Metabolism of endogenous and exogenous noradrenaline in the rabbit perfused heart

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from rabbit perfused hearts was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs and without nerve stimulation, the outflow of endogenous noradrenaline over a period of 108 min averaged 0.17 pmol×g^–1×min^–1 and the outflow of DOPEG 2.1 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.13 pmol×g^–1×min^–1). The effect of perfusion with (–)-noradrenaline 0.1, 1 or 10 mol/l for 18 min was then investigated. As the concentration of noradrenaline increased so did the outflow of DOPEG. Moreover, DOMA was found in the venous effluent during and after perfusion with noradrenaline 1 or 10 mol/l. The increase in the outflow of DOPEG and DOMA was almost abolished when cocaine 10 mol/l was present during the perfusion with noradrenaline 1 mol/l. The release of endogenous noradrenaline by sympathetic nerve stimulation or tyramine 10 mol/l, but not the release evoked by nicotine 30 mol/l, was accompanied by an increase in the outflow of DOPEG; an outflow of DOMA was not observed.It is concluded that, in the rabbit perfused heart, DOPEG is an important metabolite of endogenous noradrenaline. DOMA is at best a minor product, either when the neurones are at rest or when noradrenaline is released by sympathetic nerve stimulation, nicotine or tyramine. DOMA is formed in detectable amounts when the tissue is exposed to a high concentration of exogenous noradrenaline. Like DOPEG, it is formed intraneuronally. The results confirm and extend those obtained previously on guinea-pig incubated atria. They make it unlikely that, in these tissues at least, DOMA formation is one of the physiological pathways of noradrenaline catabolism.     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

Summary The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3mol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min^–1 (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min^– × 103) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.Members of Consejo Nacional de Investigaciones Científicas - Técnicas (CONICET), Argentina Send offprint requests to M. C. Camilión de Hurtado 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     

Selective inhibition by amezinium of intraneuronal monoamine oxidase

Summary Intra- and extraneuronal compartments of rat hearts were selectively labelled by perfusion with ³H-noradrenaline in the presence of corticosterone 87 M or cocaine 30 M, respectively. The subsequent outflow of ³H-compounds was examined. As little as 1 nM amezinium diminished the outflow of intraneuronally formed ³H-DOPEG. This effect was antagonized by cocaine. Amezinium 1 M was necessary to diminish the outflow of extraneuronally formed ³H-DOPEG. This effect was not counteracted by corticosterone. The results indicate that amezinium is both a potent and, at low concentrations, selective inhibitor of intraneuronal MAO.Abbreviations used DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - MAO monoamine oxidase (EC 1.4.3.4) - NA noradrenaline - NMN normetanephrine - OMDA O-methylated deaminated metabolites     

The influence of inhibition of catechol-O-methyl transferase or of monoamine oxidase on the extraneuronal metabolism of 3H-(−)-noradrenaline in the rat heart

Summary The extraneuronal metabolism of ³H-(–)-noradrenaline (1 nmol/l) was determined in rat hearts obtained from reserpine-pretreated animals (in the presence of 30 mol/l cocaine).Inhibition of monoamine oxidase (MAO) (by pretreatment of the animals with pargyline) increased the formation of O-methylated metabolites by nearly that amount by which the formation of deaminated metabolites declined; hence, catechol-O-methyl transferase (COMT) seemed to be able to nearly fully compensate for the loss of MAO activity. However, when COMT was inhibited (by the presence of either 1 or 10 mol/l U-O521), the increase in the formation of deaminated metabolites was smaller than the decrease in the formation of O-methylated metabolites; hence, MAO seemed to be unable to fully compensate for the loss of COMT activity.These results are discussed with regard to the hypothesis that the two extraneuronal enzymes co-exist in one compartment. As inhibition of COMT causes a much greater increase in the steady-state tissue/medium ratio for ³H-(–)-noradrenaline than does inhibition of MAO, it is suggested that it is this increase in the intracellular concentration of ³H-(–)-noradrenaline which-by promoting an efflux of the unchanged amine that is proportional to the tissue/medium ratio-actually decreases the net removal of ³H-(–)-noradrenaline from the perfusion fluid.The results are compatible with (but no evidence for) the hypothesis that the two enzymes co-exist in the same extraneuronal compartment.The following abbreviations are used here NMN normetanephrine - DOPEG dihydroxyphenylglycol - DOMA dihydroxymandelic acid - MOPEG methoxyhydroxyphenylglycol - VMA methoxyhydroxymandelic acid - OMDA MOPEG+VMA Supported by the Deutsche Forschungsgemeinschaft     

Effects of the noradrenaline metabolites on tyrosine hydroxylase activity in guinea-pig atria

Summary The effects of noradrenaline, its five metabolites and metanephrine, were studied on tyrosine hydroxylase activity in guinea-pig atria. The deaminated metabolite, (±)-3,4-dihydroxyphenylglycol (DOPEG), was equipotent with (±)-noradrenaline in its inhibitory action on tyrosine hydroxylase activity in the homogenates of guinea-pig atria. The inhibition by DOPEG was competitive with the cofactor, reduced pteridine. The deaminated acid, 3,4-dihydroxymandelic acid (DOMA) and the O-methylated deaminated acid, 3-methoxy, 4-hydroxymandelic acid (VMA) had 1/50th and 1/30th, respectively, the potency of noradrenaline in inhibiting tyrosine hydroxylase. The rest of the metabolites did not inhibit tyrosine hydroxylase in homogenates in concentrations up to 1.0 mM. In intact guinea-pig atria noradrenaline was considerably more potent than DOPEG in inhibiting tyrosine hydroxylase. Normetanephrine 1.4×10^–4 M inhibited tyrosine hydroxylase in the intact tissue but failed to inhibited the enzyme in the homogenate even in higher concentrations. The effect of normetanephrine in the intact tissue is related to the ability of this compound to release endogenous noradrenaline.A reserpine-like agent, Ro 4-1284, did not inhibit tyrosine hydroxylase activity in the homogenate but in the intact tissue the inhibition was more than 50%. This effect of Ro 4-1284 in the intact tissue appears to be related to the releasing effects of this agent and to an increase in the axoplasmic levels of DOPEG.Since the formation of the deaminated glycol, DOPEG, represents the main metabolic pathway for the neurotransmitter in adrenergic nerve endings, the present results are compatible with the view that, in addition to the pool of extravesicular noradrenaline, the cytoplasmic concentration of DOPEG could also participate in the regulation of the activity of tyrosine hydroxylase.     

Stereoselectivity of noradrenaline uptake into synaptic vesicles of the rat brain

Summary To investigate the stereoselectivity of the ATP-Mg²⁺-dependent uptake of noradrenaline, synaptic vesicles were isolated from the rat brain by differential centrifugation and incubated with ³H-(±)-, ³H-(–)- or ¹⁴C-(+)-noradrenaline in the absence and in the presence of ATP-Mg²⁺. The K _m values of the ATP-Mg²⁺-dependent uptake were found to be different for the two isomers (mol/l): ³H(±)-noradrenaline 14.9 ± 2.2 × 10^–1, ³H-(–)-noradrenaline 7.7 ± 0.5 × 10^–1, ¹⁴C-(+)-noradrenaline 17.3 ± 3.7 × 10^–1, whereas the V _maX of the racemate was identical with those of the two isomers (pmol/mg protein/min): ³H-(±)-noradrenaline 5.5 ± 0.4, ³H-(–)-noradrenaline 4.9 + 0.1, ¹⁴C-(+)-noradrenaline 5.1 ± 0.4. Moreover, (+)-noradrenaline inhibited competitively the ATP-Mg²⁺-dependent uptake of ³H-(±)-noradrenaline (K_i 19.2 + 1.0 × 10^–1 mol/l) and ³H-(–)-noradrenaline (Ki 17.7 ± 1.8 × 10^–1 mol/l), the K_i values being nearly identical with the K _m of the ATP-Mg²⁺-dependent uptake of ¹⁴C-(+)-noradrenaline. It is concluded that the ATP-Mg²⁺-dependent uptake of noradrenaline into synaptic vesicles of the rat brain is stereoselective and that both isomers share the same transport system.The experiments were carried out at the Institut für Pharmakologie and Toxikologie der Universitat Würzburg, FRG. This work was supported by the Deutsche Forschungsgemeinschaft Send offprint requests to A. Philippu at the above address     

Plasma 3,4-dihydroxyphenylglycol as a tool to assess the role of neuronal uptake in the anaesthetized rabbit

Summary (1.) The purpose of this study was to investigate the role of neuronal uptake in the appearance in plasma of the primary noradrenaline metabolite 3,4-dihydroxyphenylglycol (DOPEG). To this end, steady-state changes in mixed central-venous plasma concentrations of noradrenaline and DOPEG produced by noradrenaline infusions or by changes in sympathetic tone were determined in anaesthetized rabbits either under control conditions or after treatment with desipramine (2 mg kg^–1). The steady-state kinetics of infused DOPEG were also evaluated. (2.) Infused DOPEG (2.9 nmol kg^–1 min^–1 i.v. for 75 min) reached steady-state concentrations in plasma within less than 30 min, disappeared from plasma with a half-life of 2.3 min and showed a total-body plasma clearance of 84.0 ml kg^–1 min^–1 (3.) Constant-rate infusions of noradrenaline (1.2–5.9 nmol kg^–1). (min^–1 i.v. for 75 min) produced increases in plasma noradrenaline and DOPEG concentrations which were linearly related to the rate of noradrenaline infusion. Thus, the plasma clearance of infused noradrenaline (75.8 ml kg^–1). min^–1 as well as the increase in plasma DOPEG expressed in % of that in plasma noradrenaline (9.4%) was virtually independent of the noradrenaline infusion rate. (4.) Desipramine reduced the plasma clearance of infused noradrenaline by 35.4% and the increment in plasma DOPEG relative to that in plasma noradrenaline by 75.3%. From these results and the plasma clearance of noradrenaline and DOPEG it was calculated that the rate at which presynaptically formed DOPEG appeared in plasma amounted to 7.9% of the rate of total noradrenaline removal and to 22.3% of the rate of neuronal uptake. (5.) The rate of appearance in plasma of DOPEG originating from the neuronal re-uptake of endogenous noradrenaline was 192.3 pmol (kg^–1). min^–1 suggesting that the rate of neuronal re-uptake amounted to 862.3 pmol (kg^–1) min^–1 (6.) The slope of the regression line relating plasma DOPEG to plasma noradrenaline concentrations under conditions of noradrenaline release exceeded that of the corresponding regression line observed during noradrenaline infusion by a factor of about 10. This difference in slope suggests that, in the absence of infused noradrenaline, the average noradrenaline concentration at all noradrenergic neuroeffector junctions of the rabbit is 3.2 times as high as that in plasma.This study was supported by the Deutsche Forschungsgemeinscbaft (Gr 490/5). A preliminary account of the present results was presented to the German Pharmacological Society (Halbragge and Wölfel 1989) Send offprint requests to T. Halbragge at the above address     

Differences in the metabolic fate of noradrenaline released by electrical stimulation or by tyramine

Summary Strips of canine saphenous vein were loaded with ³H-noradrenaline (1.4 M) and perifused with Krebs solution and either subjected to field stimulation or exposed to tyramine 40 M. ³H, noradrenaline and its metabolites were determined in the perifusion fluid. Stimulation caused an increase predominantly in noradrenaline, followed by DOPEG, whereas tyramine released DOPEG in larger amounts than noradrenaline. Tyramine had more sustained effects than stimulation. Cocaine (1.6 M) drastically reduced DOPEG efflux due to stimulation, but had no effects on the pattern of release by tyramine. It is concluded that tyramine releases noradrenaline which is deaminated before it reaches the synaptic gap, whereas after stimulation deamination of the transmitter occurs after re-uptake.Results presented in part to the 8th Annual Meeting of the Portuguese Pharmacological Society (Coimbra, December 1977) and to the 3rd International Symposium on Vascular Neuroeffector Mechanisms (Louvain-Antwerpen, July 24–26, 1978). This work was supported by a grant from Instituto Nacional de Investigação Científica (FmPl)     

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     

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     

The aziridinium derivative of DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) accelerates the beating rate of isolated rat atria by enhancing the spontaneous release of noradrenaline

Summary The aziridinium derivative of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (az-DSP4) depletes endogenous noradrenaline stores and exerts neurotoxic actions on noradrenergic neurons. These effects are persistent in the central nervous system and transient in the periphery. To determine if transmitter release plays a role in the noradrenaline depletion caused by az-DSP4, the action of the compound was studied in isolated and spontaneously beating rat atria. 1. az-DSP4 enhanced atrial beating rate when present in the incubation medium at concentrations ranging from 10^–3 M to 10^–4 M but at 10^–3 s M decreased that rate below basal levels. 2. Preincubation of atria for 30 min with the noradrenaline uptake blocker desimipramine (DMI, 10^–6 M) or with the betablocker propranolol (10^–7 M), abolished the positive chronotropic action of az-DSP4. 3. The rate-accelerating effect of az-DSP4 could be prevented by pretreating the rats with reserpine (5 mg/kg i. p. 24 h) or enhanced by pargyline pretreatment (100 mg/kg i. p. 18 h). 4. az-DSP4 stimulated the spontaneous efflux of tritium from the isolated atria previously labeled with ³H-noradrenaline (4 × 10^–7 M), an increase that was mainly accounted for by DOPEG. 5. COMT and MAO activities in atria homogenates were inhibited by az-DSP4 in a concentration-dependent manner. However, MAO inhibition did not result in a change of the metabolic pattern as could be expected. 6. The results obtained indicate that az-DSP4 enhances the rate of spontaneous beating of isolated rat atria. The positive chronotropic effect of az-DSP4 requires the interaction of the compound with the noradrenaline uptake system. The mechanism of the accelerating effect of az-DSP4 most probably involves the release of noradrenaline from adrenergic nerve terminals in the atria and its subsequent interaction with adrenergic beta-receptors.Abbreviations DSP4 N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride - az-DSP4 aziridinium derivative of DSP4 - NA noradrenaline - 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) - COMT catechol-O-methyltransferase - MAO monoamineoxidase Send offprint requests to M. E. Landa     

The outward transport of axoplasmic noradrenaline induced by a rise of the sodium concentration in the adrenergic nerve endings of the rat vas deferens

Summary The adrenergic nerve endings of the rat vas deferens were loaded with ³H-(–)-noradrenaline; COMT was inhibited by the presence of 10 mol/l U-0521, and all experiments were carried out with calcium-free solution. After 100 min of wash-out a neuronal efflux of tritium was obtained which remained constant with time (when expressed as fractional rate of loss; FRL); it contained more DOPEG than noradrenaline.The in vitro administration of reserpine-like drugs (reserpine and Ro 4-1284) increased the FRL of tritium, presumably because of an increase in the leakage of noradrenaline from storage vesicles; the efflux of DOPEG increased more than that of noradrenaline, and the ratio NA/DOPEG declined.Inhibition of the membrane ATPase (by omission of potassium from the medium or by the presence of 3 mmol/l ouabain) increased the FRL of tritium, presumably because of an increase in the net leakage of noradrenaline from the storage vesicles (as a consequence of the fall in the concentration of free axoplasmic noradrenaline; see below).Veratridine also increased the FRL of tritium, partly because of its known reserpine-like effect (Bönisch et al. 1983); in the presence of 1 mol/l veratridine, the efflux of DOPEG increased.Irrespective of the presence or absence of reserpine or Ro 4-1284, inhibition of the membrane ATPase or the presence of veratridine (agents or procedures which increase the axoplasmic sodium concentration) always resulted in a brisk increase of the efflux of noradrenaline that was accompanied by a simultaneous decrease in the efflux of DOPEG (see above for one exception). In all experiments the rise in internal sodium caused the ratio NA/DOPEG to increase.These results indicate that—as long as the sodium gradient is normal—the axonal membrane functions as a barrier that largely prevents any outward movement of axoplasmic noradrenaline. Consequently, the axoplasmic amine is largely deaminated, and the ratio NA/DOPEG is low. However, when the axoplasmic sodium concentration rises, axoplasmic noradrenaline is transported out of the nerve ending at such high rates that the axoplasmic noradrenaline concentration falls; the fall in the efflux of DOPEG is indicative of a fall in the intraneuronal formation of DOPEG. The results show that changes in the efflux of DOPEG (i.e., of a highly lipophilic metabolite that easily leaves adrenergic nerve endings) can serve as an index of changes in axoplasmic noradrenaline levels.Supported by the Deutsche Forschungsgemeinschaft (Tr.96)     

Opioid receptor agonists activate pertussis toxin-sensitive G proteins and inhibit adenylyl cyclase in canine cardiac sarcolemma

Although both opioid receptors and endogenous opioids are abundant in cardiac tissues, the signal transduction pathways of opioids in cardiac sarcolemmal membranes have yet to be identified. In highly purified canine cardiac sarcolemmal membranes, binding of the opioid receptor antagonist [³H]diprenorphine and effects of , and agonists on low K_m GTPase and adenylyl cyclase were measured. Equilibrium binding of [³H]diprenorphine revealed a maximal binding capacity of 7.2 pmol/mg protein and a K_d of 1.3 nmol/1. In the presence of GTP, (D-Pen^2,5, p-Cl-Phe⁴)enkephalin and (D-Arg⁶)dynorphin A 1-13 fragment both inhibited adenylyl cyclase by 20–25% (from 206 ± 30to164 ± 28 pmol·min^{– 1}·mgprotein^–1, EC₅₀6 mol/Landfrom254 ± 109to204 ± 90 pmol·min^{– 1}·mg protein^–, EC₅₀ 8 pmol/L, respectively; P<0.001). Both substances stimulated low Km GTPase by 20%and13%,respectively(from12.7 ± 3.0 to 15.2 ± 3.7 pmol·min^–1.mgprotein^–1,EC₅₀ 12 mol/L, P<0.01, and from 9.1 ± 2.8 to 10.4 ± 3.2 pmol-min^{– 1}·mg protein^–1, EC₅₀ 6 mol/L, P<0.05, respectively). These effects were blocked by the opioid receptor antagonist naltrexone and by pretreatment of sarcolemmal membranes with pertussis toxin. The opioid receptor agonists (D-AIa², Me Phe⁴, Gly-[ol]⁵)enkephalin and morphiceptin had no effect on either cardiac adenylyl cyclase or low Km GTPase activities. These data suggest that in cardiac sarcolemma, opioid receptors are coupled to pertussis toxin sensitive G proteins and mediate inhibition of adenylyl cyclase activity.     

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     

Cultured chick sympathetic neurons: prostanoid EP1 receptor-mediated facilitation of noradrenaline release

Prostanoid EP receptor-mediated modulation of noradrenaline release from cultured chick sympathetic neurons was investigated. Transmitter release from dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline, was elicited either by electrical field stimulation (36 pulses/3 Hz) or by elevating the extracellular concentration of K⁺ (to 30 mM; for 2 min).Prostaglandin E₂ (PGE₂; 0.01–3 M) enhanced electrically evolved [³H]-noradrenaline release in a concentration-dependent manner with a maximal increase by about 50% at 1 M. Also iloprost (0.1–3 M) increased transmitter release concentration-dependently, whereas misoprostol (0.1–3 M) had no effect. Indometacin (10 M) influenced neither evoked release per se nor the enhancement caused by PGE₂. AH6809 (3 M), a selective EP₁ receptor antagonist, blocked the enhancement caused by both PGE₂ and iloprost. K⁺-evoked noradrenaline release, which was virtually insensitive to tetrodotoxin (0.3 M), was increased by PGE₂ to an extent comparable to that observed after electrical stimulation.In summary, the present data indicate that PGE₂ facilitates noradrenaline release from cultured chick sympathetic neurons by a receptor which shows the pharmacological profile of the EP₁ subtype and is probably located at the processes of the neuron.     

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     

Alterations in β-adrenoceptor number and catecholamine content of chick atria after reversible sympathetic denervation with 6-hydroxydopamine

Summary 1. The characteristics of [³H]-dihydroalprenolol (DHA) binding were determined in atria from untreated chicks. [³H]-DHA binding to atrium homogenates was rapid (k1 = 8.52 × 10⁸ 1 mol^–1 min^–1), reversible (k _–1 = 0.47 min^–1), saturable, and of high affinity (K _D = 0.61.–nmol/l). Isoprenaline competed for specific [³H]-DHA binding in a stereoselective manner; IC₅₀ values (mol/l) were: (–)isoprenaline 0.12, (+)isoprenaline 4.7. 2. The number of [³H]-DHA binding sites and catecholamine content of left and right atria were examined after injection of chicks with a single dose of 6-hydroxydopamine hydrobromide (100 mg/kg). There were transient increases in the number of [³H]-DHA binding sites in both the left and the right atrium after 6-OHDA treatment. These increases were quicker in onset and in offset in the right atrium than in the left atrium. [³H]-DHA binding was significantly increased- in the left atrium at 5 and 7 days, and in the right atrium at 3 and 5 days after 6-OHDA injection. 3. Saturation binding isotherms indicated that the increase in [³H]-DHA binding was due to an increase in -adrenoceptor number with no change in affinity for [³H]-DHA. 4. Twenty four hours after 6-OHDA treatment there was a significant (80%) decrease in noradrenaline content of left and of right atria. 5. The decrease in noradrenaline content was reversible, noradrenaline levels returning to 55% of control in left atrium and to 71% of control in right atrium by 21 days after 6-OHDA treatment. These changes are consistent with reversible sympathetic denervation of the atria. 6. Adrenaline levels in the atria were 5 to 18% of total catecholamine (noradrenaline+adrenaline) content and were not significantly altered by 6-OHDA treatment.     

[3H]Noradrenaline accumulation in cultured bovine adrenal medullary cells: modulation of accumulation by nicotine

Summary The mechanisms by which catecholamines are transported into adrenal medullary cells are not as well characterized as the high affinity catecholamine uptake system of sympathetic neurones. Therefore, we studied the accumulation of [³H]noradrenaline in 7–21 day old cultures of bovine adrenal cells. Cultured adrenal cells accumulate [³H]noradrenaline. The accumulation process has an apparent Km for noradrenaline of 0.47 mol/l, is Na⁺ -dependent, and is inhibited by desipramine, (–)-noradrenaline and (–)adrenaline (IC₅₀'s, 0.007, 0.9 and 3.8 mol/l, respectively). Other aromatic amines also inhibit accumulation with an order of potency, (+)-amphetamine dopamine > (–)-noradrenaline > (–)-adrenaline 5-hydroxytryptamine. Nicotine and other stimulants of catecholamine release (acetylcholine and depolarizing K⁺ concentrations) were found to inhibit [3H]noradrenaline accumulation. Reserpine pretreatment (72 h) or omission of calcium did not prevent nicotine's inhibitory effect on accumulation, but did reduce or inhibit nicotine-induced catecholamine release, suggesting that adrenal [3H]noradrenaline accumulation may be regulated by processes involved with stimulus-secretion coupling (e.g., sodium gradient and/or depolarization) which are independent of release. These results suggest that the catecholamine transport mechanisms of cultured bovine adrenal cells and sympathetic neurones are similar and may be regulated by secretory processes.This study has been supported in part by grants from the Ohio State University Office of Research and Graduate Studies and the National Institutes of Health (NS-24813)