Assessment of acetylcholine release from myenteric plexus of guinea-pig colon

Due to a complex mechanical tissue response to electrical stimulation, a colonic smooth muscle-myenteric plexus (SMMP) preparation, combined with radiolabelling techniques for assessing the acetylcholine release, has been developed to investigate intrinsic cholinergic nerve activity in the guinea-pig colon. Electrical field stimulation of the preparation gave reproducible release of label which was inhibited by tetrodotoxin. Release increased proportionally with the strength of stimulus (130, 150, 195 and 250 mA), but was inversely proportional to frequency of stimulation for a fixed number of pulses, 1 Hz releasing more than 10 Hz. Electrical stimulation of the tissue during incubation with [3H]choline enhanced the release by subsequent field stimulation. Release of label increased progressively towards the distal end of the colon.     

Increase by NO synthase inhibitors of acetylcholine release from guinea-pig myenteric plexus

The effects of nitric oxide (NO) synthase inhibitors on the electrically evoked release of [³H]acetylcho-line were studied in guinea-pig myenteric plexus preparations preincubated with [³H]choline. N^G-monomethyl-l-arginine (EC₅₀ 5.3 mol l^–1) and N^G-nitro-l-arginine (EC₅₀ 1.3 mol l^–1) concentration-dependently increased the evoked release of [³H]acetylcholine without affecting the basal outflow. The facilitatory effect of N^G-monomethyl-l-arginine was prevented by l-arginine but not by d-arginine. The results suggest that endogenous NO inhibits the depolarisation-evoked release of acetylcholine. Correspondence to: H. Kilbinger at the above address     

Inhibition by cannabinoid receptor agonists of acetylcholine release from the guinea-pig myenteric plexus

1. The dose-related inhibition of the twitch responses of the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine by cannabinoid (CB) agonists, (+)-WIN 55212 and CP 55940 during stimulation at 0.1 Hz with supramaximal voltage was confirmed. These agonists inhibited acetylcholine (ACh) release in the presence of physostigmine (7.7 μM) thus indicating a prejunctional site of action.
2. Inhibition of twitch responses and ACh release by CB agonists was reversed by the CB₁-selective cannabinoid receptor antagonist, SR141716A. Dose-response curves to (+)-WIN 55212 and CP 55940 were shifted to the right, with no reduction of maximal response, by pretreatment with SR141716A (31.6–1000 nM), but not its vehicle, Tween 80 (1 μM). However, at very high concentrations (25–400 μM), Tween 80 itself caused a dose-related inhibition of the twitch response which was significantly reduced in the presence of SR141716A (1 μM). The opioid receptor antagonist, naloxone (1 μM) had no significant effect on the inhibition by CP 55940 of the twitch response.
3. (+)-WIN 55212, CP 55940 and Tween 80 (50 μM) had no effect on responses to exogenous ACh, confirming that their actions were prejunctional. SR141716A (1 μM) did not increase the sensitivity of the longitudinal muscle to either ACh or histamine, but inhibited the responses to high doses of ACh.
4. The (−)-enantiomer of WIN 55212, was approximately 300 times less active than the (+) enantiomer in inhibiting the twitch response, had no CB₁ antagonist activity against the active isomer and did not inhibit the release of ACh in the presence of physostigmine.
5. The dissociation constant (K_D) values for SR 141716A against the inhibitory effect of (+)-WIN 55212 and CP 55940 on the twitch response were 12.07 nM (95% confidence intervals 8.55 and 20.83) and 6.44 nM (95% confidence intervals 4.70 and 10.24), respectively. In experiments in which the release of ACh was inhibited by (+)-WIN 55212, the K_D values were 9.21 nM and 10.53 nM at SR141716A concentrations of 31.6 nM and 100 nM, respectively. The K_D values for the antagonism by naloxone of the inhibition of the twitch responses and the inhibition of ACh release by normorphine in this preparation were found to be 2.38±0.69 nM and 2.00±0.9 nM, respectively.
6. During maximal inhibition of ACh release by (+)-WIN 55212, the addition of normorphine (400 nM) caused a further significant decrease in ACh output.
7. SR141716A alone produced a significant increase in ACh release in both the absence and presence of exogenous cannabinoid drugs, hence we conclude that it has a presynaptic site of action. We also conclude that SR141716A acts either by antagonizing the effect of an endogenous CB₁ receptor agonist or by having an inverse agonist effect at these receptors.

     

Two types of neuronal muscarine receptors modulating acetylcholine release from guinea-pig myenteric plexus

Summary Longitudinal muscle strips of the guinea-pig ileum were incubated with [³H]choline and the effects of muscarinic agonists on smooth muscle contraction and on spontaneous and electrically-evoked outflow of tritium were studied. Muscarine and pilocarpine concentration-dependently increased both muscle contraction and spontaneous outflow of [³H]ACh, and inhibited the electrically-evoked outflow of [³H]ACh. The increase in spontaneous outflow was prevented by tetrodotoxin and scopolamine, but not by hexamethonium. Oxotremorine (1–100 M) did not increase the spontaneous outflow of tritium.Pirenzepine in concentrations of 10 and 100 nM hardly affected the muscle contractions induced by pilocarpine, but significantly antagonized the pilocarpine-evoked increases in [³H]ACh outflow. Likewise, pirenzepine (100 nM) antagonized more effectively the enhancement by muscarine of spontaneous outflow than the inhibitory effect of muscarine on the electrically-evoked release of [³H]ACh. Scopolamine (1 and 10 nM) antagonized to a similar extent the effects of pilocarpine on spontaneous outflow of [³H]ACh and on muscle contraction.The results suggest that the cholinergic nerves of the myenteric plexus are endowed with excitatory (ganglionic) and inhibitory (prejunctional) muscarine receptors which modulate the release of ACh and which differ in their affinities to pirenzepine.     

Modulation of acetylcholine release from guinea-pig Ileum myenteric plexus by arachidonic acid cascade inhibitors

Effects of arachidonic acid and mepacrine on ACh release from guinea-pig ileum myenteric plexus were investigated. Mepacrine (1-8 microM) inhibited the ACh release in a concentration-dependent manner. Arachidonic acid counteracted the inhibitory effect of mepacrine, but PGE2 did not. The inhibition induced by a combination of mepacrine and indomethacin on nicotine-induced ACh release was prevented by arachidonic acid, while that on spontaneous ACh release was prevented by arachidonic acid and PGE2 added simultaneously. The roles of arachidonic acid and PGs in the ACh release will be discussed.     

Muscarinic modulation of acetylcholine release evoked by dimethylphenylpiperazinium and high potassium from guinea-pig myenteric plexus

The effects of oxotremorine and atropine on the ACh release evoked from the guinea-pig myenteric plexus by dimethylphenylpiperazinium (DMPP) or by high potassium were investigated. DMPP caused an output of ACh by stimulating nicotine receptors that are probably situated on the soma-dendritic part of the cholinergic neuron. The DMPP-induced release of ACh was dose-dependently inhibited by oxotremorine and increased by atropine. The ACh output evoked by either 45 or 108 mM potassium was enhanced by atropine. Oxotremorine did not affect the ACh release by high potassium but prevented the facilitatory effect of atropine. It is concluded that the inhibitory muscarinic mechanism modulates similarly the ACh release evoked by DMPP or high potassium and the release caused by electrical stimulation. From the experiments with high potassium it is concluded that the inhibitory muscarine receptors are localized at the site of ACh release.     

Benzimidazolones and renzapride facilitate acetylcholine release from guinea-pig myenteric plexus via 5-HT4 receptors

The effects of the 5-HT₄ receptor agonists BIMU 8, BIMU 1, renzapride and of the 5-HT_1p receptor agonist 5-hydroxyindalpine on basal and electrically evoked outflow of tritium were studied in guinea-pig longitudinal muscle myenteric plexus preparations preincubated with [³H]choline. Muscle contractions were recorded simultaneously.BIMU 8 caused a calcium dependent and tetrodotoxin sensitive increase in basal [³H]outflow that was assumed to represent release of [³H]acetylcholine. In addition, BIMU 8 enhanced the release of [³H]acetylcholine and twitch contractions evoked by submaximal electrical stimulation. Ondansetron (1 mol/l) did not change the effects of BIMU 8, but DAU 6285 and tropisetron (each 1 mol/l) competitively antagonized the various facilitatory effects of BIMU 8 with pA₂ values of 7.0–7.2 (DAU 6285) and 7.0–7.3 (tropisetron). The phosphodiesterase inhibitors IBMX and rolipram did not increase the effects of BIMU 8. BIMU 1 and renzapride also concentration-dependently increased basal release of acetylcholine, and release and contractions caused by submaximal stimulation. The effects of BIMU 1 and renzapride were competitively antagonized by 1 mol/l tropisetron (pA₂ 6.6–7.1). The EC₅₀ values for the increase in the evoked [³H]acetylcholine release and contractions were closely similar. 5-Hydroxyindalpine did not change basal release and slightly inhibited the evoked release of [³H]acetylcholine. Release of acetylcholine and contractions elicited by submaximal stimulation were strongly inhibited by ( + )-tubocurarine which indicates that nicotinic ganglionic transmission is involved in this kind of release.The results suggest that BIMU 8, BIMU 1 and renzapride stimulate 5-HT₄ receptors at cholinergic interneurones and thereby facilitate nicotinic ganglionic transmission in the myenteric plexus. Cyclic AMP is probably not involved in the 5-HT₄ receptor mediated facilitation of acetylcholine release.     

The differential contribution of endogenous prostaglandins to the release of acetylcholine from the myenteric plexus of the guinea-pig ileum.

1. Prostaglandin E (PGE) may be essential for maintaining the sensitivity of the myenteric plexus of guinea-pig ileum to nicotine. The contributions of prostaglandins to nervous activity evoked by different stimuli have now been investigated by measuring the amount of acetylcholine (ACh) released from the myenteric plexus of the guinea-pig ileum. 2. The amount of ACh released in response to dimethylphenylpiperazinium (DMPP) or substance P was depressed to about 40% of control by 2.8 microM indomethacin (Ind), whereas the release of ACh induced by 5-hydroxytryptamine (5-HT) was not affected. The inhibitory effects of Ind were overcome by 14.3 nM PGE2. 3. Mepacrine 5 microM, an inhibitor of phospholipase A2, depressed the release of ACh in response to DMPP and substance P to the same extent as Ind. These inhibitory effects of mepacrine were overcome by arachidonic acid (10 microM), but not by arachidonic acid plus Ind. The release of ACh evoked by 5-HT or electrical field stimulation (EFS) was also inhibited to about 60% of control by mepacrine but these inhibitions were overcome by arachidonic acid (10 microM) either in the absence or the presence of Ind. 4. The results suggest that endogenous prostaglandins and arachidonic acid contribute to the maintenance of the excitability of the myenteric plexus by DMPP and substance P. By contrast, the release of ACh induced by 5-HT and EFS may be regulated by arachidonic acid and not by prostaglandins.     

Factors influencing the release of acetylcholine from the myenteric plexus of the ileum of the guinea-pig and rabbit.

1 The effects of electrical stimulation, changes in external ion concentrations and various drugs on acetylcholine release from the myenteric plexus were measured by bioassay in the presence of physostigmine and by recording the responses of the longitudinal muscle. In preparations from the guinea-pig, the acetylcholine output per pulse increased with decreasing frequency of stimulation and reached its maximum at a frequency of 0.017 Hz (1/min) and thus ensured that the output per unit of time was constant at frequencies below 0.5 Hz. Spontaneous release was suppressed during stimulation at 0.017 Hz. 2 In the rabbit, the fractional acetylcholine release was lower than in the guinea-pig. The output per pulse increased with decreasing frequency of stimulation but at a lesser rate, with the effect that the output per unit decreased between 0.5 and 0.017 Hz. 3 In the guinea-pig, reduction of the Ca2+ concentration, addition to the bath fluid of Mn2+, ganglion-blocking drugs, morphine and catecholamines reduced output more at low than at high frequencies of stimulation. In the rabbit, acetylcholine output was less sensitive to changes in Ca2+ concentration and insensitive to Mn2+ and morphine. 4 In the guinea-pig, morphine and catecholamines depressed both the contractile response and acetylcholine output whereas Mn2+ in concentrations up to 125 muM, bretylium and ganglion-blocking drugs depressed only acetylcholine output. 5 In preparations from the guinea-pig, drugs blocking noradrenergic neurons or alpha-adrenoceptors, e.g. bretylium, phenoxybenzamine, thymoxamine and phentolamine, increased acetylcholine output during stimulation at high (1.5 to 10 Hz) but not at low frequencies. 6 The implications of these findings for the release of acetylcholine from different pools in the heterogeneous myenteric plexus are considered. The possible errors, introduced by the effects of physostigmine, on the size of the acetylcholine pools and on the transmission of impulses within the myenteric plexus are discussed.     

Effect of external Ca2+ on the spontaneous and the various stimuli-induced acetylcholine release from guinea-pig ileum myenteric plexus

Effect of external Ca2+ concentration ([Ca2+]o) on spontaneous and various types of stimuli-induced acetylcholine (ACh) release from guinea-pig ileum myenteric plexus was studied. Electrical field stimulation- or high-K+-induced ACh release increased with the increment of [Ca2+]o. On the other hand, the spontaneous and the nicotine-induced ACh release increased up to 0.45 mM [Ca2+]o and then declined progressively as [Ca2+]o was raised. Qualitatively similar results were obtained with dimethylphenylpirerazinium-, 5-hydroxytryptamine- and substance P-induced ACh release. These results were discussed in terms of the stabilizing effect of Ca2+.     

Actions of Ca2+ antagonists on the guinea-pig ileal myenteric plexus preparation

The guinea-pig ileal myenteric plexus preparation has been used to compare the actions of three Ca2+ channel antagonists, D 600 (methoxyverapamil), nicardipine and diltiazem, on smooth muscle contractions and acetylcholine release in response to electrical stimulation. Acetylcholine release was not affected by these agents at concentrations of 10(-6)-10(-5)M. In confirmation of previous work, however, the smooth muscle contractile responses were effectively inhibited at these concentrations. Quercetin, a non-Ca2+ channel antagonist, was approximately equieffective in blocking both acetylcholine release and smooth muscle contractions. These data suggest that differences may exist in the antagonist sensitivity of voltage-dependent Ca2+ channels.     

Effects of 5-HT4 receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus

Summary The effects of 5-methoxytryptamine and 5-hydroxytryptamine (5-HT) on both basal and electrically evoked outflow of tritium were studied in guinea-pig myenteric plexus preparations preincubated with [³H]-choline. Basal outflow. 5-Methoxytryptamine caused a transient and calcium-dependent increase in basal outflow of [³H]acetylcholine that was abolished by tetrodotoxin. Ondansetron (1 mol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 mol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PK_B value for ICS 205-930 was 6.6 suggesting an involvement of 5-HT₄ receptors. 5-HT caused an increase in basal outflow of [³H]acetylcholine and a biphasic concentration-response curve was obtained. The maximal response of the first phase to 5-HT (release of 0.98% of tissue tritium) and the maximal response to 5-methoxytryptamine (0.94% of tissue tritium) were similar but 5-methoxytryptamine (-log EC₅₀: 6.9) was less potent than 5-HT (-log EC₅₀ of the high affinity component: 7.9). ICS 205-930 (0.01–1.0 mol/1) acted as a competitive antagonist against the low affinity component of the 5-HT concentration-response curve with a pA₂ value of 8.0. It is concluded that stimulation of both 5-HT₄ receptors (by 5-methoxytryptamine and submicromolar concentrations of 5-HT) and 5-HT₃ receptors (by micromolar concentrations of 5-HT) causes a release of acetylcholine which in turn leads to smooth muscle contraction. Electrically evoked outflow. This outflow of [³H]acetylcholine was concentration-dependently inhibited by both 5-methoxytryptamine and 5-HT. ICS 205-930 (1 mol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 mol/1) 5-methoxytryptamine enhanced the evoked outflow of [³H]acetylcholine, an effect which was attenuated by 3 mol/1 ICS 205-930. These results suggest that 5-methoxytryptamine may both inhibit (via 5-HT₁ receptors) and facilitate (via 5-HT₄ receptors) the evoked release of acetylcholine from guinea-pig myenteric neurones. The facilitatory action is unmasked when the 5-HT₁ receptor is blocked by methiothepine. Send offprint requests to H. Kilbinger at the above address     

Localization of endothelin ETB receptors on the myenteric plexus of guinea-pig ileum and the receptor-mediated release of acetylcholine.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.     

Acetylcholine release from the myenteric plexus of guinea-pig ileum by prostaglandin E1

Release of acetylcholine (ACh) by prostaglandin E₁ from the nerve terminals of the guinea-pig longitudinal muscle strip was studied in order to reveal the effect of PGE₁ on myenteric plexus activity. The ACh released was collected in the presence of physostigmine (2·1 μg ml^?1) and choline (0·1 μg ml^?1) at 38° C. Five to 100 ng ml^?1 PGE₁ enhanced the release dose-dependently. The effect was maintained during the presence of PGE₁ in the organ bath, while rapid tachyphylaxis was observed with the ACh-releasing action of nicotine. Tetrodotoxin or morphine almost completely inhibited the effect of PGE₁ on ACh release. Hexamethonium, in a concentration which completely blocked the effect of nicotine, partially inhibited the effect of PGE₁. In the late phase of nicotine action, the tissue was still sensitive to PGE₁ despite the continued exposure to nicotine. These data suggest the presence in the myenteric plexus of PG receptors which can increase ACh release.     

Effects of genistein, an isoflavone isolated from Genista tridentata, on isolated guinea-pig ileum and guinea-pig ileal myenteric plexus.

The inhibitory action of the major constituent of Genista tridentata L. (Papilionaceae), 4',5,7-trihydroxyisoflavone (genistein), on contractions induced by agonists and electrical field stimulation of smooth muscle was analysed. Genistein inhibited twitches evoked by electrical-stimulation of strips of guinea-pig ileum with an IC50 value of 34 microM. Genistein (34 microM) inhibited contractions of the guinea-pig ileum by several agonists in a non-selective, antispasmodic action and had no effect on inhibition of 3H-ACh release from ileal myenteric plexus. Genistein (34 microM) produces an increase in cAMP levels of guinea-pig ileum which resulted in a smooth muscle relaxation which leads us to think that there must be a blockade of its phosphodiesterase.     

Effects of adenosine on 45Ca uptake and [3H]acetylcholine release in synaptosomal preparation from guinea-pig ileum myenteric plexus

The effects of adenosine on acetylcholine (ACh) release and calcium uptake were examined in a synaptosomal fraction prepared from guinea-pig ileum myenteric plexus-longitudinal muscle. A high concentration of potassium (40 mM) and electrical pulses (ES:10Hz) caused a marked increase in the output of [3H]ACh from [3H]choline-preloaded crude synaptosomes. This [3H]ACh output was calcium- and temperature-dependent. Adenosine reduced the high potassium-induced release significantly, and the electrically stimulated release completely. When the preparation was depolarized by high potassium or electrical pulses, the 45Ca uptake by synaptosomes was significantly enhanced. The uptake of 45Ca induced by high potassium was significantly reduced and that induced by electrical stimulation was completely abolished by adenosine. From these results, it may be suggested that adenosine inhibits neurotransmitter release by suppressing the presynaptic influx of calcium ion during depolarization of the cholinergic nerve terminals in guinea-pig ileum.     

Kinetics of morphine-sensitive [3H]-acetylcholine release from the guinea-pig myenteric plexus.

1 Longitudinal muscle-myenteric plexus preparations from the guinea-pig ileum were superfused at a constant rate while isotonic contractions were monitored. 2 The preparations were superfused with [3H]-choline while stimulated supramaximally at 0.1 Hz followed by washout in the presence of hemicholinium-3. The evoked release of the label due to a second 0.1 Hz stimulation in the absence of an anticholinesterase was measured. 3 Evoked efflux of the label was initially fast followed by a slower phase. 4 Morphine reduced the size of the pool and the rate of the initial fast efflux and the size of the pool but not the rate of the slow efflux evoked by supramaximal stimulation. 5 Submaximal stimulation reduced only the size of the pools from which the fast and slow efflux originated. 6 Naloxone reversed the depression of contractions and evoked release produced by morphine. 7 Results suggest that 0.1 Hz stimulation releases [3H]-acetylcholine simultaneous from two pools. The fast release may originate from spontaneously firing units whose rate of discharge is depressed by morphine, while the slow release originates from neurones which do not fire spontaneously and whose threshold to field stimulation is increased by morphine.