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     

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.     

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.     

Dual effects of 5-hydroxytryptamine on the release of gamma-aminobutyric acid from myenteric neurones of the guinea-pig ileum.

The effects of 5-hydroxytryptamine (5-HT) on the release of gamma-aminobutyric acid (GABA) were examined in the longitudinal muscle-myenteric plexus (LM-MP) preparation of guinea-pig ileum. 5-HT increased the spontaneous release and inhibited the electrically-evoked release of [3H]-GABA. The 5-HT-evoked release was Ca2+-dependent and tetrodotoxin-sensitive, and was antagonized by (3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930), but not by methysergide and ketanserin. The inhibitory effect of 5-HT was antagonized by methysergide, but not by ketanserin and ICS 205-930. 8-Hydroxy-2-(di-n-propylamino)tetralin mimicked the inhibitory effect of 5-HT. Thus, 5-HT may exert an excitatory effect on the enteric GABAergic neurone via the 5-HT3 receptor and an inhibitory effect via the 5-HT1A receptor.     

The effect of ascorbate on the acetylcholine release from guinea-pig ileal myenteric plexus

The effect of ascorbate on the release of acetylcholine from the longitudinal muscle-myenteric plexus preparation of the guinea-pig isolated ileum has been investigated using a bioassay and an isotopic technique. Ascorbate at 5 mM increased the spontaneous output of acetylcholine and enhanced DMPP-induced output of acetylcholine, while iso-ascorbate, 5 mM, did not. Ascorbate did not influence either the spontaneous or the DMPP-induced release of acetylcholine from synaptosomes of the ileal myenteric plexus. These results suggest that ascorbate promotes acetylcholine release from intramural cholinergic nerves.     

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     

The variation of acetylcholine release from myenteric neurones with stimulation frequency and train length

The effects of scopolamine on the release of 3H-acetylcholine (ACh) from the guinea-pig myenteric plexus were studied at different stimulation frequencies (0.03-10 Hz) and train lengths (1-180 pulses). Release of 3H-ACh was measured in the absence of cholinesterase inhibitors as the outflow of tritium from myenteric plexus-longitudinal muscle preparations preloaded with 3H-choline. In control experiments the volley output of 3H-ACh declined with increasing train length and increasing stimulation frequency. Stimulation by one pulse produced the highest volley output. Scopolamine facilitated the evoked output of 3H-ACh via blockade of presynaptic muscarine receptors. A significant increase was already observed when the preparation was stimulated with 3 pulses at 10 Hz which indicates that the inhibitory muscarinic mechanism becomes operational within 200 ms. The facilitatory effects of scopolamine depended on both train length and frequency of stimulation. Maximal increases in 3H-ACh output were seen with brief trains (3 and 6 pulses) at a high frequency (10 Hz), or with longer trains (20 and 180 pulses) at lower frequencies (0.3 and 1 Hz). Scopolamine compensated for the frequency-dependent decline of 3H-ACh volley output only during brief periods of stimulation (3 and 6 pulses). It is therefore concluded that the decline in volley output during the first few pulses of a train is due to the negative feedback mechanism which is activated by the transmitter released by the preceding impulses. With longer trains of stimulation the negative feedback mechanism plays only a minor role in regulating the output per pulse.     

Presynaptic muscarinic and alpha-adrenoceptor-mediated regulation of GABA release from myenteric neurones of the guinea-pig small intestine.

The effects of cholinomimetic and sympathomimetic drugs on the release of [3H]-gamma-aminobutyric acid ([3H]-GABA) evoked by high K+ from the isolated small intestine of the guinea-pig were investigated, in the presence of tetrodotoxin. Acetylcholine and oxotremorine, at concentrations ranging from 10(-9) to 10(-6) M inhibited the evoked release of [3H]-GABA in a concentration-dependent manner, while nicotine was without effect. Scopolamine and pirenzepine inhibited the effect of oxotremorine, while hexamethonium had no effect. The IC50 values for scopolamine and pirenzepine of the oxotremorine (3 X 10(-8) M)-induced inhibition were 1.02 X 10(-9) M and 9.78 X 10(-10) M, respectively. Noradrenaline, but not isoprenaline inhibited the evoked release of [3H]-GABA. Clonidine (10(-10)-10(-6) M) reduced the evoked release of [3H]-GABA in a concentration-dependent manner, but phenylephrine had no effect. The inhibitory effect of clonidine was antagonized by yohimbine but not by prazosin. These findings provide evidence for the localization of M1-muscarinic and alpha 2-adrenoceptors on GABAergic nerve terminals and their involvement in the presynaptic control of the release of GABA from the guinea-pig small intestine.     

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.     

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.

     

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.     

Succinylcholine-induced acceleration and suppression of electrically evoked acetylcholine release from mouse phrenic nerve-hemidiaphragm muscle preparation.

The effects of a depolarizing neuromuscular blocker on electrically evoked acetylcholine (ACh) release were studied using a mouse phrenic nerve-diaphragm muscle preparation preloaded with [3H]-choline, and the changes in muscle tension were recorded simultaneously. Succinylcholine at a low concentration (1 microM) enhanced evoked [3H]-ACh release, which tended to follow the increase in peak amplitude of tetanic tension; whereas at high concentrations (10 and 30 microM), it simultaneously reduced both release and tension. Decamethonium even at 10 and 30 microM had little effect on [3H]-ACh release despite producing a significantly greater reduction in tension compared with succinylcholine. (+)-Tubocurarine (5 microM) prevented the enhancing effect of [3H]-ACh release induced by 1 microM, but not the decreasing effect induced by 10 microM succinylcholine. These results suggest that succinylcholine induced acceleration at low concentrations due to a positive feedback mechanism through presynaptic nicotinic ACh receptors and the inhibition of ACh release at high concentration contributes in part to the neuromuscular blockade.     

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.     

Actions of gamma-aminobutyric acid on neurones of guinea-pig myenteric plexus.

The effects of gamma-aminobutyric acid (GABA) applied by ionophoresis, pressure ejection and superfusion to myenteric neurones of the guinea-pig ileum were investigated by intracellular recording techniques. Ionophoretic or pressure application of GABA (10 pC-30 nC) caused membrane depolarizations of AH neurones but not S neurones. This depolarization was associated with a conductance increase. It reversed polarity at a membrane potential of -18 mV when intracellular electrodes contained KCl, and -39 mV when electrodes contained K acetate, citrate or sulphate. The ionophoretic depolarization was antagonized by bicuculline (1-30 microM) in an apparently competitive manner. During prolonged or repeated ionophoretic application of GABA, both the depolarization and conductance increase desensitized. Superfusion of GABA (1-100 microM) caused a membrane depolarization in AH neurones, associated with an increase in membrane conductance. The increase in conductance was always smaller than that evoked by ionophoresis of GABA. Bicuculline only partially depressed the depolarization induced by superfusion of GABA, particularly slowing its rising phase. beta-p-Chlorophenyl GABA (baclofen) (10 microM) caused a depolarization similar to that observed with GABA in the presence of bicuculline. The depolarization induced by baclofen and GABA (in presence of bicuculline) superfusion did not decline during prolonged applications; superfusion of GABA but not baclofen reversibly reduced or eliminated the effects of GABA ionophoresis. It is concluded that GABA has two effects on the membrane of myenteric neurones. The first is a bicuculline-sensitive, rapidly desensitizing chloride activation: the second is a bicuculline-insensitive, non-desensitizing depolarization.     

Polydeoxyribonucleotides and nitric oxide release from guinea-pig hearts during ischaemia and reperfusion.

1. Two polydeoxyribonucleotides, produced by the controlled hydrolysis of DNA of mammalian lung (defibrotide and its lower molecular weight fraction, P.O. 085 DV), were studied for their ability to modify the release of nitrite and the coronary flow in perfusates collected from isolated, normally perfused hearts of guinea-pigs and from hearts subjected to regional ischaemia and reperfusion. 2. In guinea-pig normally perfused hearts, both defibrotide (DFT) and its fraction, P.O. 085 DV, increase the amount of nitrite appearing in perfusates in a concentration-dependent fashion. At the highest concentration studied (10(-6) M), P.O. 085 DV was more effective than DFT. A concomitant increase in the coronary flow was observed. 3. The increase in nitrite in perfusates and the increase in coronary flow induced by both DFT and P.O. 085 DV were significantly reduced by NG-monomethyl-L-arginine (L-NMMA, 10(-4) M), an inhibitor of nitric oxide synthase (NOS). 4. The endothelium-dependent vasodilator, acetylcholine (ACh), enhances the formation of nitrite and the coronary flow. Both the increase in coronary flow and in the formation of nitrite were significantly reduced by L-NMMA (10(-4) M). 5. In guinea-pig hearts subjected to ischaemia and reperfusion, the effect of both compounds in increasing the amount of nitrite in perfusates was more evident and more pronounced with P.O. 085 DV. 6. Reperfusion-induced arrhythmias were significantly reduced by both compounds to the extent of complete protection afforded by compound P.O. 085 DV. 7. The cardioprotective and antiarrhythmic effects of DFT and P.O. 085 DV are discussed.     

Muscarinic agonists and potassium currents in guinea-pig myenteric neurones.

1. Intracellular electrophysiological recordings were obtained from single neurones of the guinea-pig myenteric plexus in vitro. Using single electrode voltage clamp techniques, four distinct potassium currents were described and the effects of muscarinic agonists on these currents were studied. 2. A calcium-dependent potassium current (gKCa) was present in AH neurones at rest, and was much increased following a brief depolarization (50 ms, to 0 mV). Muscarinic agonists reduced both the resting current and the current evoked by depolarization. Pirenzepine competitively antagonized the suppression by muscarine of the calcium-dependent potassium current (or after-hyperpolarization) following an action potential. The dissociation equilibrium constant for pirenzepine was about 10 nM. 3. The conductance of AH neurones increased two to three fold when they were hyperpolarized negative to -90 mV. This inward rectification was blocked by extracellular caesium (2 mM) or rubidium (2 mM), but not by tetraethylammonium (TEA, 40 mM), 4-aminopyridine (100 microM) or cobalt (2 mM). The inward rectification was unaffected by muscarinic agonists. 4. When AH neurones were depolarized from very negative holding potentials (less than -80 mV) a brief outward current was recorded with a duration of about 200 ms. This transient or A current was completely blocked by 4-aminopyridine (100 microM) but was not affected by tetrodotoxin (300 nM), TEA (40 mM) or cobalt (2 mM). Muscarinic agonists did not affect the A current. 5. In S neurones, and in AH neurones in calcium-free solutions, the potassium conductance (in TEA and caesium) behaved according to constant field assumptions. This background conductance was suppressed by muscarinic agonists. 6. It is concluded that the depolarization by muscarinic agonists of myenteric AH neurones is due to a suppression of both a calcium-dependent potassium conductance and a background potassium conductance. Muscarinic depolarization of S neurones results only from suppression of the background potassium conductance. Effects on both conductances result from M1-receptor activation. Inward rectifying and transient outward (A) potassium currents are unaffected.