Norepinephrine N-methyltransferase Research Articles

76] Norepinephrine N-methyltransferase from rabbit adrenal glands

76] Norepinephrine N-methyltransferase from rabbit adrenal glands

Regulation of thyrotropin secretion by the central epinephrine system. Studies in the chronically cannulated rat.

In the present investigation CNS epinephrine (EPI) biosynthesis was selectively interrupted with specific norepinephrine N-methyltransferase (NMT) inhibitors, SK&F 64139 (Smith, Kline & French Laboratories) and LY 78335 (Eli Lilly & Co. Research Laboratories), to determine the effects of central EPI depletion on basal and cold, thyrotropin-releasing hormone, and hypothalamic somatostatin antiserum induced thyrotropin (TSH) secretion in chronically cannulated rats. Because these NMT inhibitors also are alpha 2-adrenergic receptor blockers, the effects of alpha 2- and alpha 1-adrenergic blockade and alpha 2-activation on plasma TSH were assessed with rauwolscine and corynanthine and B-HT 933, respectively. Serum T4 and plasma corticosterone were also measured. Blockade of CNS EPI synthesis resulted in inhibition of basal and cold and thyrotropin-releasing hormone induced TSH release, suppression of serum T4, and increased corticosterone release. The stimulatory effect of SRIF antiserum on plasma TSH was not altered by SK&F 64139. alpha 2-adrenergic blockade suppressed plasma TSH levels, but not to the same degree as the NMT inhibitors; activation of alpha 2-receptors enhanced TSH secretion. Thus, it is possible that part of the effect of the NMT inhibitors on TSH was due to alpha 2-blockade. alpha 1-adrenergic blockade also lowered plasma TSH. These results indicate that central EPI systems have a stimulatory role in TSH regulation, possibly mediated by alpha 2-adrenergic receptors, cold-induced TSH release is mediated, in part, by EPI, and central EPI systems exert an inhibitory effect on the hypothalamic-pituitary-adrenal axis.

ChemInform Abstract: IMPORTANCE OF THE AROMATIC RING IN ADRENERGIC AMINES. 8. 2‐(AMINOMETHYL)‐TRANS‐2‐DECALOLS AS INHIBITORS OF NOREPINEPHRINE N‐METHYLTRANSFERASE

In an effort to determine the surface appearance of the hydrophobic ring binding region of the norepinephrine N-methyltransferase active site, we employed some trans-decalin analogues of 1-(aminomethyl)cycloundecanol (1), a potent NMT inhibitor. These analogues [axial and equatorial 2-(aminomethyl)-trans-2-decalol, 2 and 3] closely resemble a low energy crown conformation of 1. Both compounds were as potent as 1 at inhibiting NMT (Ki = 3.6, 5.6, and 3.8 microM for 1, 2, and 3, respectively), indicating that this conformation is most likely adopted within the active site in order to optimize contact with a flat hydrophobic area. None of the compounds showed significant substrate activity for NMT, a fact that is consistent with our proposed active site binding model.

ChemInform Abstract: DIRECTIONAL PROBES OF THE HYDROPHOBIC COMPONENT OF THE AROMATIC RING BINDING SITE OF NOREPINEPHRINE N‐METHYLTRANSFERASE

AbstractDie Acetylbiphenyle (I) werden zu den Verbindungen (II) reduktiv aminiert, und in guten Ausbeuten in die Ester (III) übergeführt.

Inhibition of brain epinephrine synthesis by 3,4-dichlorophenylethanolamine, a competitive substrate for norepinephrine N-methyltransferase

3,4-Dichlorophenylethanolamine (DCPE) is a substrate for norepinephrine N-methyltransferase (NMT) from rat brain stem or from rabbit adrenal glands and competitively inhibits the methylation of (−)norepinephrine by NMT in vitro. The K i value for inhibition of rat brain NMT by DCPE was 6 × 10 −5 M. When DCPE was injected i.p. into rats at a dose of 50 mg/kg, epinephrine concentration in hypothalamus was reduced, although NMT activity measured in hypothalamic homogenates in vitro was not inhibited. The findings contrast in several ways to those with 8,9-dichloro-2,3,4,5-tetrahydro-1 H-2-benzazepine (LY134046), a competitive inhibitor of rat brain NMT with an in vitro K i of 2.4 × 10 −8 M. Although the potency of DCPE was 1/2500th that of LY134046 in vityro, it lowered hypothalamic epinephrine concentrations at similar doses in vivo. The NMT activity measured in vivo in homogenates of hypothalamic tissue from LY134046-treated rats was reduced by a greater percentage than was epinephrine concentration. The reduction of epinephrine concentration in vivo by DCPE may relate to its ability to be a substrate for NMT, which would result in conversion of S-adenosylmethionine (SAMe) to S-adenosylhomocysteine (SAH) in NMT-containing neurons. The resulting increase in the SAH/SAMe ratio may inhibit norepinephrine N-methylation, since SAH is a competitive inhibitor of NMT when SAMe is the variable substrate. This mechanism is similar to that suggested for the decrease in epinephrine concentration in rat brain following l-dopa injection. l-Dopa injection increases SAH and decreases SAMe concentration measured in hypothalamus, due presumably to extensive O-methylation of dopa and its decarboxylated metabolites. In contrast, SAH and SAMe concentrations in hypothalamus were not measurably altered after DCPE injection, a not-unexpected finding since SAH/SAMe changes would occur only in NMT-containing neurons, which make up a negligibly small percentage by weight of hypothalamic tissue. DCPE and other NMT substrates may be able to inhibit epinephrine synthesis in vivo as effectively as non-substrate, competitive inhibitors having much higher affinity for NMT because their N-methylation elevates SAH/SAMe ratios specifically within NMT-containing neurons.

Species differences in epinephrine concentration and norepinephrine n-methyltransferase activity in hypothalamus and brain stem

1. The concentration of epinephrine, norepinephrine and dopamine, and the activity of norepinephrine N-methyltransferase, the epinephrine-forming enzyme, were determined in hypothalamus and brain stem in several species. 2. Epinephrine concentration in hypothalamus, a nerve terminal region, varied in the order frog > turtle > chicken > cat > dog > pigeon > rat > ferret > hamster > mouse, with concentrations being undetectable in rabbits, horses and guinea pigs. 3. Epinephrine concentration was lower than norepinephrine concentration in all species except the frog. 4. NMT activity was detected in all species except guinea pigs. 5. Epinephrine concentration was lower in brain stem, a cell body region, than in hypothalamus in all species. Only in the frog brain stem was there more epinephrine than norepinephrine. 6. No epinephrine or NMT activity was detected in either brain region in guinea-pigs.

Probes of the active site of norepinephrine N-methyltransferase: effect of hydrophobic and hydrophilic interactions on side-chain binding of amphetamine and alpha-methylbenzylamine.

A series of omega-substituted analogues of amphetamine and alpha-methylbenzylamine were prepared and evaluated as inhibitors of norepinephrine N-methyltransferase (NMT). These included several alkyl side chain extended analogues (1-5), as well as the terminally hydroxylated derivatives phenylalanol (6a) and phenylglycinol (7a). None of the alkyl-substituted derivatives displayed appreciable activity as inhibitors; however, the hydroxylated analogues were up to twofold more potent than the parent compounds. The positive contribution of the side-chain hydroxy suggests that the terminal methyl group of the lead compounds is situated close to a hydrophilic area or hydrogen bonding functional group within the active site.

Directional probes of the hydrophobic component of the aromatic ring binding site of norepinephrine N-methyltransferase.

We investigated the directional nature of the bulk tolerance and hydrophobic binding in the aromatic ring binding region of the active site of norepinephrine N-methyltransferase (NMT) by comparing the substrate and inhibitor activities of m- and p-phenyl-substituted derivatives of amphetamine, phenylethanolamine, and alpha-methylbenzylamine. The para isomers of amphetamine and phenylethanolamine displayed significantly greater activities as inhibitor and substrate, respectively, than the meta isomers, which indicated that the bulk tolerance was near the para position. For benzylamines, the greatest inhibitory activity was observed for the meta isomer, demonstrating a significant difference in the binding requirements for phenylethylamines and benzylamines. These findings are consistent with a two-state model for the NMT active site that has been proposed elsewhere to account for its ability to bind both benzylamines and phenylethylamines in a fully extended side-chain conformation.

Importance of the aromatic ring in adrenergic amines. 8. 2-(Aminomethyl)-trans-2-decalols as inhibitors of norepinephrine N-methyltransferase.

In an effort to determine the surface appearance of the hydrophobic ring binding region of the norepinephrine N-methyltransferase active site, we employed some trans-decalin analogues of 1-(aminomethyl)cycloundecanol (1), a potent NMT inhibitor. These analogues [axial and equatorial 2-(aminomethyl)-trans-2-decalol, 2 and 3] closely resemble a low energy "crown" conformation of 1. Both compounds were as potent as 1 at inhibiting NMT (Ki = 3.6, 5.6, and 3.8 microM for 1, 2, and 3, respectively), indicating that this conformation is most likely adopted within the active site in order to optimize contact with a flat hydrophobic area. None of the compounds showed significant substrate activity for NMT, a fact that is consistent with our proposed active site binding model.

Conformational preferences of dopamine analogues for inhibition of norepinephrine N-methyltransferase. Conformationally defined adrenergic agents. 7

A series of analogues of dopamine (DA) with varying degrees of conformational flexibility have been examined as potential substrates or competitive inhibitors of the enzyme norepinephrine N-methyltransferase (NMT). A conformationally defined (rigid) analogue of the fully extended conformation of DA, 2-amino-6, 7-dihydroxybenzonorbornene hydrobromide ( 3 ; 6, 7-D2HX) proved to be a better substrate than the non-catechol parent 2-aminobenzonorbornene ( 4 ; 2HX). However, analogues 3 and 4 displayed equivalent competitive inhibitory activity toward phenylethanolamine (PEA). Neither 6, 7-ADTN (5), a DA analogue in the 2-aminotetralin (2AT) system, nor 6, 7-DTHIQ ( 7 ), a DA analogue in the tetrahydroisoquinoline (THIQ) system, showed substrate activity; 6, 7-ADTN was a poorer competitive inhibitor than the parent 2AT but 6, 7-DTHIQ was a better competitive inhibitor than its parent, THIQ ( 8 ). A tricyclic conformationally defined analogue 9 of 6, 7-ADTN was devoid of either substrate or inhibitory activity. From these results it may be concluded that a fully extended side chain conformation is required for NMT substrate activity, and the better substrate activity for 6, 7-D2HX compared to 4 is consistent with a proper catechol orientation for interaction with the norepinephrine (NE) binding site of NMT.

Depletion of epinephrine in rat hypothalamus by a dopamine agonist, pergolide.

The i.p. injection of pergolide mesylate, a dopamine agonist, at doses of 0.3-0.6 mg/kg led to a decrease in epinephrine concentration in rat hypothalamus. After a 0.6 mg/kg dose of pergolide mesylate, epinephrine concentration in hypothalamus decreased within 2 hr, reached a minimum concentration at about 8 hrs, and then returned toward control values. Norepinephrine N-methyltransferase activity was not decreased after pergolide injection in vivo nor was it inhibited by pergolide added in vitro at concentrations as high as 10(-3) M. Higher i.p. doses of less potent dopamine agonists, apomorphine (10 mg/kg) and lergotrile (3 mg/kg), also decreased epinephrine concentration in hypothalamus. The pergolide-induced decrease in hypothalamic epinephrine concentration was prevented by pretreatment with haloperidol or spiperone, antagonists of dopamine receptors. Activation of dopamine receptors appears to result in a decrease in epinephrine concentration in rat brain, possibly due to enhanced release of epinephrine.

Regulation of episodic growth hormone secretion by the central epinephrine system. Studies in the chronically cannulated rat.

Catecholamines are postulated to regulate growth hormone (GH) secretion by their influence on the release of two hypothalamic substances, somatostatin, which inhibits GH release, and GH-releasing factor, as yet unidentified. Extensive pharmacologic studies in man and animals indicate a stimulatory effect of central norepinephrine and dopamine on GH, but the function of epiphephrine (EPI) is uncertain. Furthermore, many of the agents used to study the role of catecholamines in GH regulation are not selective in that they affect adrenergic as well as nor-adrenergic and/or dopaminergic neurotransmission. In the present investigation, central nervous system (CNS) EPI biosynthesis was selectively interrupted with the specific norepinephrine N-methyltransferase inhibitors, SK & F 64139 (Smith, Kline & French Laboratories) and LY 78335, (Eli Lilly & Co. Research Laboratories) and the effects of central EPI depletion on episodic GH secretion in the chronically cannulated rat model were determined. Inhibition of CNS EPI synthesis with SK & F 64139 caused complete suppression of episodic GH secretion and concomitantly reduced the EPI level in the hypothalamus without affecting dopamine or norepinephrine. Administration of LY 78335 produced similar effects on pulsatile GH. Morphine-induced, but not clonidine-induced, GH release also was blocked by SK & F 64139. These results indicate that (a) the central EPI system has a major stimulatory function in episodic GH release, (b) morphine-induced GH release is mediated by the central EPI system, and (c) clonidine stimulates GH release by activation of postsynaptic alpha-adrenergic receptors. Drugs that affect CNS adrenergic systems have a potential role in the diagnosis and treatment of disorders of GH secretion.

α 2 andrenoreceptor affinity of some inhibitors of norepinephrine N-methyltransferase

Because certain inhibitors of norepinephrine N-methyltransferase (NMT, the epinephrine-forming enzyme) are α 2 adrenoreceptor blockers, we compared the ability of various compounds to inhibit rat brain NMT activity and the binding of tritiated clonidine to rat brain membranes in vitro . There was no correlation between potency of NMT inhibition and potency of α 2 receptor antagonism (as measured by inhibition of tritiated clonidine binding) among NMT inhibitors representing several chemical classes or among members of a single class of compounds, l-aminoindans. In addition, several potent α 2 blocking drugs were essentially inactive as NMT inhibitors. These findings indicate that NMT inhibition and α 2 blockade are dissociable activities. Future development of NMT inhibitors should include this dissociation as a goal to increase the usefulness of NMT inhibitors as pharmacologic tools.

Properties of 8,9-dichloro-2,3,4,5-tetrahydro-1H- 2-benzazepine, an inhibitor of norepinephrine N-methyltransferase

LY134046, 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, was a potent inhibitor of norepinephrine N-methyltransferase (NMT) from rat brain or rabbit adrenal glands in vitro. The inhibition was competitive with respect to the methyl-accepting substrate, (-)-norepinephrine, the K i for LY134046 being 2.4 × 10 −8 M. LY134046 inhibited the NMT activity in rat brain stem and hypothalamus in vivo at doses of 10–40 mg/kg, i.p., and lowered the epinephrine (but not norepinephrine or dopamine) concentration in these brain regions. The epinephrine reduction produced by a single 40 mg/kg, i.p. dose of LY134046 persisted at 24 hr and daily injections of 10–40 mg/kg doses for 5 days produced cumulative reductions in epinephrine concentration. LY134046 was similar to SK&F 64139 (7,8-dichloro-1,2,3,4-tetrahydroisoquinoline hydrochloride), a structurally related compound, as an inhibitor of NMT in vitro and in vivo, but the two compounds differed in their relative abilities to block α 2 receptors. SK&F 64139 was 20-to 50-fold more potent than LY134046 in antagonizing [ 3H]clonidine binding to rat brain membranes and phenylephrine-induced contractions of rat aortic strips, but it was only about twice as potent as LY134046 in inhibiting NMT activity. LY134046 seems to be more selective than other currently known inhibitors of NMT and may be useful for pharmacologic intervention in the function of epinephrine-forming neurons in brain.

Inhibition of rat brain norepinephrine N-methyltransferase by 2,3,4,5-tetrahydro-1H-indeno [1,2-c]-pyridine hydrochloride (LY87130)

2,3,4,5-Tetrahydro-1H-indeno[1,2-c]pyridine hydrochloride (LY87130) was a potent inhibitor of rat brain stem norepinephrine N-methyltransferase (NMT) in vitro. The inhibition was competitive with l-norepinephrine as the variable substrate and non-competitive with S-adenosyl- l-methionine as the variable substrate. The K i value for LY87130 calculated from a Dixon plot was 2 × 10 −7 M. In rats, LY87130 inhibited NMT in vivo when injected i.p. at 10–40 mg/kg doses. Epinephrine concentration in hypothalamus was lowered within 1 h after LY87130 injection; the lowering was maximum at 6 h and persisted at 24 but not 48 h after a 40 mg/kg dose of LY87130. The lowering of epinephrine at 6 h was dose-related over a 10–40 mg/kg dose range. Norepinephrine and dopamine concentrations were increased transiently; by 6 h the concentrations of these other catecholamines were normal whereas epinephrine was maximally depleted. MOPEG sulfate, a major metabolite of norepinephrine in rat brain, was markedly elevated by LY87130. The effects of low doses of LY87130 (5 and 10 mg/kg) were enhanced by iprindole, which increased LY87130 levels in brain, suggesting that ring hydroxylation is a major pathway of LY87130 metabolism. The ability of LY87130 to produce selective depletion of epinephrine in rat brain makes it a useful tool for manipulating epinephrine-forming neurons.

Properties of norepinephrine N-methyltransferase from pigeon brain

Epinephrine cocentration in pigeon hypothalamus was higher on an absolute basis and as a percentage of total catecholamine concentration than in other regions of pigeon brain or in rat hypothalamus. Norepinephrine N-methyltransferase (NMT; the epinephrine-forming enzyme) from pigeon brain had Km values for S-adenosyl-L-methionine and L-norepinephrine of 24 ± 1 and 101 ± 4 μM, respectively. The enzyme was inhibited by excess L-norepinephrine and by several arylalkylamines that earlier had been identified as NMT inhibitors with the enzyme from mammalian brain and adrenal medulla. These results indicate that pigeon brain NMT is similar to that in mammalian brain and adrenal glands and can be inhibited by agents previously used to deplete brain epinephrine selectively in rats. The use of pigeons as experimental subjects in studies on the possible role of epinephrine-forming neurons in behavior is suggested.

Properties of norepinephrine N-methyltransferase from rat, cat and chicken brain

1. Kinetic properties of norepinephrine N-methyltransferase (NMT) in ammonium sulfate fractions from rat, cat and chicken brain were studied. 2. For all three enzymes, K m values for S-adenosyl-methionine—the methyl-donating substrate—were lower than those for l-norepinephrine—the methyl-accepting substrate. For both substrates, K m values were lowest for cat, intermediate for rat, and highest for chicken brain NMT. 3. The enzymes from all three species were inhibited by excess concentrations of l-norepinephrine. 4. The enzymes were inhibited by various ring-substituted benzylamines and α-methylbenzylamines, and the structure-activity relationships and stereospecificity of this inhibition were similar for the enzymes from all three species. 5. Though quantitative differences exist, NMT from rat, cat and chicken brain seems to be qualitatively similar both in substrate kinetics and susceptibility to inhibitors.

Inhibition in vitro of rabbit adrenal norepinephrine N-methyltransferase by 2,3,4,5-tetrahydro-1H-2-benzazepines

Inhibition in vitro of rabbit adrenal norepinephrine N-methyltransferase by 2,3,4,5-tetrahydro-1H-2-benzazepines

Inhibition of rat brain norepinephrine N-methyltransferase by 7,8-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine

Inhibition of rat brain norepinephrine N-methyltransferase by 7,8-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine

Inhibition of norepinephrine N-methyltransferase by 1-aminoindans, conformationally rigid analogs of benzylamines

Bicyclic analogs of benzylamine (with the α carbon connected by one or more methylene units to the ortho position of the benzene ring) inhibited rabbit adrenal norepinephrine N-methyltransferase (NMT) in vitro . Inhibition was greater when the second ring contained five carbons (1-aminoindan) than when it contained four, six, or seven carbons. Substitution of chlorines on the benzene ring further enhanced the inhibition by 1-aminoindan. The most active inhibitor, 4,5-dichloro- 1-aminoindan, showed competitive kinetics with ℓ-norepinephrine as the variable substrate, and the K i for this compound as an inhibitor of adrenal NMT was 0.22 μM. 4,5-Dichloro-1-aminoindan significantly decreased epinephrine concentration in the adrenal glands of exercised rats, suggesting that it was effective in inhibiting NMT in vivo .