Cholinergic Twitch Responses Research Articles

Effect of CB1 Ligands on Neurogenic and Myogenic Contractile Responses in the Guinea-Pig Ileum

This study aimed at investigating whether the synthetic cannabinoid receptor agonist (+)-WIN 55212-2 has neurogenic and myogenic relaxant effects on the longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum. (+)-WIN 55212-2, 1–1,000 nmol/L, concentration-dependently inhibited both the electrical stimulation-induced cholinergic twitch responses as well as the myogenic smooth muscle contractions in the LMMP preparation. SR-141716A (rimonabant) 1–1,000 nmol/L, the cannabinoid CB1 receptor antagonist, being without effect on its own, antagonized the (+)-WIN 55212-2-induced effects. The allyl isothiocyanate (mustard oil, 100 µmol/L) induced a relaxant effect in the guinea-pig ileum, which can be regarded as neurogenic and myogenic, was augmented by (+)-WIN 55212-2, and inhibited by SR-141716A. (+)-WIN 55212-2 only moderately modified the 60 mmol/L KCl-evoked contractions. These results provide functional evidence that the CB1 agonist (+)-WIN 55212-2-induced inhibitory effects in the guinea-pig ileum are exerted both at the neuronal as well as at the intestinal smooth muscle cell level.

Effects of Allyl Isothiocyanate, Acetaminophen, and Dipyrone in the Guinea-Pig Ileum

Allyl isothiocyanate (AITC, mustard oil, 50-200 µmol/l), depending on specific dosages, inhibited the cholinergic twitch response in the longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum. AITC also induced short-lasting contractile responses, and decreases of the basal tone of the LMMP strip at low concentrations and increases at high concentrations. Hexamethonium, a blocker of nicotinic ganglionic transmission, was able to prevent the AITC-evoked inhibitory effect, an effect that was also observed with the opioid antagonist naloxone. The P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid and guanethidine had no significant influence on the inhibitory effect of AITC. Since AITC also reduced the electrical stimulation-induced myogenic smooth muscle contractions in the LMMP preparation, its contractile and relaxant actions can be regarded as neurogenic and myogenic in nature. The analgesics, acetaminophen (paracetamol, 100-500 µmol/l) and dipyrone (metamizole, 100-500 µmol/l), reduced both the cholinergic twitch and the myogenic contractions in the LMMP strip to the same extent; therefore, their action in the intestinal smooth muscle can be regarded as myogenic spasmolytic in nature.

Interaction of αβ-Methylene ATP with the Cholinergic Twitch Response in the Guinea-Pig Ileum

In this study, direct effects of the P2X purinoceptor agonist αβ-methylene ATP (αβ-meATP) and effects on the cholinergic twitch response of the electrically stimulated longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum, were investigated. αβ-meATP (1, 3, and 10 µmol/l) induced short-lasting contractions on its own, followed by an inhibition of the twitch response during its presence in the organ bath. The inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels, apamin (100 nmol/l), prevented the inhibitory effect of αβ-meATP on the twitch response, whereas tetraethylammonium (300 µmol/l), a blocker of voltage-gated K⁺ channels and an inhibitor at nicotinic acetylcholine receptors, augmented the inhibitory effect of αβ-meATP on the twitch response. It is concluded, that there is a functional interaction between P2X receptors and nicotinic receptors in the LMMP strip, and that a major part of the excitatory input to the cholinergic motor neuron evoking the twitch response is purinergic and not nicotinergic.

Nicotine-Induced Modulation of the Cholinergic Twitch Response in the Ileum of Guinea Pig

In the present study, the direct drug effects of nicotine and its effects on the cholinergic twitch responses of the electrically stimulated longitudinal muscle-myenteric plexus strip from the ileum of guinea pig were investigated. Nicotine dose-dependently (0.3-10 µmol/l) evoked the well-known contractile responses on its own. Whereas the interposed twitch responses remained present without a change in height at 1 µmol/l nicotine, a nicotine concentration of 3 µmol/l slightly and a concentration of 10 µmol/l markedly diminished the twitch during their presence. After the washout of 1-10 µmol/l nicotine, the height of the twitch response was also temporarily and significantly reduced by 30-77%. The P2X purinoceptor agonist αβ-methylene ATP (1-10 µmol/l) dose-dependently induced contractions on its own and reduced the twitch response during its presence in the organ bath; however, it did not diminish the twitch responses after washout of the drug as nicotine did. The P2X antagonist pyridoxalphosphate-6-azophenyl-2′-4′-disulphonic acid, the NMDA channel blocker MK-801 and the inhibitor of small conductance Ca²⁺-activated K⁺ (SK) channels apamin reduced the contractile effect of 1 µmol/l nicotine. Apamin also significantly prevented the ‘post-nicotine inhibition of the twitch' following the washout of 1-3 µmol/l nicotine. As a conclusion, we provide evidence for a functional interaction between nicotinic receptors and the P2X receptors in the ileum of the guinea pig. The ‘post-nicotine inhibition of the twitch' is not due to nicotinic acetylcholine receptor desensitization or transmitter depletion, but most probably the secondary effects of nicotine on SK channels determine the reduced cholinergic motor neuron excitability.

Smooth Muscle-Depressant Activity of AP-18, a Putative TRPA1 Antagonist in the Guinea Pig Intestine

AP-18, a putative antagonist at TRPA1 receptor/ion channel, caused smooth muscle relaxation at 10-100 µmol/l. It inhibited cholinergic twitch responses evoked by electrical field stimulation of cholinergic nerves as well as contractions in response to acetylcholine and histamine in the guinea pig small intestine. AP-18 (30 µmol/l) blocked spontaneous contractions of longitudinal strips of human jejunum. It is concluded that AP-18 may have limited value in studying TRPA1-mediated responses in smooth muscles and should probably be used with care in other preparations because of possible nonspecific effects.

Cortisone: a potent GABAA antagonist in the guinea-pig isolated ileum

In the guinea-pig isolated ileum, cortisone at 0.001-10 nM induced a non-competitive, dose-dependent antagonism of GABAA-receptor-mediated contractile responses to applied GABA, depressing the maximum contractile response to GABA (100 microM), without affecting contractile responses to acetylcholine or cholinergic twitch contractions. At higher concentrations (greater than 10 nM), cortisone depressed contractile responses to acetylcholine (10-100 nM) and cholinergic twitch responses to transmural stimulation. Cortisone is thus the most potent non-competitive antagonist at GABAA-receptor complexes in the guinea-pig ileum. From molecular modelling, sterically there appeared little difference between cortisone and cortisol, the latter being an enhancer of GABAA-receptor-mediated action in the ileum. However, there were significant differences in electrostatic potentials between the two steroids, due to the different levels of oxidation at C11 which may contribute to such opposing actions.

Stimulus-evoked opioid inhibition in guinea-pig longitudinal muscle-myenteric plexus strip is modulated by NMDA receptors

Longitudinal muscle-myenteric plexus (LMMP) strips of the guinea-pig ileum were used to investigate the stimulus-evoked endogenous opioid inhibition and its modulation by ionotropic glutamate receptors. Regular cholinergic twitch responses evoked by a short 3-s-field stimulation in intervals of 80 s were found reduced after an interposed period of prolonged 40-s-field stimulation. In the presence of a peptidase-inhibitor-cocktail, the cholinergic twitch response following the period of prolonged stimulation was even further reduced as compared to normal Tyrode solution without peptidase inhibitors. In both instances, the impairment of the cholinergic twitch response was completely abolished by naloxone thus demonstrating its opioidergic nature. This endogenous inhibitory opioid effect was significantly mitigated by the NMDA-receptor antagonist MK-801, but not by the AMPA/kainate receptor antagonist CNQX. These results demonstrate by functional experiments that there is a significant opioid-mediated inhibition in guinea-pig LMMP preparations evoked by a prolonged electrical stimulation, and that an NMDA antagonist can mitigate the opioid inhibition.

Role of the epithelium and acetylcholine in mediating the contraction to 5‐hydroxytryptamine in the mouse isolated trachea

1. The 5-HT receptor subtype that mediates bronchocontraction and the involvement of neuronal and non-neuronal acetylcholine was assessed in murine isolated tracheae. 2. Atropine (1-10 nM) caused a rightward shift of the methacholine concentration-effect curves (pA(2)=9.0) but reduced the maximum response to 5-HT, suggesting that 5-HT acts as an indirect agonist. The potency of 5-HT receptor agonists (alpha-methyl-5-HT approximately 5-HT>5-carboxamidotryptamine), together with the competitive antagonism of 5-HT by ketanserin (pA(2)=9.4), suggests the involvement of the 5-HT(2A) receptor. 3. While cholinergic twitch responses to electrical field stimulation were abolished by the fast sodium channel inhibitor tetrodotoxin (300 nM), as well as by combined blockade of N-, P- and Q-type voltage-operated calcium channels by omega-conotoxin GVIA (30 nM) and agatoxin IVA (100 nM), responses to 5-HT were unaffected. Similarly, botulinum toxin A (50 nM) inhibited EFS twitch responses, but not contractions to 5-HT. 4. Choline acetyltransferase immunoreactivity was localised to ganglia and nerve fibres as well as approximately half the epithelial cells in the preparation. Removal of the epithelial layer markedly attenuated the contractile response to 5-HT, but had no effect on contractions to either methacholine or EFS. 5. These findings suggest that 5-HT, acting at 5-HT(2A) receptors on mouse tracheal epithelial cells, stimulates these cells to release acetylcholine, which then causes contraction of airway smooth muscle. This phenomenon should be borne in mind in when interpreting studies of murine models of airway disease.

Evaluation of 3-aminopropanesulphonamide analogues of GABA as antagonists at GABA B receptors in peripheral and central preparations

Cholinergic twitch responses in the guinea-pig isolated ileum, and spontaneous discharges in rat neocortical slices, were depressed by the GABA B receptor agonist baclofen. These actions were reversibly antagonised by the sulphonamide derivatives ( R, S)-2-hydroxy-3-phthalimidopropanesulphonamide (HPIPS), 3-amino- N-benzoylpropanesulphonamide (ABPS) and 3-phthalimidopropanesulphonamide (PIPS) which produced rightwards shifts of the baclofen concentration–response curves, with p A 2 values ranging between 4.1 and 4.3 in both preparations. From these results, HPIPS, ABPS and PIPS constitute a novel class of antagonists at GABA B hetero-receptors.

Excitation and Inhibition via Adenosine Receptors of the Twitch Response to Electrical Stimulation in Isolated Guinea Pig Ileum

The effects of adenosine and its related compounds on the cholinergic twitch response were examined in electrically stimulated guinea pig ileum. Adenosine (3×10-7-10-5 M) and an adenosine A1-receptor agonist N6-cyclohexyladenosine (CHA, 10-8-10-6 M) suppressed the twitch. Conversely, the A2a-receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarboxamidoadenosine (CGS 21680, 10-9-10-7M) potentiated the twitch in half the preparations examined. The A1-antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), which per se did not affect the twitch, recovered the attenuated twitch caused by CHA (10-7 M) or adenosine (10-6 M) and converted it into a potentiated twitch. These results suggest the presence of adenosine A1- and A2a-receptors coupled negatively and positively, respectively, to acetylcholine release in the preparation.

CP-96,345, a non-peptide antagonist of substance P: I. Effects on the actions mediated by substance P and related tachykinins on the guinea-pig ileum and rabbit jejunum.

The effects of a non-peptide antagonist of substance P, CP-96,345, were investigated, in vitro, on the guinea-pig ileum and the rabbit jejunum. Contractions of the guinea-pig ileum, induced by substance P and neurokinin A, were specifically inhibited by the racemate (+/-)CP-96,345 (pIC50 7.8 and 7.3, respectively). The inhibition by (+/-)CP-96,345 of contractions evoked by neurokinin B and by bradykinin (pIC50 6.1 and 4.9, respectively) was attributed to unspecific effects of the antagonist. The inhibition of substance P-induced contractions of the rabbit jejunum required a 10 times higher concentration of (+/-)CP-96,345 (pIC50 = 6.8) than was required with the guinea-pig ileum. The plateau phase of contraction of the guinea-pig ileum induced by high concentrations of substance P, neurokinin A or neurokinin B, which is known to be mediated through tachykinin receptors on intrinsic cholinergic neurones, was inhibited by 200 nM (+/-)CP-96,345 but not by the inactive enantiomer, CP-96,344. This indicates a specific inhibition of these neuronal tachykinin receptors by (+/-)CP-96,345. Contractions known to be mediated by the release of substance P, such as those evoked by capsaicin and by mesenteric nerve or field stimulation, were partially inhibited by (+/-)CP-96,345 at concentrations of 200 to 600 nM. Unspecific inhibitory effects of CP-96,345, in concentrations of 1 microM or higher, were observed on histamine-induced contractions, and on the cholinergic twitch response to electrical stimulation, of the guinea-pig ileum.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the cholinergic twitch response in guinea-pig common bile duct: an evaluation of the effects of noradrenaline, clonidine and fentanyl.

Presynaptic inhibitory effects of noradrenaline, clonidine and fentanyl on twitch contractions evoked by transmural electrical stimulation were investigated in guinea-pig common bile duct (CBD). Antagonism of the response to fentanyl by naloxone and to noradrenaline and clonidine by yohimbine confirmed the involvement of presynaptic opiate and alpha 2-adrenoceptors respectively. Clonidine caused desensitization of presynaptic alpha 2-adrenoceptors. No cross desensitization between clonidine and fentanyl was observed. Furthermore, partial agonistic activity of clonidine at presynaptic alpha 2-adrenoceptors was also observed by the antagonism of noradrenaline response in this preparation.

Short-chain baclofen analogues are GABA B receptor antagonists in the guinea-pig isolated ileum

A new series of GABA B receptor antagonists, based on short-chain baclofen analogues has been investigated. In guinea-pig isolated ileal preparations, the GABA B receptor-mediated, baclofen-induced depression of cholinergic twitch responses was reversibly and competitively antagonised by the short-chain baclofen analogues 3-amino-3-(p-chlorophenyl)propionic acid (apparent pA 2 = 3.5), 2-amino-2-(p-chlorophenyl)ethanephosphomc acid (apparent pA 2 = 3.8), and 2-amino-2-(p-chlorophenyl)ethane acid (apparent pA 2 = 4.0). The corresponding des-chloro analogues were all less active. These compounds represent another class of GABA B receptor antagonists which may cross the blood-brain barrier.

Modulation of autonomic neuroeffector transmission by nitric oxide in guinea pig ileum

NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis, markedly enhanced tonic ("hump") responses to transmural stimulation in guinea pig ileum longitudinal muscle. The enhancement of the hump responses was probably due to a prejunctional effect on substance P-like neurotransmission, since the action of L-NMMA was exerted also in the presence of atropine, and since responses to substance P, a mimic of nerve stimulation, were unaffected by L-NMMA as were cholinergic twitch responses and the overflow of [3H]choline. Further in support, the hump responses were blocked by the substance P antagonist Spantide. All effects of L-NMMA were stereospecifically reversed by L-arginine. Endogenous nitric oxide thus selectively modulates peptidergic neurotransmission in the gut.

Differences in the effects (in vitro) of ethylenediamine on the guinea-pig and rat intestine

The effects of ethylenediamine in different regions of the rat and guinea-pig small intestine were investigated pharmacologically using isolated gut-bath preparations. In the guinea-pig, ethylenediamine caused concentration-dependent neurally mediated contractions or biphasic responses (contraction followed by relaxation). The contractions could be prevented by muscarinic and GABA A receptor antagonists. Ethylenediamine-evoked relaxations and depression of electrically evoked cholinergic twitch responses were blocked by desensitization to baclofen. However, in the rat intestine, the primary response to applied ethylenediamine was a concentration-dependent, non-desensitizing relaxation, evidently due to a direct action of ethylenediamine on the muscularis since it was unaffected by tetrodotoxin or GABAergic blockade.

Phaclofen: a peripheral and central baclofen antagonist

Phaclofen, the phosphonic acid derivative of baclofen, reversibly antagonized the depression of the cholinergic twitch response of the guinea pig ileum and distal colon by either baclofen or GABA. When administered microelectrophoretically, phaclofen reversibly blocked the presumed presynaptic reduction by baclofen of the monosynaptic excitation of spinal interneurones by impulses in primary afferent fibres of the cat but did not block the postsynaptic depressant action of baclofen on these neurones. Phaclofen may thus be useful in determining the physiological significance of central and peripheral bicuculline-insensitive receptors with which GABA and (−)-baclofen interact.

Adenosine-modulation of cholinergic and non-adrenergic non-cholinergic neurotransmission in the rabbit iris sphincter.

The characteristics of smooth muscle responses to transmural nerve stimulation in the rabbit iris sphincter were examined. Transmural stimulation elicited a composite contractile response that could be divided in two phases. Atropine abolished the phase I contraction and inhibited the phase II contraction. The atropine-resistant component of the phase II contraction which was unaltered by sympathetic denervation, was mimicked by substance P and abolished by capsaicin. Adenosine inhibited the phase I contraction. The adenosine analogue L-N6-phenylisopropyladenosine (L-PIA) was more potent than 5'-N-ethylcarboxamideadenosine (NECA) in mimicking this adenosine effect. By contrast, adenosine enhanced the phase II contraction in non-pretreated preparations, as well as the atropine-resistant capsaicin-sensitive part of this contraction. Here, NECA was more potent than L-PIA. Adenosine, NECA, L-PIA and D-PIA also enhanced the atropine-sensitive component of the phase II contraction, as well as the contractile response to exogenous acetylcholine or carbachol, but not to exogenous substance P. In this respect, L-PIA was the most powerful adenosine analogue with at least 10 fold higher potency than D-PIA. The adenosine antagonist 8-p-sulphophenyltheophylline enhanced the phase I contraction and decreased the capsaicin-sensitive non-adrenergic non-cholinergic component of the phase II contraction. We conclude that adenosine inhibited the nerve-induced cholinergic twitch (phase I) responses by action at prejunctional A1-receptors. Furthermore, adenosine enhanced the phase II contractile responses via postjunctional enhancement of the cholinergic transmission by action at A1-receptors, and via enhancement of the non-adrenergic non-cholinergic transmission by action at presumably prejunctional A2 receptors.

Evidence for the presence of P 1-purinoceptors on cholinergic nerve terminals in the guinea-pig ileum

The inhibitory effects of adenosine, ATP, 5′-adenylyl methylene diphosphonate (β, γ-meATP) and adenosine 5′-α, β-methylene triphosphonate (α, β-meATP) were compared on the cholinergic twitch responses to transmural stimulation of the guinea-pig ileum. Adenosine ATP and β, γ-meATP reduced the twitch responses in a concentration dependent manner. Theophylline antagonized and dipyridamole potentiated the inhibitory responses to adenosine, ATP and β, γ-meATP. Inhibitory responses to α, β-meATP were usually preceded by an enhancement in twitch height. Contractions of the unstimulated ileum to α, β-meATP were blocked by atropine and tetrodotoxin while those elicited by ATP were unaffected, which suggests that thetial excitatory effects of α, β-meATP may be due to its ability to release ACh from cholinergic nerve terminals. Use of high pressure liquid chromatography and bioluminescence assay techniques demonstrated the ability of the tissue to degrade ATP and β, γ-meATP and, at a much slower rate, α, β-meATP. Inhibitory responses to ATP, AMP and β, γ-meATP were reduced by adenosine deaminase, which also abolished responses to adenosine. 5′-AMP deaminase abolished responses to AMP and adenosine, and reduced those to ATP and β, γ-meATP. The results suggest that the inhibitory effect of ATP on cholinergic neurotransmission is due to its rapid breakdown to AMP or adenosine, which act on prejunctional P 1-purinoceptors.

An α-adrenoceptor inhibitory action of kynuramine

Kynuramine, an endogenously occuring amine, inhibited the vasoconstrictor responses to norepinephrine in the isolated perfused mesentric arteries of rats and blocked the relaxation of rabbit intestinal smooth muscle by phenylephrine. Similarly, kynuramine reversed clonidine-induced inhibition of the cholinergic twitch response in the guinea-pig ileum. These effcets, seen with concentrations ranging from 4 to 60 μg/ml, aer consistent with an α-adrenoceptor inhibitory action of kynuramine at both presynaptic sites. Kynuramine itself, lacked intrinsic activity on α-adrenoceptors and showed a complete lack of affinity for β-adrenoceptors in the rabbit intestine and guinea pig tracheal chain preparation. None of the effects of kynuramine could be attributed to the formation of 4-hydroxyquinoline, the deaminated metabolite of kynuramine. Additionally, no evidence was obtained for an action on muscarinic receptors or non-specific effects on vascular smooth muscle. However, a slight, transient stimulant action was evident on intestinal smooth muscle. It is concluded tha kynuramine inhibits both presynaptic and postsynaptic α-adrenoceptors in vitro and it is conceivable that the compound might function as an endogenous inhibitor of α-adrenoceptors in vivo.

Functional acetylcholinesterase of rat diaphragm muscle

Surface (external) acetylcholinesterase (AChE) and internal AChE of rat diaphragm muscle were assayed separately with radioactive acetyl-β-methylcholine (MeCh) or acetylcholine (ACh) on the intact muscle in vitro and on the muscle homogenate. Surface AChE had a K m for ACh of 1.05 mM and V max of 19.4 μM min −1 per diaphragm. External AChE activity increased 27 per cent for a doubling of muscle weight and averaged 20 per cent of total homogenate activity. Phospholine treatment (4 × 10 −7 M, 7 min, 30°) potentiated the indirectly elicited twitch response 164 per cent when inhibition of external AChE was 91 per cent and internal AChE 64 per cent, whereas DFP treatment (5 × 10 −6 M, 20 min, 30°) potentiated the twitch 257 per cent when inhibition of external AChE was 88 per cent and internal AChE 92 per cent. Inhibition by DPP was reduced by 80 per cent for surface and 32 per cent for internal AChE in the presence of edrophonium (8 × 10 − 6 M) and by 77 and o per cent respectively, in the presence of ACh (2 × 10 −3 M). Selective reactivation of DFP-inhibited surface AChE by 2-PAM correlates with recovery from twitch potentiation. The diaphragm muscle gave normal low frequency twitch response if surface AChE inhibition was less than 35 per cent, even when internal AChE was 78–92 per cent inhibited. Surface AChE activity as assayed on intact rat diaphragm muscle in vitro with 5 × 10 −6 M MeCh as substrate is primarily functional endplate AChE and correlates with cholinergic twitch responses.