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.     

Stereoselectivity of extraneuronal uptake of catecholamines in guinea-pig trachealis smooth muscle cells

The extraneuronal uptake of the (-)- and (+)-isomers of three catecholamines, isoprenaline, adrenaline and noradrenaline, were compared in guinea-pig trachealis smooth muscle cells, by a fluorescence microphotometric method. Preliminary experiments showed that the initial rates of uptake of the (-)-isomers were greater than those of the (+)-isomers in tissues incubated in 25 microM adrenaline or noradrenaline or 50 microM isoprenaline. More detailed experiments showed that the Km values of the (+)-isomers of the amines for extraneuronal uptake were not significantly different from each other, but the Km value of (-)-isoprenaline less than (-)-adrenaline less than (-)-noradrenaline. Thus, the order of the ratios of the Km values for the (+):(-)-isomers was isoprenaline greater than adrenaline greater than noradrenaline. The results showed that there is stereoselectivity of the extraneuronal uptake of catecholamines, but it is greatest for isoprenaline (4.9 fold), less for adrenaline (2.5 fold) and almost negligible (1.6 fold) for noradrenaline.     

The outward transport of catecholamines mediated by uptake2 of the rat heart

The efflux of 3H-catecholamines from the extra-neuronal tissue of the rat heart was analysed (after inhibition of vesicular and neuronal uptake, monoamine oxidase and catechol-O-methyl transferase). In most experiments, hearts were first loaded with a tracer concentration of a 3H-catecholamine and then washed out. For all four catecholamines [3H-(+/-)-isoprenaline, 3H-(+/-)-adrenaline, 3H-(-)-noradrenaline, and 3H-dopamine] the loading period resulted in virtually the same distribution pattern: most of the radioactivity distributed into "compartment III". However, the rate constants for efflux from compartment III increased in the order 3H-(-)-noradrenaline less than 3H-dopamine less than 3H-(+/-)isoprenaline = 3H-(+/-)-adrenaline. O-methyl-isoprenaline (OMI, a potent inhibitor of uptake2) caused a concentration-dependent and partial inhibition of the efflux of all 3H-catecholamines; its IC50 (half-maximal inhibition of OMI-sensitive efflux) was very close to that for half-maximal inhibition of inward transport by uptake2. It is concluded that there is not only (OMI-resistant) diffusional efflux of 3H-catecholamines, but also (OMI-sensitive) outward transport of 3H-catecholamines. The contribution by each of these processes to total efflux differed considerably from one 3H-catecholamine to the next. U-0521 (the COMT inhibitor used in this study) inhibited the OMI-sensitive efflux of 3H-noradrenaline with an IC50 of about 100 mumol/l. However, no inhibitory effect was found for 10 mumol/l U-0521. During the wash-out period (see above) various unlabelled substrates of uptake2 were added to the perfusion fluid at a concentration equalling 2 X Km.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The kinetic characteristics of the extraneuronal O-methylating system of the dog saphenous vein and the supersensitivity to catecholamines caused by its inhibition

Summary The half-saturating outside concentration and the V _max of the extraneuronal O-methylating system of the dog saphenous vein were determined in vitro for 3 catecholamines: isoprenaline, adrenaline and noradrenaline.Strips pretreated with 1 mmol/l pargyline were exposed to 30 mol/l cocaine for 30 min before and during the 30 min incubation with the amines.Two methods were used to reach our aims: a) the classical one in which the ³H-O-methylated metabolites formed from a mixture of unlabelled and labelled amine were determined by using final concentrations of the substrate ranging from 0.2 and 12.8 mol/l and b) an indirect one in which 0.2 mol/l ³H-(±)-isoprenaline was used to assess the extraneuronal O-methylation of the tracer amine, and then those concentrations of unlabelled amines were determined which reduce the O-methylation of ³H-(±)-isoprenaline by 50% (IC₅₀).The half-saturating outside concentrations and the V _max obtained by the first method were: 1.3, 2.5 and 3.4 mol/l and 241, 317 and 294 pmol/g/min for ³H-(±)-isoprenaline, ³H-(±)-adrenaline and ³H-(-)-noradrenaline, respectively. The IC₅₀s obtained by the second method used were: 1.1, 0.6, 0.7 and 1.4 mol/l for (±)-isoprenaline, (-)-isoprenaline, (-)-adrenaline and (-)-noradrenaline, respectively.It was observed that the contraction of the strips caused by adrenaline and noradrenaline distorted IC₅₀ values. In the presence of 1 mol/l phentolamine the IC₅₀ for adrenaline and noradrenaline was about 2.5 times higher than in its absence.The relationship between the ratio of half-saturating outside concentration/ED₅₀ for -adrenoceptor-mediated responses for the three amines and the supersensitivity to them caused by inhibition of COMT was also assessed. It was observed that the ratio was 37, 8 and 1.3 for isoprenaline, adrenaline and noradrenaline, respectively and the supersensitivity caused by inhibition of COMT was 11.8-, 8.9- and 1.8-fold, respectively.     

Dopamine and adrenaline,but not isoprenaline,are substrates for uptake and metabolism in isolated perfused lungs of rats

Summary The uptake and subsequent metabolism by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) of dopamine, adrenaline, isoprenaline and noradrenaline in isolated perfused lungs of rats has been examined. In lung preparations in which COMT and MAO were inhibited, the uptake of ³H-labelled dopamine, (–)-adrenaline and (–)-noradrenaline, but not (±)-isoprenaline, was reduced by cocaine (10 or 100 mol/l) The rank order of the Km values of the amines that were substrates for uptake in the lungs were: dopamine (0.246 mol/l) < noradrenaline (0.967 mol/l) < adrenaline (3.32 mol/l). These results are consistent with transport of catecholamines in rat lungs by Uptake₁.In lung preparations with COMT and MAO intact, dopamine and noradrenaline were removed from the circulation (50% and 32%, respectively) and mainly metabolized. There was very little (3.0%) removal of isoprenaline by the lungs and adrenaline was not included in this part of the study. In lung preparations in which only MAO was inhibited, the rank order of COMT activity for O-methylation of the amines was dopamine noradrenaline adrenaline (k_COMT values: 4.98 min^–1, 0.357 min^–1, and 0.234 min^–1, respectively).If dopamine or adrenaline are perfused through the pulmonary circulation in isolated lungs of the rat, they are taken up and then metabolized by COMT and MAO, as also occurs for noradrenaline. Isoprenaline is not a substrate for uptake in the lungs. There was less uptake of adrenaline than noradrenaline, indicating that uptake and metabolism in the lungs may not be a significant removal process for adrenaline in the circulation of rats in vivo. The more marked uptake of dopamine (than of noradrenaline) indicates that uptake and metabolism by the lungs, at least in the rat, may play an important role in the removal of dopamine from the circulation in vivo.Abbreviations COMT catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPAC 3,4-dihydroxyphenylacetic acid - DOPEG 3,4-dihydroxyphenylglycol - DOPET 3,4-dihydroxyphenyl ethanol - MAO monoamine oxidase - MN metanephrine - MTA 3-methoxytyramine - NMN normetanephrine - OMDA O-methylated deaminated metabolites - OMI 3-O-methylisoprenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone Some of the results of this study were presented to the Australasian Society of Clinical and Experimental Pharmacologists (Bryan and O'Donnell 1987, 1988; Bryan et al. 1989; Bryan-Lluka 1990) Send offprint requests to L.J. Bryan-Lluka at the above address     

The handling of five amines by the extraneuronal deaminating system of the rat heart

Summary The handling of five amines by the extraneuronal deaminating system was studied in perfused hearts of rats (pretreated with reserpine; COMT and neuronal uptake inhibited). Hearts were perfused with 50 nmol/l ³H-noradrenaline for 30 min, in the presence of increasing concentrations of unlabelled (–)-adrenaline, (–)-noradrenaline, dopamine, tyramine and 5-HT. IC₅₀'s were determined as those concentrations of unlabelled amines which halved the steady-state rate of deamination of ³H-noradrenaline. After correction for changes in the tissue/medium ratio for ³H-noradrenaline, half-saturating outside concentrations were obtained. They increased in the order (–)-adrenaline (15 mol/l) — tyramine — dopamine — noradrenaline —5-HT (53 mol/l). The V _max for extraneuronal deamination was determined for ³H-(–)-adrenaline, ³H-(–)-noradrenaline and ³H-dopamine, as well as (by HPLC and electrochemical detection) for tyramine and 5-HT. It was low for (–)-adrenaline, intermediate for (–)-noradrenaline, dopamine and 5-HT, high for tyramine. For the three catecholamines the half-saturating outside concentrations of the extraneuronal deaminating system clearly exceeded those for the extraneuronal O-methylating system of the same organ (see Grohmann and Trendelenburg 1985), although the two enzymes appear to co-exist in the same cells, so that the same transport system is involved.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg     

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.     

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)     

Evidence for saturation of catechol-0-methyltransferase by low concentrations of noradrenaline in perfused lungs of rats

Previous studies on the pulmonary removal and metabolism of catecholamines in rat lungs have shown that, when the lungs are perfused with a low concentration (1 nmol/1) of noradrenaline, the amine is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), but is predominantly O-methylated, and the activities of COMT and MAO are 0.357 min^–1 and 0.186 min^–1, respectively. The aim of the present study was to examine the changes in the metabolic profile of noradrenaline in rat lungs over a range of concentrations, and to examine the kinetics of the pulmonary O-methylation of noradrenaline and adrenaline.In isolated lungs perfused with ³H-noradrenaline, there was a progressive decrease in the proportion of O-methylated metabolites and a corresponding increase in the proportion of deaminated metabolites, as the noradrenaline concentration in the perfusion solution was increased from 1 to 10 to 100 to 1000 nmol/l. Experiments designed to determine the rate of uptake of noradrenaline in lungs perfused with 1 nmol/l ³H-noradrenaline, under conditions of MAO inhibited, COMT inhibited and COMT and MAO inhibited, showed that the results were compatible with co-existence of COMT and MAO in the pulmonary endothelial cells. Hence, it appeared that the changing metabolic profile with amine concentration in the previous series of experiments was not due to saturation of noradrenaline uptake into cells that contained COMT but not MAO.Further experiments to examine the kinetics of O-methylation of noradrenaline and adrenaline (MAO inhibited) showed that the O-methylation of these amines in the lungs was predominantly saturable, with half-saturation occurring at concentrations (9.8 nmol/I and 19.4 nmol/l, respectively) that were two orders of magnitude lower than those required to half-saturate uptake₁ of the amines. Saturation of O-methylation by these low concentrations of noradrenaline (1) provides the explanation for the change in the metabolic profile of noradrenaline described above and (ii) appears to occur because V_{max uptake} V_{max COMT} for the metabolizing system consisting of non-neuronal uptake₁ + COMT in the lungs, as has been described previously for the system consisting of uptake₂ + COMT in extraneuronal sites in rat heart. The results show that the metabolic profile of catecholamines in the pulmonary circulation will reflect that occurring at physiological levels only if studies are carried out with very low amine concentrations.Abbreviations COMT Catechol-O-methyltransferase - DOMA 3,4-dihydroxymandelic acid - DOPEG 3 4-dihydroxyphenylglycol - ECS Extracellular space - HSOC Half-saturating outside concentration - K_{m uptake} Half-saturation constant for uptake - k_COMT Rate constant for O-methylation - k_MAO Rate constant for deamination - k_{out NA} Rate constant for efflux of noradrenaline - MAO Monoamine oxidase - MB-COMT Membrane-bound - COMT NMN Normetanephrine - OMDA O-methylated deaminated metabolites - S-COMT Soluble COMT - T/M_NA Tissue to medium ratio of noradrenaline - U-0521 3,4-dihydroxy-2-methylpropiophenone - V_max Maximal rate of uptake or O-methylation - V_st-st Steady-state rate of metabolite formation - V_uptake Rate of uptake Preliminary results of part of this study were presented to the Seventh Meeting on Adrenergic Mechanisms, Porto, Portugal (Bryan 1990)     

Errors introduced by a tritium label in position 8 of catecholamines

The neuronal and extraneuronal disposition of 3H-7,8- and 3H-7-labelled (-)-noradrenaline and dopamine was compared in in vitro studies. In agreement with earlier studies, the present results show that the presence of a tritium label in position 8 (i.e., on the alpha-carbon) has two consequences: a) the rate of deamination declines and b) part of the deamination results in the formation of an unlabelled aldehyde plus tritium water; tritium water is recovered from the OMDA-fraction of the column chromatographic procedure of Graefe et al. (1973). Whenever the deamination of a 3H-catecholamine is reduced (by tritium in position 8), the intraneuronal 3H-catecholamine concentration is increased. This increase, in turn, partly masks the decline in neuronal deamination (rat vas deferens). Irrespective of whether one determines the spontaneous efflux, the release of 3H-noradrenaline by nerve stimulation or the release of 3H-(-)-noradrenaline by the reserpine-like compound Ro 4-1284, the presence of tritium in position 8 distorts the results (experiments with rat vasa deferentia and/or rabbit aorta). In the extraneuronal system of the rat heart, two intracellular enzymes inactivate 3H-(-)-noradrenaline and 3H-dopamine: catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO). Any hindrance of deamination (by tritium in position 8, COMT intact) leads to a shift of the metabolism of the 3H-catecholamines from the exclusively deaminated to the exclusively O-methylated metabolites. No differences between 3H-7,8- and 3H-7-labelled catecholamines were found after inhibition of MAO and COMT (extraneuronal accumulation and rate constant for efflux from the extraneuronal compartment III of the rat heart).(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Structural and functional alterations caused at the extraneuronal level by sympathetic denervation of blood vessels

Summary The lateral saphenous vein of the dog and the rabbit ear artery were surgically denervated, by clamping the vessel or by removal of the superior cervical ganglion, respectively. Both procedures resulted in denervation of the vessels.The denervated, lateral saphenous vein was supersensitive to exogenous noradrenaline and inactivation of the amine (in oil immersion experiments) was slower in denervated vein strips than in control strips treated with cocaine. Incubation experiments with ³H-noradrenaline confirmed that denervated strips formed considerably fewer metabolites than control ones (in the absence or presence of cocaine) and that O-methylation of noradrenaline was reduced by about 50%. When the strips were incubated with ³H-isoprenaline, the denervated ones accumulated and metabolized isoprenaline to a lesser degree than control strips. Hydrocortisone did not reduce the accumulation of isoprenaline in the denervated vein and had only minor effects on O-methylation. The metabolism of noradrenaline and isoprenaline gradually recovered with time.In the ear artery, denervation was accompanied by a marked reduction in O-methylation, but not in accumulation, of isoprenaline. In both vessels there was a highly significant positive correlation between noradrenaline content and O-methylating capacity; in the saphenous vein accumulation of isoprenaline was also positively correlated to noradrenaline content.Morphological changes observed in the denervated vessels consisted essentially in dedifferentiation of smooth muscle cells (which attained larger dimensions, had an indented, large nucleus, augmented euchromatin and an increased amount of ribosomes), abundance of extracellular material and fibroblasts. Mast cells were present in denervated veins (but not in controls) and the histamine content was increased in the former. Structural alterations were homogeneously distributed in the saphenous vein but restricted to the adventitio-medial area in the rabbit ear artery. Depletion of endogenous noradrenaline by reserpine pretreatment did not cause the alterations seen after denervation. On the other hand, continuous intravenous infusion of noradrenaline during 5 days did not prevent, and even worsened, the alterations caused by denervation. It was concluded that noradrenaline does not appear to be the factor the lack of which is exclusively responsible for the impairment of the extraneuronal system, in the denervated tissue.Taken together, the data show that the sympathetic innervation of blood vessels exerts a regulatory function on extraneuronal events; the disappearance of innervation results in marked impairment of the corticosteroid-sensitive O-methylating system and in morphological changes of both smooth muscle cells and fibroblasts.Part of the results were presented at 3rd Joint Meeting of the French and Spanish Pharmacological Societies in Toulouse, 1981 (Branco et al. 1982)     

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.     

Differences between the neuronal handling of 3H-7- and 3H-7,8-(−)noradrenaline: implications for the release of the labelled neurotransmitter by nerve stimulation

Neighbouring rabbit aortic strips were exposed to a tracer concentration of 3H-7- or 3H-7,8-(-)noradrenaline (bearing 30-35% of its label in position 8) and to 0.5 mumol/l unlabelled (-)noradrenaline for 60 min and then washed in amine-free Krebs solution. Catechol-O-methyl transferase and extraneuronal amine uptake were inhibited throughout. After 114 min of wash-out, the tissue contained less tritium when loaded with 3H-7-(-)noradrenaline than when loaded with 3H-7,8-(-)noradrenaline, and the fractional rate of loss of tritium was greater for the former than for the latter tissues. In the presence of cocaine (to prevent neuronal re-uptake), the same percentage of tissue tritium was released by nerve stimulation (six consecutive periods of stimulation at 1 Hz for 5 min each) in spite of the above difference between tissue tritium levels of the two differently labelled amines. In the absence of cocaine, a higher percentage of tissue tritium was released by nerve stimulation (1 or 3 Hz, 5 min each) for 3H-7- than 3H-7,8-(-)noradrenaline. Unchanged 3H-(-)noradrenaline amounted to 35% of tritium in the stimulation-evoked overflow for 3H-7- and to 50% for 3H-7,8-(-)noradrenaline (frequency of stimulation, 1 Hz). When monoamine oxidase (MAO) was inhibited, no differences were observed between the neuronal handling of 3H-7- and 3H-7,8-(-)noradrenaline, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The uptake and O-methylation of 3H-(±)-isoprenaline in rat cerebral cortex slices

Summary The O-methylation and accumulation of ³H-isoprenaline in slices of the rat cerebral cortex were studied before and after inhibition of COMT. 1. Inhibition of COMT by mol/l U-0521 virtually abolished the O-methylation and increased the accumulation of ³H-isoprenaline; hence, there is evidence for the existence of a central O-methylating system (with a transport mechanism and intracellular COMT). 2. Experiments were carried out with selective uptake inhibitors for uptake, (cocaine and desipramine) or uptake2 (corticosterone and OMI), with phenoxybenzamine (known to inhibit both carriers) and with changes in the ionic composition of the incubation medium. They revealed that the central carrier differed from both, uptake, and uptake2, although exhibiting some resemblance with uptake₂ (lack of dependence on Na⁺ and Cl^–, sensitivity to K⁺ and phenoxybenzamine, ability to transport ³H-isoprenaline). 3. Although the central carrier was rather sensitive to inhibition by beta-adrenoceptor antagonists (propranolol, carteolol), the effect of propranolol was not stereoselective; hence, beta-adrenoceptors do not seem to be involved. 4. Virtually identical IC₃₀-values were obtained for inhibitors, when determined with or without inhibition of COMT. Only OMI was found to inhibit COMT as well as the central transport system; hence it was more potent in inhibiting the O-methylation than the accumulation of ³H-isoprenaline. 5. IC₅₀-values (against initial rates of accumulation of ³H-isoprenaline; COMT inhibited) were determined for various substrates and inhibitors of peripheral uptake₂. There was no correlation with the IC₅₀-values determined earlier for uptake2 in rat heart (Grohmann and Trendelenburg 1984). 6. Unlabelled catecholamines half saturated the intracellular COMT when slices were incubated with 0.22 mol/l [(±)-dobutamine] to 4.9 mol/l [(–)-noradrenaline]. As the presence of unlabelled catecholamines increased tissue levels of ³H-isoprenaline, catecholamines are substrates of the central carrier. 7. The carrier of the central O-methylating system differs from uptake₂ of peripheral organs, although it resembles the peripheral carrier in some respects.Abbreviations COMT catechol-O-methyl transferase - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OMI 3-Omethyl-isoprenaline Supported by the Deutsche Forschungsgemeinschaft (Tr 96 and SFB 176) and by a scholarship of the Royal Society for V. G. Wilson. Some of the results were presented to the British Pharmacological Society (Trendelenburg and Wilson 1986)     

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.     

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.     

Lack of differences between the neuronal and extraneuronal handling of 3H-7- and 3H-ring-2,5,6-(−)noradrenaline

The neuronal disposition of the side chain-labelled 3H-7-(-)noradrenaline and the ring-labelled 3H-2,5,6-(-)noradrenaline of relatively high specific activity was compared in the (incubated) rabbit aorta, the extraneuronal one in the (perfused) rat heart. Furthermore, the metabolism by monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) of the labelled amines was studied in rat heart homogenates. In addition, some of the experiments were also carried out with the side chain-labelled 3H-7,8-(-)noradrenaline that is an inferior substrate of MAO. 1. In agreement with several other studies, the present results revealed the known differences between the two side chain-labelled amines brought about by the poorer deamination of 3H-7,8-(-)noradrenaline. This finding also indicated that the 3H-7-(-)noradrenaline used in this study was of the desired quality, i.e. was labelled exclusively in position 7. 2. In contrast to earlier findings, no differences between the side chain-labelled 3H-7-(-)noradrenaline and the ring-labelled 3H-2,5,6-(-)noradrenaline were observed with regard to neuronal and extraneuronal amine handling (intact tissues) and metabolism by MAO and COMT in rat heart homogenates. Hence, there is no reason to avoid the ring-labelled amine with high specific activity and, consequently, with slightly more than one tritium substituent per catecholamine molecule in experiments designed to analyse the neuronal and extraneuronal fate of (-)noradrenaline.     

Kinetics of the O-methylating system for isoprenaline in the trachea and aorta of rabbit

Segments of tracheal smooth muscle or aorta from rabbits pretreated with reserpine (1 mg/kg) were incubated in 3H-isoprenaline (0.5-60 mumol/l). Steady-state rates of O-methylation were determined by measuring the formation of 3-O-methylisoprenaline (OMI) after incubation of tracheal and aortic tissues for 30 min and 10 min, respectively. The steady-state O-methylation of isoprenaline in rabbit trachea was saturable, at least up to 60 mumol/l isoprenaline. In rabbit aorta, the O-methylation appeared to be saturable up to 30 mumol/l isoprenaline, but the rate of O-methylation increased for higher concentrations. The Km values for the saturable component of O-methylation were 11.8 mumol/l in trachea and 3.03 mumol/l in aorta. The Vmax values were 0.51 nmol X g-1 X min-1 in trachea and 0.56 nmol X g-1 X min-1 in aorta. In tissues incubated in 0.5 mumol/l isoprenaline, 100 mumol/l corticosterone caused 78% inhibition of OMI formation in trachea and 86% inhibition in aorta. There was no inhibition of OMI formation by 100 mumol/l corticosterone in tracheal or aortic tissues incubated in 60 mumol/l isoprenaline. Model calculations showed that the experimental results in trachea and aorta (3. above) were consistent with (a) entry of isoprenaline into the cells in the tissues by extraneuronal uptake and diffusion, and (b) exposure of the isoprenaline to intracellular catechol-O-methyltransferase with Vmax enzyme much less than Vmax uptake.