E. Muscholl

Die Noradrenalin-Abgabe aus dem isolierten Kaninchenherzen bei sympathischer Nervenreizung und ihre pharmakologische Beeinflussung

OUTSCHOOR~ U. VOOT wiesen 1952 nach, dab nach Reizung der Nn. accelerantes des Hundes die Konzentration yon Noradrenalin im Coronarvenenblut ansteigt. Wir suchten nach einer MSglichkeit, auch am isolierten Herzen dutch sympathische Reizung eine genfigend groBe Abgabe von Noradrenalin in die Perfusionsflfissigkeit hervorzurufen, um die Wirkung von Pharmaka auf diesen Vorgang untersuchen zu kSnnen. Das sympathisch innervierte, isolierte Iterz des Kaninchens lieB sich auf iihnliche Weise wie das sympathisch innervierte, isolierte Vorhofpri~parat (Husov~8) herstellen. Es besitzt folgende Vorteile: Technisch einfache DurchstrSmung, nach der hohen Noradrenalin-Konzentration zu urteilen eine reichliche sympathische Innervation und die MSglichkeit, den sympathischen Reizerfolg auf Kontraktionsamplitude, Herzfrequenz und CoronardurchfluB zu der abgegebenen Noradrenalin-Menge in Beziehung setzen zu kSnnen. Die Noradrenalin-Abgabe aus dem Herzen in die Perfusionsflfissigkeit entspricht jedoch nicht ganz der eigentlichen Noradrenalin-Freisetzung an den sympathischen Nervenendigungen, da ein Tell der ~bertr~gersubstanz sofort nach der Freisetzung wieder yon dem Gewebe aufgenommen wird, ohne in die Perfusionsflfissigkeit zu gelangen. Nach den Untersuchungen yon BRown u. GILLESPIE wird die bei sympathischer Reizung im Milzvenenblut der Katze auftretende Noradrenalin-Menge gesteigert, wenn die adrenergen Receptoren durch Phenoxybenzamin blockiert sind. Die

A muscarinic inhibition of the noradrenaline release evoked by postganglionic sympathetic nerve stimulation.

Summary1.The noradrenaline output from isolated rabbit hearts perfused with Tyrode solution was estimated fluorimetrically. The postganglionic sympathetic nerves of the heart were stimulated (10 shocks/sec; 1 msec) for three 1 min periods with intervals of 10 min.2.The noradrenaline output evoked by 3 consecutive stimulation periods decreased exponentially.3.Acetylcholine (10−9–10−6 g/ml) administered continuously one min before to one min after the second stimulation caused a dose-dependent reduction of the noradrenaline output evoked by the second stimulation to as low as 19% of the normal value. Acetylcholine in the concentrations applied did not cause a noradrenaline output by itself.4.The inhibitory action of acetylcholine 10−6 g/ml was fully antagonized by atropine 10−6 g/ml, whereas hexamethonium 3×10−6 g/ml had no significant antagonistic effect.5.The noradrenaline output caused by nerve stimulation was not decreased in the presence of DMPP 10−6 g/ml. DMPP 10−5 g/ml applied 3 min before electrical nerve stimulation caused an output of noradrenaline for 2 min but did not inhibit the noradrenaline release by nerve stimulation.6.Tyramine 5×10−6 g/ml was administered to the rabbit heart for two 6 min periods at an interval of 15 min. Methacholine 7.4×10−5 g/ml or atropine 1−6 g/ml if present during the second tyramine infusion did not alter the noradrenaline output produced by tyramine.7.It is concluded that low concentrations of acetylcholine by stimulating muscarinic inhibitory receptors interfere with the noradrenaline release from the postganglionic sympathetic nerve fibres evoked by electrical nerve stimulation. The possibility of a peripheral direct interaction of the cholinergic with the adrenergic nervous system is discussed.

Die Wirkung von Pharmaka auf die Elimination von Noradrenalin aus der Perfusionsflssigkeit und die Noradrenalinaufnahme in das isolierte Herz

In den letzten Jahren wurde in einer Reihe yon Arbeiten aus verschiedenen Laboratorien gezeigt, dab in die Zirkulation gelangtes Noradrenalin in sympathisch innervierte Gewebe aufgenommen wird. Definiert und experimentell bestimmt wurde diese Aufnahme entweder als Zunahme der Noradrenalinkonzentration des Gewebes gegeniiber der Konzentration des endogenen Noradrenalins nach Verabreichung yon L-Noradrenalin (MvscHOLL 1960, 1961; STROMBLAD U. NICKERSO~; CAMrOS u. Sm-DV~MAN) oder als Konzentration von tt3-Noradrenalin im Gewebe nach Verabreichung von D,L-HS-Noradrenalin (Wm~rBY, AXELROD U. WEIL-MALHERBE; HERTTING, AXELROD U. WttITBY). In einigen Versuchen mit H3-Noradrenalin wurden auch spezifische Aktivit~ten von Noradrenalin im Gewebe mitgeteilt (HV, RTTI~G U. Hv, SS; POTTS, R U. A X ~ O D ) . IVV,~SS, N mal~ am perfundierten Rattenherzen die spezifische Aktivit~t des au/genommenen Noradrenalins und erfaBte dadurch die GrSl3e des Austausches yon H3-Noradrenalin mit dem endogenen Noradrenalin. Danach dfirfte bei den Konzentrationsmessungen yon H3-Noradrenalin in Geweben in den Versuchen der Arbeitsgruppe yon AXELROD im wesentlichen eine Nettoaufnahme erfal~t worden sein. Eine zweite Methode zur Bestimmung der Noradrenalinaufnahme besteht darin, die arteriovenSse Differenz der Noradrenalinkonzentration bei einem yon Blur oder einer PerfusionslSsung durchstrSmten Organ zu messen (SI]sGs.L, GILMORV, U. SA~NOrF; Ln~DMA~ U. MUSCHOLL 1962;

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

SummaryIsolated 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.

Dopaminergic modulation of evoked vasopressin release from the isolated neurohypophysis of the rat

Summary1.Neurointermediate lobes of rat pituitaries were incubated in Locke or Krebs solution, and the vasopressin released into the medium was assayed on the blood pressure of the pithed rat or by a radioimmunological procedure. Release of vasopressin over resting levels was evoked either by incubation with 60 mM KCl (high K) solution or by electrical stimulation of the pituitary stalk. Two different kinds of electrical stimulation were carried out. Procedure A (1 ms, 10 Hz, 5 times for 1 min within 10 min) induced a vasopressin overflow which was greatly calcium-dependent but only insignificantly sensitive to tetrodotoxin (TTX). Procedure B (0.2 ms, 15 Hz, 10 s trains with 10 s intervals for 10 min) evoked a completely calcium-dependent and TTX-sensitive vasopressin overflow.2.The vasopressin output evoked by high K was unaltered in the presence of dopamine 10 μM or apomorphine 100–300 μM.3.The vasopressin overflow evoked by stimulation procedure A was decreased by 50–67% in the presence of apomorphine 10 μM. However, the dopamine antagonists sulpiride 1–100 nM and flupenthixol 10 μM also inhibited the vasopressin release by 30–40%. Nevertheless, the combination of apomorphine 10 –M with sulpiride 1 nM or flupenthixol 10 μM resulted in a significant increase of the vasopressin release if compared with apomorphine (or sulpiride) alone. Thus, apomorphine antagonized the inhibition of the vasopressin release by the dopamine antagonists, and these drugs blocked the inhibitory action of apomorphine, indicating complex but specific agonist-antagonist interactions.Naloxone 1 μM which failed to change vasopression overflow, prevented the inhibitory effect of sulpiride 100 nM. This indicates that the inhibition of vasopressin release by sulpiride may be mediated by an increased secretion of endogenous opioids probably from the intermediate lobe.4.The vasopressin overflow evoked by stimulation procedure B was decreased by 20% in the presence of apomorphine 100 nM or 1 μM. Bromocriptine had a biphasic effect on vasopressin release, stimulating it by 20% at 1 nM and inhibiting it by 35% at 3 μM. Sulpiride 10 nM to 1 μM stimulated the vasopressin release by about 20% and antagonized the inhibitory actions of apomorphine and bromocriptine in a concentration-dependent manner. However, sulpiride 100 nM had no effect on the facilitatory action of bromocriptine 1 nM.5.These findings suggest a complex dopaminergic modulation, in the neurointermediate lobe, of vasopressin release evoked by intermittent electrical stimulation, and support a physiological role for the intrinsic dopaminergic fibres described by others. We propose the existence of two dopamine receptors, one mediating inhibition of vasopressin release (activated by apomorphine, bromocriptine at high concentrations or endogenous dopamine, and blocked by sulpiride), and the other one mediating facilitation of release (activated by endogenous dopamine, and blocked by flupenthixol). It is further suggested that under the field-stimulation conditions of procedure A an endogenous opioid is secreted which inhibits vasopressin release and which itself is under inhibitory control by endogenous dopamine.

Unterschiede zwischen Tyramin und Dimethylphenylpiperazin in der Ca++-Abhängigkeit und im zeitlichen Verlauf der Noradrenalin-Freisetzung am isolierten Kaninchenherzen

On the perfused rabbit heart a constant infusion of tyramine released noradrenaline continuously and independently of the external Ca++ concentration. In contrast, noradrenaline release by DMPP was only transient and required the presence of Ca++.

The effect of dopamine on the overflow of endogenous noradrenaline from the perfused rabbit heart evoked by sympathetic nerve stimulation.

Summary1.The effects of dopamine and two dopamine receptor antagonists (pimozide, flupenthixol) on the release of endogenous noradrenaline evoked by electrical stimulation of the postganglionic sympathetic nerves and their influence on cardiac performance were measured in isolated perfused rabbit hearts.2.Dopamine 0.2μM decreased noradrenaline overflow and ventricular tension development in response to nerve stimulation.3.Dopamine 2μM increased spontaneous noradrenaline output and tension development. The noradrenaline overflow in response to nerve stimulation was greatly enhanced. This action was only partly reversed by wash out of exogenous dopamine, indicating de novo synthesis and facilitated release of noradrenaline.4.Pimozide (approximately 1μM) and flupenthixol 5μM had no effect on noradrenaline overflow and cardiac peroormance.5.In the presence of cocaine 18.3μM dopamine 2μM decreased the overflow of noradrenaline. This effect was fully antagonized by flupenthixol which on its own neither influenced transmitter overflow nor antagonized the presynaptic inhibitory effect of oxymetazoline 0.38μM.6.It is concluded that in the rabbit heart dopamine has an inhibitory effect on the release of endogenous noradrenaline which appears to be, mediated by presynaptic dopamine receptors and which differs from the prejunctional α-adrenoceptor mediated negative feedback mechanism.

The effects of several muscarinic antagonists on pre- and postsynaptic receptors in the isolated rabbit heart

SummaryIn order to reveal possible differences between pre- and postsynaptic muscarine receptors, seven antagonists were tested for their affinities on these receptor sites in the rabbit isolated perfused heart. Methacholine was used as an agonist to inhibit the noradrenaline overflow evoked by electrical stimulation (3 Hz, 3 min) of the sympathetic nerves (presynaptic parameter) and to decrease the systolic tension development of the right atrium (postsynaptic parameter). The affinity of an antagonist was expressed as pA2.A decreasing order of potency was obtained with ipratropium, scopolamine, atropine, trihexyphenidyl, amitriptyline, and gallamine, both for pre- and postsynaptic responses. The antagonists acted competitively and their effects were reversible. Furthermore, for none of the drugs did the pA2 (pre) differ from the pA2 (post).With QNB (3-quinuclidinyl benzilate) a pA2 (post) of 11.65 was obtained. However, the affinity to presynaptic receptors could not be determined as a pA2 value due to the very prolonged exposure time required for the equilibrium with QNB and for that with methacholine in the presence of QNB.It is concluded that the antagonists employed do not reveal differences between pre- and postsynaptic muscarine receptors of the rabbit heart, in spite of their greatly varying chemical structure and their individual affinities ranging over 5 orders of magnitude. The findings confirm the view of a homogeneous muscarine receptor population characterized by functional parameters.

Die Konzentration von Noradrenalin und Adrenalin in den einzelnen Abschnitten des Herzens

1. A method for the assay of noradrenaline and adrenaline in small pieces of heart tissue is described. The amines were extracted with acid ethanol and separated by paper chromatography. The regions containing the amines were eluted and the amount of amines in the eluates was determined by bioassay. Noradrenaline was assayed on the blood pressure of the pithed rat. Adrenaline was assayed on the isolated atropinized rats uterus stimulated with oxytocin. 2. The identity of the substances tested by bioassay with noradrenaline and adrenaline was confirmed by a number of chemical and pharmacological tests. 3. The concentration of catechol amines in different parts of the heart was studied in cats, rabbits, guinea pigs, and rats. In all species investigated, the noradrenaline concentration was much higher in the right atrium than in the left atrium and higher in the right ventricle than in the left ventricle. Approximately 4% of the total amines consisted of adrenaline. 4. The small percentage of adrenaline indicates that the heart of these species contains only little chromaffine tissue. The high concentration of noradrenaline in the right atrium is thought to be mainly due to the presence of adrenergic fibres.Summary1.A method for the assay of noradrenaline and adrenaline in small pieces of heart tissue is described. The amines were extracted with acid ethanol and separated by paper chromatography. The regions containing the amines were eluted and the amount of amines in the eluates was determined by bioassay. Noradrenaline was assayed on the blood pressure of the pithed rat. Adrenaline was assayed on the isolated atropinized rats uterus stimulated with oxytocin.2.The identity of the substances tested by bioassay with noradrenaline and adrenaline was confirmed by a number of chemical and pharmacological tests.3.The concentration of catechol amines in different parts of the heart was studied in cats, rabbits, guinea pigs, and rats. In all species investigated, the noradrenaline concentration was much higher in the right atrium than in the left atrium and higher in the right ventricle than in the left ventricle. Approximately 4% of the total amines consisted of adrenaline.4.The small percentage of adrenaline indicates that the heart of these species contains only little chromaffine tissue. The high concentration of noradrenaline in the right atrium is thought to be mainly due to the presence of adrenergic fibres.

Muscarinic inhibition of potassium-induced noradrenaline release and its dependence on the calcium concentration

Summary1.Noradrenaline release from the isolated rabbit heart was evoked by perfusion with a medium containing 135 mM potassium and 17 mM sodium ions (high K+-low Na+).2.The noradrenaline output in response to high K+-low Na+ was dose-dependently decreased by methacholine (0.625–320 μM) and this effect was reserved by atropine 1.44 μM.3.Lowering the calcium concentration of high K+-low Na+ from 1.8–0.1125 mM decreased the noradrenaline output by 85%. The effect of methacholine, expressed as % inhibition of noradrenaline release, was potentiated by lowering of the calcium concentration.4.Both at normal and lowered calcium concentrations the inhibitory action of methacholine was larger from 0–5 than from 5–10 min after perfusion with high K+-low Na+.5.Perfusion of hearts with media containing high K+-high Na+ or normal K+-low Na+ caused noradrenaline outputs somewhat smaller than those after high K+-low Na+. The release from 0–5 min was both calcium-dependent and inhibited by methacholine.6.High K+ and/or low Na+ solutions caused an increase in coronary perfusion pressure which was little affected by the noradrenaline released simultaneously.7.It is concluded that activation of muscarine receptors at the terminal adrenergic fibre decreases the availability of calcium for transmitter release.