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     

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     

Plasma concentrations of noradrenaline and 3,4-dihydroxyphenylethyleneglycol under conditions of enhanced sympathetic activity

Summary Antecubital venous blood was sampled at rest and during orthostasis or supine bicycle exercise. The plasma was analyzed for noradrenaline and 3,4-dihydroxyphenylethyleneglycol (DOPEG) by HPLC.Orthostasis resulted in increases in plasma concentrations of both noradrenaline and DOPEG. The magnitude of changes in both was dependent on the degree of orthostasis. In conditions of supine rest, sitting, and standing the plot of the geometric mean values of plasma DOPEG (ordinate) against those of plasma noradrenaline was linear, had a slope of about unity, and intersected the ordinate at a finite value of plasma DOPEG.After administration of desipramine (to block uptake₁), plasma concentrations of DOPEG fell both at rest and during orthostasis. Moreover, desipramine abolished the plasma DOPEG response to orthostasis without affecting the plasma noradrenaline response. Hence, changes in plasma DOPEG brought about by changes in sympathetic tone are presynaptic in origin.The plasma concentration of DOPEG observed in the presence of desipramine was virtually identical with the ordinate intercept of the regression line relating plasma DOPEG to plasma noradrenaline in the absence of desipramine. This pool of plasma DOPEG (which amounted to about 75% of that observed at supine rest in the absence of desipramine) probably stems from intraneuronal noradrenaline leaking out of the storage vesicles of peripheral sympathetic neurones and may in part also be derived from the central nervous system.Supine bicycle exercise failed to increase plasma DOPEG. This may be due to the separation of the sampling site from the site of noradrenaline release (i.e. the exercising limbs) by organs involved in DOPEG extraction. The failure of plasma DOPEG to rise under these conditions may also be a consequence of increased blood flow in the exercising limbs, resulting in a marked decrease in the proportion of the released noradrenaline being recaptured by the sympathetic nerve endings.     

Desipramine inhibits sympathetic nerve activity in the rabbit

Summary The aim of the study was to determine the sites of action of intravenously administered desipramine on the sympathetic nervous system in anaesthetized rabbits (alfadolone + alfaxalone). Renal postganglionic sympathetic nerve activity was measured in order to determine central nervous and ganglionic effects. The clearance of noradrenaline from the plasma was determined with an isotope tracer method. From the noradrenaline clearance and the plasma concentration of noradrenaline the noradrenaline spillover rate was calculated. These parameters as well as blood pressure and heart rate were measured before (basal values) and at the end of 20-min infusions of sodium nitroprusside, which was given in order to modulate efferent sympathetic nerve activity through the baroreceptors.Desipramine 0.5 mg kg-1 + 0.05 mg kg^–1 h^–1 (bolus injection followed by infusion) and 2 mg kg^–1 + 0.2 mg kg^–1 h^–1 dose-dependently inhibited basal sympathetic nerve activity and the noradrenaline clearance. Desipramine had no effect on basal blood pressure, noradrenaline spillover rate or heart rate. Nitroprusside produced hypotension and simultaneously increased sympathetic nerve activity, noradrenaline spillover rate and heart rate; the clearance of noradrenaline was reduced with decreasing blood pressure. The relationship between sympathetic nerve activity and blood pressure was shifted by desipramine in a manner indicating central sympathoinhibition. Desipramine had no effect on the relationship of the noradrenaline spillover rate to blood pressure, whereas it shifted the heart rate-blood pressure relationship in a manner indicating an enhancement of reflex cardioacceleration. In a separate series of experiments, desipramine also inhibited sympathetic nerve activity in baroreceptor-denervated animals.The results show that desipramine centrally inhibits sympathetic outflow in the rabbit. Simultaneously, it enhances the noradrenaline spillover per action potential peripherally. In our study, the two effects compensated for each other, so that desipramine had no major effect on the relationship between noradrenaline spillover and blood pressure. In the heart, the peripheral effect of desipramine outweighed the central sympathoinhibition, hence the reflex cardioacceleration. Send of fprint requests to B. Szabo at the above address     

Persistent enhancement of potassium-induced responses of the rat vas deferens by desipramine

Summary The effect of desipramine on the cumulative dose-response curves of noradrenaline and potassium (K⁺) was examined on the isolated rat vas deferens. An exposure of 10 min to 10^–7 M desipramine caused a leftward shift and an increase in the maximum response of cumulative dose-response curves of noradrenaline. Desipramine (10^–7 M), in contact with the tissue for 10 min, enhanced responses to cumulative additions of K⁺ without causing a consistent change in threshold concentrations. Wash-out of desipramine resulted in a rapid loss of enhanced maximum response to noradrenaline while the maximum response to K⁺ did not show any decrease for up to 120 min after wash-out of drug. One possible explanation for the persistent enhancement of K⁺-induced responses may be that desipramine causes postjunctional changes which selectively influence contractile responses of this tissue to K⁺.     

Disposition of endogenous adrenaline compared to noradrenaline released by cardiac sympathetic nerves in the anaesthetized dog

Summary The fate of adrenaline released from cardiac sympathetic nerves was compared with that of noradrenaline before and during two periods of electrical stimulation of the left ansa subclavia in eight anaesthetized dogs. Cardiac spillovers and extractions of both catecholamines were estimated simultaneously using infusions of ³H-labelled adrenaline and noradrenaline. Animals were studied before and after neuronal uptake blockade with desipramine.Cardiac spillover of adrenaline, detectable at rest at 1.4 ± 0.3 pmol/min, increased to 4.0 ± 1.1 and 5.3 ± 1.2 pmol/min during sympathetic stimulation. Cardiac noradrenaline spillover increased from 49 ± 12 to 205 ± 40 and 451 ± 118 pmol/min. After desipramine, cardiac spillovers of adrenaline were decreased, whereas those of noradrenaline were increased so that the ratio of adrenaline to noradrenaline spillover, meaned before and during stimulation, decreased substantially from 1:42 to 1:166. The desipramine-induced decrease in cardiac extractions of ³H-labelled catecholamines indicated adrenaline was removed 60% less efficiently than noradrenaline by neuronal uptake, whereas the extractions remaining indicated adrenaline was removed 50% more efficiently by extraneuronal uptake.The differences in removal processes indicated that 35% of the adrenaline released by cardiac sympathetic nerves was recaptured compared to 88% for noradrenaline, leaving 53% to be removed extra-neuronally compared to 6.6% for noradrenaline, so that proportionally more released adrenaline than noradrenaline escaped to spillover into plasma (12% versus 5.4%). Since extraneuronal uptake was more efficient for adrenaline than noradrenaline, proportionally less released adrenaline than noradrenaline escaped local removal to spillover into plasma when neuronal uptake was blocked (17% versus 45%). This reversed the situation before blockade so that desipramine substantially decreased the ratio of adrenaline to noradrenaline spillover. Thus, differences in the efficiencies of neuronal or extraneuronal uptake are important determinants of the amounts of locally released adrenaline and noradrenaline that escape removal processes to act at neuroeffector sites or spillover into plasma.     

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.     

Role of nitric oxide formation in the regulation of haemodynamics and the release of noradrenaline and adrenaline

Summary This study in the anaesthetized rabbit aimed at determining the role of nitric oxide (NO), the putative endothelium-derived relaxing factor, in the regulation of haemodynamics and the release into plasma of noradrenaline and adrenaline. Specific inhibition of NO formation was achieved by i.v. bolus injection of l-N^G-monomethyl-arginine (l-NMMA; 3–100 mg kg^–1). Phenylephrine was infused i.v. at constant rates (2.5–20 g kg^–1 min^–1) in order to assess baroreflex-mediated changes in release due to direct peripheral vasoconstriction. Rates of noradrenaline and adrenaline release into plasma were determined by the radio-tracer technique. l-NMMA, but not d-NMMA, dose-dependently increased mean arterial pressure and total peripheral vasular resistance, whereas both heart rate and cardiac output decreased concomitantly. The corresponding ED₅₀ values for l-NMMA ranged from 11.2 to 18.5 mg kg^–1. Inhibition of NO formation by l-NMMA as well as phenylephrine infusion caused decreases in the plasma clearance of noradrenaline and adrenaline which were correlated with the drug-induced decreases in cardiac output. Both l-NMMA and phenylephrine reduced the rate of noradrenaline release into plasma as they increased total peripheral resistance. Moreover, the curvilinear relationship between these two parameters obtained for l-NMMA was virtually identical to that produced by phenylephrine, indicating that the reduction in noradrenaline release by l-NMMA is mediated solely by the baroreflex. From the l-NMMA-induced maximum inhibition of noradrenaline release, it is concluded that the counter-regulation against peripheral vasodilation by NO accounts for 69% of basal noradrenaline release. The baroreflex-sensitive component of noradrenaline release, as determined by the maximum inhibition of release induced by phenylephrine, amounted to 83% of basal release. l-NMMA also reduced the release into plasma of adrenaline; the maximum inhibition of release was 52%. However, when related to total peripheral resistance, this inhibition of adrenaline release was more pronounced than that induced by phenylephrine, suggesting that the formation of endogenous NO facilitates the release of adrenaline.This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5-3). A preliminary account of the present results was presented to the German Pharmacological Society (Halbrügge and Lütsch 1991) Send offprint requests to T. Halbrügge at the above address     

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.     

Influence of neuronal uptake on pre- and postjunctional effects of α-adrenoceptor agonists in tissues with noradrenaline — ATP cotransmission

Summary This study was undertaken in an attempt to explain why in some of the tissues in which noradrenaline and ATP act as co-transmitters the noradrenergic component predominates, while in others the predominant component is purinergic. Four different tissues were used: the epididymal portion of the rat vas deferens and the rabbit ear artery, tissues where the noradrenergic component predominates, and the prostatic portion of the rat vas deferens and the rabbit jejunal artery, where the purinergic component predominates. The noradrenaline content as well as the electrically-evoked release of noradrenaline were determined in all tissues. To determine the evoked release, the tissues were pretreated with pargyline (1 mmol · 1^–1) and then exposed to ³H-noradrenaline, washed out and transmurally stimulated (1 Hz). In addition, the influence of inhibition of neuronal uptake by desipramine (40nmo1·1^–1) on pre- and postjunctional effects of adrenaline and -methylnoradrenaline (and/or noradrenaline) was compared.The noradrenaline content of the tissues averaged: 17.4, 23.2, 3.1, and 4.8 g·g^–1 for the epididymal and the prostatic portions of the rat vas deferens and for the ear and the jejunal arteries of the rabbit, respectively. The fractional electrically-evoked release of ³H-noradrenaline was 2.02 and 2.04 × 10^–5 for the epididymal and the prostatic portions of the rat vas deferens, respectively, and 3.33 and 3.26 × 10^–5 for the ear and the jejunal arteries of the rabbit, respectively. Desipramine enhanced much more the postjunctional effect of noradrenaline, adrenaline, and -methylnoradrenaline in the epididymal than in the prostatic portion of the rat vas deferens. Moreover, desipramine similarly enhanced the effects of noradrenaline and adrenaline in the ear artery, while it did not modify the responses to these amines in the jejunal artery. The influence of inhibition of neuronal uptake on the prejunctional effect of sympathomimetic agonists was studied in the rat vas deferens only. Desipramine caused very marked and similar enhancements of the inhibitory effect of the amines in the epididymal and the prostatic portions (72.5 and 93.5 times, respectively, for adrenaline, and 34.7 and 41.4 times, respectively, for -methylnoradrenaline).These results are compatible with the hypothesis that postjunctional adrenoceptors are situated closer to the varicosities in the epididymal than in the prostatic portion of the rat vas deferens and also closer in the rabbit ear than in the rabbit jejunal artery. Therefore, the variation in the relative importance of the noradrenergic and the purinergic components may depend on the distance between the sites from where the transmitters are released and the sites where they act. Send offprint requests to J. Gonçalves 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.     

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.     

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.     

3H]desipramine labels with high affinity the neuronal transporter for adrenaline in the frog heart

Desipramine, a tricyclic antidepressant, inhibits the neuronal uptake of adrenaline and noradrenaline and, as a radioligand, labels the noradrenaline transporter in central and peripheral tissues of the rat. To study whether [3H]desipramine also labels the neuronal adrenaline transporter in vitro, its binding was evaluated in the frog heart, a tissue with a rich adrenergic innervation but virtually devoid of noradrenergic innervation. [3H]Desipramine binding to membranes from the frog heart was of high affinity (Kd = 1.94 nM) and was potently inhibited by nisoxetine and (+)oxaprotiline. Unexpectedly, [3H]desipramine binding to the transporter for adrenaline in the frog heart was also sensitive to inhibition by imipramine and the atypical antidepressants mianserin and iprindol. This is the first study to demonstrate radioligand binding to the neuronal transporter for adrenaline. The results indicate that the pharmacological profile of the transporter for adrenaline may be different from that of the noradrenergic transporter, if species differences can be excluded. It remains to be established if the affinity of imipramine, mianserin and iprindol for the adrenaline transporter contributes to their therapeutic efficacy in depression.     

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     

Parallel increases in noradrenaline reuptake and release into plasma during activation of the sympathetic nervous system in rabbits

Summary Rates of noradrenaline reuptake and spillover into plasma were examined in conscious rabbits before and during activation of the sympathetic nervous system to determine whether neuronal reuptake varies disproportionately or in parallel with increases in noradrenaline release. The sympathetic nervous system was stimulated by nitroprusside-induced hypotension, 2-deoxyglucoseinduced glucopenia or intravenous infusion of isoprenaline before and after administration of desipramine to block neuronal uptake. Spillover of noradrenaline into plasma was estimated from the dilution of intravenously infused 3H-noradrenaline with endogenous plasma noradrenaline. The amount of dihydroxyphenylglycol (DHPG) in plasma that was derived from metabolism of recaptured noradrenaline, together with the desipramine-induced decreases in clearance from plasma of ³H-noradrenaline and appearance in plasma of ³H-DHPG, were used to estimate the rate of neuronal reuptake of noradrenaline.The mean (± SEM) resting noradrenaline reuptake rate (n = 28) was 0.62 ± 0.04 nmol kg^–1 min^–1, 5-fold greater than the rate of its spillover into plasma (0.12 ± 0.02 nmol kg^–1 min^–1). Intravenous infusion of nitroprusside at 3 rates titrated to cause graded increases in heart rate caused 74%, 129% and 240% increases in noradrenaline spillover into plasma and 66%, 104% and 198% increases in noradrenaline reuptake. At 15–30 min after intravenous injection of 2-deoxyglucose (500 mg/kg) there was a 106% increase in noradrenaline spillover and a 93% increase in noradrenaline reuptake. Infusion of isoprenaline (0.25 g kg^–1 min^–) caused a 102% increase in noradrenaline spillover and a 130% increase in noradrenaline reuptake. The proportionally similar increases in noradrenaline reuptake and spillover into plasma during activation of noradrenaline release from sympathetic nerves indicated that the efficacy of neuronal reuptake — the percentage of released noradrenaline that is recaptured — is unaltered by short-term increases in the amount of neurotransmitter liberated into the synaptic cleft. Send offprint requests to G. Eisenhofer at the above address     

β-Receptor responsiveness after desipramine treatment

To examine whether a tricyclic antidepressant affects the functional response to a -receptor agonist in man, the response of heart rate, blood pressure, and plasma cAMP to isoproterenol was measured in 14 normal controls taking 75 mg desipramine daily. Desipramine significantly increased the bolus dose of isoproterenol needed to increase heart rate by 25 bpm at 14–30 days but not at 3–8 days. During infusions of isoproterenol, the increase in systolic blood pressure was blunted at both 3–8 days and 14–30 days, while the decrease in diastolic blood pressure was unaffected. Blood pressure findings were not affected by preadministration of bethanechol. In ten controls, isoproterenol infusions increased plasma cAMP, but this was unaffected by desipramine treatment. These findings suggest a decrease in the functional response of ₁, but not ₂, receptors after treatment with desipramine.Presented in part at the 143rd Annual Meeting of the American Psychiatric Association, New York, NY, May 15, 1991.     

Stereoselectivity of catecholamines: differential effects of cocaine and desipramine on catecholamine-induced contractions of the rat isolated vas deferens

The effect of uptake inhibitors, cocaine and desipramine, on the contractile response of the rat isolated vas deferens to the enantiomers of noradrenaline and adrenaline and to the corresponding deoxy-derivatives, dopamine and epinephline, was investigated. Cocaine (10(-6)M) significantly potentiated all six agonists; the effect was most marked for the laevo-isomers (-)-noradrenaline and (-)-adrenaline. Desipramine (10(-7) M) also potentiated (-)-noradrenaline, (+)-noradrenaline, (-)-adrenaline, (+)-adrenaline and epinephline but, in contrast, antagonized dopamine. This selective antagonism of dopamine by desipramine was also observed for the separated epididymal and prostatic ends of the rat vas deferens.     

Relationship between arterial and peripheral venous catecholamine plasma catecholamine concentrations during infusion of noradrenaline and adrenaline in healthy volunteers

Summary Noradrenaline and adrenaline were infused IV at 5 different rates (0.01–0.2 μg · kg · min⁻ for 30 min to volunteers. The plasma catecholamine concentrations were determined by HPLC and electro-chemical detection. At the highest infusion rate, the arterial and venous plasma concentrations of noradrenaline increased from 1.18 to 44.1 nmol · l⁻¹and from 1.14 to 31.9 nmol · l⁻¹, respectively, and of adrenaline from 0.29 to 23.9 nmol · l⁻¹ and from 0.28 to 19.3 nmol · l⁻¹ respectively. The peripheral venous plasma concentration of noradrenaline averaged 76% of the arterial concentration, and of adrenaline it was 73%. There was a linear relationship between the peripheral venous and arterial plasma noradrenaline and adrenaline concentrations at therapeutic doses.