Atropine-resistant effects of the muscarinic agonists McN-A-343 and AHR 602 on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart

1 The effects of 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) and N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602) on cardiac performance and noradrenaline release from terminal sympathetic fibres were measured in isolated perfused hearts of rabbits.

2 In the presence of sufficient atropine to block muscarinic receptors, high concentrations of McN-A-343 and AHR 602 caused no cardiac stimulation and there was no increase in the resting output of noradrenaline into the perfusates.

3 McN-A-343 and AHR 602 increased both the mechanical responses and the transmitter overflow evoked by electrical stimulation of the sympathetic nerves (SNS) but inhibited both parameters during perfusion with 1,1-dimethyl-4-phenylpiperazinium (DMPP). The effects were atropine-resistant and qualitatively similar to those seen with cocaine. Hexamethonium inhibited DMPP, but affected neither SNS per se nor the facilitatory effects of McN-A-343 and AHR 602 on SNS.

4 McN-A-343, cocaine and desipramine (but not AHR 602 or hexamethonium) blocked the net cardiac noradrenaline uptake and increased the positive chronotropic effect of noradrenaline.

5 Prior perfusion with concentrations of cocaine and desipramine sufficient to block uptake reduced or abolished the facilitatory effects of both McN-A-343 and AHR 602 on SNS.

6 Cocaine, McN-A-343 and AHR 602 displayed local anaesthetic properties on the guinea-pig wheal and frog nerve plexus tests, and their relative potencies in this respect were similar to those for inhibition of DMPP-evoked transmitter overflow. Hexamethonium did not produce local anaesthesia.

7 The results indicate that the facilitated release of noradrenaline after SNS and the inhibition of release after DMPP produced by McN-A-343 and AHR 602 are the result of their combined local anaesthetic action and inhibition of amine uptake.

     

Diet-induced atherosclerosis inhibits release of noradrenaline from sympathetic nerves in rabbit arteries

Contractile responses to sympathetic nerve stimulation and exogenous noradrenaline were compared in aortas and pulmonary arteries of control rabbits and rabbits fed a cholesterol-rich diet (0.3%) for 16 or 30 weeks. The diet-induced atherosclerosis reduced the contractions to increasing concentrations of exogenous noradrenaline (0.1 nM to 10 μM) in both arteries, and the reduction was more pronounced after 30 weeks of the hypercholesterolemia. The contractions produced with increasing frequencies of electrical stimulation (1–32 Hz) were nearly abolished in the atherosclerotic arteries. Labeling of the aorta and the pulmonary arteries with [³H]noradrenaline resulted in accumulation of radioactivity in both control and atherosclerotic blood vessels. After mounting the labeled blood vessels for superfusion, a basal efflux of [³H]noradrenaline and of ³H-metabolites was detected. In the atherosclerotic arteries, a decreased efflux of the intraneuronal deaminated metabolites 3,4-dihydroxyphenyl glycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) was detected. Electrical stimulation at 1 Hz (pulmonary artery) or 2 Hz (aorta) caused an augmented efflux of total ³H from the control arteries; this was mostly due to release of intact [³H]noradrenaline. The electrical imp ulses evoked significantly less (16 weeks) or no (30 weeks) release of [³H]noradrenaline in the atherosclerotic arteries. These data illustrate that diet-induced atherosclerosis exerts an inhibitory action on the sympathetic nerve terminals in the aorta and the pulmonary artery of the rabbit. This effect, together with an inhibitory effect at the postjunctional level results in a loss of the responsiveness to nerve stimulation. The atherosclerotic process also inhibits the intraneuronal deamination of the sympathetic transmitter.     

Prejunctional muscarinic receptor modulation of noradrenaline release from sympathetic neurones in rabbit aorta

The prejunctional muscarinic modulation of stimulation-evoked release of 3H-noradrenaline from sympathetic neurones in rabbit aorta was examined. The role of transmitter uptake, alpha-adrenoceptor blockade, stimulation frequency and endothelium on the modulation was investigated. Rings of aorta were incubated with (-)-3H-noradrenaline and subsequently subjected to electrical-field stimulation. Fractional 3H-overflow was determined by liquid scintillation counting. Acetylcholine (10(-8)-3 x 10(-6) M) added cumulatively, reduced the stimulation-evoked 3H-overflow up to 80%. The effect of acetylcholine was the same in intact and endothelium-free aorta. The inhibitory effect of acetylcholine was inversely related to the frequency of stimulation (1-10 Hz). The maximal inhibition (%) was 80 (1 Hz), 53 (3 Hz) and 14 (10 Hz). The inhibitory effect of acetylcholine (10(-6) M) and carbachol (10(-5) M) reached a maximum 15 min. after addition and then remained almost constant. Cocaine (3 x 10(-5) M) did not alter the effect of acetylcholine. Desipramine (10(-6) M) and corticosterone (4 x 10(-5) M) attenuated the inhibition seen with low concentrations (10(-8)-10(-7) M) of acetylcholine. The acetylcholine-induced inhibition was antagonized by desipramine. Cocaine plus corticosterone attenuated the inhibition seen with high concentrations (10(-6)-3 x 10(-6) M) of acetylcholine. Rauwolscine (10(-6) M) enhanced the maximal inhibitory effect of acetylcholine. We conclude that the inhibitory effect of acetylcholine on 3H-overflow from rabbit aorta preloaded with 3H-noradrenaline is (1) inversely related to stimulation frequency; (2) independent of endothelium; (3) unaffected by neuronal and extraneuronal transmitter uptake; (4) that cocaine is not a prejunctional muscarinic antagonist; (5) that cocaine, but not desipramine, is suited as a neuronal uptake inhibitor in studies of prejunctional muscarinic receptor subtypes; and (6) and that there is an inverse interaction between prejunctional alpha2-adrenoceptors and muscarinic receptors.     

The effect of methacholine on noradrenaline release from the rabbit heart perfused with indometacin

Summary The experiments were undertaken in order to study the effect of inhibition of prostaglandin synthesis on the muscarinic inhibition of noradrenaline release evoked by sympathetic nerve stimulation. The right sympathetic nerves of the perfused rabbit heart were stimulated electrically. The noradrenaline output was enhanced after perfusion of the hearts with indometacin 3×10^–5 M indicating blockade of the prostaglandin-mediated negative feedback control. Both in the presence and in the absence of indometacin methacholine 4×10^–5 M decreased the noradrenaline output by a similar percentage. It is concluded that the muscarinic inhibition of noradrenaline release does not require the functional integrity of the prostaglandin-mediated feedback system.     

Mibefradil- and ω-conotoxin GVIA-induced inhibition of noradrenaline release from the sympathetic nerves of the human heart

Segments of human right atrial appendages preincubated with [3H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone were used to determine the effects of mibefradil, ω-conotoxin (ω-CTx) GVIA and nifedipine on tritium overflow evoked by transmural electrical stimulation. Mibefradil (which predominantly blocks T-type, and at lower potency also N-type, Ca²⁺ channels) at concentrations of 0.3–3μM reduced the electrically evoked tritium overflow in a reversible and concentration-dependent manner (IC_50%: 1μM), whereas 0.1–10μM nifedipine (a selective blocker of L-type channels) was ineffective. The evoked tritium overflow was almost abolished by 0.2μM ω-CTx GVIA (a selective blocker of N-type channels). It is concluded that noradrenaline release from cardiac sympathetic nerves is triggered by Ca²⁺-influx via N-type, but not L-type, Ca²⁺ channels and that the inhibitory effect of mibefradil at clinically relevant concentrations on noradrenaline release is probably due to its blocking action on N-type Ca²⁺ channels. This property of mibefradil is unique among the therapeutically applied Ca²⁺ channel blockers and may contribute to the slight negative chronotropic effect of the drug in vivo. Received: 24 September 1997 / Accepted: 3 November 1997     

Effects of presynaptic modulators on Ca2+-induced noradrenaline relase from cardiac sympathetic nerves

Summary In order to elucidate the mode of action of the Ca²⁺-antagonistic inhibitor nifedipine, its effect on Ca²⁺-mediated action potentials and transmembrane slow inward current in papillary muscles of guinea pigs and cats was studied.Nifedipine (0.5 mg/l1.4×10^–6M) depressed upstroke velocity and overshoot of the Ca²⁺-mediated action potential and reduced the transmembrane slow inward current by about 50%, but the kinetics of inactivation and recovery from inactivation were not affected. The decrease of upstroke velocity was accompanied by a proportional diminution of isometric contractile force. This indicates that nifedipine exerts its Ca²⁺-antagonistic effect on excitation-contraction coupling in mammalian ventricular myocardium by inhibition of the transmembrane Ca²⁺ inward current. The inhibitory action of nifedipine on contractile tension development could be neutralized by an augmentation of the extracellular Ca²⁺ concentration from 2 mM to 4 mM or by -receptor stimulation (isoproterenol) that promotes the transmembrane Ca²⁺-rich medium or under the influence of isoproterenol the upstroke velocity of the Ca²⁺-mediated action potentials rose even above the initial values which were measured prior to the nifedipine administration.     

Interactions of inhibitors of noradrenaline uptake and angiotensin on the sympathetic nerves of the isolated rabbit heart

1. The interaction of angiotensin and several inhibitors of the uptake of noradrenaline across the neuronal membrane (cocaine, desipramine, protriptyline. and pronethalol) on the output of noradrenaline produced by sympathetic nerve stimulation has been studied in the isolated perfused rabbit heart.2. Most of these drugs increased noradrenaline outflow-angiotensin, for example, by 175%. Cocaine (10(-4)M) did not change the amine overflow, probably because this very high concentration inhibited not only the re-uptake but also the liberation of noradrenaline.3. Desipramine, protriptyline, and pronethalol, although infused in concentrations which enhanced the noradrenaline output, were not able to impair the angiotensin-induced increase of transmitter overflow. In the presence of cocaine (10(-4)M) the increase elicited by angiotensin was slightly reduced, though lower concentrations of cocaine, as previously described, do not interfere with the effect of angiotensin.4. In contrast to the interaction between uptake inhibitors and angiotensin, the augmented output of noradrenaline caused by an uptake inhibitor could not be increased further by infusion of a second uptake inhibitor.5. It is concluded that the increase of the outflow of noradrenaline during sympathetic nerve stimulation by small doses of angiotensin is not caused by an inhibition of re-uptake. On the contrary, the transmitter liberation seems to be facilitated. This is a novel principle of drug action on the sympathetic nerve terminals.     

Sodium-azide-evoked noradrenaline and catecholamine release from peripheral sympathetic nerves and chromaffin cells

1. The spontaneous release of [3H]noradrenaline [( 3H]NA) has been measured from rabbit pulmonary arteries and bovine chromaffin cells in the presence of neuronal uptake blocker cocaine (3 x 10(-5) M). 2. The Na+-pump inhibitor sodium-azide (NaN3, 2mM) produced a moderate increase of [3H]NA release from both preparations and relaxed the arteries. The [3H]releasing action of NaN3 was accompanied by a 30% inhibition of 86Rb-uptake into chromaffin cells. 3. In both preparations, ouabain (10(-4) M) markedly increased the release of [3H], contracted the arteries and inhibited the 86Rb-uptake of chromaffin cells by about 75%. A combined application of NaN3 and ouabain produced a similar inhibition of 86Rb-uptake of chromaffin cells and failed to increase further the release of [3H] in comparison to that found in response to ouabain alone. 4. Removal of K+ from the external medium increased both the release of [3H]NA and the tone of pulmonary arteries. NaN3 further increased the transmitter release in "K+-free" solution but relaxed the muscle. In the absence of external K+ and in the presence of azide, ouabain further enhanced the transmitter release but failed to produce significant contraction. 5. Reactivation of the Na+-pump by readmission of K+ (5.9 mM) to the external medium abolished the transmitter releasing action of NaN3 in arteries. 6. It is concluded that in peripheral sympathetic nerves and chromaffin cells, NaN3 inhibits the Na+-pump producing NA and CA release respectively and in nerves even if NA release had already been increased by K+-removal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of receptor-mediated noradrenaline release from the sympathetic nerves of the isolated rabbit heart by anaesthetics and alcohols in proportion to their hydrophobic property

The actions of anaesthetics (diethyl ether, enflurane, chloroform, methoxyflurane) and alcohols (ethanol, 1-propanol, 1-butanol, 1-pentanol) on the noradrenaline release from (and uptake into) the sympathetic nerve terminals were studied in isolated rabbit hearts perfused with Tyrode solution at constant flow rate. The noradrenaline in the perfusate was assayed spectrofluorimetrically.

Electrical events in cardiac adrenergic nerves and noradrenaline release from the heart induced by acetylcholine and KCl

Summary The isolated cat heart with intact sympathetic nerve supply was perfused with varying concentrations of acetylcholine, DMPP and KCl. The ensuing asynchronous discharge in cardiac sympathetic nerves was recorded and the noradrenaline liberated into the perfusate was measured.The infusion of acetylcholine for one minute at a relatively low concentration (5×10^–5 to 8×10^–5 g/ml) induced asynchronous firing over the entire period of infusion and an average liberation of 44 ng/min noradrenaline. Increasing the concentration of acetylcholine or adding atropine to the perfusion fluid greatly shortened the duration of the antidromic discharge but enhanced severalfold the amount of noradrenaline liberated.Within a narrow range of very low concentrations DMPP induced continuous firing. At higher concentrations antidromic discharges were restricted to the first few seconds of infusion. Muscarinic drugs such as pilocarpine and methacholine neither induced firing nor released noradrenaline. When added to acetylcholine, pilocarpine reduced the amplitude of acetylcholine-evoked firing and the amount of noradrenaline liberated.KCl in concentrations above 50 mM induced a very short-lasting firing and a considerable noradrenaline output which was concentration-dependent.It is concluded that the acetylcholine- and DMPP-induced action potentials by themselves contribute little to the noradrenaline-releasing activity of these drugs. As in the case of KCl, the sustained depolarization of the sympathetic nerve ending by acetylcholine and DMPP seems to enhance entry of Ca⁺⁺ into the ending, an essential factor for the liberation of noradrenaline.Preliminary results of this report have been communicated to the German Pharmacological Society at the Spring Meeting 1968 in Mainz.     

Trifluoperazine and calmidazolium have multiple actions on the release of noradrenaline from sympathetic nerves of mouse atria

The aim of this study was to determine whether the calmodulin inhibitors trifluoperazine (TFP) and calmidazolium (CMZ) could decrease the action-potential-evoked release of noradrenaline from mouse isolated atria incubated with [³H]-noradrenaline in support of the hypothesis that calmodulin is involved in neurotransmitter release.TFP (10 M and 30 M) significantly enhanced stimulation-induced (S-1) outflow of radioactivity from mouse atria but had no effect at 1.0 M or 70 M. TFP (70 M) also significantly increased the spontaneous outflow of radioactivity. The facilitatory effect of TFP (10 M) on S-I outflow of radioactivity persisted in either the presence of 3-isobutyl-1-methylxanthine (100 M) or atropine (0.3 M) indicating that this effect of TFP was not mediated through either inhibition of phosphodiesterases or through interference with presynaptic muscarinic receptors, respectively. In the presence of phentolamine, the facilitatory effect of TFP (10 M) on S-I outflow was reduced but there was no effect on S-I outflow at 70 M. However, in the presence of a combination of both phentolamine (l.0 M) and the neuronal uptake blocker desipramine (1.0 M) a significant inhibitory effect of TFP (70 M) on the S-I outflow of radioactivity was observed, indicating that effects of TFP on presynaptic a-adrenoceptors and neuronal uptake had disguised an inhibitory effect on S-1 noradrenaline release. Another inhibitor of the Ca²⁺-calmodulin complex, calmidazolium (CMZ, 10 M) inhibited the S-1 outflow of radioactivity but had no effect at 1.0 M. However, CMZ (10 M) also induced a concomitant increase in the spontaneous outflow of radioactivity. In the presence of both phentolamine (1.0 M) and desipramine (1.0 M), CMZ (10 M) also decreased S-1 outflow of radioactivity. The spontaneous outflow of radioactivity by calmidazolium (10 M) was mainly attributable to a rise in unmetabolized noradrenaline.Since concentrations of both TFP and CMZ, which inhibited S-1 noradrenaline release, also caused an increase in the spontaneous outflow of radioactivity, it is possible that the inhibitory effects on S-1 noradrenaline release may be secondary to changes in spontaneous outflow. This suggests that these drugs have complex effects on transmitter release dynamics which are perhaps due to multiple roles for calmodulin within the sympathetic nerve terminal. Correspondence to: M. Barrington at the above address     

Metabolism of endogenous and exogenous noradrenaline in the rabbit perfused heart

Summary The outflow of noradrenaline, 3,4-dihydroxyphenylglycol (DOPEG) and 3,4-dihydroxymandelic acid (DOMA) from rabbit perfused hearts was studied by chromatography on alumina followed by high pressure liquid chromatography with electrochemical detection. In the absence of drugs and without nerve stimulation, the outflow of endogenous noradrenaline over a period of 108 min averaged 0.17 pmol×g^–1×min^–1 and the outflow of DOPEG 2.1 pmol×g^–1×min^–1. The outflow of DOMA was below the detection limit (<0.13 pmol×g^–1×min^–1). The effect of perfusion with (–)-noradrenaline 0.1, 1 or 10 mol/l for 18 min was then investigated. As the concentration of noradrenaline increased so did the outflow of DOPEG. Moreover, DOMA was found in the venous effluent during and after perfusion with noradrenaline 1 or 10 mol/l. The increase in the outflow of DOPEG and DOMA was almost abolished when cocaine 10 mol/l was present during the perfusion with noradrenaline 1 mol/l. The release of endogenous noradrenaline by sympathetic nerve stimulation or tyramine 10 mol/l, but not the release evoked by nicotine 30 mol/l, was accompanied by an increase in the outflow of DOPEG; an outflow of DOMA was not observed.It is concluded that, in the rabbit perfused heart, DOPEG is an important metabolite of endogenous noradrenaline. DOMA is at best a minor product, either when the neurones are at rest or when noradrenaline is released by sympathetic nerve stimulation, nicotine or tyramine. DOMA is formed in detectable amounts when the tissue is exposed to a high concentration of exogenous noradrenaline. Like DOPEG, it is formed intraneuronally. The results confirm and extend those obtained previously on guinea-pig incubated atria. They make it unlikely that, in these tissues at least, DOMA formation is one of the physiological pathways of noradrenaline catabolism.     

ACTH increases noradrenaline release in the rabbit heart

Summary The effects of ACTH on the release of noradrenaline and the increase of heart rate produced by sympathetic nerve stimulation (1 Hz) were studied in isolated perfused rabbit hearts. ACTH-(1–24) 0.1–100 nmol/l increased the stimulation-evoked overflow of noradrenaline concentration-dependently, reversibly and up to two-fold. The basal outflow of noradrenaline, the basal heart rate and the stimulation-evoked increase in heart rate were not changed. Human ACTH-(1–39) also increased the evoked overflow of noradrenaline. The effect of ACTH-(1–24) 0.3 nmol/l persisted after blockade of -adrenoceptors with propranolol and blockade of neuronal catecholamine uptake by cocaine. ACTH-(1–24) 3 nmol/l did not change the removal of noradrenaline from the perfusion fluid, when hearts were perfused with medium containing 59 nmol/l noradrenaline. The results show that ACTH increases the action potential-evoked release of noradrenaline from cardiac postganglionic sympathetic neurones, probably by activating specific presynaptic ACTH receptors. The high potency of ACTH suggests that these presynaptic receptors may be activated in vivo by circulating ACTH under certain pathophysiological conditions.Send offprint requests to B. Szabo at the above address