Negative Inotropic Effect Of Acetylcholine Research Articles

Quantitative Characteristics of Negative Inotropic Effect of Acetylcholine on the Heart of the Snail Helix pomatia

We studied the quantitative characteristics of the negative inotropic response of an isolated heart of the edible snail Helix pomatia with the use of modern methods of mathematical analysis of the results. The experiments were performed with an isolated heart ventricle of the snail washed by means of a cannula. The ventricle contracted in its own rhythm under the influence of the pressure of the solution used for perfusion. The solution contained 110 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , and 5 mM N‡cee A (pH 7.2‐7.4). The contractions of the heart muscle converted by means of a 6M-X1B isotonic mechanotransducer were recorded by an H-3021-1 recorder. The mathematical analysis of the data was performed with the use of the MathCad 7.0 pro software. In most cases, the ACh dose‐effect curves had a complex biphasic sigmoid shape (in semilogarithmic coordinates), which complicated data analysis by usual methods. A search for the most adequate mathematical model to fit the data obtained was performed with the use of the MathCad 7.0 pro software. We found that the experimental data best fit the model that included two independent pools of receptors with cooperative properties. The physiological response is described by the following equation: where C is the physiological effect (in percent) calculated as ( H – h / H ) × 100 %; c is the initial amplitude of the spontaneous contractions of the myocardium of the snail perfused with Ringer’s solution; h is the amplitude of ACh-induced myocardium contractions; is the percentage of the high-affinity receptor pool in the total receptor population; (100 ‐ ) or is the per

Endothelial cytochrome P450 contributes to the acetylcholine-induced cardiodepression in isolated rat hearts.

Acetylcholine (ACh) is known to reduce the contractility of the heart by acting on myocardial muscarinic M2 receptors. ACh induces also an endothelial-dependent vasodilatation by causing the release of nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factors from the vascular endothelium. It has been proposed that ACh elicits a hyperpolarization of the coronary endothelial cells which may be accompanied by the activation of cytochrome P450 (CYP) and the resulting release of epoxyeicosatrienoic acids (EETs). The study aims at investigating whether endothelial CYP is involved in the cardiodepression by ACh. In isolated rat hearts, cardiodepression by ACh (i.e. 25-30% reduction of developed left ventricular pressure) was partially attenuated either by inhibition of CYP with 1-aminobenzotriazole (ABT) or by endothelial dysfunction obtained with Triton X-100. No attenuation of cardiodepression was seen after nitric oxide synthase and cyclooxygenase inhibition by L-nitro-arginine methyl ester and indomethacin, respectively. The results suggest that the negative inotropic effect of ACh depends not only on a direct myocardial effect but also on the endothelial CYP activation.

In vitro pharmacological effects of S 12370 (2-[4-benzhydryloxypiperidinoethyl]isoxindole; an antibronchoconstrictor agent) in normal and sensitized tissue.

The effects of S 12370 (2-[4-benzhydryloxypiperidinoethyl]isoxindole), were studied in vitro. In guinea pig isolated tracheal rings, S 12370 induced a similar competitive inhibition of the contractile responses produced by acetylcholine, histamine and serotonin. However, it did not affect the contractions induced by leukotriene D4 (LTD4), substance P and U 46619, a stable analogue of thromboxane A2. S 12370 induced a concentration dependent inhibition of the cholinergic component of the contraction induced by electrical field stimulation, whereas it did not influence the sustained nonadrenergic noncholinergic (NANC) excitatory response observed in guinea pig isolated bronchi. S 12370 did not influence the relaxations induced by prostaglandin E2, isoprenaline and salbutamol, and did not modify the nonadrenergic noncholinergic inhibitory response induced by electrical field stimulation. In isolated left atria, the negative inotropic effect of acetylcholine was competitively inhibited by S 12370. In binding experiments, S 12370 exhibited similar affinity for M1, M2, M3, M4 muscarinic receptors and also recognized 5-HT2 serotonin and H1 histamine receptor subtypes. In ovalbumin-sensitized animals, the contractile response of isolated tracheal rings produced by exposure to the allergen was not influenced by S 12370. Tracheal rings from sensitized animals preexposed in vitro to the allergen developed a hyporesponsiveness to beta-adrenoceptor stimulation. S 12370 prevented the inhibitory effect caused by ovalbumin immune sensitization in the relaxation to isoprenaline. In rat polymorphonuclear neutrophil (PMN) cells, S 12370 up to 10(-5) M did not inhibit the arachidonic acid metabolism. These results suggest that in guinea pig tracheal smooth muscle, S 12370 is a competitive inhibitor of muscarinic, serotonin and histamine receptors and can modulate the beta-adrenergic dysfunction induced by immune sensitization. S 12370 may present some therapeutic interest in inflammatory airway diseases.

Regional differences in the negative inotropic effect of acetylcholine within the canine ventricle.

1. Regional differences in the effects of ACh on sub-epicardial, mid-wall and sub-endocardial cells of the dog left ventricle have been studied. 2. ACh produced a dose-dependent, atropine-sensitive negative inotropic effect that was greatest in sub-epicardial cells and small or absent in sub-endocardial cells. 3. In sub-epicardial (but not sub-endocardial) cells, ACh also resulted in a dose-dependent decrease in action potential duration. The inotropic effect of ACh on sub-epicardial cells was primarily the result of the decrease of action potential duration, because during trains of voltage clamp pulses the inotropic effect of ACh was reduced or abolished. At a holding potential of -80 mV, 10(-5)M ACh decreased L-type Ca2+ current by approximately 8% and this is thought to be responsible for the small inotropic effect during trains of pulses. 4. Although 4-AP, a blocker of the transient outward current (I(to)), abolished the "spike and dome' morphology of the sub-epicardial action potential, it had little or no effect on the actions of ACh on sub-epicardial cells. ACh had no effect on I(to) in sub-epicardial cells in voltage clamp experiments. 5. ACh activated a Ba(2+)-sensitive outward current (IK,ACh) in sub-epicardial cells, but little or no such current in sub-endocardial cells. In sub-epicardial cells, ACh also inhibited the inward rectifier current, IK,1. 6. It is concluded that in left ventricular sub-epicardial cells, ACh activates IK,ACh. This results in a shortening of the action potential and, therefore, a negative inotropic effect. In subendocardial cells, ACh activates little or no IK,ACh and, therefore, it has little or no negative inotropic effect. This may result from a regional variation in the expression of the muscarinic K+ channel.

Nitric oxide synthase does not participate in negative inotropic effect of acetylcholine in frog heart.

In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.

Action of acetylcholine on regional myocardial work and metabolism in vivo: association with cyclic GMP

This study was designed to test the hypothesis that in the in vivo dog heart, increases in cyclic (c) GMP and also decreases in cAMP induced by intracoronary administration of acetylcholine are associated with depressed myocardial function. In 10 open-chest anesthetized dogs, 0.5 microgram.kg-1.min-1 of acetylcholine was infused into the left anterior descending coronary artery. The intracoronary infusion of acetylcholine was continued simultaneously with 0.1 microgram.kg-1.min-1 of isoproterenol. Regional segment work was calculated as the integrated product of force (auxotonic force transducer) and segment shortening (sonomicrometry). Regional myocardial O2 consumption was calculated from blood flow measurements and regional O2 saturations. Competitive radioligand binding assays were used to determine the intracellular level of cAMP and cGMP in the myocardium. Local intracoronary infusion of acetylcholine significantly reduced regional segment work (from 36.7 +/- 6.5 to 19.1 +/- 3.7 x 10(-3) J/min) and O2 consumption (from 6.4 +/- 0.8 to 3.8 +/- 0.7 mL O2.min-1.100 g-1). This was related to a decrease in cAMP levels (from 364 +/- 25 to 262 +/- 17 pmol/100 g) and an increase in cGMP levels (from 1.34 +/- 0.06 to 1.78 +/- 0.15 pmol/100 g). When isoproterenol (0.1 microgram.kg-1.min-1) was added to the acetylcholine infusion line, cAMP levels tripled to 769 +/- 84 pmol/100 g, while O2 consumption rose to 6.6 +/- 1.4 mL O2.min-1.100 g-1. However, regional work was only partially restored (25.7 +/- 4.8 x 10(-3) J/min). Thus, both cAMP decrements and cGMP elevation occurred together with the negative inotropic effect of acetylcholine, and increased cAMP alone (produced by isoproterenol) did not fully overcome the acetylcholine effect. This was associated with elevated intracellular levels of cGMP.

Electromechanical effects of acetylcholine on the atrial tissues of the cultured tilapia (Oreochromis nilotica � O. aureus)

The effects of acetylcholine (ACh) on the action potential and twitch force of atrial tissues isolated from 15 tilapia (Oreochromis nilotica × O. aureus) were studied by means of conventional microelectrode techniques. In isolated whole atrium or sinoatrial tissue, scattered pacemaker-like cells with spontaneous diastolic depolarization were found mainly near the sinoatrial junction but also occasionally throughout the atrial wall. However, most of the atrial cells recorded were myocardial fibers as judged by a stable diastolic potential and a markedly reduced action potential duration (APD) in response to low concentrations of ACh (0.1-1 μM). The shortening in APD in atrial myocardial fibers was correlated with a significant fall in twitch force in the atrial preparations. ACh at high concentrations (10-300 μM) decreased moderately the APD and the slope of diastolic depolarization of the pacemakers and prolonged the spontaneous cycle length but did not induce hyperpolarization. The negative chronotropic action of ACh was competitively inhibited by atropine, a muscarinic antagonist. The means (± SEM) negative logarithm of the dissociation constant (pKb or pA2 value) for atropine against the ACh action on muscarinic receptors were 9.10 (± 0.13) (n = 6), similar to those values obtained in mammalian atria. The present findings indicate that while the negative inotropic effects of ACh in tilapia atria are comparable to those observed in mammalian hearts, unique electrophysiological responses to ACh exist in different types of tilapia atrial cells.

Characterization of the positive and negative inotropic effects of acetylcholine in the human myocardium

In the human isolated myocardium, acetylcholine (10 −9 to 10 −3 M) elicited a biphasic inotropic effect (a decrease in the lower and an increase in the higher concentration range) in atrial and a positive inotropic effect in ventricular trabeculae. However, under conditions of raised contractility achieved by exposure to noradrenaline (10 −5 M), only negative inotropic effects were observed in both atria and ventricles. Atropine (10 −6 M), but not propranolol (10 −6 M), antagonized both positive and negative inotropic effects of acetylcholine, thus showing that the responses were mediated by muscarinic acetylcholine receptors. The use of subtype selective muscarinic receptor antagonists (10 −7 to 10 −5 M), pirenzepine (M 1 > M 3 > M 2), AF-DX 116 (11-({2-[(diethylamino)-methyl]-1-piperidyl}acetyl)-5,11-dihydro-6 H-pyridol[2,3- b][1,4]benzodiazepine-6-one base; M 2 > M 1 > M and HHSiD ( p-fluorohexahydro-siladifenidol hydrochloride; M 3 ≥ M 1 ⪢ M 2) revealed that the negative inotropic effect of acetylcholine in atrial as well as the positive inotropic effect in ventricular trabeculae were best antagonized by AF-DX 116 and not by pirenzepine, suggesting the involvement of the muscarinic M 2 receptor subtype, possibly linked to different second messenger systems. On the other hand, the positive inotropic effect of acetylcholine (10 −6 to 10 −3 M) in the atrial tissue, observed only in preparation with depressed contractility, was not effectively antagonized by either AF-DX 116 or HHSiD, but was significantly reduced by pirenzepine. Furthermore, the selective muscarinic M 1 receptor agonist McN-A-343 (4-( m-chlorophenylcarbamoyloxy)-2-butynyltrimethyl ammonium chloride; 10 −9 to 10 −3 M), which failed to significantly change the baseline contractility in either atrial or ventricular trabeculae, produced a positive inotropic effect in atrial preparations when contractility had been depressed by prior treatment with acetylcholine (10 −9 to 10 −7 M). This effect of McN-A-343 was effectively antagonized by pirenzepine (10 −5 M). These data show that, besides the muscarinic M 2 receptor mediating both negative (atria) and positive (ventricle) inotropic effects, muscarinic M 1 receptors, capable of reversing depressed atrial contractility, are present in the human heart.

A direct negative inotropic effect of acetylcholine on rat ventricular myocytes

Acetylcholine (ACh) decreased the contraction of rat ventricular cells within 20 s. ACh (3.1 x 10(-8) M) produced a half-maximal effect and 10(-6) M ACh produced a maximal effect (a 23.8 +/- 5.4% decrease; mean +/- SE, n = 11). During a 3-min exposure to ACh, the inotropic effect faded. Parallel changes were observed in action potential duration: ACh caused an immediate shortening of the action potential, but then the effect faded with time. The changes in action potential duration were the cause of the changes in contraction, because ACh had no effect on contraction when the contractions were triggered by voltage-clamp pulses of constant duration. The changes in action potential duration were the result of the activation of a K+ current (iK,ACh) by ACh. During an exposure to ACh, this current faded as a result of desensitization. iK,ACh was 6.3 times smaller in ventricular than in atrial cells. This may explain why the negative inotropic effect of ACh on atrial cells was greater: 1.0 x 10(-8) M ACh produced a half-maximal effect on atrial cells, and 10(-6) M ACh produced a near maximal effect (a 74.5 +/- 9.5% decrease; n = 4).

Doxorubicin: an antagonist of muscarinic receptors in guinea pig heart

While studying the mechanisms of doxorubicin-induced cardiotoxicity, we observed that doxorubicin inhibited the negative inotropic effect of acetylcholine in isolated heart muscle preparations. We therefore examined the effects of doxorubicin on muscarinic acetylcholine receptors. In left atrial muscle preparations isolated from guinea pig heart and stimulated at 2 Hz at 30°C, doxorubicin caused a parallel right-ward shift of the dose-response curves for the negative inotropic effects of acetylcholine. The inhibitory action was reversed by an additional incubation in the absence of doxorubicin. Doxorubicin reversed the carbachol-induced inhibition of developed tension: a high concentration of doxorubicin brought the force back to its original strength. Doxorubicin inhibited specific [ 3H]quinuclidinyl benzilate (QNB) binding to membrane preparations obtained from ventricular muscle of guinea pig hearts. The pA 2 value for doxorubicin obtained in the inotropic study corresponded to the IC 50 value for doxorubicin observed in the [ 3H]QNB binding assay. These results indicate that doxorubicin acts as a weak competitive antagonist on muscarinic acetylcholine receptors.

Species differences in the negative inotropic effect of acetylcholine and soman in rat, guinea pig, and rabbit hearts

1. Acetylcholine reduced atrial contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The EC 50 values for the negative inotropic effect of acetylcholine were 3.3 × 10 −7 M in rat and guinea pig atria and 4.1 × 10 −6 M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atrial acetylcholinesterase with soman reduced the ec 50of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine. 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy.

Cholinergic Vasodilatation of Coronary Resistance Vessels in Dogs, Baboons and Goats

Intracoronary infusion of acetylcholine produces a prompt increase in coronary blood flow in dogs, but a paradoxical decrease has been reported in baboons and cattle. The action of acetylcholine was reexamined in anesthetized dogs, baboons and goats, with the coronary circulation pump perfused at constant pressure and the heart rate held constant with electrical pacing. Intracoronary infusions of low doses of acetylcholine produced coronary vasodilatation and an increase in coronary venous oxygen tension without a change in cardiac contractility (dP/dt) or myocardial oxygen consumption in all three species. High doses of acetylcholine produced coronary vasodilatation only in dogs, but resulted in a decrease in cardiac contractility and myocardial oxygen consumption accompanied by a decrease in coronary flow in baboons and goats. It is concluded that low doses of acetylcholine produce coronary vasodilatation in all three species, and that the decrease in coronary blood flow observed in baboons and goats at high doses is probably due to local metabolic vasoconstriction secondary to the negative inotropic effects of acetylcholine.

Interaction between the inhibitory action of acetylcholine and the alpha-adrenoceptor autoinhibitory feedback system on release of [3H]-noradrenaline from rat atria and rabbit ear artery.

Stimulation-induced increases in the efflux of radioactivity (S-I efflux) were measured in the bathing medium following labelling of the noradrenergic transmitter pools of rat atria and rabbit artery preparations with [3H]-noradrenaline. In atria stimulated with trains of 16 or 60 pulses at 2 Hz, phentolamine enhanced, whereas acetylcholine inhibited S-I efflux. With trains of 16 pulses phentolamine had a smaller enhancing effect than with trains of 60 pulses, whereas the inhibitory effect of acetylcholine was more pronounced with 16 pulses of stimulation. The inhibitory effect of acetylcholine was markedly enhanced by phentolamine when stimulation was with 60 pulses. With 16 pulses of stimulation the effect of acetylcholine was unaltered by phentolamine and abolished by the alpha 2-adrenoceptor agonist 3,4-dihydroxyphenylimino-2-imidazolidine (DPI). Phentolamine had no effect on the negative inotropic effect of acetylcholine on driven left atrial preparations. In arterial preparations stimulated with trains of 30 pulses at 1 Hz, both acetylcholine and clonidine inhibited S-I efflux, whereas yohimbine and idazoxan enhanced S-I efflux. Combining acetylcholine with clonidine did not alter the inhibitory effect of clonidine but the combination of acetylcholine with yohimbine or idazoxan abolished the marked enhancing effects of yohimbine or idazoxan on S-I efflux. These findings indicate that there may be a reciprocal interaction between prejunctional alpha-adrenoceptors and prejunctional muscarinic cholinoceptors.

Interactions of amantadine with the cardiac muscarinic receptor

The antiviral drug amantadine has anticholinergic effects in the guinea-pig atrium at concentrations greater than 1 X 10(-4)M. It is a competitive inhibitor of [3H]-quinuclidinyl benzilate binding to the muscarinic receptor, but antagonizes the negative inotropic effect of acetylcholine in a non-competitive manner. It increases the duration of the atrial action potential and also increases the force of atrial contraction. These effects are evident at approximately 10 times lower concentrations than the antimuscarinic effects. The increase in contractility can be reversed by propranolol (5 X 10(-7)M) but the increase in action potential duration is potentiated by propranolol. Shortening of the action potential duration by acetylcholine was reversed by amantadine, but at approximately ten times lower levels than were needed to reduce the negative inotropic effect. Interactions between beta adrenoceptor binding of [3H]-dihydroalprenolol and amantadine could not be demonstrated. Similarly, binding of [3H]-nitrendipine to the calcium channel is not influenced. It is suggested that amantadine may exert its positive inotropic effect by interaction with the potassium channel, causing a delay in outward current.

The inotropic and chronotropic effects of physostigmine and neostigmine on guinea-pig isolated atria.

1 Physostigmine (0.1-1 mM) induced dose-dependent negative and neostigmine (0.1-1 mM) positive inotropic effects on driven guinea-pig isolated atria. Physostigmine decreased and neostigmine increased cardiac frequency of spontaneously beating atria. 2 The depression of amplitude of contraction by physostigmine cannot be attributed to its inhibitory effect on tissue cholinesterase. The negative inotropic action of physostigmine is not influenced by preincubation with atropine. 3 The positive inotropic effect of neostigmine is not caused by a nicotine-like action of the drug. Neostigmine (1 mM) counteracts the negative inotropic effect of acetylcholine. The possible mechanisms of action are discussed.

A negative inotropic effect of acetylcholine in the presence of several phosphodiesterase inhibitors.

The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine (IBMX) reveal a negative inotropic effect of acetylcholine in cat ventricular heart muscle. This effect in unrelated to beta-adrenoceptor stimulation and possibly mediated by the accumulation of cyclic GMP.

Changing negative inotropic effect of acetylcholine in maturing canine cardiac muscle.

The effect of maturation on the contractile response to acetylcholine was studied in canine right atrial and right ventricular trabeculae. At any age the only inotropic response observed was negative. The decrease in contractile performance was always significantly greater in the atrial trabecula. Between 3 and 9 mo of age (12 dogs each group) there was a significant increase in the amount of negative inotropic action exerted by identical concentrations of acetylcholine. These data suggest that either the number of muscarinic receptors or the target cell response to acetylcholine increases during maturation.

Papaverine enhances the negative inotropic effect of acetylcholine in rat auricles

The negative inotropic effect of acetylcholine in rat left auricles is enhanced in the presence of the phosphodiesterase inhibitor papaverine. This result favours the idea of a cyclic GMP-mediated action of acetylcholine in the heart.

Are increases in cyclic GMP levels responsible for the negative inotropic effects of acetylcholine in the heart?

It has been suggested that increases in cyclic GMP levels are responsible for the negative inotropic effects of acetylcholine in the heart. This hypothesis was tested by monitoring the effects of acetylcholine and sodium nitroprusside on tension and cyclic nucleotide levels in strips of cat atrial appendage. Sodium nitroprusside markedly increased atrial cyclic GMP levels but did not decrease the twitch tension developed by the atrial strips. Low concentrations of acetylcholine, on the other hand, decreased twitch tension without increasing myocardial cyclic GMP levels. No significant change in cyclic AMP levels was observed in any of these experiments. These results are not consistent with the proposed role for cyclic GMP as the mediator of the negative inotropic effects of acetylcholine.

Does cyclic GMP mediate the negative inotropic effect of acetylcholine in the heart?

DURING vagal stimulation the pacemaker activity of the heart is diminished. The reduction in heart rate is due to a release of acetylcholine (ACh) from the parasympathetic nerve terminals that increases the permeability of the myocardial cell membrane for potassium ions (for review see ref. 1). This is accompanied by a shortening of the action potential duration in atrial muscle and a diminished calcium uptake2, which in turn results in a negative inotropic effect. Voltage clamp experiments in mammalian atrial muscle have shown that with higher concentrations of ACh not only is the potassium current augmented but also the slow inward current of calcium is reduced3. It is not clear how the current changes brought about by ACh are mediated. I have described the negative inotropic action of 8-bromo-cyclic GMP in the mammalian heart4, and these findings were confirmed in ventricular fibre fragments from mouse hearts5. If cyclic GMP levels in the myocardial cell are linked to the effects of ACh, as George et al. proposed6, then 8-bromo-cyclic GMP in addition to its negative inotropy should produce changes in the ion movements similar to those described for ACh. I report here the effects of 8-bromo-cyclic GMP on the action potential and the presumed underlying ion movements in rat atrial muscle.