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     

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.     

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     

Evidence for uptake1-mediated efflux of catecholamines from pulmonary endothelial cells of perfused lungs of rats

Previous pharmacological studies have demonstrated that pulmonary endothelial cells and noradrenergic neurones possess the same transporter for inward transport of catecholamines, uptake₁. In noradrenergic neurones, it has been shown that uptake₁ is also involved in the carrier-mediated outward transport, or efflux, of noradrenaline and dopamine. The aim of the present study was to examine the efflux of noradrenaline and dopamine from perfused lungs of rats to determine whether uptake₁, in addition to diffusion, mediates efflux of catecholamines from pulmonary vascular endothelial cells.The effects of reducing the cellular sodium gradient and of substrates and inhibitors of uptake₁ on the efflux of ³H-noradrenaline and ³H-dopamine from rat lungs were measured. Isolated; perfused lungs of rats (monoamine oxidase and catechol-0-methyltransferase inhibited) were loaded with ³H-(–)noradrenaline or ³H-dopamine for 10 min followed by perfusion with either (1) a low sodium, amine-free: Krebs solution, in which NaCl was replaced by either Tris.HCl or LiCl, for 15 or 10 min, respectively or (2) amine-free Krebs solution for 30 min in the absence or presence of a substrate or inhibitor of uptake₁ for the last 15 min. The rate constants for spontaneous efflux of noradrenaline and dopamine from the lungs were 0.0163 min^–1 and 0.0466 min^–1, respectively. When NaCl was replaced by Tris.HCl during efflux, the rate constants for efflux of noradrenaline and dopamine were increased 2.5-fold and 3-fold, respectively, whereas, when NaCl was replaced by LICl, the rate constants were increased 8-fold and 4-fold, respectively. The uptake₁ substrates, dopamine (1 and 3 mol/l) and adrenaline (40 mol/l), both caused a rapid and marked increase in the efflux of noradrenaline, while noradrenaline (4 mol/l) had a similar effect on the efflux of dopamine. The ^uptake 1 inhibitors, imipramine (3 and 10 mol/l) and nisoxetine (50 nmol/l), caused small and gradual increases in the efflux of noradrenaline and dopamine from rat lungs.These results demonstrate that efflux of noradrenaline and dopamine from rat lungs is affected by alterations in the normal sodium gradient across the cell and by drugs that interact with the uptake₁ transporter. Thus, it can be concluded that the spontaneous efflux of catecholamines from pulmonary vascular endothelial cells is mediated predominantly by uptake₁. In addition, efflux of catecholamines from the lungs has a diffusional component, which, combined with inhibition of reuptake, accounts for the small increase in amine efflux by inhibitors of uptake₁.Abbreviations COMT Catechol-O-methyltransferase - FRL Fractional rate of loss - K _m Michaelis or half-saturation constant - t _out rate constant for efflux - k _uptake rate constant for uptake - MAO monoamine oxidase - t _/12 half-time for efflux - U-0521 3,4-dihydroxy-2-methylpropiophenone - V _max maximal rate of uptake Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Scarcella et al. 1993).     

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake₁, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake₁. In addition, another drug that is both a substrate of uptake₁ and a MAO inhibitor, debrisoquine, was investigated in the study.The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake₁ in rat lungs. After loading of isolated perfused lungs with ³H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of ³H-noradrenaline was measured for 30 min. When 1 mol/l amezinium or 15 mol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of ³H-noradrenaline, which was reduced by about 70% when 1 mol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l ³H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l ³H-noradrenaline under conditions where the amine was accumulated by uptake₂ (COMT, uptake₁ and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline.The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake₁. Amezinium is much less potent as a MAO inhibitor in cells with the uptake₂ transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.Abbreviations COMT catechol-O-methyltransferase - DOMA 3, 4-dihydroxy-mandelic acid - DOPEG 3, 4–'dihydroxyphenylglycol - ECS extracellular space - FRL fractional rate of loss - IC ₅₀ inhibitor concentration that causes 50% inhibition - K _{m uptake} Michaelis or half-saturation constant for uptake - k _{M AO} rate constant for deamination - k _{out NA} rate constant for efflux of noradrenaline - MAO monoamine oxidase - MAO-Aa type A monoamine oxidase - MAO-B type B monoamine oxidase - T/M _NA tissue to medium ratio of noradrenaline - U-0521 3, 4-dihydroxy-2-methylpropiophenone - V _max maximal rate - v _st–st steady-state rate of metabolite formation Preliminary results of this study were presented to the 1993 Meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Bryan-Lluka 1993).     

Evidence for an extraneuronal location of monoamine oxidase in renal tissues

Summary (1) Homogenates of renal cortex and renal medulla of control and 6-hydroxydopamine-denervated cat kidneys were prepared. (2) Monoamine oxidase (MAO) activity was determined with [³H]-5-hydroxytryptamine ([³H]-5HT) and [¹⁴C]--phenylethylamine ([¹⁴C]--PEA) as preferential substrates for MAO-A and MAO-B, respectively. (3) The endogenous dopamine and noradrenaline tissue contents of control and chemicallydenervated kidneys were compared with the MAO activities. (4) The results show that a 70% depletion of monoamine content by chemical denervation resulted only in a 23% reduction of MAO-A activity in the renal cortex, whereas MAO-13 was unaffected either in the cortical or the medullary zones; in the renal medulla MAO-A activity was not changed by denervation. Most of the MAO activity in the cat kidney is of the B type (74%) and is located in the renal cortex.On leave from Faculdade de Farmácia, Universidade de Coimbra, Portugal Send offprint requests to P. Soares-da-Silva at the above address     

The interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hindlimb

The vascular response to bolus injections of 5-hydroxytryptamine (5HT) over the range 0.01-10.0 microgram was measured in isolated perfused rat hindlimbs. The hindlimb vasculature displayed a vasoconstrictor response to 5HT, which was potentiated by the addition of 0.01 microgram/ml noradrenaline (NA) to the perfusate at all doses tested. The response to 5HT was also enhanced by the monoamine oxidase (MAO) inhibitor, tranylcypromine (0.05 mg/ml), suggesting that MAO located in the vascular wall is involved in the termination of the response to 5HT in the peripheral circulation. MAO inhibition abolishes the potentiation of 5HT by NA, indicating that this potentiation results from competition for MAO by both amines, leaving more amine intact to activate membrane receptors. Corticosterone (0.01 mg/ml), catecholamine extraneuronal uptake inhibitor, did not alter the response to 5HT, nor the increase of this response by NA, it thus appears that 5HT and NA have different membrane transport systems in the peripheral vasculature.     

Reversal of noradrenaline denervation-induced increase of beta-adrenoreceptor binding in rat neocortex by noradrenaline infusion

The effect of intraventricular infusion of (-)-noradrenaline (NA) on beta-receptor binding in vitro to homogenates from 6-hydroxydopamine (6-OH-DA)-denervated and from normal rat cerebral cortex was studied. NA was infused with osmotic minipumps connected to cannulas placed in the right lateral ventricle, delivering 1 or 5 microgram (-)-NA/h continuously for 9 days. One day later the rats were sacrificed and cortical tissue taken for beta-receptor (using [3H]dihydroalprenolol ([3H]DHA) as radioligand) and NA assay. The NA level in the cerebral cortex of 6-OH-DA treated rats was decreased to 70-80% of that of controls. No substantial change in the NA level was observed after infusion of 1 microgram (-)-NA/h, whereas infusion of 5 microgram/h led to a 40-60% increase compared to that of control rats infused with vehicle alone. Infusion of vehicle alone into control rats did not cause any change in [3H]DHA binding, whereas in denervated rats there was a 30-50% increase in [3H]DHA binding compared to that of controls. This increase was completely counteracted by infusion of 1 or 5 microgram (-)-NA/h. Infusion of 1 microgram (-)-NA/h to control rats did not cause any change, while infusion of 5 microgram (-)NA/h led to a significant decrease (-24%) in [3]DHA binding. The present results further support the view that the availability of NA at the receptors controls the number of beta-adrenergic receptors, thereby probably regulating the NA sensitivity of cells with beta-receptors.     

Disprocynium24, a novel inhibitor of the extraneuronal monoamine transporter, has potent effects on the inactivation of circulating noradrenaline and adrenaline in conscious rat

The role of extraneuronal uptake in terminating the actions of catecholamines has been difficult to evaluate in vivo, largely because of lack of suitable inhibitors. The compound, 1,1-diisopropyl-2,4-cyanine iodide or disprocynium24 (D24), is a novel inhibitor of extraneuronal uptake with a high degree of potency in vitro. This study examined the actions of D24 on the inactivation and metabolism of circulating noradrenaline and adrenaline in conscious rats.Animals received i.v. infusions of ³H-labelled noradrenaline and adrenaline, and their extraneuronal O-methylated metabolites, normetanephrine and meta nephrine. Plasma concentrations of endogeneous and ³H-labelled catecholamines and metanephrines were measured before and after D24. D24 caused large increases in plasma concentrations of noradrenaline and adrenaline, effects due to both decreases in their plasma clearances and increases in their rates of release into plasma. Plasma concentrations of normetanephrine and metanephrine also increased due to their decreased clearance from plasma. Increased release of normetanephrine into plasma did not contribute to increased plasma concentrations of normetanephrine. In fact, the contribution of extraneuronal O-methylation to noradrenaline clearance decreased substantially after D24.The data indicate that D24 is a potent inhibitor of the extraneuronal catecholamine transporter in vivo and that this process contributes importantly to the removal of circulating catecholamines and their O-methylated amine metabolites. Increased release of noradrenaline into plasma may reflect an increase in the proportion of transmitter that escapes from sites of release into the circulation. However, increased adrenaline release indicates that the drug also causes sympathoadrenal activation.     

Possible physiological significance of the initial step in the catabolism of noradrenaline in the central nervous system of the rat

Summary The hypothalamus, the cerebral cortex and the cerebellar cortex of the rat were labelled in vitro with ³H-noradrenaline (³H-NA) and the metabolism of the tritiated transmitter was studied during spontaneous outflow and under conditions of release elicited by exposure to 20 mM K⁺.In the three areas of the central nervous system of the rat, ³H-NA accounted for approximately 40% of the total radioactivity in spontaneous outflow while the ³H-O-methylated deaminated fraction (³H-OMDA) and ³H-3,4-dihydroxyphenylglycol (³H-DOPEG) were the main metabolites. Exposure to the reserpine-like agent, Ro 4-1284 induced a selective increase in the spontaneous outflow of ³H-DOPEG, while the contribution of the ³H-OMDA metabolites to the release induced by Ro 4-1284 was very small.During ³H-transmitter release elicited by exposure to 20 mM K⁺, approximately 80% of the radioactivity was collected as unmetabolized ³H-NA, while ³H-DOPEG was the main metabolite formed under these experimental conditions. Exposure to cocaine prevented ³H-DOPEG formation from ³H-NA released by K⁺, indicating that ³H-DOPEG was formed after neuronal reuptake of the transmitter released by K⁺.After in vitro labelling with ³H-NA, the unmetabolized transmitter represented approximately 70% of the total radioactivity retained in the tissue. However, when ³H-NA was administered in vivo, by intraventricular injection, only 30% of the total radioactivity retained by the tissue was accounted for by ³H-NA, and 60% of the radioactivity corresponded to the ³H-OMDA fraction, most of which was retained as ³H-MOPEG sulfate.When the rats were pretreated with pyrogallol, free ³H-DOPEG accounted for nearly 50% of the radioactivity retained in the three areas of the central nervous system after in vivo labelling with ³H-NA. When monoamine oxidase was inhibited by pargyline and ³H-NA was administered by intraventricular injection, ³H-NMN accounted for approximately 50% of the total radioactivity retained in the three areas of the central nervous system of the rat.The results obtained are compatible with the view that formation of the deamined glycol is the first step in the metabolism of ³H-NA in the rat central nervous system. In addition, it is concluded that the determination of the levels of some NA metabolites retained in the central nervous system does not necessarily represent an accurate reflection of the degree of central noradrenergic activity or of selective metabolic pathways. Consequently, in studies on the metabolism of NA it is important to take into account not only the transmitter and its metabolites in the tissue but also in the outflow from the structures studied either under in vivo or in vitro conditions.     

Conclusive evidence for distinct transporters for 5-hydroxytryptamine and noradrenaline in pulmonary endothelial cells of the rat

The aims of this study were to obtain conclusive evidence about the roles of a 5-hydroxytryptamine [5-HT] transporter and uptake, in the dissipation of 5-HT in the lungs of the rat and to compare the properties of the 5-HT transporter in rat lungs with that in other tissues, including brain and platelets. In the first part of the study, the IC₅₀ values of a range of selective inhibitors and substrates of the 5-HT transporter or uptake₁ were determined for inhibition of uptake of 5-HT or noradrenaline in intact perfused lungs of rats. Monoamine oxidase was inhibited and, in experiments with noradrenaline, catechol-O-methyltransferase was also inhibited. Initial rates of uptake of 5-HT or noradrenaline were measured in lungs perfused with 2 nmol/l ³H-5-HT or ³H-noradrenaline for 2 min, in the absence or presence of at least three concentrations of paroxetine, citalopram, fluoxetine, 7-methyltryptamine, tryptamine, nisoxetine, imipramine, 5-HT, desipramine, (+)-oxaprotiline, cocaine or tyramine. The results showed that pharmacologically distinct transporters are involved in the uptake of 5-HT and noradrenaline in rat lungs, since there was no significant correlation between the IC₅₀ values for inhibition of 5-HT and noradrenaline uptake in the lungs. However, there were significant correlations between the IC₅₀ values for (a) inhibition of 5-HT uptake in rat lungs and of uptake by the 5-HT transporter in rat brain and (b) inhibition of noradrenaline uptake₁ in rat lungs and of uptake, in rat phaeochromocytoma PC-12 cells. The results support the conclusion that 5-HT uptake in rat lungs occurs, at least predominantly, by a 5-HT transporter which is very similar to or the same as that in other tissues, such as the brain, and provide further evidence for transport of noradrenaline by uptake₁.Further experiments were carried out to determine whether there is any transport of 5-HT by uptake₁ or of noradrenaline by the 5-HT transporter in rat lungs. Lungs were perfused with 2 nmol/1 ³H-5-HT or ³H-noradrenaline for 2 min in the absence or presence of 1 mol/l citalopram, desipramine, or citalopram and desipramine. The results showed that there was no evidence of any transport of 5-HT in the lungs by uptake₁ or of noradrenaline by the 5-HT transporter, in that desipramine had no effect on 5-HT uptake (in the absence or presence of citalopram) and citalopram had no effect on noradrenaline uptake (in the absence or presence of desipramine).The final series of experiments was carried out to determine whether, at high concentrations of the amine, there is any interaction of 5-HT with uptake₁ or of noradrenaline with the 5-HT transporter. Noradrenaline, at a concentration of 10 mol/l, did not affect 5-HT uptake in lungs perfused with 2 nmol/l ³H-5-HT for 2 min (uptake₁ inhibited), but 50 mol/l 5-HT inhibited noradrenaline uptake by 56% in lungs perfused with 2 nmol/l ³H-noradrenaline for 2 min (5HT transporter inhibited). These and the above results show that the 5-HT transporter appears to be exclusively responsible for 5-HT uptake in rat lungs, despite the possible interaction of 5-HT at high concentrations with the uptake, transporter in the cells. On the other hand, noradrenaline is transported exclusively by uptake₁ in the lungs, and there is no evidence that it interacts with the 5-HT transporter, even at high concentrations.Preliminary results of this study were presented to the December 1993 meeting of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (Paczkowski and Bryan-Lluka 1993).     

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.     

Diminished pressor response to noradrenaline of the perfused tail artery of pregnant rats

A reduced pressor response of the perfused rat tail artery to various doses of noradrenaline (NA) occured during pregnancy and was most pronounced at day 7–8 of gestation. Progesterone pretreatment caused a similar reduction in non-pregnant rats, while oestradiol had no effect. It is suggested that the increased circulating progesterone level during pregnancy is responsible for the reduced NA response in vitro.     

Influence of chronic administration of nicotine on the turnover and metabolism of noradrenaline in the rat brain

Chronic administration of nicotine (0.5 mg/kg, subcutaneously, 3 to 5 times a day for 6 weeks) accelerated the rate of disappearance of intraventricularly administered ³H-noradrenaline from rat brain. This was associated with normal levels of ³H-normetanephrine suggesting an increase in intraneuronal deamination.The rate constant of amine decline (k) in animals chronically treated with nicotine was significantly greater than that of controls, while the steady state level of brain noradrenaline was about equal in both groups of rats. Amphetamine, reserpine, acetylcholine, histamine, pheniprazine, pargyline, and nicotine affected the catecholamine levels in the rat brain treated with nicotine to the same degree as they did in the controls. It is concluded that chronic administration of nicotine may increase noradrenaline turnover in the brain and possibly increase the deamination of this amine.     

Vascular uptake of catecholamines in perfused lungs of the rat occurs by the same process as Uptake1 in noradrenergic neurones

Summary The aim of the study was to determine whether the uptake process for catecholamines in rat lungs is Uptake₁, Uptake₂ or a distinct process with some properties of both Uptake₁ and Uptake₂. The initial rate of uptake of noradrenaline was measured in isolated lungs of rats perfused with 2 nmol/l ³H-(–)-noradrenaline for 2 min with monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited, in the absence or presence of drugs that are substrates or inhibitors of Uptake₁ or Uptake₂ or of alterations in the ionic composition of the Krebs solution. The rank order of the IC₅₀ values for inhibition of uptake of noradrenaline in the lungs by drugs that are substrates or inhibitors of Uptake₁ or Uptake₂ is compatible with the conclusion that uptake of catecholamines in rat lungs occurs by Uptake₂, and not by a process with the properties of Uptake₂. Additional evidence was provided by the marked inhibition of uptake in the lungs when the Na⁺ concentration in the Krebs solution was decreased from 143 to 25 mmol/l and by the lack of inhibition when the K⁺ concentration was increased from 5.9 mmol/l to either 10.9 or 20.9 mmol/1.Further experiments were included in the study to obtain data additional to histological evidence (Hughes et al. 1969; Nicholas et al. 1974) regarding the site of Uptake₁ in rat lungs. Pretreatment of rats with either 6-hydroxydopamine (to destroy noradrenergic neurones) or reserpine (to inhibit synaptic vesicle uptake) had no effect on the deamination or accumulation of noradrenaline in lungs perfused with ³H-noradrenaline (COMT inhibited). In a further series of experiments, efflux of noradrenaline from rat lungs, after loading with ³H-noradrenaline (MAO and COMT inhibited), could be described by a single compartment with a half-time for efflux of 42 min and with no bound fraction. These results provide further evidence that no significant uptake of noradrenaline in the lungs occurs into noradrenergic neurones and are compatible with histological evidence that the endothelial cells of the lung microvasculature are the site of noradrenaline uptake.The study has shown that the uptake of catecholamines in the lungs, at least in the rat, occurs by Uptake₁, and hence the pulmonary endothelial cells are a nonneuronal site where catecholamine transport occurs by Uptake₁.Some of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists (Bryan and O'Donnell 1988; Bryan et al. 1988a) and the British Pharmacological Society (Bryan et al. 1988) Send offprint requests to L. J. Bryan-Lluka at the above address     

Inhibition of rat brain monoamine oxidase enzymes by fluoxetine and norfluoxetine

Fluoxetine and its primary metabolite, norfluoxetine, are inhibitors of neuronal uptake of 5-hydroxytryptamine. While fluoxetine has also been reported to inhibit monoamine oxidase (MAO) in vitro at concentrations much lower than those measured in brain following chronic fluoxetine treatment, neurochemical profiles are not consistent with substantial MAO inhibition in vivo. In an attempt to explain this inconsistency, we have examined the interactions of fluoxetine and norfluoxetine with rat brain MAO-A and -B by a radiochemical assay method.Fluoxetine and norfluoxetine were competitive inhibitors of MAO-A in vitro, with K_i values of 76.3 M and 90.5 M, respectively. Both compounds were non competitive or uncompetitive inhibitors of MAO-B in vitro. Inhibition of MAO-B was time-dependent and was very slowly reversible by dialysis. IC₅₀ values versus metabolism of 50 M, -phenylethylamine were 17.8 M (fluoxetine) and 18.5 M (norfluoxetine). Analysis of the time-dependence of MAO-B inhibition by fluoxetine revealed that an initial competitive interaction between the enzyme and the inhibitor (K_i 245 M) was followed by tight-binding enzyme inactivation (k_inact 0.071 min^–1).Following administration of fluoxetine (20 mg kg^–1 day^–1]) for 7 days, the cortical concentration of fluoxetine + norfluoxetine was estimated by gas-liquid chromatography to be 700 M. Such drug treatment reduced MAO-A activity by 23% in 1:8 (w/v) cortical homogenates, but not in 1:80 homogenates. Inhibition of MAO-B in 1:8 homogenates was modest (12%) and was not significantly reduced by homogenate dilution. The concentration of 5-hydroxyindole-3-acetic acid, measured by high pressure liquid chromatography, was reduced by 47% in cortices from drug-treated rats, while concentrations of 5-hydroxytryptamine, noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were unchanged. These results suggest that, following chronic drug administration leading to relatively high tissue concentrations of fluoxetine and norfluoxetine, inhibition of either form of MAO would be restricted by competition for the enzyme with intraneuronal amine substrates.     

Early intraneuronal mobilization and deamination of noradrenaline during global ischemia in the isolated perfused rat heart

Summary Isolated rat hearts were perfused according to the Langendorff technique and both extraneuronal uptake of noradrenaline and COMT were inhibited. The noradrenergic neurones were first prelabelled with ³H-(–)-noradrenaline (13 nmol/1). Thereafter the hearts were submitted to global ischemia (perfusion rate reduced from 5 up to 0.5 ml/min) for 60 min and subsequently reperfused for 5 min. The coronary effluent was continuously collected and analyzed for the appearance of ³H-noradrenaline and its metabolites. 1. Global ischemia was associated with an early release of ³H-noradrenaline. At reperfusion a brisk increase in the FRL of ³H-noradrenaline was observed which may indicate that, on severe restriction in coronary flow, perfusion of the tissue became heterogenous and thus partially masked the amount of ³H-noradrenaline released from the noradrenergic nerve terminals. Gradual reduction in coronary flow also progressively reduced (but did not abolish) the total formation of ³H-DOPEG. 2. The maximal efflux of ³H-noradrenaline was observed during the 1st min of reperfusion whereafter the efflux declined rapidly, indicating a wash-out of transmitter trapped in the extracellular space. The efflux of the lipophilic metabolite ³H-DOPEG, on the other hand, continuously increased during the reperfusion. This was due to both new formation and wash-out of ³H-DOPEG retained and/or distributed into the tissue during the period of restricted flow. 3. Neither a reduction of the extracellular calcium concentration (from 2.6 mmol/l to 0.1 mmol/1) nor the presence of the calcium entry blocker verapamil (250 nmol/l) reduced the efflux of ³H-noradrenaline seen during ischemia and reperfusion. 4. Desipramine (100 nmol/l) markedly reduced the ischemia-induced release of ³H-noradrenaline and simultaneously attenuated the formation of ³H-DOPEG. 5. A moderate reduction in the ischemia-induced mobilization of ³H-noradrenaline was seen in hearts perfused with 1ol/l reserpine, whereas the formation of ³H-DOPEG from such hearts was markedly higher than in corresponding controls. Only minor deviations from this pattern was observed when desipramine was present in addition to reserpine. It is concluded that a severe restriction in myocardial perfusion rate is associated with an enhanced net leakage of vesicular noradrenaline. This results in a rise of the free axoplasmic noradrenaline concentration which, in combination with an altered transmembrane sodium gradient, induces an increased local release of noradrenaline partly mediated by a calcium-independent, carrier-mediated outward transport. Desipramine, which inhibits this transport mechanism, may have, in addition to its effect on the membrane carrier, an additional effect in reducing the net leakage of transmitter from storage vesicles. Furthermore, despite severe restriction in coronary flow, and thus oxygen delivery, DOPEG is still formed, possibly as a consequence of the elevated axoplasmic noradrenaline concentration which may in part compensate for a reduced monoamineoxidase activity.Abbreviations DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MAO monoamineoxidase - COMT catechol-O-methyltransferase - OMI 3-O-methoxy-(±)-isoprenaline - FRL fractional rate of loss This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5-1) and AB Hässle, Mölndal, Sweden     

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.     

Time-dependent changes in neuronal net uptake of noradrenaline after pretreatment with pargyline and/or reserpine

Summary Isolated rabbit hearts were perfused with 20 to 200 ng/ml of (–)-noradrenaline and arterio-venous differences were determined at various times to measure the rate of net removal of the amine from the perfusion fluid. Animals were untreated or pretreated with reserpine and/or pargyline to block vesicular retention and/or intraneuronal monoamine oxidase (MAO).The arterio-venous difference (in percent of the arterial concentration) remained rather constant during prolonged perfusions of untreated hearts with (–)-noradrenaline, the magnitude of the difference being inversely related to the arterial concentration. After block of MAO the rate of net removal declined exponentially with time; the rate of decline increased with increasing arterial concentration of the amine and also after the additional pretreatment with reserpine. The time-dependent decline in the rate of net removal was shown to be due to an increased efflux of the amine from the nerve endings. The net removal of noradrenaline-H³ at the 5th min of perfusion of pargyline-pretreated hearts was mainly due to neuronal net uptake, since a) O-methylation accounted for only 5% of the removal, and b) cocaine (10–30 (g/ml) virtually abolished net removal.Initial rates of removal were not affected by the various pretreatments.In untreated hearts retention of exogenous (–)-noradrenaline increased linearly with the duration of the perfusion but the increase was exponential after block of MAO. Apparently, the storage capacity becomes exhausted during prolonged perfusions of pargyline-pretreated hearts.The ratio noradrenaline retained by the heart/noradrenaline removed by the heart was quite small in untreated (0.16), very small in reserpine-pretreated (0.03) and nearly unity in pargyline-pretreated hearts.It is concluded that any impairment of the intraneuronal mechanisms of inactivation (vesicular storage and MAO) leads to an increase in the axoplasmic concentration of free noradrenaline which causes an increased efflux of the amine, while the influx remains unchanged. The axoplasmic concentration of free noradrenaline seems to rise more after block of MAO than after pretreatment with reserpine and is most pronounced after both. Changes in the sensitivity of the pacemaker to (–)-noradrenaline were found to be correlated with changes in the rate of removal of the amine from the perfusion fluid.Part of this study was supported by the Deutsche Forschungsgemeinschaft.     

Subchronic milnacipran treatment increases basal extracellular noradrenaline concentrations in the medial prefrontal cortex of rats

In this study, we investigated the acute effects of milnacipran, a serotonin–noradrenaline reuptake inhibitor, following subchronic treatment with milnacipran (30 mg/kg periorally for 7 days) on extracellular noradrenaline, dopamine and serotonin concentrations in the medial prefrontal cortex. Subchronic administration of milnacipran produced significantly higher basal levels of extracellular noradrenaline. Acute milnacipran administration following subchronic milnacipran treatment for 7 days produced a greater increase in extracellular noradrenaline than a single dose of milnacipran alone. The present results suggest that subchronic milnacipran treatment enhances noradrenergic neural transmission beyond that achieved with acute administration of milnacipran alone, but has no effect on serotonergic or dopaminergic neural transmission.