. for 12 d) increased muscle PNMT activity seven-fold but did not change NMT activity. Immunotitration with an anti-PNMT antibody indicated that the PNMT elevation was due to increased numbers of PNMT molecules. Dexamethasone rapidly increased PNMT activity and this elevation was largely maintained 6 d after glucocorticoid treatment stopped. Muscle epinephrine levels were transiently elevated by dexamethasone. Dexamethasone-treated rats had elevated insulin levels after a glucose load, and chronic administration of the PNMT inhibitor SKF 64139 reversed this increase. Chronic SKF 64139 improved glucose tolerance in normal rats. Dexamethasone induced muscle synthesis of the epinephrine-forming enzyme PNMT. A PNMT inhibitor lowered insulin levels in glucocorticoid-treated rats and glucose levels in untreated rats. These findings are compatible with antagonism of insulin-mediated glucose uptake by epinephrine synthesized in skeletal muscle.

Cardiac epinephrine synthesis. Regulation by a glucocorticoid.

The heart can synthesize epinephrine. Homogenates of rat heart, which contain the enzymes phenylethanolamine N-methyltransferase (PNMT) and nonspecific N-methyltransferase (NMT), methylate norepinephrine to form epinephrine. The cardiac atrium contains primarily PNMT and the cardiac ventricle contains both PNMT and NMT. Rats were given the glucocorticoid dexamethasone at doses ranging from 0.2 to 20 mg/kg. Twenty-four hours later, cardiac atria, ventricle, skeletal muscle, and adrenal had increases in PNMT activity to as much as 230% of baseline. NMT activity was unchanged. Longer-term treatment with 1 mg/kg dexamethasone daily for 12 days increased cardiac PNMT activity about fivefold and also increased atrial epinephrine levels. Dexamethasone did not alter ventricular epinephrine levels but increased levels of both PNMT and catechol-O-methyltransferase, the major catabolic enzyme for epinephrine. After dexamethasone treatment, greater volumes of anti-PNMT antiserum were needed to decrease PNMT enzymatic activity, indicating that dexamethasone treatment resulted in greater amounts of PNMT and did not just activate existing PNMT molecules. Denervation of the masseter muscle of rats by unilateral superior cervical ganglionectomy markedly diminished tissue norepinephrine and epinephrine levels but had no effect on masseter PNMT or NMT levels. We have previously shown that chemical sympathectomy with 6-hydroxydopamine increases cardiac PNMT levels. These findings suggest that PNMT is an extraneuronal enzyme in both cardiac and skeletal muscle. Glucocorticoids have several cardiovascular effects, including increased cardiac output and blood pressure. Enhanced cardiac epinephrine synthesis may mediate some of these glucocorticoid effects.

Lung epinephrine synthesis

We studied in vitro and in vivo epinephrine (E) synthesis by rat lung. Nine days after removal of the adrenal medullas, circulating E was reduced to 7% of levels found in sham-operated rats but 30% of lung E remained. Treatment of demedullated rats with 6 hydroxydopamine plus reserpine did not further reduce lung E. In the presence of S-[3H]adenosylmethionine lung homogenates readily N-methylated norepinephrine (NE) to form [3H]E. The rate of E synthesis by lung homogenates was progressively more rapid with increasing NE up to a concentration of 3 mM, above which it declined. The rate of E formation was optimal at an incubation pH of 8 and at temperatures of approximately 55 degrees C. We compared the E-forming enzyme(s) of lung homogenates with those of adrenal and cardiac ventricle. The adrenal contains mainly phenylethanolamine N-methyltransferase (PNMT), which is readily inhibited by SKF 29661 and methylates dopamine (DA) very poorly. Cardiac ventricles contain mainly nonspecific N-methyltransferase (NMT), which is poorly inhibited by SKF 29661 and readily methylates both DA and NE. Lung homogenates were inhibited by SKF 29661 about half as well as adrenal but more than ventricle. We used the rate of E formation from NE as an index of PNMT-like activity and deoxyepinephrine synthesis from DA as an index of NMT-like activity. PNMT and NMT activity in rat lung homogenates were not correlated with each other, displayed different responses to change in temperature, and were affected differently by glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Dog liver N-methyltransferase : A drug-metabolizing enzyme

A non-specific N-methyltransferase was demonstrated in dog liver. This enzyme is different from other N-methylating systems, especially in terms of substrate and species specificity. The enzyme catalyzes the methylation of a variety of endogenous and exogenous amines; of the compounds studied, SK&F 64139 (7,8-dichloro-1,2,3,4-tetrahydroisoquinoline) was found to be the best substrate. The enzyme utilized S-adenosylmethionine but not 5-methyltetrahydrofolate as a methyl donor, and it had a pH optimum at 8.0. Study of SK&F 64139 with the partially purified enzyme indicated that this dog liver N-methyltransferase had very low K m and high V max values for SK&F 64139. Methylation of SK&F 64139 was not observed with the monkey or rat liver enzyme preparation. This finding is in accordance with the fact that SK&F 64139 is methylated extensively in the dog, but not in other species. The ability of this enzyme to methylate a number of arylalkylamines suggests its possible importance in drug biotransformation.

A highly sensitive assay for non-specific N-methyltransferase activity in rat tissues by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for non-specific N-methyltransferase activity in rat tissues by high-performance liquid chromatography with electrochemical detection

Neonatal corticosteroid permanently alters brain activity of epinephrine-synthesizing enzyme in stressed rats

(Accepted December 14th, 1978) The enzyme responsible for the conversion of norepinephrine to epinephrine, phenylethanolamine N-methyltransferase (PNMT; E. C. 2.1.1.28), is present in the adrenal medulla 1, other chromaffin tissues, sympathetic ganglia 4, and the central nervous systemlL Immunofluorescence and radioenzymatic assays have localized PNMT in discrete brain regions including the hypothalamus 10,15. Cell bodies contai- ning the enzyme are restricted to two cell groups in the rostral medulla of the brain stem, the ventral C1 region and the dorsal Cz region, areas which have established roles in cardiovascular regulation. The activity of non-specific N-methyltransferase is negligible in the brain stem 14. Epinephrine levels are reported to vary directly with PNMT activity in both adrenal and brainlZ, 19. Glucocorticoids are required for maximal activity of adrenal PNMT. In intact rats the physiological level of circulating corticosteroid appears to be virtually optimally effective, since supernormal levels of exogenous steroid do not result in increased activity of PNMT 3,~0. In perinatal rats, treatment with the potent synthetic glucocorticoid, dexameth- asone, produces a marked rise in PNMT activity in the superior cervical ganglion and other ganglia 8,11. This increased is transitory, with enzyme activity falling rapidly after cessation of treatment. Neonatal dexamethasone administration has also been shown to increase PNMT activity in both hypothalamus and brain stem medullalL In brain, the effect of glucocorticoid on PNMT activity has been examined only acutely, at the termination of steroid treatment. We wished to determine whether the neonatal enhancement of brain PNMT activity by dexamethasone might be a permanent effect. Such a finding would suggest that perinatal stress, with its concomitant rise in circulating glucocorticoids, as well as exposure to exogenous corticoids, can affect the level of synthesis of epinephrine in the adult animal. Pregnant Sprague-Dawley rats from Charles River were received in mid- gestation. We pooled the pups on the day following parturition, and randomly * To whom all correspondence should be sent at the following address: Departments of Psychology and Biology, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061, U.S.A.

Phenylethanolamine- N-methyl transferase (PNMT) activity and catecholamine storage and release from chromaffin tissue of the spiny dogfish, Squalus acanthias

1. The activity and characteristics of phenylethanolamine- transferase (PNMT) was studied in the large anterior chromaffin bodies (axillary bodies) of the spiny dogfish, Squalus acanthias. 2. The content of adrenaline and noradrenaline in plasma and various tissues was determined. Strees, induced by keeping the fish in air for 10 min, significantly increased plasma values of both adrenaline and noradrenaline. 3. Electrical stimulation for 1 min of the anterior part of the spinal cord caused release of endogneous adrenaline into the perfused posterior cardinal sinus. 4. The activity of the N-methylating enzyme measured in the large anterior axillary bodies was PNMT and not due to a non-specific N-methyl transferase. 5. It is concluded that adrenaline is synthetized, stored and released from the large anterior chromaffin bodies in the posterior cardinal sinus. The present study indicates that these chromaffin cells are innervated by nerve fibers originating from the central nervous system.

A peptide-like inhibitor of N-methyltransferase in rabbit brain

After pretreatment with pheniprazine, rabbits were administered C-14-tryptamine i.v. and the lung was assayed for the N-methylated derivatives. Unoxidized tryptamine was present, but no N-methyl or N, N-dimethyltryptamine was found in this tissue, which contains high levels of N-methyltransferase. It appears that the indolamine-N-methyltransfer reaction is inhibited in the intact tissue. Our investigation of the possible inhibitory mechanism has led to the purification and characterization of a dialysable factor which inhibits the enzyme in vitro . The factor, which is present in most tissues, was purified from newborn rabbit brain. It is present in two forms, one having approximate mol. wt. 1,500 and one mol. wt. 1,300. Both were inactivated by crystalline trypsin. The 1,300 form was digested by carboxypeptidase A to a smaller, but still active form. It is suggested that these peptides may control in vivo the activity of the non-specific N-methyltransferase against tryptamine and serotonin.

The distribution and properties of the nonspecific N-methyltransferase in brain.

Abstract— The distribution and properties of a nonspecific N‐methyltransferase in the rat brain are described. The enzyme N‐methylates tryptamine and N‐methyltryptamine as well as β‐phenylethylamine, phenylethanolamine, tyramine and octopamine. The enzyme exhibits a pH optimum of 7·9 with phosphate buffer and has a Km for tryptamine of 28 μM. There are potent inhibitors to the enzyme that can be removed by dialysis. Enzymatic activity is present in the brains of a number of species including man, rat, mouse, guinea‐pig and frog. Its activity is unevenly distributed in the brain with the highest activity in the cerebral cortex and striaturn of the rat and in the subcortical white matter in man. Studies of its subcellular distribution indicate that most of the N‐methylation activity is released into the soluble fraction. Enzymatic activity is also present in a number of peripheral tissues of the rat.