Metabolism of endogenous and exogenous noradrenaline in guinea-pig atria

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from guinea-pig isolated atria was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs, the outflow of endogenous noradrenaline over a period of 3 h averaged 1.6 pmol×g^–1×min^–1 and the outflow of DOPEG 17 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.31 pmol×g^–1×min^–1). Tyramine greatly increased the outflow of noradrenaline and DOPEG, and the reserpine-like compound Ro 4-1284 selectively increased the outflow of DOPEG; DOMA remained below the detection limit. When atria were exposed to (–)-noradrenaline 1.7 or 17 M, the subsequent outflow of noradrenaline and DOPEG was enhanced. Moreover, substantial amounts of DOMA were now found. This outflow of DOMA was prevented when atria were exposed to (–)-noradrenaline in the presence of cocaine or after an initial incubation with amezinium. Exposure to (–)-noradrenaline 1.7 M mainly enhanced the formation of DOPGE, while exposure to (+)-noradrenaline 1.7 M mainly enhanced the formation of DOMA.Our experiments confirm some and qualify other conclusions drawn from studies in which exogenous ³H-noradrenaline had been used to examine the metabolism of noradrenaline in guinea-pig atria. In agreement with the isotope studies, DOPEG is a major metabolite of endogenous noradrenaline. In contrast to what the isotope studies had suggested, however, endogenous DOMA is a very minor product, at least as long as the neurones are at rest. DOMA is only formed when the tissue is exposed to high concentrations of exogenous noradrenaline. In further contrast to previous conclusions, DOMA is then formed intra- and not extraneuronally.     

Effects of the noradrenaline metabolites on tyrosine hydroxylase activity in guinea-pig atria

Summary The effects of noradrenaline, its five metabolites and metanephrine, were studied on tyrosine hydroxylase activity in guinea-pig atria. The deaminated metabolite, (±)-3,4-dihydroxyphenylglycol (DOPEG), was equipotent with (±)-noradrenaline in its inhibitory action on tyrosine hydroxylase activity in the homogenates of guinea-pig atria. The inhibition by DOPEG was competitive with the cofactor, reduced pteridine. The deaminated acid, 3,4-dihydroxymandelic acid (DOMA) and the O-methylated deaminated acid, 3-methoxy, 4-hydroxymandelic acid (VMA) had 1/50th and 1/30th, respectively, the potency of noradrenaline in inhibiting tyrosine hydroxylase. The rest of the metabolites did not inhibit tyrosine hydroxylase in homogenates in concentrations up to 1.0 mM. In intact guinea-pig atria noradrenaline was considerably more potent than DOPEG in inhibiting tyrosine hydroxylase. Normetanephrine 1.4×10^–4 M inhibited tyrosine hydroxylase in the intact tissue but failed to inhibited the enzyme in the homogenate even in higher concentrations. The effect of normetanephrine in the intact tissue is related to the ability of this compound to release endogenous noradrenaline.A reserpine-like agent, Ro 4-1284, did not inhibit tyrosine hydroxylase activity in the homogenate but in the intact tissue the inhibition was more than 50%. This effect of Ro 4-1284 in the intact tissue appears to be related to the releasing effects of this agent and to an increase in the axoplasmic levels of DOPEG.Since the formation of the deaminated glycol, DOPEG, represents the main metabolic pathway for the neurotransmitter in adrenergic nerve endings, the present results are compatible with the view that, in addition to the pool of extravesicular noradrenaline, the cytoplasmic concentration of DOPEG could also participate in the regulation of the activity of tyrosine hydroxylase.     

The effects of labetalol (AH 5158) on metabolism of 3H(-)-noradrenaline released from the cat spleen by nerve stimulation

The competitive α and β adrenoceptor antagonist labetalol, in concentrations up to 10^?4 M, produced a dose dependent increase in overflow of ³H and [³H]noradrenaline in the isolated blood perfused cat spleen following stimulation of the splenic nerves at a frequency of 10 Hz. Labetalol had no effect on the pattern of overflow of label following stimulation. In experiments in which the metabolism of [³H]noradrenaline released on nerve stimulation was examined, labetalol produced a concentration dependent increase in the percentage of [³H]noradrenaline and a decrease in the percentage of [³H]DOPEG in the venous blood following nerve stimulation. Production of [³H]COMT metabolites and [³H]DOMA was not affected. It is suggested that in the isolated blood perfused cat spleen labetalol produces the elevation of overflow and effects on noradrenaline metabolism by inhibition of neuronal uptake of noradrenaline. The drug has no detectable effects on the enzymes MAO or COMT or on extraneuronal uptake.     

Effects of catechol-O-methyltransferase inhibition on the plasma clearance of noradrenaline and the formation of 3,4-dihydroxyphenylglycol in the rabbit

Summary The purpose of this study was to elucidate the finding of Friedgen et al. (1993b) that catechol-O-methyltransferase (COMT) inhibition is much more effective in increasing the plasma concentration of endogenous dihydroxyphenylglycol (DOPED) than in increasing the plasma concentration of infused DOPED. To this end, reserpine-pretreated rabbits were anaesthetized and infused with noradrenaline and/or DOPED, and the plasma clearances of infused noradrenaline (Cl_NA) and DOPED (Cl_DOPEG) as well as the plasma DOPED response to noradrenaline infusion [as reflected by the ratio of the steady-state increase in plasma DOPED (DOPEG) to that in plasma noradrenaline (NA)] were determined before and after blockade of neuronal uptake by desipramine. Experiments were carried out either under control conditions or after COMT inhibition by i.v. administration of 3,4-dihydroxy-4-methyl-5-nitrobenzophenone (Ro 40-7592). On the assumption that rates of neuronal noradrenaline uptake equal steady-state rates of neuronal DOPED formation, the desipramine-sensitive components of Cl_NA and DOPEG/NA were used to estimate the apparent plasma clearance of DOPED formed intraneuronally (Cl_f-DOPEG) in response to noradrenaline infusion.Cl_NA was 83.6 ml kg-^–1 min^–1 in the absence and 48.1 ml kg ^–1min^–1 in the presence of desipramine. Neither the former nor the latter value was altered after COMT inhibition. However, the COMT inhibitor reduced Cl_DOPEG from 47.6 to 28.5 ml kg ^–1 min^–1 (indicating a 1.7-fold increase in the plasma DOPED response to DOPED infusion) and Cl_f-DOPEG from 106.2 to 43.3 ml kg^–1 min^–1 (indicating a 2.5-fold increase in the neuronal component of the plasma DOPED response to noradrenaline infusion). DOPEG/NA increased following treatment with Ro 40-7592 from 0.381 to 1.294 in the absence and from 0.036 to 0.391 in the presence of desipramine. These increases were more pronounced than the Ro 40-7592-induced change in Cl_f-DOPEG would predict. Therefore, the values of Cl_f-DOPEG were used to calculate rates of DOPED formation. The results show that the increases in DOPEG/NA induced by COMT inhibition were not due only to the block of O-methylation of DOPED, but also to a pronounced increase in the extraneuronal DOPED formation.Hence, COMT inhibition affects neither the neuronal nor the extraneuronal removal of circulating noradrenaline from plasma. By contrast, COMT inhibition reduces the clearance of infused DOPED by about 40% and that of neuronally formed DOPEG by about 60%. The observation of Ro 40-7592 being more effective in increasing the plasma DOPEG response to noradrenaline infusion than in increasing the plasma DOPED response to DOPEG infusion is due to the fact that newly formed DOPEG is O-methylated not only while being removed from plasma, but also on the way to plasma, and that COMT inhibition increases the DOPEG formation at extraneuronal sites.This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5). A preliminary account of the results was presented to the German Society for Pharmacology and Toxicology (Halbrügge et al. 1992)Correspondence to K. H. Graefe at the above address     

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--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 (k_MAO) 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 k_MAO 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 k_MAO-A and k_MAO-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.Abbreviations COMT Catechol-O-methyltransferase - DBH Dopamine-\-hydroxylase - DOPEG 3,4-dihydroxyphenylglycol - DOMA 3,4-dihydroxyman delic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPET 3,4-dihydroxphenylethanol - ECS Extracellular space - K_m Michaelis or half-saturation constant - k_COMT Rate constant for O-methylation by COMT - k_deam Rate constant for total deamination - k_MAO Rate constant for deamination by MAO - MAO Monoamine oxidase - MB-COMT Membrane-bound COMT - SSAO Semicarbazidesensitive amine oxidases - S-COMT Soluble COMT - T/M Tissue to medium concentration ratio of dopamine or noradrenaline - V_max Maximal rate - V_{st - st} Steady-state rate of metabolite formation     

Metabolism of endogenous and exogenous noradrenaline in the rabbit perfused heart

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from rabbit perfused hearts was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs and without nerve stimulation, the outflow of endogenous noradrenaline over a period of 108 min averaged 0.17 pmol×g^–1×min^–1 and the outflow of DOPEG 2.1 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.13 pmol×g^–1×min^–1). The effect of perfusion with (–)-noradrenaline 0.1, 1 or 10 mol/l for 18 min was then investigated. As the concentration of noradrenaline increased so did the outflow of DOPEG. Moreover, DOMA was found in the venous effluent during and after perfusion with noradrenaline 1 or 10 mol/l. The increase in the outflow of DOPEG and DOMA was almost abolished when cocaine 10 mol/l was present during the perfusion with noradrenaline 1 mol/l. The release of endogenous noradrenaline by sympathetic nerve stimulation or tyramine 10 mol/l, but not the release evoked by nicotine 30 mol/l, was accompanied by an increase in the outflow of DOPEG; an outflow of DOMA was not observed.It is concluded that, in the rabbit perfused heart, DOPEG is an important metabolite of endogenous noradrenaline. DOMA is at best a minor product, either when the neurones are at rest or when noradrenaline is released by sympathetic nerve stimulation, nicotine or tyramine. DOMA is formed in detectable amounts when the tissue is exposed to a high concentration of exogenous noradrenaline. Like DOPEG, it is formed intraneuronally. The results confirm and extend those obtained previously on guinea-pig incubated atria. They make it unlikely that, in these tissues at least, DOMA formation is one of the physiological pathways of noradrenaline catabolism.     

Plasma 3,4-dihydroxyphenylglycol as a tool to assess the role of neuronal uptake in the anaesthetized rabbit

Summary (1.) The purpose of this study was to investigate the role of neuronal uptake in the appearance in plasma of the primary noradrenaline metabolite 3,4-dihydroxyphenylglycol (DOPEG). To this end, steady-state changes in mixed central-venous plasma concentrations of noradrenaline and DOPEG produced by noradrenaline infusions or by changes in sympathetic tone were determined in anaesthetized rabbits either under control conditions or after treatment with desipramine (2 mg kg^–1). The steady-state kinetics of infused DOPEG were also evaluated. (2.) Infused DOPEG (2.9 nmol kg^–1 min^–1 i.v. for 75 min) reached steady-state concentrations in plasma within less than 30 min, disappeared from plasma with a half-life of 2.3 min and showed a total-body plasma clearance of 84.0 ml kg^–1 min^–1 (3.) Constant-rate infusions of noradrenaline (1.2–5.9 nmol kg^–1). (min^–1 i.v. for 75 min) produced increases in plasma noradrenaline and DOPEG concentrations which were linearly related to the rate of noradrenaline infusion. Thus, the plasma clearance of infused noradrenaline (75.8 ml kg^–1). min^–1 as well as the increase in plasma DOPEG expressed in % of that in plasma noradrenaline (9.4%) was virtually independent of the noradrenaline infusion rate. (4.) Desipramine reduced the plasma clearance of infused noradrenaline by 35.4% and the increment in plasma DOPEG relative to that in plasma noradrenaline by 75.3%. From these results and the plasma clearance of noradrenaline and DOPEG it was calculated that the rate at which presynaptically formed DOPEG appeared in plasma amounted to 7.9% of the rate of total noradrenaline removal and to 22.3% of the rate of neuronal uptake. (5.) The rate of appearance in plasma of DOPEG originating from the neuronal re-uptake of endogenous noradrenaline was 192.3 pmol (kg^–1). min^–1 suggesting that the rate of neuronal re-uptake amounted to 862.3 pmol (kg^–1) min^–1 (6.) The slope of the regression line relating plasma DOPEG to plasma noradrenaline concentrations under conditions of noradrenaline release exceeded that of the corresponding regression line observed during noradrenaline infusion by a factor of about 10. This difference in slope suggests that, in the absence of infused noradrenaline, the average noradrenaline concentration at all noradrenergic neuroeffector junctions of the rabbit is 3.2 times as high as that in plasma.This study was supported by the Deutsche Forschungsgemeinscbaft (Gr 490/5). A preliminary account of the present results was presented to the German Pharmacological Society (Halbragge and Wölfel 1989) Send offprint requests to T. Halbragge at the above address     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

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

Summary 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 mol/l) The rank order of the Km values of the amines that were substrates for uptake in the lungs were: dopamine (0.246 mol/l) < noradrenaline (0.967 mol/l) < adrenaline (3.32 mol/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 (k_COMT 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.Abbreviations COMT catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPEG 3,4-dihydroxyphenylglycol - DOPET 3,4-dihydroxyphenyl ethanol - MAO monoamine oxidase - MN metanephrine - MTA 3-methoxytyramine - NMN normetanephrine - OMDA O-methylated deaminated metabolites - OMI 3-O-methylisoprenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone Some of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists (Bryan and O'Donnell 1987, 1988; Bryan et al. 1989; Bryan-Lluka 1990) Send offprint requests to L.J. Bryan-Lluka at the above address     

Effects of flow-stop on the metabolism of noradrenaline released by nerve stimulation in the perfused spleen

Summary The metabolic pathway of ³H-noradrenaline released spontaneously and by nerve stimulation was studied in the isolated perfused spleen of the cat. The deaminated glycol, DOPEG, (3,4 dihydroxyphenylglycol) was the main metabolite in spontaneous outflow, accounting for 62.5±1.6% of the total radioactivity (n=13). Of the total increase in radioactive products elicited by nerve stimulation at 5 Hz or 10 Hz around 30% was accounted for by the noradrenaline metabolites, particularly DOPEG and the O-methylated fraction. In the presence of 2.9×10^–6 M of cocaine the total overflow of radioactivity induced by stimulation was unchanged but DOPEG formation from released noradrenaline was abolished. These findings indicate that DOPEG formation results from the recapture of the released transmitter by adrenergic nerve endings and subsequent intraneuronal deamination. The total overflow of noradrenaline was reduced by flow-stop while the metabolism of the released transmitter was increased significantly. Cocaine, 2.9×10^–6 M, prevented the increase in DOPEG when stimulation was applied under flow-stop conditions. The decrease in noradrenaline overflow induced by flow-stop is partly due to the increase in the metabolism of the released transmitter.     

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 V_{max uptake} V_{max 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.Abbreviations COMT Catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3 4-dihydroxyphenylglycol - ECS Extracellular space - HSOC Half-saturating outside concentration - K_{m uptake} Half-saturation constant for uptake - k_COMT Rate constant for O-methylation - k_MAO Rate constant for deamination - k_{out NA} Rate constant for efflux of noradrenaline - MAO Monoamine oxidase - MB-COMT Membrane-bound - COMT NMN Normetanephrine - OMDA O-methylated deaminated metabolites - S-COMT Soluble COMT - T/M_NA Tissue to medium ratio of noradrenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone - V_max Maximal rate of uptake or O-methylation - V_st-st Steady-state rate of metabolite formation - V_uptake Rate of uptake Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990)     

Plasma normetanephrine for examination of extraneuronal uptake and metabolism of noradrenaline in rats

The importance of neuronal reuptake for terminating the actions of noradrenaline is well established, but the role of extraneuronal uptake is less clear. This study used plasma concentrations of the extraneuronal noradrenaline metabolite, normetanephrine, to estimate rates of extraneuronal removal of noradrenaline in rats. Animals received infusions of ³H-noradrenaline,. with and without inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), to examine the extraneuronal removal of noradrenaline and formation of normetanephrine from infused and endogenous noradrenaline. Infusions of ³H-normetanephrine were also carried out to examine the plasma kinetics of normetanephrine before and after inhibition of MAO.Normetanephrine was cleared rapidly from the circulation and had a short plasma halflife (1 min). Spillover of normetanephrine into plasma (79 pmol kg^–1min^–1) was a third that of noradrenaline, but increased 2.8-fold after inhibition of MAO; noradrenaline spillover remained unchanged. Combined inhibition of MAO and COMT decreased the plasma clearance of ³H-noradrenaline by 38070, reflecting removal of ³H-noradrenaline by extraneuronal uptake. Division of the rate of extraneuronal removal of ³H-noradrenaline by the specific activity of plasma ³H-normetanephrine during the ³H-noradrenaline infusion indicated that the rate of extraneuronal removal of endogenous noradrenaline was 250 pmol kg^–1min^–1; this was close to the spillover of normetanephrine into plasma after inhibition of MAO (219 pmol kg^–1 min^–1).Forty-five % of plasma normetanephrine was derived from circulating noradrenaline and 55% from noradrenaline before entry into the circulation. Assuming that these proportions reflected the sources of noradrenaline metabolized extraneuronally indicated that the rate of extraneuronal metabolism of noradrenaline before entry into the circulation was 138 pmol kg^–1min n^–1. Comparison of this with the rates at which noradrenaline was recaptured by sympathetic nerves (2540 pmol kg^–1min^–1) or spilled over into plasma (228 pmol kg^–1min^–1), indicated that 87% of the noradrenaline released by sympathetic nerves was recaptured, 5% was metabolized extraneuronally and 8% escaped into plasma. Thus, extraneuronal uptake removes much less of the noradrenaline released by sympathetic nerves than neuronal reuptake.     

The influence of inhibition of catechol-O-methyl transferase or of monoamine oxidase on the extraneuronal metabolism of 3H-(−)-noradrenaline in the rat heart

Summary The extraneuronal metabolism of ³H-(–)-noradrenaline (1 nmol/l) was determined in rat hearts obtained from reserpine-pretreated animals (in the presence of 30 mol/l cocaine).Inhibition of monoamine oxidase (MAO) (by pretreatment of the animals with pargyline) increased the formation of O-methylated metabolites by nearly that amount by which the formation of deaminated metabolites declined; hence, catechol-O-methyl transferase (COMT) seemed to be able to nearly fully compensate for the loss of MAO activity. However, when COMT was inhibited (by the presence of either 1 or 10 mol/l U-O521), the increase in the formation of deaminated metabolites was smaller than the decrease in the formation of O-methylated metabolites; hence, MAO seemed to be unable to fully compensate for the loss of COMT activity.These results are discussed with regard to the hypothesis that the two extraneuronal enzymes co-exist in one compartment. As inhibition of COMT causes a much greater increase in the steady-state tissue/medium ratio for ³H-(–)-noradrenaline than does inhibition of MAO, it is suggested that it is this increase in the intracellular concentration of ³H-(–)-noradrenaline which-by promoting an efflux of the unchanged amine that is proportional to the tissue/medium ratio-actually decreases the net removal of ³H-(–)-noradrenaline from the perfusion fluid.The results are compatible with (but no evidence for) the hypothesis that the two enzymes co-exist in the same extraneuronal compartment.The following abbreviations are used here NMN normetanephrine - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid - OMDA MOPEG+VMA Supported by the Deutsche Forschungsgemeinschaft     

Desensitization by noradrenaline of responses to stimulation of pre- and postsynaptic adrenoceptors

1 The effect of exposing isolated preparations of rat aortic strip, rat atria and mouse vas deferens to perfusions of Krebs solution containing various concentrations of noradrenaline on their sensitivity to the drug has been determined.

2 The responses evoked by stimulation of postsynaptic adrenoceptors in all the tissues and presynaptic α-adrenoceptors in the mouse vas deferens were diminished by the perfusion of noradrenaline through the organ bath for 30 min.

3 The concentration of noradrenaline required to produce desensitization was higher in the mouse vas deferens than in the other tissues and more was required to desensitize the chronotropic responses than the inotropic responses in rat isolated atria.

4 The inclusion of cocaine (10^-5 M) in the bathing solution to block uptake₁ increased the sensitivity of most tissues to noradrenaline. With the possible exception of the response to stimulation of presynaptic receptors in the mouse vas deferens, desensitization was somewhat increased in its presence.

5 When uptake₂ was blocked by oestradiol (10^-5 M), it was not possible to desensitize the contractor responses of the aortic strip and vas deferens to exogenous noradrenaline, nor the inotropic response of the atria to the drug. However, oestradiol failed to block the desensitization of chronotropic responses and responses to stimulation of presynaptic receptors in the vas deferens.

6 Blockade of monoamine oxidase (MAO) with iproniazid (7.2 × 10^-4 M) or with pargyline (5 × 10^-4 M) did not affect the desensitization process in the aortic strip.

7 Blockade of catechol-O-methyltransferase (COMT) with U-0521 (5.3 × 10^-5 M) greatly increased desensitization in the aortic strip and desensitization of inotropic responses in the atria. It had no effect on desensitization of chronotropic responses. Its effect on responses in the mouse vas deferens was not determined.

8 The perfusion of methoxamine at concentrations about 1000 times higher than those of noradrenaline also produced desensitization in the aortic strip.

9 The desensitization of presynaptic receptors in the mouse vas deferens was shown to be specific and that of the responses to postsynaptic receptor stimulation to be non-specific.

10 It is concluded that responses to adrenoceptor stimulation may be desensitized by accumulation of noradrenaline inside the cells bearing the receptors and that the desensitization is caused by noradrenaline itself not by a metabolite. Desensitization may also be caused without accumulation of noradrenaline in uptake₂ and for some receptors these may not be alternative mechanisms.

     

The contribution by monoamine oxidase and catechol-O-methyltransferase to the total-body and pulmonary plasma clearance of catecholamines

To study the effects of inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) on the removal of circulating catecholamines, anaesthetized rabbits were infused for 120 min with ³H-labelled noradrenaline, adrenaline and dopamine. Total-body plasma clearances (Cl_tot) and pulmonary fractional extractions (ER_P) of the infused amines and the cardiac output of plasma (CO_P) were determined under steady-state conditions at the end of each of two consecutive 60-min treatment periods. MAO and COMT were inhibited by treatment with pargyline (40 mg/kg)and tolcapone (3 mg/kg followed by 1.5 mg/kg given every 30 min), respectively. Two groups of animals were studied. Group I involved animals treated with tolcapone throughout and given pargyline at the beginning of the second treatment period. In group II, pargyline was given at the beginning of the first, and the treatment with tolcapone was started at the beginning of the second treatment period. As previous experiments had shown that COMT inhibition alone is without any effect on Cl_tot, of the three catecholamines considered here, the results obtained in the first treatment period of group I can be taken to reflect control results.At the end of the first treatment period, Cl_tot of noradrenaline, adrenaline and dopamine (expressed as a percentage of CO_P) was 88%, 85% and 142%, respectively, in group I (COMT inhibition) and 67%, 77% and 115%, respectively, in group II (MAO inhibition; P < 0.05 for the group difference regarding Cl_tot of noradrenaline and dopamine). MAO inhibition on top of COMT inhibition (group I) lowered Cl_tot of noradrenaline, adrenaline and dopamine by 23%, 12% and 26%, respectively, and COMT inhibition on top of MAO inhibition (group II) reduced Cl_tot of these catecholamines by 13%, 20% and 17%, respectively. At the end of the first treatment period, the pulmonary plasma clearance (Cl_p = ER_P x CO_P) of noradrenaline and dopamine was 13 and 25 ml kg^–1 min ^–, respectively, in group I and 12 and 28 ml kg^–1 min^–, respectively, in group II. Cl_P of adrenaline did not differ from zero in either group. Cl_P of noradrenaline and dopamine was reduced by 74% and 70%, respectively, when both enzymes were inhibited in group I and by 70% and 67%, respectively, when both enzymes were inhibited in group II.Hence, inhibition of either MAO or COMT alone had little, if any, effect on the removal of noradrenaline, adrenaline and dopamine on passage through the systemic and pulmonary circulation. Combined inhibition of both MAO and COMT was highly effective in reducing the pulmonary clearance of noradrenaline and dopamine, but produced only minor decreases in the total-body clearance of all three catecholamines.     

Acute cardiovascular toxicity of thallium (I) ions

The acute cardiovascular toxicity of thallium (I)-ions was studied in anesthetized rats and also in some isolated preparations. In pentobarbital-anesthetized rats, intravenously applied Tl₂SO₄ (range 3 to 100 mg/kg) caused a significant, dose-dependent decrease in mean arterial blood pressure and heart rate. In spontaneously beating guinea pig isolated atria, thallium (I)-ions (10^–5 to 10^–3 M) caused a concentration-dependent decrease in frequency, which was not influenced by atropine, cocaine, or by considerable changes in the potassium concentration of the medium. A direct influence of Tl⁺ on the sinus node is presumed. Thallium decreases contractile force probably as a result of its negative chronotropic properties, since the amplitude of contraction remained uninfluenced in electrically driven atria. Thallium (I)-ions were readily taken up by guinea pig isolated atria. At a bath concentration of 5 × 10^–5 M Tl⁺ a tissue/medium ratio of approximately 45 was achieved. Probably, an exchange against cellular potassium takes place. Relaxation of vascular smooth muscle by Tl⁺ was demonstrated both in isolated aortic strips (rabbit) and in the isolated perfused rabbit ear preparation. Both the vascular smooth muscle relaxation and the decrease in heart rate would explain the acute hypotensive effect of thallium.Some of the results were communicated at the Congress of the European Society for Toxicology at Montpellier, France (June 1975).     

Presence of muscarinic inhibitory and absence of nicotinic excitatory receptors at the terminal sympathetic nerves of chicken hearts

Summary Nicotine (2×10^–4 M) or acetylcholine (5.5×10^–4 M) in the presence of 3×10^–6 M atropine did not increase the rate or amplitude of contraction in isolated atria or ventricular strips of the chicken heart; both drugs also did not cause an output of noradrenaline or adrenaline and did not evoke antidromic discharges in the right sympathetic nerves of isolated perfused chicken hearts. In contrast, high K⁺-solutions evoked an output of noradrenaline and adrenaline and caused a burst of antidromic discharges. Dimethylphenylpiperazine (DMPP; 3.1×10^–4 M), by a tyramine-like action, elicited a small output of noradrenaline and increased rate and amplitude of contraction, but did not evoke antidromic discharges. The noradrenaline output caused by DMPP was not reduced by lowering the extracellular Ca²⁺ concentration from 1.8 to 0.1125 mM.-Acetylcholine (10^–5 and 10^–4 M) inhibited the noradrenaline and adrenaline outputs evoked by electrical stimulation of the right sympathetic nerves (3 Hz, 1 ms, 30 V); the inhibition was blocked by 3×10^–6 M atropine. —It is concluded that the terminal parts of sympathetic nerves of the chicken heart posses muscarinic inhibitory receptors but lack nicotinic excitatory receptors. Thus prejunctional nicotinic receptors are not an integral part of the terminal sympathetic neurone otherwise they would be present at this neurone in all species.This work was part of the M. D. thesis of U. Engel     

The outward transport of axoplasmic noradrenaline induced by a rise of the sodium concentration in the adrenergic nerve endings of the rat vas deferens

Summary The adrenergic nerve endings of the rat vas deferens were loaded with ³H-(–)-noradrenaline; COMT was inhibited by the presence of 10 mol/l U-0521, and all experiments were carried out with calcium-free solution. After 100 min of wash-out a neuronal efflux of tritium was obtained which remained constant with time (when expressed as fractional rate of loss; FRL); it contained more DOPEG than noradrenaline.The in vitro administration of reserpine-like drugs (reserpine and Ro 4-1284) increased the FRL of tritium, presumably because of an increase in the leakage of noradrenaline from storage vesicles; the efflux of DOPEG increased more than that of noradrenaline, and the ratio NA/DOPEG declined.Inhibition of the membrane ATPase (by omission of potassium from the medium or by the presence of 3 mmol/l ouabain) increased the FRL of tritium, presumably because of an increase in the net leakage of noradrenaline from the storage vesicles (as a consequence of the fall in the concentration of free axoplasmic noradrenaline; see below).Veratridine also increased the FRL of tritium, partly because of its known reserpine-like effect (Bönisch et al. 1983); in the presence of 1 mol/l veratridine, the efflux of DOPEG increased.Irrespective of the presence or absence of reserpine or Ro 4-1284, inhibition of the membrane ATPase or the presence of veratridine (agents or procedures which increase the axoplasmic sodium concentration) always resulted in a brisk increase of the efflux of noradrenaline that was accompanied by a simultaneous decrease in the efflux of DOPEG (see above for one exception). In all experiments the rise in internal sodium caused the ratio NA/DOPEG to increase.These results indicate that—as long as the sodium gradient is normal—the axonal membrane functions as a barrier that largely prevents any outward movement of axoplasmic noradrenaline. Consequently, the axoplasmic amine is largely deaminated, and the ratio NA/DOPEG is low. However, when the axoplasmic sodium concentration rises, axoplasmic noradrenaline is transported out of the nerve ending at such high rates that the axoplasmic noradrenaline concentration falls; the fall in the efflux of DOPEG is indicative of a fall in the intraneuronal formation of DOPEG. The results show that changes in the efflux of DOPEG (i.e., of a highly lipophilic metabolite that easily leaves adrenergic nerve endings) can serve as an index of changes in axoplasmic noradrenaline levels.Supported by the Deutsche Forschungsgemeinschaft (Tr.96)     

Release of vesicular noradrenaline in the rat tail artery induced by cocaine

Summary The effects of cocaine on overflows of endogenous noradrenaline and DOPEG from isolated rat tail arteries were examined. 1. Both overflows increased progressively with increasing concentration of cocaine, while the (NA overflow)/(DOPEG overflow) ratio first increased and then decreased. The changes in the overflows induced by cocaine (0.1 mmol/l) appeared reversible. 2. Exposure of the tissue for 30 min to cocaine, 1 mmol/l, resulted in a significant decrease in the proportion of storage vesicles containing electron-dense cores. 3. The changes in overflows of noradrenaline and DOPEG induced by cocaine (0.1 mmol/l) were unaffected by the presence of desipramine (0.1 mol/l) or removal of extracellular Ca²⁺. The effect of cocaine on the overflow of noradrenaline was potentiated by prior inhibition of MAO with clorgyline. 4. Exposure of segments to a Ca²⁺-free, high K, low Na incubation medium was accompanied by increased overflow of noradrenaline. Cocaine (0.1 mmol/l) reduced the overflow of noradrenaline to about a half, and substantially increased the overflow of DOPEG. 5. The increase in the overflow of DOPEG from segments bathed in HEPES-buffered solutions, the pH of which ranged from 6.80 to 7.38, was approximately proportional to the calculated concentration of unprotonated (uncharged) cocaine. 6. Quantitatively similar changes in the overflows were observed when norcocaine was substituted for cocaine. Ecgonine methyl ester was much less potent than cocaine, and O-benzoyl ecgonine was ineffective. 7. The small increases in the overflow of noradrenaline observed at relatively low concentration (<30 mol/l) of cocaine can be attributed primarily to inhibition of reuptake of the released transmitter by the cocaine- and desipramine-sensitive amine carrier. The overflows of NA and DOPEG in the presence of higher concentrations of the alkaloid exhibit features compatible with the following hypothesis: (A) Cocaine is translocated across the axonal membrane mainly in the form of the unprotonated species, a large fraction of which is reprotonated upon the entry into the axon. (B) Cocaine releases noradrenaline from storage vesicles into the extravesicular space, where the bulk of the amine is converted to DOPEG. (C) Efflux of the remaining noradrenaline from the axon is not mediated by the Na⁺-dependent, cocaine- and desipramine-sensitive neuronal amine carrier. It seems to represent uncoupled efflux of the protonated form of noradrenaline.Abbreviations DOPEG 3,4-dihydroxyphenylethylene glycol - DOMA 3,4-dihydroxymandelic acid - HEPES N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid - MAO monoamine oxidase - MOPEG 3-methoxy-4-hydroxyphenylethylene glycol - NA (–)noradrenaline - pH_j pH in the extravesicular space of the axon - pH_o pH of the bathing solution - pK_a negative logarithm of the dissociation constant This study was supported by the British Columbia Heart Foundation Send of fprint requests to V. Palaty at the above address     

The handling of five amines by the extraneuronal deaminating system of the rat heart

Summary The handling of five amines by the extraneuronal deaminating system was studied in perfused hearts of rats (pretreated with reserpine; COMT and neuronal uptake inhibited). Hearts were perfused with 50 nmol/l ³H-noradrenaline for 30 min, in the presence of increasing concentrations of unlabelled (–)-adrenaline, (–)-noradrenaline, dopamine, tyramine and 5-HT. IC₅₀'s were determined as those concentrations of unlabelled amines which halved the steady-state rate of deamination of ³H-noradrenaline. After correction for changes in the tissue/medium ratio for ³H-noradrenaline, half-saturating outside concentrations were obtained. They increased in the order (–)-adrenaline (15 mol/l) — tyramine — dopamine — noradrenaline —5-HT (53 mol/l). The V _max for extraneuronal deamination was determined for ³H-(–)-adrenaline, ³H-(–)-noradrenaline and ³H-dopamine, as well as (by HPLC and electrochemical detection) for tyramine and 5-HT. It was low for (–)-adrenaline, intermediate for (–)-noradrenaline, dopamine and 5-HT, high for tyramine. For the three catecholamines the half-saturating outside concentrations of the extraneuronal deaminating system clearly exceeded those for the extraneuronal O-methylating system of the same organ (see Grohmann and Trendelenburg 1985), although the two enzymes appear to co-exist in the same cells, so that the same transport system is involved.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg