Metabolism of endogenous and exogenous noradrenaline in guinea-pig atria

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from guinea-pig isolated atria was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs, the outflow of endogenous noradrenaline over a period of 3 h averaged 1.6 pmol×g^–1×min^–1 and the outflow of DOPEG 17 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.31 pmol×g^–1×min^–1). Tyramine greatly increased the outflow of noradrenaline and DOPEG, and the reserpine-like compound Ro 4-1284 selectively increased the outflow of DOPEG; DOMA remained below the detection limit. When atria were exposed to (–)-noradrenaline 1.7 or 17 M, the subsequent outflow of noradrenaline and DOPEG was enhanced. Moreover, substantial amounts of DOMA were now found. This outflow of DOMA was prevented when atria were exposed to (–)-noradrenaline in the presence of cocaine or after an initial incubation with amezinium. Exposure to (–)-noradrenaline 1.7 M mainly enhanced the formation of DOPGE, while exposure to (+)-noradrenaline 1.7 M mainly enhanced the formation of DOMA.Our experiments confirm some and qualify other conclusions drawn from studies in which exogenous ³H-noradrenaline had been used to examine the metabolism of noradrenaline in guinea-pig atria. In agreement with the isotope studies, DOPEG is a major metabolite of endogenous noradrenaline. In contrast to what the isotope studies had suggested, however, endogenous DOMA is a very minor product, at least as long as the neurones are at rest. DOMA is only formed when the tissue is exposed to high concentrations of exogenous noradrenaline. In further contrast to previous conclusions, DOMA is then formed intra- and not extraneuronally.     

Metabolic fate of 3H-noradrenaline released from the mouse hypothalamus

Summary In slices of mouse hypothalamus labelled in vitro with ³H-noradrenaline (³H-NA), the deaminated metabolite ³H-3,4-dihydroxyphenylglycol (³H-DOPEG), represented 40.2±2.6% of the total outflow of radioactivity and was the main fraction in the sponteneous efflux. Inhibition of neuronal monoamine oxidase by exposure to 60 M bretylium, reduced the outflow of ³H-DOPEG to 9.7±0.3%. At the same time, the proportion of ³H-normetanephrine (³H-NMN) was significnatly increased. On the other hand, an increased outflow of ³H-DOPEG and a lower proportion of ³H-NMN was obtained in the presence of 2.9 M of the reserpine like agent Ro 4-1284.It is suggested that in the mouse hypothalamus, the deaminated metabolite, DOPEG, is formed inside the nerve terminals, while the O-methylated metabolite, NMN, might result from the activity of extraneuronal catechol O-methyltransferase.     

Effects of flow-stop on the metabolism of noradrenaline released by nerve stimulation in the perfused spleen

Summary The metabolic pathway of ³H-noradrenaline released spontaneously and by nerve stimulation was studied in the isolated perfused spleen of the cat. The deaminated glycol, DOPEG, (3,4 dihydroxyphenylglycol) was the main metabolite in spontaneous outflow, accounting for 62.5±1.6% of the total radioactivity (n=13). Of the total increase in radioactive products elicited by nerve stimulation at 5 Hz or 10 Hz around 30% was accounted for by the noradrenaline metabolites, particularly DOPEG and the O-methylated fraction. In the presence of 2.9×10^–6 M of cocaine the total overflow of radioactivity induced by stimulation was unchanged but DOPEG formation from released noradrenaline was abolished. These findings indicate that DOPEG formation results from the recapture of the released transmitter by adrenergic nerve endings and subsequent intraneuronal deamination. The total overflow of noradrenaline was reduced by flow-stop while the metabolism of the released transmitter was increased significantly. Cocaine, 2.9×10^–6 M, prevented the increase in DOPEG when stimulation was applied under flow-stop conditions. The decrease in noradrenaline overflow induced by flow-stop is partly due to the increase in the metabolism of the released transmitter.     

The 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     

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.     

Neuropeptide Y differentiates between exocytotic and nonexocytotic noradrenaline release in guinea-pig heart

Summary The overflow of neuropeptide Y (NPY; radioimmunoassay), noradrenaline and dihydroxyphenylethylenglycol (DOPEG; high pressure liquid chromatography) from guinea-pig perfused hearts was investigated in relationship to exocytotic and nonexocytotic release mechanisms. Exocytotic release: Electrical stimulation of the left stellate ganglion (12 Hz; 1 min) evoked a calcium-dependent overflow of noradrenaline and NPY, that was accompanied by a minor and prolonged increase in DOPEG overflow. This increase in DOPEG overflow was attenuated by blockade of neuronal amine re-uptake. In the presence of calcium, a closely related co-release of noradrenaline and NPY was also observed during administration of veratridine (10 M); it was completely prevented by tetrodotoxin (1 M). Nonexocytotic release: In the absence of extracellular calcium, veratridine (30 M) induced noradrenaline overflow only when combined with the reserpine-like agent Ro 4-1284 (10 M). This overflow was accompanied by efflux of DOPEG, but not of NPY. Similarily, tyramine (1–100 M) induced a calcium-independent concomitant overflow of both noradrenaline and DOPEG, but not of NPY. During anoxic and glucose-free perfusion a predominantly calcium-independent overflow of noradrenaline was observed; only in the presence of extracellular calcium was this overflow accompanied by a minor overflow of NPY. Noradrenaline overflow, induced by veratridine plus Ro 4-1284 (in the absence of calcium), by tyramine, or by anoxia, was suppressed by blockade of neuronal amine re-uptake, and was, therefore, mediated by reversed transmembrane amine transport by the neuronal uptake₁ carrier.The results indicate that NPY is co-released with noradrenaline only during calcium-dependent exocytosis. On the other hand, whenever, noradrenaline is released by non-exocytotic (calcium-independent and carrier-mediated) release mechanisms, no substantial NPY overflow is observed. The simultaneous determination of noradrenaline and NPY overflow, therefore, allows a differentiation between exocytotic and nonexocytotic noradrenaline release, and NPY may be utilized as a marker of exocytotic noradrenaline release.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 320 — Herzfunktion und ihre Regulation)Presented in part at the 62nd Scintific Sessions of the American Heart Association, New Orleans/USA, November 1989     

Effects of TRH on hypothalamic catecholaminergic and serotoninergic neurons

The in vivo and in vitro effects of TRH on tyrosine hydroxylase activity and on endogenous levels of noradrenaline as an index of noradrenergic neurons activity were studied in rat hypothalamus. The hydroxylation of tryptophan and the liberation of 5-hydroxyindole acetic acid were analyzed as an index of serotoninergic activity. TRH had no effect on tyrosine hydroxylase from hypothalamic homogenates in concentrations up to 100 ng/ml. Neither was the hydroxylation of tyrosine in intact hypothalamus. The TRH (4 micrograms/kg) decreased 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine accumulation after 10 min, and no effect on serotonin and noradrenaline concentrations was observed. TRH decreased endogenous levels of 5-hydroxyindole acetic acid by 35%. Thus, the results show that TRH or TSH might play a role in a short-loop feedback action involving catecholaminergic and serotonergic nerve terminals that regulate the liberation of TRH.     

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)     

Effects of the noradrenaline metabolites on the adrenergic receptors

Summary The effects of the five noradrenaline (NA) metabolites and of the O-methylated metabolite of adrenaline, metanephrine, were studied in a tissue with adrenergic beta-receptors (guinea-pig atria) and in a tissue with predominance of alpha-receptors (cat's nictitating membrane). In atria, normetanephrine and metanephrine elicited positive chronotrophic effects which were mediated through the beta-receptors. Both O-methylated metabolites had only 1/1000th of the potency of NA. In the normally innervated nictitating membrane normetanephrine and metanephrine elicited maximal responses of the same magnitude as NA. While normetanephrine had one half of the potency of NA, metanephrine was even more potent than NA. The effects of normetanephrine or metanephrine were mediated through the activation of the alpha-receptors and were not potentiated by either cocaine or denervation. Neither the deaminated nor the deaminated-O-methylated metabolites of NA had activity as agonists on alpha-or beta-receptors in concen-of up to 1×10^–4M. These metabolites did not elicit alpha-or beta-receptor block in concentrations of up to 1×10^–4M.     

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220.

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Release of vesicular noradrenaline in the rat tail artery induced by cocaine

Summary The effects of cocaine on overflows of endogenous noradrenaline and DOPEG from isolated rat tail arteries were examined. 1. Both overflows increased progressively with increasing concentration of cocaine, while the (NA overflow)/(DOPEG overflow) ratio first increased and then decreased. The changes in the overflows induced by cocaine (0.1 mmol/l) appeared reversible. 2. Exposure of the tissue for 30 min to cocaine, 1 mmol/l, resulted in a significant decrease in the proportion of storage vesicles containing electron-dense cores. 3. The changes in overflows of noradrenaline and DOPEG induced by cocaine (0.1 mmol/l) were unaffected by the presence of desipramine (0.1 mol/l) or removal of extracellular Ca²⁺. The effect of cocaine on the overflow of noradrenaline was potentiated by prior inhibition of MAO with clorgyline. 4. Exposure of segments to a Ca²⁺-free, high K, low Na incubation medium was accompanied by increased overflow of noradrenaline. Cocaine (0.1 mmol/l) reduced the overflow of noradrenaline to about a half, and substantially increased the overflow of DOPEG. 5. The increase in the overflow of DOPEG from segments bathed in HEPES-buffered solutions, the pH of which ranged from 6.80 to 7.38, was approximately proportional to the calculated concentration of unprotonated (uncharged) cocaine. 6. Quantitatively similar changes in the overflows were observed when norcocaine was substituted for cocaine. Ecgonine methyl ester was much less potent than cocaine, and O-benzoyl ecgonine was ineffective. 7. The small increases in the overflow of noradrenaline observed at relatively low concentration (<30 mol/l) of cocaine can be attributed primarily to inhibition of reuptake of the released transmitter by the cocaine- and desipramine-sensitive amine carrier. The overflows of NA and DOPEG in the presence of higher concentrations of the alkaloid exhibit features compatible with the following hypothesis: (A) Cocaine is translocated across the axonal membrane mainly in the form of the unprotonated species, a large fraction of which is reprotonated upon the entry into the axon. (B) Cocaine releases noradrenaline from storage vesicles into the extravesicular space, where the bulk of the amine is converted to DOPEG. (C) Efflux of the remaining noradrenaline from the axon is not mediated by the Na⁺-dependent, cocaine- and desipramine-sensitive neuronal amine carrier. It seems to represent uncoupled efflux of the protonated form of noradrenaline.Abbreviations DOPEG 3,4-dihydroxyphenylethylene glycol - DOMA 3,4-dihydroxymandelic acid - HEPES N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid - MAO monoamine oxidase - MOPEG 3-methoxy-4-hydroxyphenylethylene glycol - NA (–)noradrenaline - pH_j pH in the extravesicular space of the axon - pH_o pH of the bathing solution - pK_a negative logarithm of the dissociation constant This study was supported by the British Columbia Heart Foundation Send of fprint requests to V. Palaty at the above address     

Effects of nitroglycerin on release, synthesis and metabolism of norepinephrine and activation of tyrosine hydroxylase in guinea-pigs.

Nitroglycerin produced concentration-dependent increases in outflow of norepinephrine (NE) in perfused guinea-pig hearts. These effects of nitroglycerin were inhibited by guanethidine, but were not blocked by desmethylimipramine or rauwolscine. Nitroglycerin-induced increases in NE outflow correlated with its positive chronotropic responses. Higher concentrations of nitroglycerin also increased outflow of 3,4-dihydroxyphenylglycol (DOPEG) which were inhibited by desmethylimipramine. Nitroglycerin-induced maximal outflow of NE occurred during the first 2 min, but maximal outflow of DOPEG was 2-4 min. In addition, the 3,4-dihydroxyphenylalanine formation in atrial and striatal slices were enhanced by nitroglycerin in the presence of m-hydroxybenzylhydrazine dihydrochloride (an inhibitor of aromatic L-amino acid decarboxylase). The results suggest that nitroglycerin increases release of NE which correlates with cardiac stimulation by the drug. The drug also stimulates tyrosine hydroxylase activity resulting in increased synthesis of NE.     

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     

Tyrosine hydroxylase activity increases in pineal sympathetic nerves after depletion of neuronal serotonin

Summary The injection of p-chlorophenylalanine (PCPA) acutely reduced serotonin in the pineal gland of the rat and selectively elevated the noradrenaline (NA) content during the subsequent 24 h. The activity of tyrosine hydroxylase (TH) also increased in intact glands during the first 6 h after PCPA injection but returned to normal at 24 h. This enhancement of enzyme activity was only observed in the presence of a non-saturating concentration of the cofactor. Serotonin depletion by PCPA cannot directly account for the increased enzyme activity, because the amine does not modify TH activity. Moreover, this increase is restricted to the pineal, since in other sympathetically innervated organs, such as the atria, PCPA produced an acute but transient reduction in TH activity. The elevation described here is not due to a net increase in the amount of enzymatic protein, because TH activity is similar in pineal homogenates from treated and control rats when a saturating concentration of the cofactor was used. The availability of storage sites in pineal nerve vesicles due to serotonin depletion, seems to release TH activity from the negative control exerted by cytoplasmic catecholamines. Enzyme activity in the pineal is acutely enhanced until a new steady state is reached at a higher concentration of endogenous NA.     

Preferential decarboxylation of l-threo-3,4-dihydroxyphenylserine in rat renal tissues

• 1.1. Administration of l-threo-3,4-dihydroxyphenylserine (l-threo-DOPS; 3, 10 and 30 mg/kg, i.p.) produced a dose-dependent increase in the tissue levels of both noradrenaline and its deaminated metabolite 3,4-dihydroxyphenylglycol (DOPEG) in the rat jejunum, liver and renal cortex, but not in the left ventricle.
• 2.2. The accumulation of noradrenaline and DOPEG after the administration of l-threo-DOPS (30 mg/kg, i.p.) was also found to be a time-dependent effect, reaching its maximum 15 min after the injection and then declining progressively.
• 3.3. The accumulation of noradrenaline and DOPEG after l-threo-DOPS (30 mg/kg, i.p.) was found to be similar in control and 6-OHDA treated rats and completely prevented by previous treatment with benserazide.
• 4.4. Administration of l-threo-DOPS (30 mg/kg) produced an increase in plasma levels of noradrenaline and DOPEG; this effect was maximum, for both noradrenaline (6.2-fold increase) and DOPEG (3.4-fold increase), at 30 min after the injection of l-threo-DOPS.
• 5.5. The results presented here support the view that most l-threo-DOPS is decarboxylated into noradenaline by non-neuronal AAAD, a reaction occurring predominantly in renal tissues.

     

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     

Effect of a reserpine-like agent on the release and metabolism of [3H]NA in cell bodies and terminals

1. The reserpine-like agent, Ro 4-1284 (2-hydroxy-2ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11b-[H]benzo (a)quinolizine) releases [³H]noradrenaline ([³H]NA) from prelabelled superior cervical ganglion (cell bodies) and nictitating membrane (nerve endings) of the cat.2. The potency of Ro 4-1284 29.0 μM was higher in the cell bodies than in the nerve endings.3. In both tissues, exposure to the reserpine-like agent Ro 4-1284 induced a selective increase in the spontaneous outflow of [³H]DOPEG, while the [³H]OMDA metabolites to the release induced by Ro 4-1284 was very small.4. The desamination is the preferential way of the metabolic inactivation of the [³H]NA released by the reserpine-like agent in both parts of the noradrenergic neuron.     

Effects of catechol-O-methyltransferase inhibition on the plasma clearance of noradrenaline and the formation of 3,4-dihydroxyphenylglycol in the rabbit

Summary The purpose of this study was to elucidate the finding of Friedgen et al. (1993b) that catechol-O-methyltransferase (COMT) inhibition is much more effective in increasing the plasma concentration of endogenous dihydroxyphenylglycol (DOPED) than in increasing the plasma concentration of infused DOPED. To this end, reserpine-pretreated rabbits were anaesthetized and infused with noradrenaline and/or DOPED, and the plasma clearances of infused noradrenaline (Cl_NA) and DOPED (Cl_DOPEG) as well as the plasma DOPED response to noradrenaline infusion [as reflected by the ratio of the steady-state increase in plasma DOPED (DOPEG) to that in plasma noradrenaline (NA)] were determined before and after blockade of neuronal uptake by desipramine. Experiments were carried out either under control conditions or after COMT inhibition by i.v. administration of 3,4-dihydroxy-4-methyl-5-nitrobenzophenone (Ro 40-7592). On the assumption that rates of neuronal noradrenaline uptake equal steady-state rates of neuronal DOPED formation, the desipramine-sensitive components of Cl_NA and DOPEG/NA were used to estimate the apparent plasma clearance of DOPED formed intraneuronally (Cl_f-DOPEG) in response to noradrenaline infusion.Cl_NA was 83.6 ml kg-^–1 min^–1 in the absence and 48.1 ml kg ^–1min^–1 in the presence of desipramine. Neither the former nor the latter value was altered after COMT inhibition. However, the COMT inhibitor reduced Cl_DOPEG from 47.6 to 28.5 ml kg ^–1 min^–1 (indicating a 1.7-fold increase in the plasma DOPED response to DOPED infusion) and Cl_f-DOPEG from 106.2 to 43.3 ml kg^–1 min^–1 (indicating a 2.5-fold increase in the neuronal component of the plasma DOPED response to noradrenaline infusion). DOPEG/NA increased following treatment with Ro 40-7592 from 0.381 to 1.294 in the absence and from 0.036 to 0.391 in the presence of desipramine. These increases were more pronounced than the Ro 40-7592-induced change in Cl_f-DOPEG would predict. Therefore, the values of Cl_f-DOPEG were used to calculate rates of DOPED formation. The results show that the increases in DOPEG/NA induced by COMT inhibition were not due only to the block of O-methylation of DOPED, but also to a pronounced increase in the extraneuronal DOPED formation.Hence, COMT inhibition affects neither the neuronal nor the extraneuronal removal of circulating noradrenaline from plasma. By contrast, COMT inhibition reduces the clearance of infused DOPED by about 40% and that of neuronally formed DOPEG by about 60%. The observation of Ro 40-7592 being more effective in increasing the plasma DOPEG response to noradrenaline infusion than in increasing the plasma DOPED response to DOPEG infusion is due to the fact that newly formed DOPEG is O-methylated not only while being removed from plasma, but also on the way to plasma, and that COMT inhibition increases the DOPEG formation at extraneuronal sites.This study was supported by the Deutsche Forschungsgemeinschaft (Gr 490/5). A preliminary account of the results was presented to the German Society for Pharmacology and Toxicology (Halbrügge et al. 1992)Correspondence to K. H. Graefe at the above address     

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     

Effects of db cAMP on tyrosine hydroxylase activity of ganglia and nerve endings

Summary Preincubation of intact superior cervical ganglia or nictitating membrane for 2 h with dibutyryl cyclic AMP (db cAMP) increased the hydroxylation of tyrosine. This effect was not blocked by the protein synthesis inhibitor, cycloheximide. The K _m of tyrosine hydroxylase for the substrate, tyrosine, and for the cofactor, reduced pteridine, were decreased by db cAMP. There were no changes in the V _max of the enzyme. The inhibitory potency of noradrenaline on the hydroxylation of tyrosine was also decreased. Thus an inductive effect may be ruled out. The activation of the enzyme was only observed when the tissues were preincubated with the db cAMP and not when the cyclic nucleotide was added to the isolated enzyme.Preincubation of cervical ganglia for 4 h with db cAMP increased activity of decarboxylase and monoamine oxidase in tissue homogenates without changing the tyrosine hydroxylase activity.