Evidence for saturation of catechol-0-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/1) 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. Further experiments to examine the kinetics of O-methylation of noradrenaline and adrenaline (MAO inhibited) showed that the O-methylation of these amines in the lungs was predominantly saturable, with half-saturation occurring at concentrations (9.8 nmol/I and 19.4 nmol/l, respectively) that were two orders of magnitude lower than those required to half-saturate uptake1 of the amines. Saturation of O-methylation by these low concentrations of noradrenaline (1) provides the explanation for the change in the metabolic profile of noradrenaline described above and (ii) appears to occur because Vmax uptake ≫ Vmax COMT for the metabolizing system consisting of non-neuronal uptake1 + COMT in the lungs, as has been described previously for the system consisting of uptake2 + COMT in extraneuronal sites in rat heart. The results show that the metabolic profile of catecholamines in the pulmonary circulation will reflect that occurring at physiological levels only if studies are carried out with very low amine concentrations

A kinetic investigation of the pulmonary metabolism of dopamine in rats shows marked differences compared with noradrenaline

The aim of this study was to investigate the deamination of dopamine in the intact pulmonary circulation of isolated lungs of the rat. The first part of the study showed that dopamine is not converted to noradrenaline by dopamine-β-hydroxylase (DBH) when dopamine is perfused through isolated lung preparations with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited. Hence, it was not necessary to inhibit DBH in subsequent experiments. The metabolite profile for deamination of dopamine in the lungs was examined by determining whether MAO and semicarbazide-sensitive amine oxidases (SSAO) contribute to the deamination of dopamine (and noradrenaline), and by determining the activity of MAO (kMAO) for the metabolism of dopamine. Lungs were perfused with I nmol/l 3H-dopamine or 3H-noradrenaline with COMT inhibited and, in experiments to determine the contribution of SSAO to deamination, with MAO inhibited. Inhibition of MAO reduced the deamination of dopamine and noradrenaline by 99.8% and 98.6%, respectively, indicating that MAO, and not SSAO, was responsible for deamination of the catecholamines in the lungs. The kMAO value for deamination of dopamine was 3.89 min-1. Further experiments were carried out to determine the contributions of MAO-A and MAO-B to the deamination of dopamine in lungs perfused with 1 nmol/l 3H-dopamine and 100 nmol/1 lazabemide or 300 nmol/I Ro41-1049, respectively. The values of kMAO-A and kMAO-B were 3.05 min-1 and 0.626 min-1, respectively. It was concluded that, in rat lungs, MAO-A contributed 78-84% and MAO-B 16-22% to the total deamination of dopamine and SSAO had no significant role in its pulmonary metabolism. These relative contributions of MAO-A and MAO-B to the deamination of dopamine are very similar to those that have been determined previously for noradrenaline, but the rate constant for deamination of dopamine is 26-fold greater than that for noradrenaline in rat lungs

Evidence from guinea-pig trachealis that Uptake2 of isoprenaline is enhanced by hyperpolarization of the smooth muscle

Previous studies (Bönisch et al. 1985; Trendelenburg 1986, 1987) have provided evidence that Uptake2 of catecholamines is inhibited by depolarization of cells. The aim of this study was to further examine the relationship between Uptake2 and membrane potential by testing the hypothesis that Uptake2 is, conversely, stimulated by hyperpolarization of cells. The effects of β-adrenoceptor agonists (isoprenaline and salbutamol) and β-adrenoceptor antagonists (propranolol and ICI 118,551) on Uptake2 of isoprenaline were examined in guinea-pig trachealis muscle, in which stimulation of β-adrenoceptors mediates hyperpolarization of the smooth muscle cells (Allen et al. 1985), and in rat heart, in which β-adrenoceptor agonists do not cause hyperpolarization. In guinea-pig trachealis muscle segments, propranolol and ICI 118,551 reduced Uptake2 (as measured by the steady-state rate of corticosterone-sensitive formation of 3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in 2.5–250 nmol/l 3H-isoprenaline (in the range over which isoprenaline causes hyperpolarization of the muscle), but not in 1 nmol/l 3H-isoprenaline (which does not hyperpolarize the muscle). The normalized rates were greater in tissues incubated in 25 nmol/l than 1 nmol/l isoprenaline, and were enhanced by 2.5 gmol/l salbutamol in tissues incubated in 1 nmol/l isoprenaline. In rat hearts perfused with 1 or 25 nmol/l 3H-isoprenaline and U-0521 to inhibit catechol-O-methyltransferase, the rate of Uptake2 of isoprenaline, normalized for the isoprenaline concentration, was unaffected by the isoprenaline concentration or the presence of propranolol, ICI 118,551 or salbutamol. The results of the study suggest that, in guinea-pig trachealis muscle, isoprenaline and salbutamol enhance Uptake2 due to β-adrenoceptor-mediated hyperpolarization of the muscle and that propranolol and ICI 118,551 decrease Uptake2 of isoprenaline by preventing this hyperpolarization of the smooth muscle, and not by directly inhibiting the Uptake2 transport process

Dopamine and adrenaline, but not isoprenaline, are substrates for uptake and metabolism in isolated perfused lungs of rats

The uptake and subsequent metabolism by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) of dopamine, adrenaline, isoprenaline and noradrenaline in isolated perfused lungs of rats has been examined. In lung preparations in which COMT and MAO were inhibited, the uptake of 3H-labelled dopamine, (−)-adrenaline and (−)-noradrenaline, but not (±)-isoprenaline, was reduced by cocaine (10 or 100 μmol/l) The rank order of the Km values of the amines that were substrates for uptake in the lungs were: dopamine (0.246 μmol/l) < noradrenaline (0.967 μmol/l) < adrenaline (3.32 μmol/l). These results are consistent with transport of catecholamines in rat lungs by Uptake1. In lung preparations with COMT and MAO intact, dopamine and noradrenaline were removed from the circulation (50% and 32%, respectively) and mainly metabolized. There was very little (3.0%) removal of isoprenaline by the lungs and adrenaline was not included in this part of the study. In lung preparations in which only MAO was inhibited, the rank order of COMT activity for O-methylation of the amines was dopamine ≪ noradrenaline ≥ adrenaline (kCOMT values: 4.98 min−1, 0.357 min−1, and 0.234 min−1, respectively). If dopamine or adrenaline are perfused through the pulmonary circulation in isolated lungs of the rat, they are taken up and then metabolized by COMT and MAO, as also occurs for noradrenaline. Isoprenaline is not a substrate for uptake in the lungs. There was less uptake of adrenaline than noradrenaline, indicating that uptake and metabolism in the lungs may not be a significant removal process for adrenaline in the circulation of rats in vivo. The more marked uptake of dopamine (than of noradrenaline) indicates that uptake and metabolism by the lungs, at least in the rat, may play an important role in the removal of dopamine from the circulation in vivo