Comparison between the effects produced by chronic denervation and by cocaine on the sensitivity to,and on the disposition of,noradrenaline in isolated spleen strips

Summary A comparison was made between the effects of cocaine and denervation on the sensitivity to, on the rate of inactivation of, and on the roles played by iproniazid and tropolone in the inactivation of noradrenaline by cat spleen strips. For studying the rate of inactivation of noradrenaline the oil immersion technique was used. Cocaine was used in four different concentrations. In all concentrations did it enhance the sensitivity to noradrenaline. When cocaine was used in concentrations of 10 and 50×10^–6 M, the enhancement was significantly higher than that caused by denervation (11.61 and 14.81 vs. 6.42, respectively: p<0.001). Since denervation produces an enhancement of the effect of noradrenaline which is smaller than that caused by cocaine, the blockade of neuronal uptake cannot fully account for all supersensitivity induced by cocaine. On the other hand, cocaine produces no further enhancement of the effect of noradrenaline in denervated strips. It is assumed that cocaine acts in normal (control) preparations by two mechanisms: 1. blockade of neuronal uptake accounting for an enhancement like that caused by denervation (about 6 times); 2. facilitation of the action of noradrenaline by interference with a hypothetical postjunctional structure which depends on the presence of intact adrenergic nerves and which accounts for the remaining degree of enhancement (about 2 times).Strips treated with cocaine (50×10^–6 M) required for half-relaxation 7.42 times the time of the controls, whereas denervated strips only required 4.84 times the control time. On the other hand in denervated strips cocaine (50×10^–6 M) produced a further 1.55-fold prolongation of the half-relaxation time. Thus, the effect of cocaine on this parameter is concluded to be due primarily to blockade of neuronal uptake and secondarily to another factor, which could be related to uptake₂.The influence of cocaine and denervation on the role played by iproniazid and tropolone on the inactivation of noradrenaline was not significantly different; apparently, in this preparation, the monoamine oxidase involved in terminating the action of noradrenaline is predominantly if not entirely situated intraneuronally, whereas catechol-O-methyltransferase seems to be situated intra- and extraneuronally almost in equal proportions.     

The termination of action of exogenous noradrenaline in the isolated cat spleen tissue

Summary The influence of cocaine, iproniazid and tropolone on the inactivation of exogenous noradrenaline was studied in cat spleen strips. The results show that, in this preparation, neuronal uptake represents the most important pathway of inactivation, oxidative deamination and O-methylation being of lesser importance. The influence of cocaine on the sensitivity to noradrenaline and on the inactivation of noradrenaline in this preparation correlates well with that observed in other preparations, showing that there is a causal relationship between rate of inactivation and supersensitivity, though the additional influence of other factors cannot be excluded.Supported in part by a grant from the Instituto de Alta Cultura (PMC-2).     

Mechanism of cocaine potentiation of responses to amines

1. Effects of cocaine on the magnitude of responses to several biologically active amines and on their rates of inactivation were studied in strips of rabbit thoracic aorta in vitro.

2. Although cocaine both potentiated responses to noradrenaline, adrenaline and phenylephrine and slowed their inactivation, the correlation between these two parameters under various experimental conditions was poor, and in all cases the delay in intrinsic inactivation was inadequate to account for the observed potentiation.

3. Potentiation of responses to noradrenaline by cocaine was little decreased in strips stored at 6° C for up to 10 days, although the response to low doses of tyramine was abolished much earlier. Similarly, cocaine clearly potentiated responses to noradrenaline for at least 28 hr at 37° C, at which time responses to noradrenaline alone were markedly decreased.

4. Cocaine potentiated responses to phenylephrine as well after 60 as after 10 min exposure to the amine in strips in which all intra-neuronal disposition of this amine had been eliminated by treatment with reserpine and iproniazid.

5. Cocaine effectively potentiated responses to histamine, but had only a slight and variable effect on those to 5-hydroxytryptamine (5-HT). It did not alter the tissue inactivation of histamine, but did significantly slow the inactivation of 5-HT.

6. Procaine slowed amine inactivation in the same way and to the same extent as did cocaine, but did not potentiate responses or affect the potentiation produced by cocaine added in its presence.

7. Cocaine potentiated responses to methoxamine to approximately the same degree as it did those to noradrenaline, although studies by the oil immersion technique clearly demonstrated that the aortic strips were entirely incapable of inactivating methoxamine.

8. The observations reported and discussed are incompatible with the hypothesis that cocaine potentiates responses to sympathomimetic amines because it prevents their inactivation by nerve uptake and storage and thus diverts larger amounts of agonist to tissue receptors. It is concluded that potentiation and inhibition of amine inactivation reflect two largely independent actions of cocaine in this vascular smooth muscle preparation, and probably in other organs, and that potentiation is a generally unreliable criterion of the blockade of processes inactivating sympathomimetic amines or of the importance of these processes in terminating the action of the amines.

     

Mechanism of potentiation of contractor responses to catecholamines by methylxanthines in aortic strips

1. Caffeine and theophylline increased the amplitude of contractor esponses of untreated and reserpine pretreated rabbit aortic strips to catecholamines (adrenaline, alpha-methylnoradrenaline, noradrenaline). Responses to amines without both the 3- and 4-OH groups in the benzene ring (methoxamine, phenylephrine, Synephrine) were not increased by theophylline and only those to Synephrine were slightly enhanced by caffeine.2. Compounds which inhibit catechol-O-methyltransferase (pyrogallol, tropolone, U-0521) potentiated responses to catecholamines and abolished the enhancing effect of theophylline and caffeine. Also, the potentiation produced by inhibitors of O-methylation was significantly reduced in the presence of the methylxanthines.3. Experiments done with the aid of the technique of oil immersion, to eliminate the diffusion of drug from the tissue into the bathing medium, showed that theophylline and caffeine decreased the rate of inactivation of adrenaline by O-methylation.4. These findings indicate that methylxanthines potentiate the contractor responses to catecholamines in aortic strips by inhibiting their extraneuronal inactivation.     

Preparation and pharmacological activity of the condensation product of adrenaline with acetaldehyde

Summary In vitro incubation of adrenaline with acetaldehyde resulted in the formation of an amorphous product (MA 3) which gave origin to two spots on chromatography plates. Preparative TLC allowed us to separate the corresponding substances, MA 4 and MA 5. Gas chromatography-mass spectrometry, ultraviolet and infrared spectra of MA 4 agree with the structure corresponding to 1,2-dimethyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline. MA 5 is very unstable and was not further characterized.Pharmacological experiments were carried out with MA 3 and MA 4 (and in some cases, MA 5) on isolated saphenous vein strips and isolated guinea-pig atria; responses of the nictitating membrane, blood pressure and hind-limb perfusion pressure were obtained in the anaesthetized dog. There were only quantitative differences between the effects of MA 3, MA 4 and MA 5 (where tested). Therefore, these effects are described as effects of TIQs (tetrahydroisoquinolines).TIQs contracted isolated saphenous vein strips, behaving as total agonists; the dose-response curves were displaced to the right by phentolamine and to the left by cocaine (potentiation factor: 2.7±0.1). In the dog, contractions of the nictitating membrane, rises of blood pressure and of the perfusion pressure (after i.a. injection) were observed. On isolated guinea-pig atria, weak beta adrenergic receptor activation was found. With higher concentrations, beta receptor blockade was observed, for both cardiac and vascular smooth muscle receptors.The effects of TIQs were short-lasting, showing that a rapid inactivation occurred both in vitro and in vivo; neuronal uptake appears to play an important role in inactivation, since cocaine was able to block about 70% of the inactivation capacity of isolated vein strips.The effects of nerve stimulation on the vein strips or on the nictitating membrane were reduced by TIQs; however, this did not affect responses to noradrenaline and enhanced those to tyramine or DMPP. Simultaneously with reduction of the effects of electrical stimulation, blockade of inactivation of endogenous and exogenous noradrenaline was induced by TIQs.Marked depletion of noradrenaline levels in the heart, hypothalamus and aorta of the guinea pig was caused by MA 3 (1–3 mg/kg).It is concluded that the condensation products of adrenaline with acetaldehyde are not devoid of pharmacological activity, are taken up by adrenergic nerve terminals and may act as false transmitters. The similarity of effects of TIQs and acetaldehyde suggests that formation of TIQs may occur in vivo, after acetaldehyde dehyde (or ethanol( administration, both in the adrenal gland and in sympathetic nerve terminals. These TIQ alkaloids could play an important role in alcoholic intoxication and in the ethanol withdrawal syndrome.Part of the results were presented to the 2nd Meeting on Adrenergic Mechanisms, University of Porto, October 3–6, 1973 and to the 4th Meeting of the Protuguese Pharmacological Society (Oeiras, November 26–27, 1973).Supported by grants from Instituto de Alta Cultura, Research Projects PMC-2 and PFR-6.     

Effects of a haloalkylamine on responses to and disposition of sympathomimetic amines

1. The mechanisms by which a haloalkylamine (GD-131) alters the inactivation of and potentiates responses to certain sympathomimetic amines, and the relationship of these actions to the similar effects of cocaine were investigated in rabbit aortic strips. The technique of oil immersion was used to assess rates of amine inactivation.2. Exposures to GD-131, which produced no detectable alpha-adrenergic blockade, markedly slowed the inactivation of noradrenaline. It was concluded that it is unnecessary to postulate a role of adrenergic receptors in the inactivation of catecholamines to account for the reported effects of haloalkylamines on amine output during adrenergic nerve stimulation.3. The reduction in the rate of noradrenaline inactivation produced by moderate exposure to GD-131 was approximately equivalent to that due to inhibition of both monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT). Addition of GD-131 did not further slow inactivation in preparations in which MAO and COMT had been inhibited, but the effects of both GD-131 and of enzyme inhibition on noradrenaline disposition were additive with that of cocaine.4. Cocaine consistently inhibited and GD-131 markedly potentiated responses to tyramine. The augmentation of responses by GD-131 was much greater than could be accounted for by the slight release of endogenous catecholamine by this agent. Thus the principal effect of the haloalkylamine appears not to involve inhibition of nerve cell membrane transport of amine.5. Maximal exposure to GD-131 short of that which produced alpha-adrenergic blockade sometimes slowed the inactivation of noradrenaline as much as did inhibition of both MAO and COMT plus the maximal effect of cocaine.6. These results seem best explained by postulating that GD-131 and other haloalkylamines inhibit the passage of sympathomimetic amines through biological membranes. Passage to sites of enzymatic inactivation, predominantly in non-neuronal tissue, is most readily inhibited. The "cocaine-sensitive mechanism," transport to sites of binding and storage, can also be inhibited, but is considerably less sensitive.7. GD-131 potentiated responses to noradrenaline more than did the maximally effective concentration of cocaine. Cocaine produced very little additional potentiation when added in the presence of GD-131, whereas the latter had a reduced, but still significant effect in the presence of cocaine. Most of the effect of cocaine and at least half of that of GD-131 was due to a common action on effector cells, which is unrelated to any alteration of amine disposition. The balance of the potentiation by GD-131 may be due to inhibition of access of amine to sites of enzymatic inactivation, perhaps involving a reduction in the volume of distribution in intracellular water, and a very small part of the potentiation by cocaine may be secondary to inhibition of transport of amine to sites of binding and storage.8. On the basis of the present observations, it is postulated that a major part of the noradrenaline released by adrenergic nerve activity is involved in the activation of tissue receptors and has its action terminated by movement away from the region of the receptors. A small portion of the mediator is removed by the circulation, some is taken up by adrenergic nerves, but the major part enters non-nervous cells and is distributed in intracellular water. The capacity of this intracellular compartment appears to be limited and enzymatic inactivation is essential to maintain its function. O-methylation is the dominant primary enzymatic process in the inactivation of physiological amounts of noradrenaline, but MAO appears to function "in series" as an effective alternate pathway of disposition.     

The effects of drugs and denervation on removal and accumulation of noradrenaline in the perfused hind-limb of the dog

Summary The removal of noradrenaline by the autoperfused hind-limb of dogs anaesthetized with pentobarbital, as well as the accumulation of noradrenaline in the saphenous vein were studied. Sensitivity of the perfused vascular area was determined by the response of the perfusion pressure to infusions of noradrenaline.The removal of noradrenaline declined very slowly during infusions lasting for up to 2 h, but edema of the perfused limb occurred after 45 to 60 min; therefore, the duration of infusion was limited to 30 min. During this period, noradrenaline was infused in rates increasing by a factor of 2 and ranging from 0.5 to 16 g/kg per minute. Accumulation capacity was saturated at 1 g/kg · min^–1, but the amount removed increased until a four-to eightfold rate was reached and then levelled off.At a rate of 1 g/kg · min^–1 the influence of drugs and of surgical denervation was investigated in other experimental series. Cocaine, nialamide, phenoxybenzamine and pretreatment with reserpine reduced removal (by 50, 45, 40 and 35%, respectively). Cortexone had no detectable influence on removal with this rate of infusion, but blocked it effectively when 4 g/kg · min^–1 were infused. Accumulation of noradrenaline in the vein was prevented by cocaine or reserpine, slightly reduced by phenoxybenzamine and enhanced by nialamide. The effects of nialamide plus cocaine did not differ significantly from those of cocaine alone, but cortexone plus cocaine completely blocked removal and accumulation. Surgical denervation reduced removal by about 70% and abolished accumulation; reserpine plus nialamide had similar effects. In the case of nialamide, removal progressively diminished during the infusion period and this time dependence of effects was accompanied by a prolongation of noradrenaline washout.Cocaine, reserpine and denervation caused supersensitivity of the perfused vessels to noradrenaline, whereas nialamide and cortexone had no such effect and phenoxybenzamine caused subsensitivity.The pronounced ability of the perfused vessels of the hind-limb to remove noradrenaline from the circulating blood is attributed primarily to neuronal uptake and intraneuronal oxidative deamination; extraneuronal uptake and inactivation seem to play an important role when neuronal mechanisms are saturated (infusion of higher noradrenaline doses) or impaired (after cocaine or denervation).Supported by Instituto de Alta Cultura (Research Project PMC/2). Part of this work was presented at the Fifth International Congress on Pharmacology (S.Francisco, July 23–28, 1972).     

Effects of reserpine on the disposition of sympathomimetic amines in vascular tissue

1. The effects of reserpine pretreatment on the intrinsic inactivation of low concentrations of phenylephrine and noradrenaline in strips of rabbit thoracic aorta were assessed by measuring the rates of relaxation, after oil immersion to prevent loss of active amine by diffusion into the surrounding medium.2. Reserpine pretreatment considerably augmented the amplitude of responses to low concentrations of phenylephrine, noradrenaline and nordefrine (Cobefrine).3. Reserpine pretreatment did not reduce the overall rate of inactivation of either phenylephrine or noradrenaline, but it did appear to decrease the contribution of uptake and storage, measured as an increased effect of enzyme inhibition and a decreased effect of cocaine on the rate of inactivation.4. The role of catechol-O-methyl transferase (COMT), but not that of monoamine oxidase (MAO), in terminating the action of noradrenaline was increased in strips from animals pretreated with reserpine. Thus it appears that interference with intraneuronal storage diverts active amine to inactivation by COMT in vascular tissue, rather than by MAO as has been previously suggested.5. As in preparations not treated with reserpine, inhibition of MAO alone had little effect on the rate of inactivation of noradrenaline, and this enzyme appears to function predominantly as an alternate pathway of little importance as long as COMT activity is unimpaired. Enzymatic processes accounted for about 85 and 70% of the inactivation of a low concentration of noradrenaline in reserpine pretreated and untreated preparations, respectively.6. Cocaine potentiated responses to noradrenaline and phenylephrine as effectively in reserpine pretreated as in untreated preparations, and inhibition of the pathways of enzymatic inactivation did not appreciably decrease the potentiation produced by this agent.7. The present results cannot be explained by the hypothesis that interference with amine inactivation by nerve uptake and storage is responsible for the potentiation of responses to noradrenaline or phenylephrine by either reserpine or cocaine, and emphasize the unrealiability of potentiation as an index of interference with mechanisms involved in terminating the action of sympathomimetic amines.     

The effects of ageing on neuronal uptake of noradrenaline in the rat

Summary We have examined the effects of ageing on the physiological function of the neuronal noradrenaline uptake system by comparing responses to cocaine in young adult (5 month) and aged (22 month) male Sprague-Dawley rats. In rat atria pre-incubated with [³H]-noradrenaline, cocaine (3–30 mol/l) significantly augmented the 2 Hz stimulation-evoked release of noradrenaline in tissues from young but not from old rats. Cocaine (1 mg/kg) produced a greater increase of the pressor response to noradrenaline in young than in old pithed rats. Cocaine significantly increased the tachycardia to noradrenaline only in young pithed rats, but in old pithed rats the duration of the response to noradrenaline was significantly increased. It is concluded that ageing in the rat is associated with a decreased function of the neuronal noradrenaline uptake system, at least in the cardiovascular system. Send offprint requests to J. R. Docherty at the above address     

The role played by the extraneuronal system in the disposition of noradrenaline and adrenaline in vessels

Summary The role played by extraneuronal sites in the disposition of noradrenaline and adrenaline was studied in the saphenous vein and in the mesenteric artery of the dog, taking as parameters the influence of blockade of these sites on the sensitivity to and on the time for half-relaxation (t ₅₀) (both in oil and in Krebs solution) of these agonists. Preliminary experiments have shown that the t ₅₀ values are not significantly changed by the changes in the height of the contraction provided the contraction is caused by the same concentration of the agonist.The results obtained permit us to conclude that in both vessels the removal of amines depends on the concentrations used. In low (0.023 and 0.23 M) or in moderately high (2.3 M) concentrations, adrenaline is removed preferentially by extraneuronal sites, whereas noradrenaline preferred neuronal sites.The selectivity of adrenaline for extraneuronal sites was present for such low concentrations that a possible physiological role of these sites in the inactivation of circulating adrenaline must be considered.The results obtained by studying the relaxation in oil or in Krebs solution and by using cortexone (60 M) or U-0521 (dihydroxy-2-methyl propiophenone; 0.1 mM) support the view that, at least in the vein, adrenaline may accumulate in extraneuronal cells and diffuse back into the biophase during the relaxation, thereby slowing the latter.Both in the veins and in the arteries noradrenaline was inactivated more rapidly than adrenaline. The difference in the rate of inactivation of these amines, already observed in controls (when all inactivation pathways are operative) became more marked when both neuronal and extraneuronal sites were blocked. The existence of an important pathway not blocked by cocaine + cortexone + iproniazid which may preferentially inactivate noradrenaline cannot be ruled out.This study was supported by a grant from the Instituto de Alta Cultura (PMC-2)Preliminary results were presented at the VI International Congress of Pharmacology (Helsinki, July 1975)     

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.     

Protection against induction of supersensitivity to catecholamines by cocaine

1. Adrenaline, noradrenaline, isoprenaline, tyramine, phentolamine, pronethalol, histamine and acetylcholine were each tested for their ability to prevent cocaine from causing supersensitivity to catecholamines in cat spleen strips in vitro. A high concentration of one of these drugs was added to the bath 5 min before cocaine hydrochloride (10 mug/ml). The effect on subsequent responses to catecholamines was compared with the effect of cocaine in control strips in the absence of an interfering drug.2. Phentolamine completely abolished the potentiating effect of cocaine. Large doses of adrenaline or noradrenaline reduced, but did not completely prevent, potentiation. Tyramine, isoprenaline, pronethalol, histamine and acetylcholine did not prevent potentiation.3. The ability of these drugs to interfere with potentiation does not correlate well with their ability to interfere with uptake of noradrenaline. Interference with uptake by cocaine is therefore unlikely to account fully for potentiation.     

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.     

Predominance of oxidative deamination in the metabolism of exogenous noradrenaline by the normal and chemically denervated human uterine artery

Summary Longitudinal strips were prepared from human uterine arteries obtained at hysterectomy. The artery had a low content of noradrenaline and dopamine, contrasting with a high content of the deaminated catechols, dihydroxyphenylglycol (DOPEG) and dihydroxymandelic acid (DOMA), which together represented 98% of endogenous catechols.When incubated with ³H-noradrenaline (0.1 mol/l), the uterine artery removed, accumulated and metabolized noradrenaline. Deaminated metabolites predominated, DOMA being the most abundant metabolite.Cocaine markedly reduced the accumulation of ³H-noradrenaline and abolished ³H-DOPEG formation, but did not change ³H-DOMA. Selective monoamine oxidase (MAO) inhibitors (clorgyline, selegiline and 2-amino ethyl carboxamide derivatives) caused a marked decrease in the amounts of ³H-DOPEG, ³H-DOMA and ³H-O-methylated and deaminated metabolites (OMDA) formed by the tissue and an increase in ³H-normetanephrine (NMN) formation. Inhibition of catechol-O-methyltransferase suppressed NMN formation and reduced that of OMDA; hydrocortisone slightly depressed the formation of DOMA and OMDA.Homogenates of the uterine artery deaminated ³H-5-HT, ¹⁴C-phenylethylamine and ³H-tyramine; inhibition curves of the deamination of ³H-tyramine by clorgyline and selegiline were compatible with the presence of both MOA A and MOA B.Exposure of the strips to 6-hydroxydopamine (1.5 mmol/l for 20 min; 3 exposure periods followed by washout periods of 15,15 and 30 min) resulted in complete and selective chemical denervation of the arterial tissue. This chemical denervation had effects which were similar to those of cocaine. The 2-amino ethyl carboxamide derivatives markedly reduced the formation of deaminated metabolites by the denervated strips.The semicarbazide-sensitive amine oxidase inhibitor semicarbazide reduced the formation of ³H-DOMA and ³H-DOPEG in intact strips, but was devoid of action in the denervated ones.It is concluded that, in the human uterine artery, deamination predominates over O-methylation and that extraneuronal deamination, leading to the formation of DOMA (and of OMDA) plays a major role in the metabolism even of low concentrations of exogenous noradrenaline.Abbreviations COMT Catechol O-methyltransferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - HPLC-ED high pressure liquid chromatography with electrochemical detection - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase - NMN normetanephrine - 6-OHDA 6-hydroxydopamine - OMDA O-methylated and deaminated metabolites of noradrenaline (3-methoxy-4-hydroxyphenylglycol and 3-methoxy-4-hydroxymandelic acid) - Ro 01-2812 3,5-dinitropyrocatechol - Ro-19-6327 N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide hydrochloride - Ro 41-1049 N-(2-aminoethyl)-5-(mfluorophenyl)-4-thiazole carboxamide hydrochloride Supported by Instituto Nacional de Investigação Científica (INIC, FmP1) and Junta Nacional de Investigação Científica e Tecnológica (JNICT). Fatima Martel is a PhD student with a grant from JNICT Send offprint requests to W. Osswald at the above address     

Effect of phentolamine on noradrenaline uptake and release

Summary The influence of phentolamine on the uptake of exogenous noradrenaline infused into the aortic cannula and on the overflow of endogenous noradrenaline caused by sympathetic nerve stimulation was investigated in the isolated perfused rabbit heart. 10^–6 M phentolamine doubled the overflow of endogenous noradrenaline, but did not change noradrenaline uptake. 10^–5 M phentolamine increased the stimulation-induced overflow of noradrenaline 4-fold and inhibited amine uptake by about 50%. 10^–4 M phentolamine elevated the overflow of noradrenaline less than 10^–5 and 3×10^–5 M did. The augmentation of transmitter overflow was only partly reversed by 13 min perfusion with drug-free medium.Pretreatment of hearts with 1.5×10^–5 M cocaine or with 10^–7 or 10^–6 M desipramine did not change the effect of phentolamine on the overflow of noradrenaline evoked by nerve impulses. Pretreatment of hearts with 10^–5 M, but not with 10^–6 M, phentolamine prevented the increase of transmitter overflow by cocaine.It is concluded that low concentrations of phentolamine potentiate the overflow of noradrenaline during nerve stimulation by a mechanism different from that of cocaine, i.e. different from inhibition of neuronal re-uptake. The nature of this mechanism is discussed.This work was supported by the Deutsche Forsehungsgemeinschaft. We have the pleasure to thank Mrs. Ch. Arts, Miss B. Piel and Mr. E. Hagelskamp for skilful technical assistance.     

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.     

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.     

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     

Effects of hypoxia on noradrenaline release and neuronal reuptake in isolated rabbit thoracic aortic strips

Summary To clarify the effects of hypoxia on stimulus-evoked noradrenaline release and on neuronal reuptake of the released noradrenaline, we examined the effects of hypoxia on contraction responses of rabbit thoracic aortic strips to transmural electrical stimulation and on the stimulation-evoked overflow of total [³H] and [³H]noradrenaline from the strips prelabelled with [³H]noradrenaline. This was done in the presence or absence of an inhibitor of neuronal uptake (cocaine). In a medium equilibrated with a gas mixture of 95% O₂/5% CO₂ (control), cocaine doubled the stimulation-evoked overflow of total [³H] and [³H]noradrenaline; there was a concomitant increase (130%) in contractions to electrical stimulation. At 0% O₂ (95% N₂/5% CO₂, hypoxia), cocaine had no significant effects on either the stimulation-evoked overflow of total [³H] and [³H]noradrenaline or contractions. In the absence of the drug, hypoxia decreased the stimulation-evoked overflow of total [³H] and [³H]noradrenaline to 47% and 43%, respectively, of the control values, whereas these values were 31% and 28%, respectively, after exposure to cocaine. The inhibition by hypoxia of contraction responses to electrical stimulation was greater in the presence of cocaine than in its absence. These results show that hypoxia inhibits both noradrenaline release evoked by a given stimulus and neuronal uptake.This work was supported by a Grant-in-Aid for New Drug Development from the Ministry of Health and Welfare of Japan and by a Grant from Smoking Research Foundation, Japan Send offprint requests to S. Miwa at the above address     

1,1′-Diisopropyl-2,4′-cyanine (disprocynium24), a potent uptake2 blocker, inhibits the renal excretion of catecholamines

1,1′-Diisopropyl-2,4′-cyanine (disprocynium24), a potent inhibitor of the extraneuronal monoamine transport system (uptake₂), was previously shown to reduce the clearance of catecholamines from plasma not only by blocking uptake₂ but presumably also by blocking organic cation transport. To provide more direct evidence for the latter conclusion, the present study was carried out in anaesthetized rabbits. It aimed at determining the effect of disprocynium24 on the renal excretion of catecholamines which is known to be, at least in part, a consequence of organic cation transport in the kidney. To this end, the plasma clearance due to renal excretion (Cl_u) of endogenous as well as infused ³H-labelled adrenaline, noradrenaline and dopamine was determined for 60-min periods of urine collection in rabbits treated either with disprocynium24 (270 nmol kg^-1 i.v followed by i.v. infusion of 80 nmol kg^-1 min^-1) or vehicle. Two groups of animals were studied: group I (monoamine oxidase and catechol-O-methyltransferase intact) and group II (monoamine oxidase and catechol-O-methyltransferase inhibited). A third group of animals with intact monoamine oxidase and catechol-O-methyltransferase was used to study the effect of disprocynium24 on the glomerular filtration rate (as determined by measuring the plasma clearance of inulin). In vehicle controls, Cl_u of endogenous adrenaline, noradrenaline and dopamine was 7.2, 5.2 and 153.6 ml kg^-1 min^-1, respectively, in group I and 10.4, 7.0 and 134.3 ml kg^-1 min^-1, respectively, in group II. Similar control values of Cl_u were obtained for infused ³H-adrenaline and ³H-noradrenaline, but not for infused ³H-dopamine; Cl_u of ³H-dopamine (4.9 ml kg^-1 min^-1 in group I and 15.4 ml kg^-1 min^-1 in group II) was considerably smaller than Cl_u of endogenous dopamine, indicating that most of the dopamine in urine (i.e., 98% in group I and 92% in group II) was derived from the kidneys rather than from the circulation. By contrast, only about one quarter of the noradrenaline in urine (32% in group I and 24% in group II) and none of the urinary adrenaline were of renal origin. In both groups, disprocynium24 markedly reduced the Cl_u of endogenous catecholamines (by 72-90%) and of infused ³H-catecholamines (by 49-69%). Moreover, it preferentially inhibited the renal excretion of those components of urinary dopamine and noradrenaline which were derived from the kidney. Therefore, disprocynium24 inhibits the tubular secretion of catecholamines and, hence, organic cation transport in the kidney. This conclusion was substantiated by the observation that disprocynium24 did not alter the glomerular filtration rate. Received: 6 February 1997 / Accepted: 12 April 1997