Inhibition by vasodilators of noradrenaline and vasoconstrictor-mediated, but not skeletal muscle contraction-induced oxygen uptake in the perfused rat hindlimb; implications for non-shivering thermogenesis in muscle tissue

1. The effect of noradrenaline as well as of vasopressin and angiotensin II to increase oxygen uptake and perfusion pressure by the isolated perfused rat hindlimb were completely inhibited by the vasodilators, nitroprusside (0.5 mM), nifedipine (2.5 microM) and isoprenaline (50 nM). 2. Oxygen uptake due to sciatic nerve stimulation of skeletal muscle contraction was not inhibited by 0.5 mM nitroprusside but was found to increase further that produced by a maximum dose of either noradrenaline or angiotensin II. 3. Analysis of high energy phosphates in samples of freeze-clamped hindlimb muscle showed no difference before and after vasoconstrictor addition or with muscle sampled in vivo. 4. It is concluded that norepinephrine mediated increase in oxygen uptake by the perfused rat hindlimb results from its vasoconstrictor action.     

Further study of the adrenoceptors of the saphenous vein of the dog: Influence of factors which interfere with the concentrations of agonists at the receptor level

In the present study the affinities of some sympathomimetic amines for α- and β-adrenoceptors of the dog saphenous vein tissue were determined after all known factors interfering with the concentration of these agonists at the receptor level had been assessed and excluded. It was observed that in control experiments the relative potencies of sympathomimetic agonists for inducing contractions were: adrenaline (1.6) > noradrenaline (1.0) > phenylephrine (0.38) > isoprenaline (0.009).The elimination of neuronal uptake by cocaine, 4 × 10^?6M, enhanced predominantly the effects of noradrenaline (by a factor of 7.5), whereas block of catechol-O-methyl transferase (COMT) by U-0521, 10^?4 M, only enhanced those of adrenaline (by a factor of 2.6) and block of β-adrenoceptors by propranolol, 5 × 10^?7 M, enhanced those of isoprenaline (by a factor of 3) and adrenaline (by a factor of 1.8).Block of COMT enhanced the effects of adrenaline approximately as much as did the blockade of neuronal uptake; this seems to indicate that the affinity of adrenaline for extraneuronal and neuronal uptake processes is approximately the same.Regarding the relaxation-inducing capacity of sympathomimetic agents it was observed that isoprenaline, adrenaline and noradrenaline are full agonists, whereas phenylephrine was not able to produce relaxation amounting to more than 5% of the maximum. Denervation did not modify the relaxant effects of isoprenaline. After elimination of all known factors interfering with the concentration of the sympathomimetic agonists in the biophase, the ratios between the ED₅₀'s of each amine for α- and β-adrenoceptors were: adrenaline = 34, noradrenaline = 109 and isoprenaline = 0.0041.     

The effects of thyroxine treatment of rats on neuronal and extraneuronal uptake and metabolism of catecholamines in the heart.

The effects of thyroxine (T4) treatment of rats on the neuronal and extraneuronal uptake and subsequent metabolism of catecholamines in the heart were examined, to determine whether changes in the local dissipation of catecholamines might contribute to the enhanced sympathetic cardiac responses that occur when thyroid hormone levels are elevated. T4-treated rats were injected subcutaneously with L-thyroxine sodium 1 mg kg(-1) on days 1, 3 and 5, and controls were injected with the normal saline vehicle on the same days. The experiments on isolated, perfused hearts were carried out on day 8. The T4-treated rats had only 50% of the growth rate of the controls and their heart weights were 18% greater than the controls. The experimental data were adjusted to allow for the increase in heart weight caused by the T4 treatment. The initial rates of neuronal uptake of noradrenaline and of extraneuronal uptake of noradrenaline and isoprenaline in the hearts from T4-treated rats were not significantly different from those in hearts from control rats. The steady-state rates of extraneuronal O-methylation of isoprenaline and of extraneuronal deamination of noradrenaline in hearts from T4-treated rats were not significantly different from those in hearts from control rats. The steady-state rate of neuronal deamination of noradrenaline was significantly lower and the accumulation of unmetabolized noradrenaline in the hearts was significantly greater in T4-treated rats than in the controls. These findings could be explained by a decrease in neuronal monoamine oxidase activity or by an increase in intraneuronal binding of noradrenaline in hearts from T4-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

DENERVATION AND β-ADRENORECEPTORS OF THE CAT NICTITATING MEMBRANE

• 1 Dose-response curves to the relaxant effects of isoprenaline, salbutamol, adrenaline and noradrenaline were determined on the partially contracted smooth muscles of the cat nictitating membrane following α-adrenoreceptor blockade in order to test the hypothesis of a causal relationship between the inhibition of neuronal uptake and denervation super-sensitivity.
• 2 Denervation did not produce super-sensitivity of the nictitating membrane to the β-adrenoreceptor-mediated relaxant effects of any of the four agents studied.
• 3 The sensitivity of the inferior muscle was greater than that of the medial muscle to agents which were good substrates for neuronal uptake (adrenaline and noradrenaline) as well as to those which were not (isoprenaline and salbutamol).
• 4 Denervation did not alter the antagonist activity of β-adrenoreceptor blockers propranolol (nonselective) and metoprolol (β₁-blocker) against isoprenaline or of H 35/25 (β₂-blocker) against salbutamol.
• 5 These results do not support the hypothesis of a causal relationship between inhibition of neuronal uptake and super-sensitivity to sympathomimetic amines.

     

A comparison of vasopressin and noradrenaline on oxygen uptake by perfused rat hindlimb, kidney, intestine and mesenteric arcade suggests that it is in part due to contractile work by blood vessels

1. The rat hindlimb, kidney and intestine were each perfused in a nonrecirculating mode at 25 degrees C using an artificial perfusate (initial pressure 85 +/- 5 mmHg) and the effects of vasopressin and noradrenaline on oxygen uptake and perfusion pressure determined. 2. Both vasopressin (K0.5 = 0.1 nM) and noradrenaline (K0.5 = 2 nM) increased oxygen uptake as well as perfusion pressure by the perfused hindlimb; changes in oxygen uptake were closely matched by changes in pressure. The maximum increase in oxygen uptake was approx. 9 mumol/hr per g wet wt of hindlimb. 3. The perfused kidney also responded to vasopressin and noradrenaline with parallel increases in oxygen uptake and perfusion pressure for each agent. The largest increase in oxygen uptake was approx. 30 mumol/hr per g wet wt but this was not maximal. 4. Vasopressin increased oxygen uptake and pressure by the perfused intestine over the range 0.01-2 nM, but the changes in pressure only became significant at doses greater than 0.1 nM. 5. Noradrenaline inhibited oxygen uptake and increased perfusion pressure in a dose-dependent manner at pharmacological concentrations (greater than 30 nM) when shunting of perfusate may have contributed to unperfused regions. 6. A network of mesenteric blood vessels estimated to contain approx. 6% vascular tissue by weight, with the remainder white fat cells, lymphatics and connective tissue, was also perfused. 7. Vasopressin (K0.5 = 0.3 nM) and noradrenaline (K0.5 = 30 nM) each increased oxygen uptake and perfusion pressure in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of uptake inhibitors on responses of sheep coronary arteries to catecholamines and sympathetic nerve stimulation.

1. Transmural stimulation of intrinsic sympathetic nerves and exogenous catecholamines produce beta 1-adrenoceptor mediated relaxant responses in strips of contracted sheep coronary artery. 2. The neuronal uptake inhibitors, metaraminol, cocaine and desipramine and the extraneuronal uptake inhibitor, cortisol, failed to potentiate responses to noradrenaline or sympathetic stimulation; responses to isoprenaline were enhanced by cortisol. 3. Oxytetracycline, which inhibits binding to connective tissue fibres, did not affect responses to noradrenaline or nerve stimulation. 4. 17 beta-Oestradiol, caffeine and U0521 proved to be unsuitable compounds for studying catecholamine inactivation since they non-selectively potentiated responses to noradrenaline and isoprenaline. 5. It is concluded that catecholamine inactivation processes do not modify transmitter function in sheep coronary arteries.     

The inactivation of noradrenaline and isoprenaline in dogs

1. The removal of infused noradrenaline and isoprenaline from the circulation of the dog has been studied, using the blood-bathed organ technique.2. Both catecholamines were removed in peripheral vascular beds; in all organs studied, noradrenaline was removed to a greater degree than isoprenaline.3. The hind legs removed an average of 60% of the noradrenaline passing through, but only 34% of the isoprenaline. With noradrenaline, the degree of removal decreased as the concentration increased, but with isoprenaline, the degree of removal increased with concentration.4. After phenoxybenzamine, the proportion of isoprenaline removed was unchanged, whereas that of noradrenaline was decreased. The change in removal with the concentration of noradrenaline was also abolished.5. The results are consistent with the concept that a small fraction of infused noradrenaline is removed from the circulation by Uptake₁ and that this is blocked by phenoxybenzamine. Isoprenaline, and the rest of the noradrenaline, are removed by another process (Uptake₂?) followed by intracellular metabolism. This inactivation process is unaffected by phenoxybenzamine in concentrations sufficient to give α-adrenoceptor blockade.6. After isoprenaline infusions, a substance sometimes appeared in the circulation which contracted the blood-bathed organs.7. The systemic pressure response to vaso-active hormones is not a reliable indicator of the concentration of hormone in the arterial circulation.     

Beta 1-adrenoceptors mediate smooth muscle relaxation in mouse isolated trachea.

1. The relaxant effects to the beta-adrenoceptor agonists isoprenaline, adrenaline, noradrenaline, RO363, procaterol and fenoterol were investigated in carbachol-contracted mouse isolated tracheal preparations. 2. The order of potencies for those beta-adrenoceptor agonists that induced full relaxation of carbachol-contracted mouse tracheal preparations was isoprenaline greater than RO363 greater than noradrenaline = adrenaline greater than fenoterol. The EC50 value of isoprenaline for relaxation was 46 nM. The beta 1-adrenoceptor-selective agonist, RO363 was ten times more potent than the beta 2-adrenoceptor-selective agonist, fenoterol. The highly beta 2-adrenoceptor-selective agonist procaterol was a partial relaxant and induced only 28 +/- 4% relaxation. 3. Relaxations induced by noradrenaline and isoprenaline were not significantly affected by the neuronal uptake inhibitor, cocaine (10 microM) or by the extraneuronal uptake inhibitor, deoxycorticosterone acetate (25 microM) respectively. The alpha-adrenoceptor agonist methoxamine induced no observable elevation of mouse tracheal smooth muscle tone. 4. Schild plots for the beta-adrenoceptor antagonists, atenolol and betaxolol (beta 1-adrenoceptor-selective) and ICI 118,551 (beta 2-adrenoceptor-selective) were linear, with slope values approaching unity. Mean pA2 values derived for atenolol, betaxolol and ICI 118,551 for antagonism of beta-adrenoceptor-mediated relaxation were 7.1, 8.4 and 7.2, respectively. These data were independent of the use of isoprenaline or noradrenaline as the agonist. 5. These findings indicate that beta-adrenoceptor-mediated relaxations of mouse isolated trachea occur predominantly through activation of beta 1-adrenoceptors.     

ASSESSMENT OF NEURONAL UPTAKE OF NORADRENALINE IN HUMANS: DEFECTIVE UPTAKE IN SOME PATIENTS WITH ESSENTIAL HYPERTENSION

1. Disappearance of tritiated noradrenaline from plasma, after infusion to steady-state, was studied to assess neuronal uptake of noradrenaline in essential hypertension. 2. Plasma tritiated noradrenaline disappearance was biexponential. Rapid removal was dependent on neuronal uptake, being slowed both in normal subjects after desipramine, and in patients with sympathetic nerve dysfunction (autonomic insufficiency). 3. In nine of thirty-eight hypertensive patients the t1½ similarly was prolonged. Endogenous noradrenaline escaping uptake after release, and spilling over into plasma, was increased in these patients. 4. Defective neuronal uptake of noradrenaline, by exposing adrenergic receptors to high local transmitter concentration, may be important in the pathogenesis of essential hypertension in some patients.     

Effect of cocaine and related drugs on the uptake of noradrenaline by heart and spleen

Noradrenaline uptake by heart and spleen after intravenous infusion of noradrenaline was measured in the pithed rat. Cocaine, given before the infusion, inhibited the noradrenaline uptake in relation (a) to the dose administered and (b) to the amount of noradrenaline infused. There was an association between increase in the pressor response to a test dose of noradrenaline and inhibition of the uptake by the heart. Drugs related chemically to cocaine, such as α-cocaine, amethocaine, and atropine, did not alter the noradrenaline uptake or potentiate the blood pressure response to noradrenaline. The noradrenaline uptake by the heart was unchanged after dibenamine, but blocked by the dichloro-analogue of isoprenaline. It was concluded that cocaine specifically prevented the uptake of noradrenaline by tissues, thus increasing the amount of noradrenaline available for combination with adrenergic receptors. The dichloro-analogue of isoprenaline appeared to block both uptake by the heart and the combination with receptors.     

Dissociation of potentiation of isoprenaline by cocaine from inhibition of uptake in cat spleen

1. Cocaine hydrochloride, 10 mug/ml, potentiated isoprenaline and noradrenaline in isolated spleen strips from normal cats and from cats treated with reserpine 24 h previously.2. Isoprenaline was taken up almost as well as noradrenaline by spleen strips from cats treated with reserpine.3. Cocaine blocked uptake of noradrenaline but did not reduce uptake of isoprenaline. Drug concentrations used in these studies were the same as in potentiation experiments.4. It is concluded that inhibition of uptake is not the mechanism by which cocaine potentiates the effect of isoprenaline on the spleen and might be only a contributory factor in the case of noradrenaline potentiation.     

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     

Disposition of endogenous adrenaline compared to noradrenaline released by cardiac sympathetic nerves in the anaesthetized dog

Summary The fate of adrenaline released from cardiac sympathetic nerves was compared with that of noradrenaline before and during two periods of electrical stimulation of the left ansa subclavia in eight anaesthetized dogs. Cardiac spillovers and extractions of both catecholamines were estimated simultaneously using infusions of ³H-labelled adrenaline and noradrenaline. Animals were studied before and after neuronal uptake blockade with desipramine.Cardiac spillover of adrenaline, detectable at rest at 1.4 ± 0.3 pmol/min, increased to 4.0 ± 1.1 and 5.3 ± 1.2 pmol/min during sympathetic stimulation. Cardiac noradrenaline spillover increased from 49 ± 12 to 205 ± 40 and 451 ± 118 pmol/min. After desipramine, cardiac spillovers of adrenaline were decreased, whereas those of noradrenaline were increased so that the ratio of adrenaline to noradrenaline spillover, meaned before and during stimulation, decreased substantially from 1:42 to 1:166. The desipramine-induced decrease in cardiac extractions of ³H-labelled catecholamines indicated adrenaline was removed 60% less efficiently than noradrenaline by neuronal uptake, whereas the extractions remaining indicated adrenaline was removed 50% more efficiently by extraneuronal uptake.The differences in removal processes indicated that 35% of the adrenaline released by cardiac sympathetic nerves was recaptured compared to 88% for noradrenaline, leaving 53% to be removed extra-neuronally compared to 6.6% for noradrenaline, so that proportionally more released adrenaline than noradrenaline escaped to spillover into plasma (12% versus 5.4%). Since extraneuronal uptake was more efficient for adrenaline than noradrenaline, proportionally less released adrenaline than noradrenaline escaped local removal to spillover into plasma when neuronal uptake was blocked (17% versus 45%). This reversed the situation before blockade so that desipramine substantially decreased the ratio of adrenaline to noradrenaline spillover. Thus, differences in the efficiencies of neuronal or extraneuronal uptake are important determinants of the amounts of locally released adrenaline and noradrenaline that escape removal processes to act at neuroeffector sites or spillover into plasma.     

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.     

Errors in the measurement of agonist potency-ratios produced by uptake processes: a general model applied to beta-adrenoceptor agonists.

1. The sensitization of guinea-pig atria and trachea to noradrenaline, isoprenaline, and salbutamol, produced by an inhibitor of neuronal (cocaine) and extraneuronal (metanephrine) uptake, was studied quantitatively. The data were compared to a theoretical model. 2. Cocaine produced near maximal sensitization to noradrenaline in guinea-pig atria (5 fold) at concentrations which produced only partial sensitization in guinea-pig trachea (4.7 fold sensitization of a maximum 11 fold). These results agreed with the model which predicts that there is a direct relationship between the amount of uptake inhibitor required to produce full sensitization and the magnitude of maximal sensitization demonstrable in the tissue. This makes extrapolation of uptake inhibition concentrations from tissue to tissue a potentially erroneous practice. 3. In normal trachea, salbutamol is 20 times more potent than noradrenaline but this difference is abolished (to 0.9 times) by cocaine (100 microM). This reduction of potency-ratio is due to the selective cocaine-induced sensitization of trachea to noradrenaline and raises a serious objection to the classification of salbutamol as a beta 2 selective agonist. 4. Metanephrine produced very little sensitization of trachea to isoprenaline. Experiments with salbutamol showed metanephrine to be a simple competitive antagonist of beta-adrenoceptors (pKb = 4.3) and that this receptor antagonism masked sensitization to isoprenaline. 5. A theoretical model indicates that an inhibitor of agonist uptake requires a remarkable degree of selectivity for the uptake mechanism (i.e. Kb for receptors 10(4) x KI for uptake sites) to demonstrate tissue sensitization to the agonist. This analysis and the data with metanephrine indicate that a sinistral shift of the concentration-response curve is a poor indicator of the importance of uptake mechanisms in an isolated tissue. 6. An alternate method to determine the importance of agonist-uptake effects on concentration-response curves is described which utilizes agonist potency ratios. Agonist potency ratios in guinea-pig atria and trachea showed that the bronchoselectivity demonstrated by salbutamol (with respect to isoprenaline) is reduced from 54 to 7.8 by metanephrine reflecting the importance of extraneuronal uptake in trachea.     

Modulation of the concentration of noradrenaline at the neuro-effector junction in human saphenous vein.

We studied the importance of neuronal and extraneuronal uptake and of the pre-junctional alpha-adrenergic feed-back mechanism for the junctional noradrenaline concentration in the human saphenous vein. All major metabolites of the enzymatic breakdown of noradrenaline were detected in the overflow of superfused veins loaded with [3H]-noradrenaline. The efflux of 3,4-dihydroxyphenylglycol (DOPEG) was drastically reduced in preparations labelled after neuronal uptake blockade indicating its neuronal origin; the other metabolites are formed extraneuronally since they behaved distinctly differently from DOPEG under several experimental conditions. Extraneuronal uptake followed by enzymatic breakdown removes the same amount of noradrenaline from the biophase during nerve activity as that diffusing intact out of the tissue, whereas neuronal uptake appears only half as effective since the overflow of intact noradrenaline increases by only 48% in the presence of desmethylimipramine (DMI). However, in preparations mounted for isometric tension recording, neuronal uptake blockade potentiated contractions to alpha-adrenergic activation, emphasizing the functional importance of the neuronal disposition mechanism. By contrast, no evidence was found for a hydrocortisone-sensitive extraneuronal uptake compartment, suggesting that extraneuronal removal may have little, if any, functional importance. During nerve stimulation, yohimbine increased the amount of labelled noradrenaline present in the superfusate, while exogenously added noradrenaline decreased it in the presence of cocaine. Thus, prejunctional alpha-adrenoceptors can modulate the junctional concentration of neurotransmitter in the human saphenous vein.     

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.     

The neuronal and extraneuronal uptake and metabolism of 3H-(−)-noradrenaline in the perfused rat heart

1. Hearts were obtained from reserpine-pretreated rats and perfused with 0.95 micron 3H(-)-noradrenaline. The rate of removal of 3H-noradrenaline from the perfusion fluid was measured (from the arterio-venous difference) as well as the rate at which the 3H-metabolites appeared in the venous effluent. 2. When either 30micron corticosterone was added under steady-state conditions during perfusion with 3H-noradrenaline (to inhibit neuronal and extraneuronal uptake, respectively), each inhibitor reduced the removal of noradrenaline by about 50%; in the presence of both inhibitors removal was abolished. 3. Dihydroxymandelic acid (DOMA) was of neuronal, normetanephrine (NMN) of extraneuronal origin; dihydroxyphenylglycol (DOPEG) and the OMDA fraction (containing methoxyhydroxyphenylglycol-MOPEG-and methoxyhydroxymandelic acid-VMA) were formed both neuronally and extra-neuronally. 4. The extraneuronal metabolism of 3H-noradrenaline was in quick equilibrium with the 3H-noradrenaline in the perfusion fluid; most of the total formation of DOPEG, MOPEG and NMN was recovered from the venous effluent. 5. Extraneuronally formed DOPEG, MOPEG and NMN distributed in the tissue with half times corresponding to their half time for efflux. 6. Inhibition of monoamine oxidase (MAO) by pargyline increased the extraneuronal formation of NMN; MAO and catechol-O-methyl transferase (COMT) appear to be contained in the same extraneuronal compartment. 7. The extraneuronal accumulation of 3H-noradrenaline required 30 min or more to reach a steady state; inhibition of one or both enzymes slowed this process. Inhibition of MAO increased the extra-neuronal accumulation of 3H-noradrenaline; inhibition of COMT failed to do so, since the enzyme inhibitor (U-0521) was a weak inhibitor of extra-neuronal uptake. 8. The rate constants for the efflux of the metabolites of noradrenaline decreased in the order of MOPEG greater than DOPEG greater than NMN greater than DOMA greater than VMA.     

Reversible inhibition of neuronal uptake by benextramine, an irreversible presynaptic alpha-adrenoceptor antagonist

Benextramine, a covalently binding alpha-adrenoceptor blocking agent, potentiated the action of noradrenaline but not isoprenaline in guinea pig isolated right atria. This potentiation was probably caused by inhibition of neuronal uptake. When the benextramine was washed from the tissues for 60 min, no potentiation of the action of noradrenaline was observed. This easily reversed inhibition of neuronal uptake by benextramine contrasts with the effects of desipramine and phenoxybenzamine because the potentiating effect of these drugs was unaffected by 60 min of washing. The presence of benextramine also caused a small tachycardia in both rabbit and guinea pig right atria which was probably due to the release of endogenous noradrenaline. Clonidine a presynaptic alpha 2-adrenoceptor agonist, inhibited the responses to electrical field stimulation. Pretreatment with benextramine greatly diminished the effect of clonidine. This alpha 2-adrenoceptor antagonism was not reversed by washing the benextramine from the tissue for 240 min. We conclude that benextramine is a readily reversible inhibitor of neuronal uptake and an irreversible antagonist of presynaptic alpha 2-adrenoceptors.     

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