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).     

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).     

Extraneuronal uptake of noradrenaline in rabbit dental pulp: evidence of identity with uptake

Summary The extraneuronal removal and disposition of noradrenaline in rabbit dental pulp was examined in view of earlier evidence that the tissue possessed an extra-neuronal uptake process resembling neuronal uptake₁. Pulp, which had been depleted of sympathetic nerves by homolateral superior cervical ganglionectomy, was incubated in vitro with ³H-noradrenaline in low concentrations (0.025 or 0.18 mol/l). When the metabolising enzymes (monoamine oxidase, catechol-O-methyl transferase) were active, ³H retention by the denervated pulp, as indicated by the ³H content after the tissue had been washed for 30 min following incubation with ³H-noradrenaline, was less than 30% of that of the innervated pulp. When the enzymes were inhibited, retention rose to approximately 30% of that of the innervated pulp. Analysis of the time course of the ³H efflux indicated that the ³H-noradrenaline in the denervated pulp had accumulated in a single compartment characterised by a t_1/2 for efflux of several hours. Accumulation did not occur under Na⁺-free conditions, and was inhibited by desipramine (IC₅₀ < 0.03 mol/l) and by substrates of neuronal uptake₁. Mean IC₅₀, values of the latter were very similar to those for inhibition of neuronal uptake₁ and comprised (in mol/l): (+)amphetamine (0.29), dopamine (0.31), tyramine (0.39), (–)noradrenaline (0.70), (–)adrenaline (1.50), 5-hydroxytryptamine (20) and bretylium (35). Uptake₂ inhibitors were less active (O-methyl isoprenaline, IC₅₀ = 60 mol/l) than uptake₁ inhibitors, or were without inhibitory effects at the concentrations tested (hydrocortisone, 210 mol/l; 2-methoxy oestrone, 10 mol/l).The effects of Na⁺ omission, of (+)amphetamine, and of O-methylisoprenaline on ³H-normetanephrine formation (measured in the absence of catechol-O-methyl transferase inhibition) matched their effects on ³H-noradrenaline accumulation. The results provide strong support for the presence in rabbit dental pulp of extraneuronal uptake₁ which is linked with catechol-O-methyl transferase in the removal of noradrenaline. Send offprint requests to D. A. S. Parker at the above address     

Release of noradrenaline from isolated rat tail artery induced by bafilomycin A1

The macrolide antibiotic bafilomycin A₁, a selective inhibitor of the vesicular H⁺-transporting ATPase, increased irreversibly the overflow of 3,4-dihydroxyphenylethylene glycol from isolated segments of the rat tail artery. Maximum increase in the overflow was produced by exposing the tissues to 0.5 mol/l bafilomycin As. Unless the Na^–-dependent neuronal amine carrier (uptake₁) was inhibited, overflow of noradrenaline was below the detection limit. The bafilomycin As-induced increase in overflow of noradrenaline from tissues with inhibited uptakes was accompanied by a significant decrease in the (noradrenaline overflow:glycol overflow) ratio. Unlike reserpine and tetrabenazine, the antibiotic did not alter the (noradrenaline overflow:glycol overflow) ratio in arteries incubated in Ca²⁺-free, 120 mmol/1 K⁺ medium.Bafilomycin A₁ increased overflow of noradrenaline and normetanephrine from tissues with inhibited monoamine oxidase. Inhibitors of extraneuronal catecholamine transport (uptake₂), corticosterone, 3-O-methylisoprenaline and 1,1-diethyl-2,2-cyanine, suppressed overflow of normetanephrine while increasing that of noradrenaline. Further increase in overflow of noradrenaline was produced by concomitant inhibition of uptake₁. A similar effect was observed in tissues previously exposed to phenoxybenzamine. After exposure to bafilomycin As, tyramine and (+) amphetamine (10 mol/l) were equally effective in increasing overflow of noradrenaline from tissues with inhibited monoamine oxidase into corticosterone-containing medium.Bafilomycin A₁ promotes leakage of noradrenaline from storage vesicles without affecting its conversion to 3,4-dihydroxyphenylethylene glycol. When uptake₁ is inhibited, axoplasmic noradrenaline can be translocated effectively across the axonal membrane by the diffusional efflux. When uptakes is inhibited, spontaneous quantal release contributes significantly to overflow of noradrenaline into normal media. The diffusional efflux of noradrenaline is unaffected by inhibitors of uptake₂. Even at highly elevated concentrations of axoplasmic noradrenaline, the uptake₁-mediated influx of noradrenaline exceeds the uptake₁-mediated efflux. Enhancement of noradrenaline overflow from tissues with inhibited monoamine oxidase by indirectly acting sympathomimetic amines depends primarily on their ability to induce leakage of the transmitter from storage vesicles rather than its translocation across the axonal membrane.     

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)     

Extraneuronal noradrenaline transport (uptake2) in a human cell line (Caki-1 cells)

Summary This study describes for the first time an experimental system for the extraneuronal transport mechanism of noradrenaline (uptake₂) which is based on a clonal cell line (Caki-1). Caki-1 cells were originally derived from a human renal cell carcinoma. The conclusion that these cells express uptake₂ is supported by several experimental findings. (1) The initial rate of ³H-noradrenaline uptake in Caki-1 cells is saturable, the K _m being 450 mol/l. (2) Inhibitors of uptake₂ such as corticosterone (1 mol/l) and O-methyl-isoprenaline (100 Emol/l) largely inhibit ³H-noradrenaline uptake in Caki-1 cells. Whereas inhibitors of the neuronal transport mechanism for noradrenaline (uptake₁) such as desipramine (1 mol/l) and cocaine (10 mol/l) do not reduce it. (3) Depolarization of Caki-1 cells by the elevation of extracellular potassium inhibits ³H-noradrenaline uptake. (4) There is a highly significant correlation between the IC₅₀'s of various compounds for the inhibition of ³H-noradrenaline uptake in Caki-1 cells and rabbit aorta known to possess uptake₂.Interestingly enough, uptake₂ in Caki-1 cells and rabbit aorta is inhibited by cimetidine, quinidine and procainamide which are substrates of the renal transport mechanism for organic cations. Moreover, ³H-cimetidine is shown to be a substrate of uptake₂ in the isolated perfused rat heart. These results indicate a striking similarity between uptake₂ and the renal transport mechanism for organic cations. Send offprint requests to E. Schömig at the above addressSupported by the Deutsche Forschungsgemeinschaft (SFB 176, Scho 373) and the Dr. Robert Pfleger Stiftung     

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     

The effect of (±)-dobutamine on adrenergic nerve endings

Summary Possible effects of (±)-dobutamine on adrenergic nerve endings were determined in experiments with ghosts of bovine chromaffin granules, with rat phaeochromocytoma (PC-12) cells and with the rat vas deferens. Dobutamine inhibited the vesicular uptake of a mixture of 70% adrenaline + 30% ³H-noradrenaline into ghosts, with an IC₅₀ of 1.7 mol/l. Dobutamine inhibited uptake, of ³H-noradrenaline in PC-12 cells (with an IC₅₀ of 0.38 mol/l) without being a substrate. However, dobutamine easily entered PC-12 cells by diffusion. After inhibition of MAO, COMT and vesicular uptake dobutamine (15 and 45 mol/l) released tritium from rat vasa deferentia preloaded with ³H-noradrenaline. Equi-inhibitory concentrations of dobutamine and desipramine (against uptake₁) were equireleasing. On the other hand, when MAO and vesicular uptake were intact, dobutamine (15 mol/l) increased the efflux of tritium from preloaded vasa deferentia much more than did an equi-inhibitory concentration of desipramine. Most of the released tritium was then ³H-DOPEG.Dobutamine is a potent inhibitor of uptake₁ as well as of vesicular uptake; moreover, it easily diffuses into adrenergic nerve endings. Hence, it blocks the neuronal and the vesicular re-uptake of noradrenaline; consequently, when MAO and vesicular uptake are intact, dobutamine increases the net leakage of noradrenaline from the storage vesicles, thereby leading to an efflux of deaminated metabolites. However, dobutamine is virtually unable to release noradrenaline into the extracellular space.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (Gr 490/5 and SFB 176) Send offprint requests to P. Fischer at the above address     

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     

Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein by presynaptic histamine H3 receptors

Summary The human saphenous vein was used to examine whether presynaptic histamine receptors can modulate noradrenaline release and, if so, to determine their pharmacological characteristics. Strips of this blood vessel were incubated with [³H]noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. Electrically (2 Hz) evoked ³H overflow was inhibited by histamine and the H₃ receptor agonist R-(–)--methylhistamine. Histamine-induced inhibition of electrically evoked tritium overflow was not affected by ₂-adrenoceptor blockade by rauwolscine. S-(+)--methylhistamine (up to 10 mol/l) as well as the histamine H₁ and H₂ receptor agonists 2-(2-thiazolyl)ethylamine (up to 3 mol/l) and dimaprit (up to 30 mol/l), respectively, were ineffective. The selective histamine H₃ receptor antagonist thioperamide abolished the inhibitory effect of histamine. The histamine H₂ and H₁ receptor antagonists ranitidine and pheniramine, respectively, did not affect the histamine-induced inhibition of evoked tritium overflow. The present results are compatible with the suggestion that the sympathetic nerves of the human saphenous vein are endowed with inhibitory presynaptic histamine receptors of the H₃ class. Send offprint requests to M. Gothert at the above address     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Accumulation of 3H-(±)-noradrenaline by isolated rat liver cells

Summary Using hepatocytes isolated by collagenase perfusion, we studied the accumulation of ³-noradrenaline. Cells incubated during 15 min in the presence of 0.4 mol/l ³H-noradrenaline (without inhibition of noradrenaline metabolism) accumulated 8.32 ± 1.77 pmol/10⁶ cells (n = 3). The accumulation of ³H-noradrenaline in isolated parenchymal liver cells was sensitive to 10 mol/l cocaine (inhibition 36.6 ± 7.9%, n = 3) and 1 mol/l desipramine (inhibition 27.2 ± 6.9, n = 3). Accumulation of ³H-noradrenaline was temperature and sodium dependent (inhibition 33.2 ± 9.4%, n = 9, when Na⁺ was replaced by Tris⁺) and was influenced by the inhibition of the membrane Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-ATPase) by 150 mol/l ouabain (34.7 ± 6.9% inhibition, n = 3). Accumulation of 3H-noradrenaline in the hepatocytes was not affected by the presence of uptake₂ inhibitors, normetanephrine (30 mol/l) and corticosterone (30 mol/l), but was reduced by 30 mol/l isoprenaline (76.3 ± 5.0% inhibition, n = 6). Thus, the system that takes up and accumulates noradrenaline in the isolated rat liver cells possesses some characteristics of both, uptake₁ and uptake₂ systems and appears to be different from other extraneuronal cocaine-sensitive systems, such as the one reported for pulmonary endothelial cells. Send offprint request to M. I. Masana at the above address     

Expression of the extraneuronal monoamine transporter (uptake2) in human glioma cells

Tritiated methylphenylpyridinium ([³H]MPP⁺), a substrate of the neuronal and extraneuronal noradrenaline transporter (uptake₁ and uptake₂, respectively) and of the organic cation transporter (OCT1), was used to characterize the amine transport system of the established human glioma cell line SK-MG-1.Uptake of [³H]MPP⁺ (25 nM) into SK-MG-1 cells increased linearly with time for up to 15 min. Selective uptake₁ inhibitors (e.g. (+)oxaprotiline) or omission of Na⁺ or Cl^– ions did not affect [³H]MPP⁺ uptake, whereas uptake₂ inhibitors such as O-methyl-isoprenaline (OMI) or corticosterone as well as depolarizing concentrations of K⁺ or Ba²⁺ strongly reduced [³H]MPP⁺ uptake. Initial rates of OMI(100 M)-sensitive [³H]MPP⁺ uptake were saturable, with a K_m of about 17 M and a maximal rate of about 50 pmol/ (min × mg protein). IC₅₀ (or K_i) values for inhibition of [³H]MPP⁺ uptake by substrates and inhibitors of uptake₂ or OCTI were highly significantly correlated with published IC₅₀ values for inhibition of uptake₂ but not with corresponding values for inhibition of OCT1.The results presented here clearly demonstrate that human glioma cells express an uptake₂ transporter. Thus, glial cells in the human central nervous system endowed with this transporter are likely to contribute to the inactivation of neuronally released noradrenaline.     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

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

Summary The uptake and metabolism of ³H-noradrenaline has been examined in the FL cell-line derived originally from human amnion. Cell cultures metabolised ³H-noradrenaline (1.0 mol/l) to ³H-normetanephrine and, to a lesser extent, to metabolites (not distinguished) of the O-methylated deaminated fraction; primary deaminated metabolites were not detected. ³(H-normetanephrine formation a) was not saturable in the noradrenaline concentration range 0.2–150 mol/l, b was decreased to 20%–30% of control levels by uptake₂ inhibitors (O-methylisoprenaline, 20 and 100 mol/l; cimetidine, 10 mol/l; hydrocortisone, 200 mol/l) and c, was almost insensitive to uptake₁ inhibitors (cocaine, 30 mol/l; desipramine, 3 mol/l).Uptake of noradrenaline was manifested after 30 minutes as a 6-fold increase in the cell content of the amine following inhibition of catechol-O-methyl transferase, either alone or in conjunction with inhibition of monoamine oxidase. Uptake was decreased maximally to 40% of control levels by O-methylisoprenaline. IC₅₀ values for inhibition of the O-methylisoprenaline-sensitive component of uptake were (in mol/l): corticosterone (0.3), papaverine (1.1), O-methylisoprenaline (3.0), cimetidine (6.0), (–)noradrenaline (460), and tetraethylammonium (2230). Except for the last agent, for which a comparative value is not available, the IC₅₀'s are in good agreement with those for inhibition of uptake₂ in the Caki-1 cell-line reported by other investigators.The component of uptake resistant to O-methylisoprenaline was depressed by papaverine (a 50% decrease at 50 mol/l), but was not affected by the other uptake₂ inhibitors or by cocaine (30 mol/l).It is concluded that the FL cell possesses an extraneuronal metabolising system very similar to the system in tissues such as heart and smooth muscle where transport of noradrenaline into the cell by uptake₂ is followed by rapid O-methylation via catechol-O-methyl transferase. The only difference appears to be the absence of saturation of ³H-normetanephrine formation in the FL cell at low micromolar concentrations of ³H-noradrenaline. The presence of a second uptake process is suggested by the inhibitory effect of papaverine on uptake resistant to O-methylisoprenaline; lack of effect of cocaine implies that this second process is not uptake,.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylethylene glycol - MAO monoamine oxidase - NMN normetanephrine - OMDA O-methylated and deaminated metabolite fraction - OMI 3-O-methylisoprenaline - TEA tetraethylammonium Correspondence to I. S. de la Lande at the above address     

The role of extraneuronal amine transport systems for the removal of extracellular catecholamines in the rabbit

As selective inhibitors of the extraneuronal monoamine uptake system (uptake₂) suitable for in-vivo studies were not available, the question of whether uptake₂ plays a definite role in vivo is largely unresolved. We attempted to resolve the question by using 1,1-diisopropyl2,4-cyanine iodide (disprocynium24), a novel agent that blocks uptake₂ in vitro with high potency. Anaesthetized rabbits were infused with ³H-labelled noradrenaline, adrenaline and dopamine, and catecholamine plasma clearances as well as rates of spillover of endogenous catecholamines into plasma were measured before and during treatment with either disprocynium24 or vehicle. Four groups of animals were studied: group I, no further treatment; group II, monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) inhibited; group III, neuronal uptake (uptake,) inhibited; group IV, uptake₁ as well as MAO and COMT inhibited.Disprocynium24 (270 nmol kg^–1 i.v. followed by an i.v. infusion of 80 nmol kg^–1 min^–1) did not alter heart rate and mean arterial blood pressure, but increased cardiac output by 22% and decreased the total peripheral vascular resistance by 16% with no difference between groups. When compared with vehicle controls, catecholamine clearances (normalized for the cardiac output of plasma) were decreased and spillover rates increased in response to disprocynium24. Although there were statistically significant between-group differences in baseline clearances (which decreased in the order: group I > group II > group III > group IV), the drug-induced clearance reductions relative to vehicle controls were similar in groups I to IV and amounted to 29–38% for noradrenaline, 22–31% for adrenaline and 16–22% for dopamine. Hence, there was still a significant % reduction in catecholamine clearances even after the combined inhibition of MAO and COMT, and there was no increase in the % reduction of clearances after inhibition of uptake₁. Noradrenaline spillover increased in response to disprocynium24 in all four groups by 1.6- to 1.9-fold, whereas a 1.5- to 2.0-fold increase in adrenaline and dopamine spillover was observed in groups II and IV only.The results indicate that disprocynium24 interferes with the removal of circulating catecholamines not only by inhibiting uptake₂, but also by inhibiting related organic cation transporters. As disprocynium24 increased the spillover of endogenous catecholamines into plasma even after inhibition of MAO and COMT, organic cation transporters may also be involved in the removal of endogenous catecholamines before they enter the circulation.     

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     

Catecholamine uptake sites in the rat heart after 6-hydroxydopamine treatment and in a genetically hypertensive strain

Summary Uptake of ³H-NA was measured in hearts from genetically hypertensive and chemically sympathectomised rats. The endogenous cardiac NA content and NA uptake₁ were depressed in genetically hypertensive animals, although NA uptake₂ per heart was normal. After treatment of normal rats with 6-hydroxydopamine (chemical sympathectomy) endogenous cardiac NA, uptake₁ and uptake₂ were markedly lowered. Uptake₂ inhibition by corticosterone varied in the two conditions tested, whereas no change in the potency of clonidine was detected in the genetically hypertensive rats.     

The membrane potential of vascular smooth muscle appears to modulate uptake2 of 3H-isoprenaline

Summary Segments of the rabbit main pulmonary artery and of its two branches were exposed for 10 min to 50 nmol/l ³H-(±)-isoprenaline, and the accumulation of tritium in the tissue was determined; COMT was inhibited in all experiments. 1. The accumulation of the tritium label was sensitive to 3-O-methyl-isoprenaline (OMI), showing that this vascular smooth muscle possesses uptake₂. 2. In the presence of 0.01 to 1 mol/l (–)-noradrenaline, the accumulation of tritium was depressed (in a concentration-dependent manner). This decline involved the OMI-sensitive accumulation of ³H-isoprenaline. 3. The presence of any one of three selective alpha₁-adrenoceptor antagonists (1 gmol/l prazosin, 1 mol/l WB4101, 10 gmol/l corynanthine) prevented the effect of 1 mol/l (–)-noradrenaline on the accumulation of tritium. However, in the absence of (–)-noradrenaline, the three antagonists failed to affect the accumulation of tritium. 4. 10 mol/l nicorandil caused the accumulation of tritium to increase. 5. As stimulation of alpha₁-adrenoceptors is known to result in depolarization, and as nicorandil is known to hyperpolarize this smooth muscle, it is concluded that the resting membrane potential modulates uptake₂. Send offprint requests to U. Trendelenburg at the above address     

The extraneuronal transport mechanism for noradrenaline (uptake2) avidly transports 1-methyl-4-phenylpyridinium (MPP+)

Summary The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake₂) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake₂ has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake₂ in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP⁺).Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake₂. The initial rate of ³H-noradrenaline transport [k_in = 0.58 l/(mg protein · min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (k_{O-methylation} = 0.54 min^–1). The neurotoxin MPP⁺ was found to be a good substrate of uptake₂. Initial rates of specific ³H-MPP⁺ transport into Caki-1 cells were saturable, the K_m being 24 mol/l and the V_max being 420 pmol/(mg protein · min). The rate constant of specific inward transport was 34 times higher [19.6 mol/l (mg protein · min)] than that of ³H-noradrenaline. The ratio specific over non-specific transport was considerably higher for ³H-MPP⁺ (12.6) than for ³H-noradrenaline (3.0). ³H-MPP⁺ transport into Caki-1 cells was inhibited by various inhibitors of uptake₂. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC₅₀'s for the inhibition of the transport of ³H-noradrenaline and ³H-MPP⁺, respectively, proves the hypothesis that MPP⁺ enters Caki-1 cells via uptake₂. ³H-MPP⁺ is taken up via uptake₂ not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake₂.Tritiated MPP⁺ is a new and convenient tool for the investigation of uptake₂. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for ³H-MPP⁺ than for ³H-noradrenaline. In uptake studies with ³H-MPP⁺ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake₂ carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake₂ can be expected to be greatly facilitated by the use of ³H-MPP⁺.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to H. Russ at the above address