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.     

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.     

On the reduced retention of extravesicular noradrenaline in rabbit atria produced by potassium

The effects of KCl on the retention of extravesicular (?)-[³H]noradrenaline were investigated. Atria, from reserpine-pretreated rabbits, were exposed to tropolone and pargyline before incubation with (?)-[³H]noradrenaline. After efflux for 50–60 min, exposure of tissues to media containing 65 mM KCl resulted in increased efflux of (?)-[³H]noradrenaline. This effect was not Ca²⁺-dependent and was not altered by oxymetazoline, phentolamine, indomethacin or methacholine. The KCl-induced reduction in retention of (?)-[³H]noradrenaline was, however, inhibited by cocaine and desipramine, but not by lidocaine. Efflux was not increased by RbCl and CsCl. It is suggested that KCl may have accelerated the efflux of extravesicular (?)-[³H]noradrenaline by increasing a cocaine- and desipramine-sensitive carrier-mediated efflux process.     

The influence of ethanol administered to pregnant rats on tyrosine hydroxylase activity of their offspring

Pregnant rats were fed a regimen of ethanol and metrecal from the tenth day of pregnancy until delivery. Control females received metrecal and an isocaloric replacement of ethanol with sucrose. Tyrosine hydroxylase activity was measured in the caudate nuclei of the offspring at 1, 2 and 3 weeks of age. The activity of the enzyme was found to be significantly increased in pups whose mothers were exposed to ethanol.     

Trans-synaptic regulation of tyrosine hydroxylase activity in adrenergic neurones: Effect of potassium concentration on cultured sympathetic ganglia

Summary Superior cervical ganglia from 6-day and 4-week-old mice showed increased tyrosine hydroxylase activity when cultured for 48 h in media containing 5 or 15 times the normal potassium concentration. Increased sodium concentration had no such effect. These results suggest that depolarisation of adrenergic neurones may be important in the trans-synaptic regulation of enzyme activity in both adult and neonatal ganglia.     

Potentiation of drug-initiated adrenal tyrosine hydroxylase induction by diazepam

Summary The increase in in vitro adrenal tyrosine hydroxylase (TH) activity caused by drugs which produce an increase in preganglionic nerve activity as a result of actions outside the central nervous system was increased by the administration of diazepam. However, other central nervous system depressants such as pentobarbital, phenobarbital and diphenylhydantion were not found to share this property. Adrenal enzyme induction provoked by isoproterenol and 6-hydroxydopamine was increased by diazepam administration, whereas that produced by reserpine or insulin was not altered. The enhanced adrenal TH activity produced by diazepam pretreatment and isoproterenol administration could not be attributed to an increase in intensity or to an altered body temperature. Administration of 5-hydroxytryptophan can antagonize the induction of adrenal TH produced by isoproterenol, however, it did not block the TH induction produced by concurrent administration of diazepam and isoproterenol. The effect of diazepam on adrenal TH induction may be the result of its ability to interfere with central nervous system amine containing nerve functions which modulate preganglionic nerve activity, but the particular neurotransmitter system involved has not been determined.     

Involvement of 3′,5′-cyclic adenosine monophosphate in the increase of tyrosine hydroxylase activity elicited by cold exposure

Summary We studied the relationship between changes of 3,5-cyclic adenosine monophosphate (cAMP) and tyrosine hydroxylase (TH) activity in adrenal medulla of rats exposed to cold stress. Exposure of rats to 4° C produced a ten-fold increase of the cAMP content of adrenal medulla in about 30 min. This increase persisted for about one hour; the levels of cAMP returned to control value within 120 min in spite of the continued exposure to 4° C. In rats with monolaterally denervated (splanchnicotomized) adrenal, the exposure to 4° C produced only insignificant changes of cAMP concentration. During the exposure to 4° C we also observed an increase (about two times) of catecholamine turnover rate as measured by ³H-dopamine efflux from adrenal glands. This increased efflux persisted for 6 h of exposure to cold suggesting that the efflux of ³H-dopamine can increase without a simultaneous increase of cAMP concentrations. Exposure of rats to 4° C for two hour also increases (about two times) the TH activity as measured 24 h later. Exposure of the animals to 4° C for a time period longer than two hour (4 or 24 h) failed to produce further increases of TH activity. These results support the concept that the increase of cAMP concentrations in adrenal medulla may play a central role in initiating the chain of biochemical events modulating the synthesis of TH.     

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.     

Evidence for M1 muscarinic cholinoceptors mediating facilitation of noradrenaline release in guinea-pig carotid artery

Summary The muscarinic agonists acetylcholine (150 mol/l), carbachol (1–10 mol/l) and McN-A-343 (1–50 mol/l, selective for M₁ receptors) increased, in a concentration-dependent manner, the electrically-evoked tritium overflow from guinea-pig carotid arteries preincubated with [³H]-noradrenaline. The increase caused by acetylcholine was not modified by hexamethonium (300 mol/l) but was reduced by the muscarinic receptor antagonists methylatropinium (0.5 and 1 nmol/l, nonselective), pirenzepine (1 and 5 mol/l, M₁-selective), methoctramine (1 and 5 mol/l, M₂-selective) and pfluoro-hexahydro-sila-difenidol (0.1–1 mol/l, M₃-selective). The order of potencies (expressed as negative logarithms of concentrations that reduced by 50% the facilitatory effect of acetylcholine) was: methylatropinium (9.93) > pirenzepine (8.83) > p-fluoro-hexahydro-siladifenidol (6.81) methoctramine (6.20). These results demonstrate the existence of facilitatory M₁ receptors modulating noradrenaline release in blood vessels. Correspondence to M. Salaices at the above address     

Some observations on the bacterial decarboxylation of metatyrosine

Summary Suspensions of Streptococcus faecalis R decarboxylate l-tyrosine and l-Dopa; there is little or no decarboxylation of metatyrosine. After treatment of the suspensions with toluene an ability of the decarboxylase to act upon metatyrosine is unmasked.This work has been supported by a grant from the Medical Research Council.     

Effect of triamterene on tyrosine hydroxylase activity

Summary Triamterene, which is structurally similar to the natural cofactor of tyrosine hydroxylase, (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, inhibited tyrosine hydroxylase in rat adrenal gland homogenates and was found to be a competitive inhibitor of the synthetic cofactor 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin. When triamterene (30 mg/kg i.p.) was administered to rats, a significant decrease in the adrenal, whole brain and kidney enzyme activities was observed after 90 min; if the drug was given orally, the diuretic affected only adrenal tyrosine hydroxylase. In both cases the drug decreased potassium excretion to 1/5 of control values and increased the excretion of sodium. Catecholamine levels in atria, kidneys, adrenal glands and whole brain were not affected in acute experiments. Chronic treatment (triamterene 30 mg/kg p.o. twice daily during 4 days) inhibited tyrosine hydroxylase and decreased catecholamine levels in the kidneys. Low potassium excretion was observed throughout the 4 days of treatment. In these chronic experiments the inhibition of the adrenal enzyme seen in acute treatments was not observed. This recovery cannot be explained by increase in the adrenal biopterin levels. It could be mediated by an induction of the adrenal tyrosine hydroxylase.     

Prejunctional effects of a purified toxin from the scorpion Tityus serrulatus

Summary The effects of a purified fraction of the venom of the Brazilian scorpion, Tityus serrulatus, were studied in isolated guinea-pig atria previously labelled with ³H-noradrenaline. Exposure to 0.3 and 1.0 /ml of the scorpion toxin resulted in a long lasting positive chronotropic effect which was concentration-dependent. The increase in atrial rate coincided with an enhancement in spontaneous outflow of radioactivity. The increase in outflow of radioactive products elicited by exposure to 1.0 g/ml of the scorpion toxin was approximately 3-fold. ³H-noradrenaline accounted for 60% of the total increase in outflow of radioactivity elicited by the scorpion toxin and the ³H-deaminated glycol (3,4-dihydroxyphenylglycol) represented the main metabolite formed, accounting for approximately 35% of the total release. 20 min after exposure to 1.0 g/ml of the scorpion toxin the overflow of the labelled transmitter elicited by accelerans nerve stimulation (4 Hz, during 60 sec, supramaximal voltage) was increased 8-fold. This effect of the scorpion toxin appears to be unrelated to inhibition of neuronal uptake, block of -adrenoceptors or stimulation of -adrenoceptors. Consequently, in addition to releasing noradrenaline, the scorpion toxin enhances transmitter overflow elicited by nerve stimulation through a prejunctional effect which appears to reflect a nove mechanism of action.     

Papaverine enhances the effect of adenosine in guinea-pig atria

Summary Papaverine, while enhancing the force of contraction of guinea-pig atria, remarkably and dose-dependently enhanced the negative inotropic response of the atria to adenosine. It also enhanced the actions of ATP and other adenine nucleotides, but not those of 2-chloroadenosine and ACh. At similar concentrations, papaverine inhibited the uptake of adenosine by the atrial tissue during incubation with adenosine. Adenosine in the medium was degraded to inactive inosine during incubation with the atrial tissue, and papaverine reduced its degradation.The enhancing effect of papaverine on the action of adenosine on guinea-pig atria was like those of dipyridamole, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine and cinepazide. The effect seemed to be due mainly to inhibition of adenosine uptake into the tissue. Inhibition of adenosine degradation may also have contributed to the action of papaverine, but this action was probably much less important than inhibition of adenosine uptake.     

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     

Formation of deaminated metabolites of dopamine in noradrenaline neurons

Summary The deaminated monoamine metabolites 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), 3,4-di-hydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined electrochemically following organic solvent extraction and reverse-phase, high performance, liquid chromatography in four regions of the mouse brain. In the noradrenaline (NA)-predominant regions (hemispheres, brain stem), the ratio of the concentrations of DOPAC plus HVA to NA plus dopamine (DA) was approximately the same as in the DA-predominant regions (corpus striatum, limbic system). Yohimbine and reserpine elevated the concentrations of DOPAC and HVA both in the NA-and the DA-predominant regions. The effect of yohimbine was somewhat enhanced by the ₁-receptor blocking agent prazosin in the NA-predominant regions. The concentration of MOPEG was increased by yohimbine and decreased by reserpine.The concentrations of DOPAC and HVA were lowered by clonidine, but not by apomorphine in the NA-predominant regions of reserpine-treated mice. In, the DA-predominant regions, apomorphine, but not clonidine, reduced the concentrations of DOPAC and HVA. The effects of clonidine and apomorphine were reversed by yohimbine and haloperidol, respectively.The results indicate that the concentrations of the acid DA metabolites DOPAC and HVA in the NA-predominant regions reflect the rate of synthesis of DA in the NA neurons.     

Effects of isolation and fighting in mice on the rate of synthesis of noradrenaline,dopamine and 5-hydroxytryptamine in the brain

Male mice were isolated or housed in groups for 6–8 weeks. Some of the isolated mice were thereafter brought together in groups of 25–30 animals for 30 min, during which period they fought each other intensively. The brain concentration of 5-HIAA was lower in isolated than in grouped animals. 30 min after administration of the aromatic amino acid decarboxylase inhibitor 3-hydroxybenzyl-hydrazine HCl (NSD 1015) the amount of Dopa accumulated in the brain was lower in isolated than in grouped animals. There was no corresponding difference in the accumulation of 5-HTP. The accumulation of Dopa and 5-HTP after NSD 1015 was accelerated during fighting. The effects of isolation and fighting on DOPA accumulation were similar in NA- and DA-dominated areas of the brain. The brain concentrations of tryptophan and tyrosine were increased and the concentration of NA was decreased in fighting NSD 1015 treated animals. The brain concentrations of HVA was lower in isolated and higher in fighting animals than in grouped animals.The present results indicate that the rate of synthesis of brain CA is lower in isolated than in grouped animals and that intensive fighting rapidly accelerates the synthesis of CA and 5-HT in the brain.     

Changes in dopa decarboxylase mRNA but not tyrosine hydroxylase mRNA levels in rat brain following antipsychotic treatment

The effects of antipsychotic administration (1–32 days, twice per day) on the levels of mRNA coding for dopa decarboxylase (DDC) and tyrosine hydroxylase (TH) in rat brain has been assessed by a procedure utilising solution hybridisation with oligonucleotides. Saline and sulpiride (20 mg/kg/day) had no apparent effect on DDC mRNA levels. Haloperidol (3 mg/kg/day) elicited increases in DDC mRNA levels of 240% after 32 days and loxapine (4 mg/kg/day) elicited increases of 180% in DDC mRNA levels. None of the drugs affected TH mRNA levels. These results indicate that DDC may be more important than TH in the long term regulation of dopamine production.     

Effects of some psychotropic drugs on tyrosine hydroxylase activity in different structures of the rat brain

M.J. BESSON, A. CHERAMY, C. GAUCHY and J. MUSACCHIO, Effects of some psychotropic drugs on tyrosine hydroxylax activity in different structures of the rat brain, European J. Pharmacol. 22 (1973) 181–186.The effect of amphetamine, rcserpins and thioproperazine treatments on CA synthesis and tyrosine hydroxylase activity were investigated in some structure of the rat brain. Striatal and cortical CA synthesis was estimated on dices incubated with a physiolosical medium containing 1–3,5-³H-tyrosine; ³H-H₂O formed during the hydroxylatioa of ³H-tyrosine to ³H-DOPA was measured. Striatal DA synthesis was decreased in all cases 24 hr after the last injection of a chronic treatment with reserpine, thioproperazine or amphetamine; cortical CA synthesis was decreased only after reserpine treatment. Tyrosine hydroxylase activity was measured on a partially purified enzyme preparation. Enzyme activity was not affected in the striatum by repeated treatments with these different drugs. Repeated treatment with reserpine increased tyrosine hydroxylase activity in the brain stem and the cortex; in contrast, repeated treatment with thioproperazine decreased the enzyme activity in these two structures.     

Failure of tyramine to release neuronal ATP as a cotransmitter of noradrenaline in the guinea-pig vas deferens

Contractions, release of noradrenaline and r elease of ATP elicited by the indirectly acting sympathomimetic amine tyramine and responses elicited by exogenous noradrenaline were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique.In tissues pretreated with pargyline 1 mM, tyramine 300 M, when added to the superfusion medium for 2 min, elicited contraction and an overflow of tritium (mainly [³H]-noradrenaline) and ATP. Contraction and ATP overflow responses were prevented and tritium overflow was greatly reduced by desipramine 10 M Prazosin 0.3 M abolished contractions and evoked ATP overflow without changing tritium overflow. Blockade of postjunctional P₂-purinoceptors by suramin 300 M caused a marked decrease of tyramine-evoked contractions and a slight reduction of tritium overflow whereas evoked ATP overflow was markedly increased. The effect on contraction was not shared by two other P₂-purinoceptor antagonists, namely pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS) 32 M and diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) 32 M: PPADS increased contractions about fourfold, whilst DIDS had no effect at all. When the vas deferens was superfused for 24 min with medium containing tyramine 300 M, evoked contractions and tritium overflow continued throughout whereas ATP overflow faded rapidly to basal values. In the presence of prazosin 0.3 M, tyramine 300 M again failed to elicit contractions as well as an overflow of ATP. Application of noradrenaline 10 M instead of tyramine also resulted in prolonged contraction and an overflow of ATP that declined rapidly.It is concluded that all ATP released by tyramine is non-neuronal in origin, secondary to the activation of postjunctional ₁-adrenoceptors by released noradrenaline. The non-neural ATP does not seem to play a functional role in smooth muscle contraction and derives from a postjunctional source which is subject to a rapid depletion upon sustained ₁-adrenoceptor activation.