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.     

Selective inhibition by hydrocortisone of 3H-normetanephrine formation during 3H-transmitter release elicited by nerve stimulation in the isolated nerve-muscle preparation of the cat nictitating membrane

Summary The metabolism of ³H-noradrenaline released by nerve stimulation in the isolated nerve-muscle preparation of the cat nictitating membrane was determined under control conditions and in the presence of hydrocortisone, 28 M, a concentration which inhibits the high affinity extraneuronal uptake of noradrenaline in this tissue. in the controls the main fraction in the overflow elicited by stimulation at 10 Hz during 2 min was the deaminated glycol, ³H-DOPEG (3,4-dihydroxyphenylglycol), which accounted for 45.2±2.96% of the total radioactivity. Under these conditions, ³H-noradrenaline represented 30.8±1.92%, while ³H-normetanephrine accounted for 14.5±0.94% of the total overflow of radioactivity. During exposure to hydrocortisone there was a selective inhibition in ³H-normetanephrine formation from ³H-noradrenaline released by stimulation while the other fractions were not affected significantly. In contrast to these results, there were no changes in the spontaneous outflow of ³H-normetanephrine during exposure to hydrocortisone. The results obtained support the view that ³H-normetanephrine in sponteneous release originates from the activity of prejunctional catechol-O-methyltransferase. On the other hand, ³H-normetanephrine formed during transmitter release elicited by nerve stimulation is due to the activity of extraneuronal catechol-O-methyltransferase. Access of ³H-noradrenaline released by nerve stimulation to extraneuronal catechol-O-methyltransferase is mediated through the high-affinity, hydrocortisone-sensitive extraneuronal uptake mechanism.     

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.     

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

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

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.     

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.     

Inhibition by parasympathetic nerve stimulation of the release of the adrenergic transmitter

Summary Isolated rabbit atria were perfused with Tyrode solution containing (+)-amphetamine. Electrical stimulation of the right postganglionic sympathetic fibres caused an output of noradrenaline which was significantly decreased by simultaneous stimulation of the vagus nerves.This work was supported by the Deutsche Forschungsgemeinschaft.     

Regulation of adrenergic receptor number following chronic noradrenaline infusion in the rabbit

In order to study noradrenaline-induced regulation of alpha- and beta-adrenoceptors, groups of male New Zealand White rabbits (n = 8) were treated with intravenous noradrenaline (0.09 mol/kg × h) or ascorbate (0.1 %) for I0 days via osmotic minipumps implanted in the femoral vein, and the number of cardiac, lung and lymphocyte beta-adrenoceptors as well as renal and platelet alpha₂-adrenoceptors were determined.

Plasma normetanephrine for examination of extraneuronal uptake and metabolism of noradrenaline in rats

The importance of neuronal reuptake for terminating the actions of noradrenaline is well established, but the role of extraneuronal uptake is less clear. This study used plasma concentrations of the extraneuronal noradrenaline metabolite, normetanephrine, to estimate rates of extraneuronal removal of noradrenaline in rats. Animals received infusions of ³H-noradrenaline,. with and without inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), to examine the extraneuronal removal of noradrenaline and formation of normetanephrine from infused and endogenous noradrenaline. Infusions of ³H-normetanephrine were also carried out to examine the plasma kinetics of normetanephrine before and after inhibition of MAO.Normetanephrine was cleared rapidly from the circulation and had a short plasma halflife (1 min). Spillover of normetanephrine into plasma (79 pmol kg^–1min^–1) was a third that of noradrenaline, but increased 2.8-fold after inhibition of MAO; noradrenaline spillover remained unchanged. Combined inhibition of MAO and COMT decreased the plasma clearance of ³H-noradrenaline by 38070, reflecting removal of ³H-noradrenaline by extraneuronal uptake. Division of the rate of extraneuronal removal of ³H-noradrenaline by the specific activity of plasma ³H-normetanephrine during the ³H-noradrenaline infusion indicated that the rate of extraneuronal removal of endogenous noradrenaline was 250 pmol kg^–1min^–1; this was close to the spillover of normetanephrine into plasma after inhibition of MAO (219 pmol kg^–1 min^–1).Forty-five % of plasma normetanephrine was derived from circulating noradrenaline and 55% from noradrenaline before entry into the circulation. Assuming that these proportions reflected the sources of noradrenaline metabolized extraneuronally indicated that the rate of extraneuronal metabolism of noradrenaline before entry into the circulation was 138 pmol kg^–1min n^–1. Comparison of this with the rates at which noradrenaline was recaptured by sympathetic nerves (2540 pmol kg^–1min^–1) or spilled over into plasma (228 pmol kg^–1min^–1), indicated that 87% of the noradrenaline released by sympathetic nerves was recaptured, 5% was metabolized extraneuronally and 8% escaped into plasma. Thus, extraneuronal uptake removes much less of the noradrenaline released by sympathetic nerves than neuronal reuptake.     

Effects of piribedil on noradrenaline and MOPEG-SO4 levels in the rat brain

I.p. administration of piribedil methansulphonate to male Sprague-Dawley rats resulted in a remarkable increase of brain MOPEG-SO₄ which was consistent over time. In parallel experiments brain content of NA was found significantly reduced 30 and 60 min after drug administration. The effect seems to be dose dependent. These data cast some doubts on the relative specificity of action of piribedil as a dopaminergic activator.     

Development of degeneration contraction and supersensitivity in the cat's nictitating membrane after 6- hydroxydopamine

Summary After subcutaneous injection of 30 mg/kg of 6-hydroxydopamine into the conscious cat the denervated and the decentralized nictitating membranes contracted immediately. While the response of the denervated membrane is of short duration (1.5 h), the decentralized membrane is half relaxed after 11 h. The duration of the latter contraction is similar to that of the degeneration contraction seen after surgical denervation. It is proposed that the response of the decentralized nictitating membrane to 6-hydroxydopamine corresponds to the degeneration contraction. Treatment of the cats with pargyline prolongs both types of degeneration contractions. However, while beta-TMlO {[2-(2,6-dimethylphenoxy)propyl] trimethylammonium chloride had profound effects on the degeneration contraction seen after surgical denervation, it failed to affect that after 6-hydroxydopamine. Indirectly acting sympathomimetic amines prevented the usual effect of beta-TMlO on the degeneration contraction seen after surgical denervation. It is proposed that the failure of beta-TMlO to affect the response of the decentralized nictitating membrane to 6-hydroxydopamine is due to a similar preventive effect of 6-hydroxydopamine.The development of supersensitivity to (-)-noradrenaline was determined at various time intervals after the injection of 6-hydroxydopamine; pithed cats and isolated nictitating membranes were used. Under both conditions the membranes were in a state of sustained contraction, if the experiment was begun before the response of the decentralized nictitating membrane to 6-hydroxydopamine had worn off. After 6-hydroxydopamine the development of supersensitivity coincided with the falling phase of the degeneration contraction. Very similar results had been obtained earlier during the degeneration contraction after surgical denervation; the temporal relation between degeneration contraction and development of supersensitivity is very similar after surgical and chemical denervation.This study was supported by the Deutsche Forschungsgemeinschaft.     

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.     

The effect of cocaine on uptake of and sensitivity to noradrenaline in isolated nictitating membranes before and after storage in the cold

Summary Experiments were carried out on fresh isolated cat nictitating membranes as well as on muscles stored in the cold for 7 days. Storage reduced the cocaine-induced supersensitivity to (–)-noradrenaline but did not abolish it it also reduced responses to tyramine, and about halved the noradrenaline content of the tissue. Cocaine failed to potentiate responses of fresh or of stored muscles to the methoxamine (which is not taken up by adrenergic nerves).The incubation with 2.5 ml of 100 ng/ml of (–)-noradrenaline (in the presence of the inhibitor of catechol-O-methyl transferase), fresh muscles removed noradrenaline from the incubation medium at a rate of about 70 ng per gram of tissue per min; 10 g/ml of cocaine reduced rate of removal by 81%. Muscles stored in the cold removed less noradrenaline from the medium (about 45 ng/g×min^–1) than fresh ones, and cocaine reduced the rate of removal by 56%.The neuronal uptake mechanism of the nictitating membrane does not seem to be stereoselective, since the rate of removal of (+)-noradrenaline from the incubation medium was similar to that of the (–)-isomer.It is concluded that cold storage of the muscle abolishes neither the neuronal uptake of noradrenaline nor the ability of cocaine to impair this uptake; however, both parameters were reduced. Since the sensitizing action of cocaine is similarly reduced, there is no reason to doubt the causal relation between impairment by cocaine of neuronal uptake and ensuing supersensitivity to (–)-noradrenaline.with the Technical Assistance of Miss Roneen D. HobbsPart of this work was supported by the Deutsche Forschungsgemeinschaft.The authors gratefully acknowledge the excellent technical assistance of Miss Helga Damm. We are grateful to Dr. E. A. Maxwell of Burroughs Wellcome and Co., Tuckahoe, N.Y.,for the methoxamine, to Dr. A. J. Plummer of CIBA Pharmaceutical Products, Inc., Summit, N.J., for the reserpine, and to Dr. G. A. Johnson of Upjohn Comp., Kalamazoo, Mich. for the U-0521.     

Effects of prolonged ethanol administration on the noradrenaline levels of rat heart.

The effect of prolonged administration of ethanol on the noradrenaline concentration of the heart was investigated in rats. After 4 weeks there was no difference in the catecholamine levels between control and ethanol consuming rats. After 12 and 24 weeks there was a highly significant increase in heart noradrenaline concentration in hearts of experimental rats. These findings may indicate a delay in the effect of ethanol in cardiac tissue. It is further suggested that continued exposure to high levels of catecholamine may play a role in the development of cardiomyopathy in chronic alcoholism.     

Effects of dopamine on veins in humans: comparison with noradrenaline and influence of age

Objective: To compare the venoconstricting effect of dopamine with that of noradrenaline and to investigate the influence of age on the responsiveness to dopamine in human subjects. Methods: In eight young and eight elderly male subjects, increasing doses of dopamine or noradrenaline were infused into a dorsal hand vein and its diameter was measured using a linear variable differential transformer. Results: There was no significant difference between the maximum venoconstriction (E_max) for dopamine and that for noradrenaline. The infusion rate to induce 50% of E_max (ED₅₀) for dopamine in the young and elderly subjects was 363 ng · min⁻¹ and 352 ng · min⁻¹, and the ED₅₀ for noradrenaline was 40.7 ng · min⁻¹ and 43.8 ng · min⁻¹, respectively. Neither in the E_max nor in the ED₅₀ for these drugs were there significant differences between the young and elderly subjects. Conclusion: The venoconstricting effect of dopamine is 5–20 times less than that of noradrenaline, and aging does not influence the responsiveness to dopamine and noradrenaline in human subjects. Received: 29 August 1997 / Accepted in revised form: 5 February 1998     

The cocaine-insensitive component of non-exocytotic efflux of noradrenaline from adrenergic axons in the isolated rat tail artery

Summary The working hypothesis was that the cocaine-insensitive component of non-exocytotic efflux of noradrenaline represents diffusion of the unprotonated amine across the axonal membrane. It was tested by examination of the effect of changing axoplasmic pH —and thus the fraction of extravesicular noradrenaline in the unprotonated form — on the overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from rat tail arteries. The catechols were assayed by liquid chromatography with amperometric detection. To dissipate the H⁺ gradient across the axonal membrane, the tissues were incubated in media of different pH, in which Na⁺ was completely replaced with K⁺ and which were HCO₃ ^– - (and Ca²⁺-)free. Exposure of the tissues to these media produced substantial, but reversible increases in the overflow of noradrenaline. Subsequently, the overflows of both noradrenaline and the glycol kept rising, but their ratio did not change.Cocaine (0.1 mmol/1) lowered the (noradrenaline overflow: glycol overflow) ratio significantly. The ratio observed in its presence increased steeply with decreasing external and, presumably, axoplasmic pH. Addition of valinomycin and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mol/1 each) to the cocaine-containing media more than doubled the overflows without altering significantly the ratio. Under identical conditions, the overflow of noradrenaline from preparations with inactive neuronal monoamine oxidase did not decrease with decreasing pH.Since, in the presence of cocaine, the overflow ratio increased — rather than decreased — with decreasing pH, and because the overflow or noradrenaline from preparations with inactive monoamine oxidase did not decline with pH, the cocaine-insensitive component of noradrenaline efflux does not seem proportional to the axoplasmic concentration of the unprotonated amine. The data can, however, be accounted for by postulating that the component reflects the concentration of the protonated form of noradrenaline.     

Effects of adrenergic drugs on sheep oviduct motility.

Recordings of electrical activity of the oviduct in conscious ewes fitted with an extracellular multi-electrode assembly confirm the presence of alpha- and beta-adrenergic receptors in both the ampullary and isthmic portions. Regional differences in response to noradrenaline and isoprenaline were observed particularly during oestrus. In all cases the lowest threshold of sensitivity was exhibited by the isthmic portion of the oviduct.     

The effect of chronic captopril therapy on adrenergic receptors,plasma noradrenaline and the vascular responses to infused noradrenaline

Summary Adrenergic receptors (alpha₂, beta₂), plasma noradrenaline, heart rate and the pressor responsiveness to infused noradrenaline were examined in ten healthy male volunteers before and after 2 weeks of placebo or captopril therapy in a double blind cross-over study. No significant differences in these measurements were observed between the captopril and placebo treated groups. The study shows that in sodium replete normotensive subjects, long-term angiotensin converting enzyme inhibition does not lead to changes in adrenoceptor density. There is also no alteration in plasma noradrenaline levels nor in the pressor responsiveness to infused noradrenaline. These data suggest that the known interaction between the renin-angiotensin system and the sympathetic nervous system observed in animals is probably of little significance in man.     

Binding of exogenous noradrenaline by the proteins of dog plasma

Summary On addition to dog blood or plasma about 40% of exogenous nor-adrenaline escaped detection by photofluorimetric methods, when noradrenaline was present in concentrations ranging from 10 to 1000 ng/ml. As shown by bioassay the missing noradrenaline was not inactivated, but rather bound to plasma proteins; this was shown by experiments using labelled noradrenaline and precipitation of proteins, or Sephadex gel filtration. Cellulose acetate electrophoresis demonstrated binding by all protein fractions, alpha 1 and 2 globulins showing the greatest avidity for noradrenaline. Drugs known to be highly bound by proteins did not affect the binding capacity for noradrenaline. It is concluded that plasma noradrenaline values found after injection or infusion of noradrenaline may be only about half of the real values.Supported by Instituto de Alta Cultura (Research Project PMC-2).     

Experimental variables in the effects of postsession injections of strychnine sulphate on a classically conditioned response

Summary Postsession strychnine injections have been shown to retard acquisition rates of the classically conditioned nictitating membrane response (CCNMR) of rabbits trained at a CS-US interval of 1000 msec. The particular interval value employed in CCNMR acquisition was found to be important in the magnitude of the postsession strychnine effect. Strychnine Ss trained at a CS-US interval=1250 msec (nonoptimal) were significantly depressed in acquisition rates relative to saline controls, while strychnine Ss trained at a CS-US interval=250 msec (optimal) were not. Interpretations are based upon the presumed effects of CNS excitants on memory consolidation and possible neurological correlates of the CS-US interval parameter.