Localization of monoamine oxidase of type A and B in blood vessels with different innervation patterns

Summary Homogenates of dog mesenteric artery, dog saphenous vein, denervated saphenous vein, rabbit ear artery, denervated ear artery and human umbilical artery were prepared in phosphate buffer. Monoamine oxidase activity was determined with [³H]-5-hydroxytryptamine ([³H]-5-HT) as a preferential substrate for MAO type A and [¹⁴C]--phenylethylamine as a preferential substrate for MAO type B. The endogenous noradrenaline content was compared with the MAO activities in these blood vessels. The results show that there is a good relationship of MAO type A content with the density of adrenergic innervation, but it is evident that this type of MAO is present even in vessels devoid of adrenergic innervation. Thus, MAO type A in these vascular structures has both intraneuronal and extraneuronal locations whereas MAO type B is predominantly or exclusively extraneuronal.Some of the results were presented to the 11th Annual Meeting of the Portuguese Pharmacology Society (Lisboa, 1980) and to the 8th International Congress on Pharmacology (Tokyo, 1981)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

The handling of five catecholamines by the extraneuronal O-methylating system of the rat heart

In a comparative study, the handling of five catecholamines by the extraneuronal O-methylating system of the rat heart was determined; all rats were pretreated with reserpine, monoamine oxidase and neuronal uptake were inhibited in all experiments. Hearts were perfused for 7 min with a tracer concentration of 3H-(+/-)-isoprenaline, either in the absence or in the presence of unlabelled catecholamines (which reduced the O-methylation of the tracer amine). IC50's were determined for unlabelled catecholamines and then converted to "half-saturating outside concentrations", i.e., to those concentrations in the perfusion fluid that half-saturate the intracellular catechol-O-methyl transferase (COMT). The values for the (-)-isomers of dobutamine, isoprenaline, adrenaline and noradrenaline and that for dopamine were low and rather similar (between 0.67 and 2.7 mumol/l). Stereoselectivity for isoprenaline probably reflected the preference of uptake2 for the (-)-isomer. The effects of (-)- and (+)-dobutamine indicated that both isomers are a) transported by uptake2 and b) good substrates of COMT. The Vmax for O-methylation [determined for 3H-(+/-)-isoprenaline, 3H-(+/-)-adrenaline, 3H-(+/-)-noradrenaline and 3H-dopamine] was rather similar for all four catecholamines. It is concluded that the extraneuronal O-methylating system of the rat heart handles the five catecholamines in a similar manner, although the Km for uptake2 had been found to increase substantially in the order: dobutamine less than isoprenaline less than adrenaline less than noradrenaline less than dopamine (Grohmann and Trendelenburg 1984b).     

Purine agonists prevent trophic changes caused by sympathetic denervation

Surgical denervation of the lateral saphenous vein of the dog causes marked extraneuronal changes, both of a morphological and functional type. In an attempt to investigate the factor(s) responsible for the trophic effects exerted by the sympathetic innervation on the dog saphenous vein we studied the effects of noradrenaline, adenosine, inosine and N-ethylcarboxamidoadenosine (NECA) on vascular tissue after sympathetic denervation. The saphenous vein was denervated using either surgical or chemical (6-hydroxydopamine, 6-OHDA) methods. Noradrenaline (0.1 microgram/kg per h), adenosine (10 micrograms/kg per h), inosine (10 micrograms/kg per h) or NECA (0.1 microgram/kg per h) were delivered continuously for 5 days through Alzet minipumps connected to the vein. 6-OHDA-induced denervation resulted in morphological changes similar to those described for surgical denervation. Smooth muscle cells and fibroblasts showed ultrastructural signs of increased synthetic activity and their size was significantly increased. In confirmation of earlier studies, constant i.v. infusions of noradrenaline did not prevent the morphological changes induced by denervation. Adenosine prevented the morphological changes induced by chemical or surgical denervation. Similarly to adenosine, infused NECA prevented the structural consequences of denervation. In contrast, inosine did not prevent the changes caused by surgical denervation. The results are compatible with an involvement of purines in the trophic effects of sympathetic innervation. Moreover, the effects of adenosine do not appear to be mediated by inosine.     

The termination of action of catecholamines in the isolated venous tissue of the dog

Summary The termination of responses to noradrenaline and adrenaline was studied, using the oil immersion technique, in helically cut strips obtained from the lateral saphenous vein of the dog. Inhibitors of catechol-O-methyltransferase (tropolone), monoamine oxidase (iproniazid) or neuronal uptake (cocaine and imipramine-N-oxide) slowed the relaxation rate of strips contracted by noradrenaline or adrenaline (10^–5 and 10^–6 M) and immersed in oil. From these experiments it is concluded that, at the concentrations used, oxidative deamination represents the major inactivation pathway for both catecholamines, and that O-methylation plays only a minor role in the disposition of the amines; however, the inactivation of adrenaline is more dependent on catechol-O-methyltransferase than that of noradrenaline. Tissue uptake and storage occupied an intermediate position between the enzymatic pathways referred to: iproniazid inhibited 79%, cocaine 54% and tropolone 27% of the inactivation capacity when 10^–5 M noradrenaline was used. However, the combination of the three drugs inhibited only 88% of the inactivation capacity. This is due to the marked overlap of the effects of cocaine and iproniazid, which is attributed to the blockade by cocaine of the access of the amine to intraneuronal sites of oxidative deamination.Cocaine caused marked potentiation of responses to noradrenaline and moderate prolongation of relaxation time, while iproniazid, which had much more marked effects on the termination of action of noradrenaline, induced only a slight augmentation of the contractile responses. It is concluded that potentiation is an unreliable index of altered disposition of noradrenaline in this preparation.Experiments with perfused venous segments showed that, following the concentration gradient, noradrenaline diffuses freely through the vein wall, and that there is no marked difference in responses to adventitial or intimal application of the amine. Radioautographic studies gave evidence of two different types of accumulation (neuronal and extraneuronal), which appeared to be in equilibrium.Supported in part by a grant from III Plano de Fomento (Actividades, 1970).     

Influence of inhibition of extraneuronal uptake and of O-methylation on the hyperglycaemia caused by sympathomimetic amines in depancreatized rats.

This study aimed at testing whether the O-methylating system (extraneuronal uptake + O-methylation) modulates, in-vivo, beta-adrenoceptor-mediated responses. The influences of U-0521 (3,4-dihydroxymethylpropiophenone, an inhibitor of catechol-O-methyl transferase (COMT)) and hydrocortisone (an inhibitor of extraneuronal uptake) on the hyperglycaemia evoked by isoprenaline and adrenaline were compared. Both inhibitors enhanced the increase of the plasma glucose level induced by either isoprenaline (0.36 nmol kg-1 min-1) or adrenaline (0.55 nmol kg-1 min-1). The enhancement caused by U-0521 developed faster than that caused by hydrocortisone, but was of the same magnitude. This is the first report of supersensitivity to sympathomimetic amines caused by inhibition of either COMT or extraneuronal uptake in-vivo and for a response not involving smooth muscle cells.     

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     

The effect of isoprenaline on adrenoceptors in human saphenous vein

1. The order of potency of sympathomimetic amines in causing contraction of strips of human saphenous vein was (-)-adrenaline>(-)-noradrenaline>(-)-phenylephrine>(+/-)-isoprenaline.2. The competitive alpha-adrenoceptor blocking drugs tolazoline, phentolamine, and thymoxamine and the irreversible blocking drug phenoxybenzamine all blocked noradrenaline and isoprenaline contractions.3. Contractions produced by 5-hydroxytryptamine were also blocked by phentolamine and thymoxamine. Fenfluramine-induced contractions were not blocked by thymoxamine or phenoxybenzamine.4. ED50 contracting doses of isoprenaline did not cause consistent relaxation of noradrenaline-contracted strips.5. It is concluded that human saphenous vein contains a dominant population of alpha-adrenoceptors which can be stimulated by high doses of isoprenaline, but the occurrence of beta-adrenoceptors mediating relaxation is rare.     

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).     

Sympathetic denervation caused by long-term noradrenaline infusions; prevention by desipramine and superoxide dismutase.

1. The effects of continuous intravenous infusion of noradrenaline (0.01 and 0.1 microgram kg-1 h-1) were studied in both the infused lateral saphenous vein and the contralateral saphenous vein of normal dogs. Noradrenaline, saline, noradrenaline + desipramine or noradrenaline + superoxide dismutase were infused using Alzet osmotic minipumps. 2. After a 5 day infusion period, the noradrenaline content in plasma and in both saphenous veins was determined, and the venous tissues submitted to light microscope morphometry and ultrastructural study and used for the determination of their O-methylation capacity (with [3H]-isoprenaline as a substrate). 3. Noradrenaline caused dose-dependent damage to the sympathetic nerve endings of the lateral saphenous veins. Concomitant changes in extraneuronal structure and function were observed (hypertrophy of smooth muscle cells, nuclear dysmorphy, thickening of the vessel wall, impairment in O-methylation capacity). 4. Desipramine and superoxide dismutase prevented or reduced the effects of noradrenaline on both the morphological and the biochemical parameters; the protection afforded by superoxide dismutase was more marked than that by desipramine. 5. It is concluded that moderately high doses of noradrenaline exert a 6-hydroxydopamine-like effect and that this chemical sympathectomy is partially or totally prevented by desipramine or superoxide dismutase. The data suggest that a substance derived from noradrenaline, in the formation of which free oxygen radicals are involved and which is subject to neuronal uptake, is the chemical entity responsible for the neurotoxic effect observed.     

Comparison of the uptake and O-methylation of isoprenaline by cardiac and respiratory tissues of guinea-pigs

An attempt was made to correlate the extraneuronal uptake and O-methylation of isoprenaline in cardiac and respiratory tissues with the predominant beta-adrenoceptor subtype known to mediate the sympathetic responses of these tissues. Papillary muscles, left atria, trachealis muscles and lung parenchymal strips of guinea-pigs were incubated with [3H]isoprenaline ([3H]ISO) (0.1 microM) for 60 min. Levels of total radioactivity and of separated [3H]ISO and [3H]O-methylisoprenaline ([3H]OMI) in each tissue and of [3H]OMI in the medium were determined. U-0521 (10(-4) M) inhibited tissue O-methylation and caused an elevation of unchanged [3H]ISO in the tissues. The latter effect was attributed to the fact that the normal conversion of [3H]ISO to [3H]OMI did not occur. Metanephrine (10(-5) and 10(-4) M) did not affect tissue levels of unchanged [3H]ISO, but reduced tissue levels of [3H]OMI. Levels of [3H]OMI in tissue and medium were only slightly reduced, indicating possible extracellular sites of O-methylation. In the presence of U-0521, metanephrine (10(-4) M) reduced the accumulation of [3H]ISO, indicating that metanephrine was an inhibitor of the extraneuronal uptake of isoprenaline. It is concluded that two cellular compartments for O-methylation exist, access to one being dependent upon metanephrine-sensitive extraneuronal uptake. The extraneuronal uptake capacities of the tissues (when O-methylation was inhibited) was in the order papillary muscle less than lung = atria less than trachea. Cellular O-methylating capabilities, measured from tissue [3H]OMI, was in the order papillary muscle less than atria = trachea less than lung. These orders are discussed in relation to the beta-adrenoceptor mediating the response of each tissue and to the reported degree of sympathetic innervation.     

The fate of isoprenaline in the isolated rabbit aorta

Summary The fate of isoprenaline (ISO) was studied in the intact rabbit aorta, as well as in the isolated adventitia and isolated media, by means of liquid scintillation counting of ³H-ISO and ³H-O-methylisoprenaline (OMI) as well as by autoradiography of tissues incubated with 2M ISO. The 3 preparations accumulated and O-methylated ISO; O-methylation was proportionally higher in the intact vessel than in its constituents. The isolated adventitia differed from the other preparations in reaching a steady-state of cortexone-resistant accumulation after 8 min of incubation and in exhibiting an O-methylating capacity which was partly resistant to the COMT inhibitor, U-0521. The effect of cocaine gave evidence for a participation of neuronal uptake in the accumulation of the amine in the intact aorta. In the media, most of the accumulation occurred in smooth muscle cells and was reduced by cortexone and increased by U-0521; elastin and collagen, present both in the media and the adventitia, showed accumulation which was not affected by the inhibitory drugs studied.The results show that in the rabbit aorta there is a corticosteroid-resistant uptake mechanism (which predominates in the adventitia) which involves structures devoid of COMT activity. Furthermore, smooth muscle cells represent, in the media, the extraneuronal metabolizing site of loss. The adventitia is a complex layer, with different types of cells which may intervene in accumulation and metabolism of ISO. Therefore, it is concluded that the isolated media represents an acceptable model for the study of both corticosteroidsensitive and-resistant extraneuronal mechanisms, whereas the isolated adventitia is characterized by the presence of neuronal and extraneuronal mechanisms.Some of the results were presented to the 3rd Meeting on Adrenergic Mechanisms, Porto, July 12–13, 1978 (Proceedings, p. 93) and to the 1st Joint Meeting of the Spanish and Portuguese Pharmacological Societies (Santiago de Compostela, 21–23.5.79). Supported by Instituto Nacional de Investigação Científica (Centro de Farmacologia e Biopatologia Quimica-FmPl)     

The effects of thyroxine treatment of rats on neuronal and extraneuronal uptake and metabolism of catecholamines in the heart.

The effects of thyroxine (T4) treatment of rats on the neuronal and extraneuronal uptake and subsequent metabolism of catecholamines in the heart were examined, to determine whether changes in the local dissipation of catecholamines might contribute to the enhanced sympathetic cardiac responses that occur when thyroid hormone levels are elevated. T4-treated rats were injected subcutaneously with L-thyroxine sodium 1 mg kg(-1) on days 1, 3 and 5, and controls were injected with the normal saline vehicle on the same days. The experiments on isolated, perfused hearts were carried out on day 8. The T4-treated rats had only 50% of the growth rate of the controls and their heart weights were 18% greater than the controls. The experimental data were adjusted to allow for the increase in heart weight caused by the T4 treatment. The initial rates of neuronal uptake of noradrenaline and of extraneuronal uptake of noradrenaline and isoprenaline in the hearts from T4-treated rats were not significantly different from those in hearts from control rats. The steady-state rates of extraneuronal O-methylation of isoprenaline and of extraneuronal deamination of noradrenaline in hearts from T4-treated rats were not significantly different from those in hearts from control rats. The steady-state rate of neuronal deamination of noradrenaline was significantly lower and the accumulation of unmetabolized noradrenaline in the hearts was significantly greater in T4-treated rats than in the controls. These findings could be explained by a decrease in neuronal monoamine oxidase activity or by an increase in intraneuronal binding of noradrenaline in hearts from T4-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of uptake inhibitors on responses of sheep coronary arteries to catecholamines and sympathetic nerve stimulation.

1. Transmural stimulation of intrinsic sympathetic nerves and exogenous catecholamines produce beta 1-adrenoceptor mediated relaxant responses in strips of contracted sheep coronary artery. 2. The neuronal uptake inhibitors, metaraminol, cocaine and desipramine and the extraneuronal uptake inhibitor, cortisol, failed to potentiate responses to noradrenaline or sympathetic stimulation; responses to isoprenaline were enhanced by cortisol. 3. Oxytetracycline, which inhibits binding to connective tissue fibres, did not affect responses to noradrenaline or nerve stimulation. 4. 17 beta-Oestradiol, caffeine and U0521 proved to be unsuitable compounds for studying catecholamine inactivation since they non-selectively potentiated responses to noradrenaline and isoprenaline. 5. It is concluded that catecholamine inactivation processes do not modify transmitter function in sheep coronary arteries.     

Uptake and Retention of 14C-Bretylium in Degenerating Postganglionic Sympathetic Nerves of the Rat

Abstract The uptake and retention of ¹⁴C-bretylium was studied in rat salivary glands and irides in vivo at different time intervals after sympathetic denervation or decentralization. The uptake of bretylium was increased on the denervated side during a time period which precedes the degeneration release of sympathetic transmitter, but was later on reduced. On the other hand, the retention of bretylium on the denervated side was already markedly reduced during the time period preceding the onset of the degeneration transmitter release. In experiments with chronically denervated salivary glands, or glands atrophied by means of excretory duct ligation, a pronounced extraneuronal accumulation was observed. The extraneuronal accumulation of bretylium may partly mask the changes in disposition of the drug induced by the sympathetic denervation. The results are in accordance with the hypothesis that bretylium must be associated with special sites at the adrenergic nerve terminals in order to exert its degeneration delaying effect.     

The effects of nifedipine on alpha 2-adrenoceptor-mediated contractions in several isolated blood vessels from the rabbit.

1. The effects of the dihydropyridine calcium channel blocker, nifedipine, on noradrenaline-induced contractile responses have been examined in several isolated blood vessels from the rabbit, with particular emphasis on responses mediated via postjunctional alpha 2-adrenoceptors. 2. In the isolated renal vein, ear vein, distal saphenous artery, saphenous vein and plantaris vein, 0.1 microM and 1 microM nifedipine reduced responses elicited by 54 mM KCl by more than 70%. The remaining responses were abolished by alpha-adrenoceptor blockade, suggesting the involvement of noradrenaline released from neurones activating a dihydropyridine-resistant mechanism. 3. In the renal vein (alpha 1-), ear vein (predominantly alpha 2-), distal saphenous artery (alpha 1- greater than alpha 2-), saphenous vein and plantaris vein (alpha 2- greater than alpha 1-), 0.01 microM and 0.1 microM nifedipine produced concentration-related reductions in the maximum response to noradrenaline. However, 1 microM nifedipine was no more effective than 0.1 microM nifedipine and the reduction in the maximum varied from 10-25% of the control response. Thus, a sizeable component of the alpha-adrenoceptor-mediated response in all blood vessels is resistant to dihydropyridine calcium channel blockers and this appears to be unrelated to the alpha-adrenoceptor subtype involved. 4. Following irreversible inactivation of alpha 1-adrenoceptors and isolation of functional alpha 2-adrenoceptors in the saphenous vein, plantaris vein and distal saphenous artery (the latter requiring the presence of angiotensin II), the effect of nifedipine on responses to noradrenaline was increased. However, a component of the alpha 2-adrenoceptor response in each preparation was present even after the concentration of nifedipine was increased to 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further study of the adrenoceptors of the saphenous vein of the dog: Influence of factors which interfere with the concentrations of agonists at the receptor level

In the present study the affinities of some sympathomimetic amines for α- and β-adrenoceptors of the dog saphenous vein tissue were determined after all known factors interfering with the concentration of these agonists at the receptor level had been assessed and excluded. It was observed that in control experiments the relative potencies of sympathomimetic agonists for inducing contractions were: adrenaline (1.6) > noradrenaline (1.0) > phenylephrine (0.38) > isoprenaline (0.009).The elimination of neuronal uptake by cocaine, 4 × 10^?6M, enhanced predominantly the effects of noradrenaline (by a factor of 7.5), whereas block of catechol-O-methyl transferase (COMT) by U-0521, 10^?4 M, only enhanced those of adrenaline (by a factor of 2.6) and block of β-adrenoceptors by propranolol, 5 × 10^?7 M, enhanced those of isoprenaline (by a factor of 3) and adrenaline (by a factor of 1.8).Block of COMT enhanced the effects of adrenaline approximately as much as did the blockade of neuronal uptake; this seems to indicate that the affinity of adrenaline for extraneuronal and neuronal uptake processes is approximately the same.Regarding the relaxation-inducing capacity of sympathomimetic agents it was observed that isoprenaline, adrenaline and noradrenaline are full agonists, whereas phenylephrine was not able to produce relaxation amounting to more than 5% of the maximum. Denervation did not modify the relaxant effects of isoprenaline. After elimination of all known factors interfering with the concentration of the sympathomimetic agonists in the biophase, the ratios between the ED₅₀'s of each amine for α- and β-adrenoceptors were: adrenaline = 34, noradrenaline = 109 and isoprenaline = 0.0041.     

Effects of clorgyline and (−)deprenyl on the deamination of normetanephrine and noradrenaline in strips and homogenates of the canine saphenous vein

Summary The influence of specific inhibitors of MAO A (clorgyline) and MAO B [(–)deprenyl] on the metabolism of normetanephrine (NMN), in strips of canine saphenous vein was studied, both in the absence and in the presence of inhibitors of neuronal (cocaine) and extraneuronal (hydrocortisone) uptake. Moreover, the formation of metabolites of noradrenaline and of NMN by saphenous vein homogenates and the influence of clorgyline or (–)deprenyl on this formation are described.Clorgyline reduced to the same degree (by about 70%) the formation of methoxy-hydroxy-phenylglycol (MOPEG) and of vanillylmandelic acid (VMA) in strips incubated with NMN, whereas (–)deprenyl reduced by about 50% the formation of MOPEG and had no effect on VMA production. Hydrocortisone had effects very similar to those of (–)deprenyl.Saphenous vein, homogenates ()-methylation inhibited), deaminated both noradrenaline and NMN; clorgyline and (–)deprenyl reduced the formation of metabolites of both noradrenaline and NMN.It is concluded that both MAO A and B are able to deaminate noradrenaline and NMN, but that in the intact tissue the former has no access to MAO B. Even in intact tissues MAO B may play a role in the metabolism (but not in the inactivation) of noradrenaline by deaminating the NMN formed from noradrenaline and giving preferentially origin to MOPEG.Supported by Instituto Nacional de Investigação Científica (INIC, FmPl)On leave from Faculdade de Farmácia, Universidade de Coimbra, with a grant from Instituto Nacional de Investigação Científica     

Cocaine-sensitive O-methylation of noradrenaline in dental pulp of the rabbit: Comparison with the rabbit ear artery

Summary Incisor pulp from the rabbit metabolises exogenous noradrenaline in concentrations between 0.12 and 1.2 mol/l mainly to NMN.Effects of chronic sympathetic denervation indicated that in incisor pulp the NMN is extraneuronal in origin, and that DOPEG and DOMA formation, as well as a major part of the noradrenaline which accumulates in the tissue, are associated with the sympathetic nerves.NMN formation was unaffected by hydrocortisone 210 mol/l, but was strongly inhibited by cocaine 30 mol/l. These effects contrasted with those in the rabbit ear artery, where NMN formation was increased by cocaine 30 mol/l and decreased by hydrocortisone 210 mol/l.In COMT-inhibited denervated pulp, cocaine inhibited the accumulation of noradrenaline.Monoamine fluorescence histochemistry of pulp exposed to noradrenaline 50 mol/l indicated that cocaine-sensitive uptake occurred in fibroblasts.It is concluded that O-methylation of noradrenaline in dental pulp involves prior uptake of the amine by a process resembling uptake, but which is distinguished from uptake₁ by its extraneuronal location.Abbreviations DOMA 3,4-dihydroxy mandelic acid - DOPEG 3,4-dihydroxyphenylethyleneglycol - NMN normetanephrine - OMDA O-methyl deaminated metabolite fraction, comprising vanillyl-mandelic acid (VMA) plus the 3-methoxy derivative of DOPEG (MOPEG) - MAO monoamine oxidase - COMT catecholO-methyl transferase Send offprint requests to I. S. de la Lande at the above address     

Kinetic constants for uptake and metabolism of 3H-(—)noradrenaline in rabbit aorta

1. The neuronal uptake of 3H-(-)noradrenaline into aortic rings from reserpine-pretreated animals was a saturable process with a Km of 2.3 mumol X l-1 and a Vmax of 0.5 nmol X g-1 X min-1. Similar constants were obtained when the neuronal deamination of noradrenaline was taken as an index for neuronal uptake. When the tissue was incubated in the usual way, i.e., when the amine was allowed to enter the aortic rings via both the intimal and the adventitial surface, then there was no initial delay (tlag) for the neuronal uptake of noradrenaline (MAO and COMT inhibited). On the other hand, when the amine entry was restricted to the intimal surface, there was a tlag of 2 min, probably due to the slow equilibration of the extracellular space of the media with the incubation medium. Furthermore, for low amine concentrations the rate of uptake in the latter situation was about 10 times lower than that in the former one. Thus, the rate of uptake clearly depended on the way the amine entered the tissue. 2. The corticosterone-sensitive extraneural uptake of 3H-(-)noradrenaline determined in nerve-free aortic rings was characterised by a high Km (490 mumol X l-1) and Vmax (35 nmol X g-1 X min-1). For uptake2 a tlag of 1 min was observed. 3. The analysis of the extraneuronal O-methylating system in nerve-free aortic rings yielded a Km of 3.6 mumol X l-1 and a Vmax of 0.6 nmol X g-1 X min-1. The tlag for the O-methylation of noradrenaline was in the same order of magnitude as that for uptake2. At low amine concentrations the corticosterone-sensitive accumulation of noradrenaline was prevented by COMT, but not by MAO. The latter enzyme reduced the steady-state accumulation of noradrenaline by about 50%. When the amine entry was restricted to one surface only, the results indicated that the extraneuronal O-methylation of noradrenaline generated a steep concentration gradient of the parent amine within the extracellular space of the aorta. 4. All saturable processes fitted the Michaelis-Menten equation. However, kinetic constants determined in incubated organs may be falsified by poor diffusion of the substrate through the extracellular space.