O-methylation Of Noradrenaline Research Articles

Evidence for saturation of catechol-O-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats.

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/l) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min-1 and 0.186 min-1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline. In isolated lungs perfused with 3H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l 3H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for uptake2-mediated O-methylation of noradrenaline in the human amnion FL cell-line

The uptake and metabolism of 3H-noradrenaline has been examined in the FL cell-line derived originally from human amnion. Cell cultures metabolised 3H-noradrenaline (1.0 mumol/l) to 3H-normetanephrine and, to a lesser extent, to metabolites (not distinguished) of the O-methylated deaminated fraction; primary deaminated metabolites were not detected. 3H-normetanephrine formation a) was not saturable in the noradrenaline concentration range 0.2-150 mumol/l, b) was decreased to 20%-30% of control levels by uptake2 inhibitors (O-methylisoprenaline, 20 and 100 mumol/l; cimetidine, 10 mumol/l; hydrocortisone, 200 mumol/l) and c), was almost insensitive to uptake1 inhibitors (cocaine, 30 mumol/l; desipramine, 3 mumol/l). Uptake of noradrenaline was manifested after 30 minutes as a 6-fold increase in the cell content of the amine following inhibition of catechol-O-methyl transferase, either alone or in conjunction with inhibition of monoamine oxidase. Uptake was decreased maximally to 40% of control levels by O-methylisoprenaline. IC50 values for inhibition of the O-methylisoprenaline-sensitive component of uptake were (in mumol/l): corticosterone (0.3), papaverine (1.1), O-methylisoprenaline (3.0), cimetidine (6.0), (-)noradrenaline (460), and tetraethylammonium (2230). Except for the last agent, for which a comparative value is not available, the IC50's are in good agreement with those for inhibition of uptake2 in the Caki-1 cell-line reported by other investigators. The component of uptake resistant to O-methylisoprenaline was depressed by papaverine (a 50% decrease at 50 mumol/l), but was not affected by the other uptake2 inhibitors or by cocaine (30 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

The uptake and O-methylation of noradrenaline by systemic vascular endothelial cells

The uptake and O-methylation of noradrenaline by systemic vascular endothelial cells

The neuronal and extraneuronal uptake and metabolism of 3H-(-)-noradrenaline in the rabbit iris.

The disposition of 3H-(-)-noradrenaline (NA) was studied in the iris isolated from albino and pigmented rabbits. Pigment cells bind some NA, but do not metabolize it. Cocaine-sensitive neuronal uptake leads either to an extensive neuronal deamination of NA (when vesicular uptake is inhibited by pretreatment of the animals with reserpine) or to a pronounced accumulation of NA in the axoplasm (if, additionally, neuronal monoamine oxidase is inhibited). Corticosterone-sensitive extraneuronal uptake leads to O-methylation of NA by extraneuronal catechol-O-methyl-transferase. In the rabbit iris, there is no extra-neuronal deamination of NA. In contrast to findings with other tissues, the extraneuronal O-methylation of NA in the rabbit iris is not saturable.

Effect of ovarian steroids on the metabolism of noradrenaline in rabbit uterus.

The metabolism of (-)-3H-noradrenaline was examined in uterine slices from ovariectomized rabbits which were either untreated or treated with 17 beta-oestradiol, alone or in combination with progesterone. 17 beta-oestradiol caused uterine enlargement which was not accompanied by a change in the formation per g of O-methylated metabolites (3H-NMN, 3H-VMA, 3H-MOPEG). Accumulation of unchanged 3H-noradrenaline and the formation of deaminated catechols (3H-DOMA and 3H-DOPEG) were decreased per g tissue, but increased per uterine horn. Progesterone produced further enlargement of the oestrogen-dominated uteri which was accompanied by (a) a decrease in deaminated catechol formation and (b) an increase in 3H-NMN formation per unit mass of tissue. In all uteri (control and hormone-treated), cocaine inhibited the formation of deaminated catechols, but not that of the O-methylated metabolites. It is suggested, therefore, that, per unit of uterine mass, the neuronal deamination of (-)-3H-noradrenaline is decreased by 17 beta-oestradiol and further decreased by progesterone, and that these changes reflect failure of the intraneuronal deaminating system in the whole uterus to increase in proportion to the increase in uterine mass. Since other agents which decreased the deamination of (-)-3H-noradrenaline (cocaine and nialamide) did not affect 3H-NMN formation in oestrogen-dominated uteri, it is suggested that stimulation of 3H-NMN formation represents a direct effect of progesterone on the extraneuronal O-methylation of noradrenaline.

Structural and functional alterations caused at the extraneuronal level by sympathetic denervation of blood vessels.

The lateral saphenous vein of the dog and the rabbit ear artery were surgically denervated, by clamping the vessel or by removal of the superior cervical ganglion, respectively. Both procedures resulted in denervation of the vessels. The denervated, lateral saphenous vein was supersensitive to exogenous noradrenaline and inactivation of the amine (in oil immersion experiments) was slower in denervated vein strips than in control strips treated with cocaine. Incubation experiments with 3H-noradrenaline confirmed that denervated strips formed considerably fewer metabolites than control ones (in the absence or presence of cocaine) and that O-methylation of noradrenaline was reduced by about 50%. When the strips were incubated with 3H-isoprenaline, the denervated ones accumulated and metabolized isoprenaline to a lesser degree than control strips. Hydrocortisone did not reduce the accumulation of isoprenaline in the denervated vein and had only minor effects on O-methylation. The metabolism of noradrenaline and isoprenaline gradually recovered with time. In the ear artery, denervation was accompanied by a marked reduction in O-methylation, but not in accumulation, of isoprenaline. In both vessels there was a highly significant positive correlation between noradrenaline content and O-methylating capacity; in the saphenous vein accumulation of isoprenaline was also positively correlated to noradrenaline content. Morphological changes observed in the denervated vessels consisted essentially in dedifferentiation of smooth muscle cells (which attained larger dimensions, had an indented, large nucleus, augmented euchromatin and an increased amount of ribosomes), abundance of extracellular material and fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of the brown fat respiratory response to noradrenaline by catechol- O-methyltransferase

The possibility that O-methylation of noradrenaline (NA)tonormetanephrine (NM) by catechol- O-methyltransferase (COMT: EC 2.1 : 1.6) may limit the concentration of NA at the receptors, and therefore control the adrenergic response, was evaluated from measurements of both the respiratory rate (MO 2) and the rate of NM formation (MNM) in brown adipose tissue from the rat. MO 2 and its increase (MO 2 response) induced in tissue slices by endogenous or exogenous NA were measured in a stop flow respirometer perfused with Krebs-Ringer bicarbonate medium. MNM was measured in identical tissue slices incubated in the same medium, from exogenous radiolabelled NA and endogenous S-adenosyl methionine. This rate was taken as the apparent activity of the COMT system. It reached maximum values of about 100 pmole/min/g wet wt and was cut down to blank value by tropolone, a specific inhibitor of COMT. For any given sustained NA stimulus, the MO 2 response reached a steady-state value. MNM was constant during one hour at concentra tions of added NA up to 3 μM, and during 30 min at higher ones. The MO 2 response was potentiated (100–200 per cent) through inhibition of COMT by 0.2 mM tropolone. This effect was only transitory, with a maximum at 30 min followed by a 100 per cent inhibition of the MO 2 response to exogenous NA 60 to 90 min later. Exposure of the stimulated preparations to 5· 10 −5 M DOPA, which competitively inhibits the extraneuronal uptake-COMT system and whose decar☐ylation product competitively inhibits not only COMT but also the neuronal uptake (uptake 1), induces a sustained increase of the MO 2 response. Under blockage of uptake 1 by desmethyl-imipramine (DMI) 10 −6 M, potentiation of the MO 2 response was quantitatively the same with tropolone as with DOPA, for both endogenous and exogenous NA stimuli, and it was sustained. Quantitative analysis of the data suggests that COMT can control the local NA concentration at the site of hormone receptor interaction, not only by the (NA) gradient to the COMT system as a sink, but also indirectly via active induction of neuronal uptake. Large metabolic responses to nanomolar (NA), observed only when both COMT and uptake 1 were blocked, suggested that such a control might be operative even under basal conditions.

The kinetic constants for the extraneuronal uptake and metabolism of 3H-(-)-noradrenaline in the perfused rat heart.

1. Rat hearts were perfused with 50–2000 μM 3H-(−)-noradrenaline in the presence of 14C-sorbitol (to measure the extracellular distribution of the amine) and of 100 μM cocaine (to inhibit neuronal uptake). Initial rates of removal of noradrenaline were determined. Extraneuronal uptake determined in this way consisted of two components: a saturable and a non-saturable one. 2. When monoamine oxidase (MAO) was inhibited, the kinetic constants for saturable extraneuronal uptake of noradrenaline were: Km 60 μM, Vmax 50 nmoles · g−1 · min−1. U-0521, an inhibitor of catechol-O-methyl transferase (COMT) acted like a competitive inhibitor of extraneuronal uptake: the Km increased, Vmax remained unaffected. 3. In separate experiments either MAO or COMT was intact. Rat hearts were perfused with various concentrations of 3H-(−)-noradrenaline until the metabolites appeared at a constant rate in the venous effluent. These steady-state rates of metabolite formation were used to determine the kinetic parameters of the metabolizing systems. 4. O-methylation of noradrenaline in the intact heart was a saturable process and obeyed Michaelis-Menten kinetics; it had very low Km (1.7 μM) and Vmax (1.2 nmoles · g−1 · min−1). 5. Also the deamination of noradrenaline by the perfused heart was a saturable process, but Km and Vmax were considerably higher than for O-methylation (140 μM and 25 nmoles · g−1 · min−1, respectively). 6. It is concluded that “extraneuronal uptake and subsequent metabolism” of noradrenaline represents a site of loss that is highly effective at very low substrate concentrations (i.e., below the Km of the two metabolizing systems).

O-methylation of noradrenaline and demethylation in rat blood

Rat blood cells O-methylate noradrenaline to normetanephrine without detectable formation of norparanephrine. In contrast, a partially purified blood enzyme O-methylates the noradrenaline in both the p- and m-positions; the normetanephrine/norparanephrine ratio (12 ± 3 at pH 7.5) in the reaction products is similar to that obtained with purified rat liver catechol O-methyltransferase ( S-adenosylmethionine: catechol O-methyltransferase, EC 2.1.1.6). Incubation with whole rat blood leads to the disappearance of both normetanephrine and norparanephrine; part of the normetanephrine is demethylated to noradrenaline. The absence of p-O-methylated products when noradrenaline is incubated with whole blood can probably be accounted for by a preferential action of the degrading enzymes on norparanephrine.