Endogenous tachykinins cause bradycardia by stimulating cholinergic neurons in the isolated guinea pig heart

The purpose of this study was to determine if endogenous tachykinins can cause bradycardia in the isolated perfused guinea pig heart through stimulation of cholinergic neurons. Capsaicin was used to stimulate release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac afferents. A bolus injection of 100 nmol capsaicin increased heart rate by 26 +/- 7% from a baseline of 257 +/- 14 beats/min (n = 6, P < 0.01). This positive chronotropic response was converted to a minor bradycardic effect in hearts with 1 microM CGRP-(8-37) present to block CGRP receptors. The negative chronotropic response to capsaicin was markedly potentiated in another group of hearts with the further addition of 0.5 microM neostigmine to inhibit cholinesterases. In this group, capsaicin decreased heart rate by 30 +/- 10% from a baseline of 214 +/- 6 beats/min (n = 8, P < 0.05). This large bradycardic response to capsaicin was inhibited by 1) infusion of neurokinin A to desensitize tachykinin receptors or 2) treatment with 1 microM atropine to block muscarinic receptors. The latter observations implicate tachykinins and acetylcholine, respectively, as mediators of the bradycardia. These findings support the hypothesis that endogenous tachykinins could mediate axon reflexes to stimulate cholinergic neurons of the intrinsic cardiac ganglia.     

Substance P evokes bradycardia by stimulation of postganglionic cholinergic neurons.

Substance P (SP) evokes bradycardia that is mediated by cholinergic neurons in experiments with isolated guinea pig hearts. This project investigates the negative chronotropic action of SP in vivo. Guinea pigs were anesthetized with urethane, vagotomized and artificially respired. Using this model, IV injection of SP (32 nmol/kg/50 microl saline) caused a brief decrease in heart rate (-30+/-3 beats/min from a baseline of 256+/-4 beats/min, n = 27) and a long-lasting decrease in blood pressure (-28+/-2 mmHg from baseline of 51+/-5 mmHg, n = 27). The negative chronotropic response to SP was attenuated by muscarinic receptor blockade with atropine (-29 +/- 9 beats/min before vs -8 +/- 2 beats/min after treatment, P = 0.0204, n = 5) and augmented by inhibition of cholinesterases with physostigmine (-23 +/- 6 beats/min before versus -74 +/- 20 beats/min after treatment, P = 0.0250, n = 5). Ganglion blockade with chlorisondamine did not diminish the negative chronotropic response to SP. In another series of experiments, animals were anesthetized with sodium pentobarbital or urethane and studied with or without vagotomy. Neither anesthetic nor vagotomy had a significant effect on the negative chronotropic response to SP (F3,24 = 1.97, P = 0.2198). Comparison of responses to 640 nmol/kg nitroprusside and 32 nmol/kg SP demonstrated that the bradycardic effect of SP occurs independent of vasodilation. These results suggest that SP can evoke bradycardia in vivo through stimulation of postganglionic cholinergic neurons.     

The chronotropic action of neurotensin in the guinea pig isolated heart

Neurotensin (NT) infusions into isolated, perfused, spontaneously beating hearts of guinea pigs evoked a concentration-dependent, positive chronotropic effect which was preceded in some hearts by transient bradycardia. The tachycardia caused by NT was not affected by propranolol, cimetidine, indomethacin, a mixture of methysergide and morphine or by atria removal. The incidence and amplitude of bradycardia caused by NT were increased by neostigmine but reduced by atropine. Neostigmine and atropine also tended to decrease and increase respectively, the tachycardia caused by NT. These results suggest that the positive chronotropic effect of NT in guinea pig isolated heart results from a direct effect on the specialized conduction system of the heart while its negative chronotropic effect is likely to reflect the activation by NT of cardiac vagal cholinergic neurons.     

Significance of cardiac cannabinoid receptors in regulation of cardiac rhythm,myocardial contractility,and electrophysiologic processes in heart

Intravenous administration of cannabinoid (CB) receptor agonists (HU-210, 0.1 mg/kg; ACPA, 0.125 mg/kg; methanandamide, 2.5 mg/kg; and anandamide, 2.5 mg/kg) induced bradycardia in chloralose-anesthetized rats irrespective of the solubilization method. Methanandamide, HU-210, and ACPA had no effect on the electrophysiological activity of the heart, while anandamide increased the duration of the QRS complex. The negative chronotropic effect of HU-210 was due to CB1 receptor activation since it was not observed after CB1 receptor blockade by SR141716A (1 mg/kg intravenously) but was present after pretreatment with CB2 receptor antagonist SR144528 (1 mg/kg intravenously). CB receptor antagonists SR141716A and SR144528 had no effect on cardiac rhythm or ECG indices. Hence, in the intact heart, endogenous CB receptor agonists are not involved in the regulation of cardiac rhythm and electrophysiological processes. The chronotropic effect of CBs was independent of the autonomic nervous system because it remained significant after autonomic ganglion blockade by hexamethonium (1 mg/kg intravenously). CB receptor activation by HU-210 (0.1 and 1 μM) in vitro decreased the rate and force of isolated heart contractions, the rates of contraction and relaxation, and end diastolic pressure. The negative chronotropic effect of HU-210 was less pronounced in vitro than in vivo. The maximum inotropic effect of HU-210 was reached at the concentration of 0.1 μM.     

Capsaicin-evoked bradycardia in anesthetized guinea pigs is mediated by endogenous tachykinins

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (− 37 ± 7 beats/min, n = 6) that was followed by a more prolonged increase (+ 44 ± 10 beats/min). Blood pressure was lowered by − 11 ± 2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 µmol/kg) nearly abolished the bradycardic response (− 8 ± 3 beats/min, n = 7). Combined blockade of NK₂ and NK₃ receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (− 14 ± 3 beats/min, n = 4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.     

Sodium channel enhancer restores baroreflex sensitivity in conscious dogs with heart failure

We compared the cardiac inotropic, lusitropic, and chronotropic responses to the Na(+) channel enhancer LY-368052 in conscious dogs before and after development of congestive heart failure (CHF). We also examined the effect of LY-368052 on baroreflex sensitivity and the efferent neural mechanisms of the bradycardic response in heart failure. Dogs were chronically instrumented, and heart failure was induced by right ventricular pacing at 240 beats/min for 3-4 wk. LY-368052 dose-dependently increased left ventricular contractile performance before and after the development of CHF to a similar extent. The inotropic effect of LY-368052 in heart failure was not altered by either ganglionic or beta-adrenergic receptor blockade. LY-368052 improved cardiac relaxation and induced bradycardia in dogs with heart failure but not in normal dogs. The negative chronotropic effect of LY-368052 was eliminated by ganglionic blockade but not beta-adrenergic blockade, suggesting that the bradycardia was mediated by the autonomic nervous system via enhanced parasympathetic tone. Baroreflex sensitivity was assessed as the pulse interval-mean arterial pressure slope in response to temporary pharmacological (nitroglycerin or phenylephrine) and mechanical (brief occlusion of inferior vena cava) alterations of arterial pressure in conscious dogs before and after development of heart failure. Baroreflex sensitivity was significantly depressed in heart failure and restored completely by acute treatment with LY-368052. Thus the Na(+) channel enhancer LY-368052 maintains its beta-receptor-independent inotropic effect in chronic CHF and specifically improves ventricular relaxation and depressed baroreflex function.     

Negative chronotropic effect of endothelin-1 (ET-1) in the cardiac pacemaker tissue.

The aim of this study was to asses the direct effect of ET-1 on spontaneous discharge rate of the pacemaker tissue in the presence of isoproterenol. The experiments were performed on pacemaker tissue of the isolated right auricle of the right heart atrium of a two-day-old rat. The spontaneous discharge rate of the pacemaker tissue was recorded on the ECG apparatus and analyzed by the computer. ET-1 alone did not significantly affect the discharge rate of the pacemaker tissue. Isoproterenol rapidly increased the discharge rate of the pacemaker tissue. ET-1 had negative chronotropic effect in the presence of isoproterenol. JKC-301, a blocker of ET(A) receptors, significantly reduced the negative chronotropic effect of ET-1 in the presence of isoproterenol, whereas IRL-1038, a blocker of ET(B) receptors, did not significantly affect the negative chronotropic effect of ET-1 in the presence of isoproterenol. In conlusion, the negative chronotropic effect of ET-1 in the presence of beta-adrenergic stimulation the pacemaker tissue of the right auricle of the right heart atrium of the two-day-old rat is mediated by ET(A) receptors.     

Sensitivity of cultured chick embryo heart cells to acetylcholine. Evidence for nicotinic and muscarinic receptors

Sensitivity of cultured chick embryo heart cells to acetylcholine changes with time in culture. In 24 h cultures, about 25% of the cells exhibit a positive chronotropic response to acetylcholine. This effect is no longer observed after 48 h in culture. Positive and negative chronotropic effects of acetylcholine can be related to the presence of nicotinic and muscarinic receptors evidenced by autoradiography. Some data suggest a possible relationship between the type of sensitivity to acetylcholine and the changes in cell membrane properties occurring in culture.     

Muscarinic receptor subtypes: M1 and M2 biochemical and functional characterization

The heterogeneity of muscarinic receptors was examined in sympathetic ganglia and atria by “in vitro” binding techniques and functional studies. As tools we have used the classical antagonist atropine, the selective antagonist pirenzepine and the unique muscarinic agonist McN-A-343. In binding studies atropine showed similar affinities to muscarinic sites in ganglionic and atrial membranes with dissociation constants of 1.1 and 3.2 nM, respectively. In contrast, pirenzepine displayed a distinctly different binding profile. In atria it bound to an homogenous population of low affinity sites (diss. const. 620 nM) while in ganglia it revealed the presence of two sites: a major population of high affinity sites (diss. const. 11 nM) and a minor one of lower affinity (diss. const. 280 nM). The functional correlate of the receptor properties in the two tissues was studied in the pithed rat by measuring A) the increase of arterial pressure evoked by McN-A-343 through selective activation of muscarinic receptors in ganglia and B) the bradycardia elicited by acetylcholine release in the heart through vagal stimulation. Mirroring the “in vitro” binding data atropine inhibited both muscarinic responses in the same narrow range of doses (2–30 μg/kg i.v.) whereas pirenzepine showed similar potency to atropine in inhibiting ganglionic stimulation (ED50 4.1 μg/kg i.v.) but was almost two orders of magnitude weaker in blocking vagal bradycardia (ED50 172 μg/kg i.v.). These data suggest that McN-A-343 and pirenzepine act selectively on a common muscarinic receptor subtype, a finding which agrees with the view that muscarinic receptors are heterogenous and that excitatory ganglionic receptors (Ml) are distinguishable from those (M2) present in effector organs like smooth muscle and heart.     

Inhibition of beta-1 receptor but not vagotomy can abolish the L-NAME evoked bradycardia in anesthetized rat

We reported previously that the nitric oxide synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) decreases cardiac output. Several studies have shown that inhibition of nitric oxide synthesis decreases the heart rate. In the present study, we investigated the effect of a single bolus administration of L-NAME on blood pressure and heart rate monitored for one hour in anesthetized rats and the influence of vagotomy and beta1-receptor blocker metoprolol on the L-NAME induced bradycardia. After L-NAME treatment, the blood pressure rose immediately after the injection of the drug (peak response in the third minute: +24%, p<0.001) and fell to the control level in the 20th minute. The heart rate decreased immediately after L-NAME administration, the lowest value being reached in the 10th minute (-14%, p<0.001). However, bradycardia was sustained even after the blood pressure had returned to the control level. Bilateral vagotomy failed to influence the negative chronotropic effect of L-NAME, but bradycardia was completely abolished by metoprolol pretreatment. We concluded that the bradycardia evoked by L-NAME is mainly due to the withdrawal of sympathetic tone upon the heart rate. However, the cause of sustained bradycardia after normalization of blood pressure cannot be elucidated.     

Pharmacological evidence for cardiac muscarinic receptor subtypes

The chronotropic and inotropic effects of muscarinic receptor agonists (Acetylcholine, Arecoline, Carbachol, Furtrethonium) and antagonists (Atropine, N-methyl and N-butyl scopolammonium, pirenzepine) on isolated guinea-pig atria were studied. All had a greater affinity constants for muscarinic receptors as assessed in terms of inotropic effects than in terms of chronotropic effects. This difference, well correlated with the pharmacological effect, suggests the occurrence of cardiac muscarinic receptor subtypes, one mediating heart rate and the other contractile force. The ratio of chronotropic to inotropic potencies for each agent shows that the physiological mediator. Acetylcholine, differentiates best between the two subtypes, while atropine is the least discriminatory.     

Studies on bradycardia mechanism in the moderately hypothermic dog.

Chronotropic action of isoprenaline on the heart was studied in anesthetized dogs, in euthermic and moderate hypothermic conditions, before and after intravenous administration of atropine and oxprenolol or a cervical bilateral vagotomy. In moderate hypothermia we observed: i) larger duration of the positive chronotropic response to isoprenaline with a delayed and slightly lesser intensity in its maximum; ii) relating to euthermic conditions, delayed but superimposed potentiation of the chronotropic isoprenaline response in atropinized or vagotomized dogs; iii) a small negative chronotropic response to isoprenaline 15 min after oxprenolol, that diminished after atropine; iiii) oxprenolol induced a marked bradycardia nearly twice as intense as in euthermic dogs, almost completely blocked subsequently by atropine. It is concluded that progressive bradycardia in the moderately hypothermic dog is due, among other factors, to a cholinergic action but not to a lesser ability of beta-adrenergic cardiac effectors to chronotropic responses.     

Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels

Summary P19 embryonal carcinoma cells were differentiated via embryolike aggregates (embryoid bodies) into spontaneously beating myocytes. During the whole process of differentiation the functional expression of cardiac-specific receptors and ionic channels was characterized by measuring the chronotropic reactivity, action potentials, and ionic currents in response to various cardioactive drugs. Positive chronotropic effects obtained at different maximal effective concentrations of adrenoceptor-mediated agonists indicated differential adrenoceptor expression during the in vitro development of cardiomyocyte-like cells. No cardiac-specific response was obtained with the muscarinic cholinoceptor agonist carbachol. Single beating cells were enzymatically isolated and investigated by the patch-clamp technique. Pacemaker action potentials similar to those of embryonal cardiomyocytes exhibited amplitudes ranging from 50 to 85 mV. The action potentials were synchronous to the mechanical contractions and, comparable to the chronotropic effects, were modulated by BayK 8644, isradipine, and adrenaline. The functional expression of L-type Ca²⁺ channels was demonstrated by the Ca²⁺ channel blockers isradipine, nisoldipine, gallopamil, and diltiazem causing negative chronotropic responses, as well as by the Ca²⁺ channel activator BayK 8644 causing positive chronotropic responses. These effects gradually increased with time of differentiation. The expression of L-type Ca²⁺ channels and of nicotinic acetylcholine receptors was confirmed in voltage-clamp experiments. The study demonstrates that P19 embryonal carcinoma cells can be induced to differentiate into cardiomyocyte-like cells comparable to embryonal and neonatal heart cells lacking the muscarinic cholinoceptor response only.     

Action of pilocarpine on the normal frog heart and in pathology

Experiments on frogs were performed to examine the effect of the M-cholinomimetic pilocarpine on the heart. It was discovered that at concentrations of 10(-15)--10(-5) g/ml pilocarpine exerted only an adverse chronotropic effect on the perfused heart. When applied at a concentration of 10(-4) g/ml the drug produced a negative as well as a positive chronotropic effect. The latter occurred spasmodically (without progressive rise in the heart rate) in association with a slow heart rate. In some experiments such effects were preceded by a certain deceleration of the heart. In experiments with positive chronotropic effects, arrhythmias and sinoatrial dissociation were observed sometimes. Experiments with recording of the electrograms of the sinuses and lower parts showed that such effects were caused not by pacemaker acceleration but by the removal of the blockade of conduction, between the pacemaker and the atria. As far as the pacemaker is concerned, pilocarpine exerted only a negative chronotropic effect.     

Motor reactivity of isolated heart of the grass-snake (Natrix natrix L.)

Stimulation of the vagus nerve with a volley of electric impulses changed the action of grass-snake heart producing a negative chronotropic and inotropic effect. The effect of vagal stimulation was not different from the effect of acetylcholine administration and it was absent in the presence of atropine and hexamethonium. It was not possible to demonstrate sympathetic nervous fibres in the stimulated segment of the vagus nerve and trials of finding a separate nerve increasing the heart rate were unsuccessful. Parasympathicotonic agents caused bradycardia and a fall in the amplitude of cardiac contractions, and in sufficiently high doses they arrested the heart in diastole. The action of muscarine-like agents was stronger than that of nicotine, and the anticholinergic action of tubocurarine was weaker than that of atropine. Catecholamines exerted a positive inotropic and chronotropic effect which was completely blocked by propranolol in some tests only.     

Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments

Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.     

Cardioselective profile of AF-DX 116, a muscarine M2 receptor antagonist

AF-DX 116 (see chemical name below) is a competitive antagonist of muscarine receptors in peripheral organs. In contrast to pirenzepine, its behaviour in functional experiments indicates selectivity for the M2 muscarinic subtype. In pithed rats AF-DX 116 inhibits vagally-induced bradycardia, an M2 response, (ED50 32 micrograms/kg i.v.) in preference to the M1-mediated pressor response to McN-A-343 (ED50 211 micrograms/kg i.v.). AF-DX 116 further discriminates among M2 receptors, showing a high affinity for the cardiac muscarine receptors. In isolated preparations, AF-DX 116 has a tenfold higher affinity for the muscarine receptors of the heart (pA2 7.33) than for those in smooth muscles (pA2 6.39-6.44). The same profile appears from animal studies, where the compound is a more potent antagonist of either endogenously or exogenously activated cardiac muscarine responses as compared to vascular, smooth muscle or secretory responses. In general, the ratios of potencies (ED50) observed in cardiac vs. other muscarine mediated functions ranged between 30 and 50. Atropine showed no discrimination, inhibiting all muscarine responses in the same range of doses. In the conscious dog intravenous AF-DX 116 increased basal heart rate, and completely reversed the reflex bradycardia induced by clonidine. Tachycardia was dose-related (ED50 79 micrograms/kg i.v.), and occurred independently of background sympathetic tone. AF-DX 116 clearly distinguishes between M1- and M2-mediated responses; it also emphasizes the long-recognized heterogeneity among the peripheral M2 subtypes. AF-DX 116, for its pronounced cardioselectivity, may have a therapeutic potential in the treatment of sinus bradycardia.     

Interaction of the neuromuscular blocking drug atracurium with muscarinic acetylcholine receptors.

On isolated rat heart atria, atracurium competitively antagonized the negative chronotropic effect of methylfurmethide, shifting the concentration-response curve to the right without diminishing the agonist's maximal effect; Kd calculated from dose ratios was 3.0 mumol/l. On the longitudinal muscle of rat ileum, atracurium antagonized the effect of methylfurmethide in a non-competitive manner; at 50 mumol/l atracurium, the maximum response to methylfurmethide was diminished by about 50%. Atracurium antagonized the binding of (3H)quinuclidinyl benzilate [3H)QNB) to muscarinic binding sites in the atria, ileal longitudinal muscle and cerebellum with IC50 values of 5-8 mumol/l, and in brain cortex of 25 mumol/l. Atracurium was little efficient, however, in antagonizing the binding of N-(3H-methyl) scopolamine [3H)NMS) to muscarinic binding sites. Complete blockade was not achieved at concentrations up to 1 mmol/l. Concentrations required to diminish the binding by 50% were 10 - 1000 times higher for (3H)NMS than for (3H)QNB. Atracurium brought about the dissociation of (3H)QNB-receptor complexes, but its effect was considerably stronger at a concentration of 30 mumol/l than at 1 mmol/l. Atracurium slowed down the dissociation of (3H)QNB-receptor complexes observed after the addition of atropine. The effects of atracurium on the dissociation of (3H)NMS-receptor complexes were similar to those on (3H)QNB-receptor complexes, but a high concentration of atracurium (1 mmol/l) produced a transient increase in (3H)NMS binding preceding its subsequent dissociation. Although the observations of the antagonism by atracurium of the effect of methylfurmethide on the heart atria, and of the inhibition of the specific binding of (3H)QNB to the atria, ileal smooth muscle, cerebellum and brain cortex are compatible with the assumption of a competitive interaction, the discrepancy between the effects of atracurium on the binding of (3H)QNB and (3H)NMS indicates that atracurium does not bind to the same binding site as (3H)QNB and (3H)NMS. It appears that most effects of atracurium on muscarinic receptors are allosteric and that both negative and positive cooperatives play a role in interactions between atracurium and muscarinic ligands.     

Double-stranded RNA causes airway hyperreactivity and neuronal M2 muscarinic receptor dysfunction.

Viral infection causes dysfunction of inhibitory M2 muscarinic receptors (M2Rs) on parasympathetic nerves, leading to airway hyperreactivity. The mechanisms of M2R dysfunction are incompletely understood. Double-stranded RNA (dsRNA), a product of viral replication, promotes the expression of interferons. Interferon-gamma decreases M2R gene expression in cultured airway parasympathetic neurons. In this study, guinea pigs were treated with dsRNA (1 mg/kg ip) on 2 consecutive days. Twenty-four hours later, anesthetized guinea pigs had dysfunctional M2Rs and were hyperresponsive to electrical stimulation of the vagus nerves, in the absence of inflammation. DsRNA did not affect either cholinesterase or the function of postjunctional M3 muscarinic receptors on smooth muscle. M2Rs on the nerves supplying the heart were also dysfunctional, but M2Rs on the heart muscle itself functioned normally. Thus dsRNA causes increased bronchoconstriction and bradycardia via increased release of ACh from the vagus nerves because of loss of M2R function on parasympathetic nerves in the lungs and heart. Production of dsRNA may be a mechanism by which viruses cause dysfunction of neuronal M2Rs and airway hyperreactivity.     

Blood pressure responses of conscious rats to intravenous administration of enkephalin derivatives (D-ala methionine and leucine enkephalinamide, and methionine and leucine enkephalinamide)

The present study was designed to determine the blood pressure (BP) responses of conscious rats given intravenous (IV) injections of enkephalin derivatives (D-ala²-methionine enkephalinamide, DAMEA; D-ala²-leucine enkephalinamide, DALEA; methionine enkephalinamide, MEA; leucine enkephalinamide, LEA) and the receptor mechanisms mediating the resultant change in BP. IV injection of 1.6–16.0 nmoles of DAMEA or DALEA caused a transient but potent decrease in mean arterial pressure (MAP) and mean heart rate (MHR). LEA and MEA (16.0 nmoles) given IV produced slight pressor responses, which were not associated with concomitant tachycardia whereas 48 nmoles of MEA elicited a hypotensive effect accompanied by a fall in MHR. Pretreatment studies whereby various receptor antagonists (naloxone, diprenorphine, phentolamine, D-L-propranolol or atropine) were given IV 5 min before subsequent IV administration of DAMEA, DALEA, MEA or LEA (16 nmoles) showed that naloxone, diprenorphine and atropine blocked the depressor and bradycardic effects of DALEA and DAMEA. Naloxone and phentolamine suppressed the pressor reponse of both MEA and LEA (16.0 nmoles) while diprenorphine blocked the rise in MAP to only MEA. The results show that DAMEA and DALEA mediate their depressor actions in conscious rats via a negative chronotropic effect through an interaction of muscarinic cholinergic receptors on the myocardium. It is suggested that the pressor response of MEA and LEA may be produced via an -receptor mediated effect on the peripheral vasculature to cause vasoconstriction.