Isolation of Cholinergic Synaptic Vesicles from the Myenteric Plexus of Guinea-Pig Small Intestine

The acetylcholine-rich myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine has been subjected to subcellular fractionation using modifications of both classical methods and that originally devised for bulk isolation of cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo marmorata by means of density gradient centrifugation in a zonal rotor. The latter method gave a vesicle fraction with the highest acetylcholine content so far recorded for a mammalian particulate fraction, 30.9 +/- S.E.M. 1.8 (5) nmol of acetylcholine . mg of protein-1. Electron-microscopical examination showed that it consisted of a homogeneous preparation of vesicl…

Determination of acetylcholine, nitrostigmine and acetylcholinesterase activity in four patients with severe nitrostigmine (E 605 forte) intoxication.

Concentrations of acetylcholine and nitrostigmine as well as acetylcholinesterase activity were determined in 4 patients with severe nitrostigmine intoxication. In the acute phase of the intoxication, acetylcholine levels up to 36 and 55 ng/ml were found in blood plasma and cerebrospinal fluid, respectively. At the same time no activity of acetylcholinesterase in plasma and erythrocytes was detected. Acetylcholinesterase activity gradually recovered at the end of the second week. In the first 3 days after the ingestion plasma nitrostigmine concentrations ranged from 409 to 86 ng/ml. At the end of the third week nitrostigmine was still found in a concentration of about 1 ng/ml plasma. Redist…

Muscarinic modulation of acetylcholine release: Receptor subtypes and possible mechanisms

The release of acetylcholine from central and peripheral neurones can be inhibited and facilitated by muscarine autoreceptors, i.e. receptors located on the cholinergic neurone. In the last few years evidence has accumulated that muscarine receptors are heterogeneous. This chapter describes attempts that have been made to classify the muscarine autoreceptors. In addition, some possible mechanisms behind the neuronal muscarine receptors are examined.

Inhibition of human detrusor contraction by a urothelium derived factor.

Stimulating muscarinic receptors in pig bladder urothelium causes the release of a diffusable factor that inhibits contractions of the underlying detrusor muscle. We investigated whether the contractions of human detrusor strips elicited by the muscarinic agonist carbachol, electrical field stimulation, KCl or the neurokinin receptor agonist neurokinin A are affected by the urothelium.Paired intact and urothelium denuded muscle strips were placed in modified gassed Tyrode's solution at 37C. Cumulative concentration-response curves to carbachol or KCl were constructed. In other tissues the strips were stimulated electrically (1 to 40 Hz) with trains of square wave pulses 20 seconds in durati…

Tissue Differences in the Effect of Atropine on the Evoked Release of Acetylcholine

The effect of atropine on the release of acetylcholine (ACh) from different peripheral parasympathetically innervated tissues was investigated. My enteric plexus The longitudinal muscle-myenteric plexus preparation of the guinea-pig was incubated in eserinecontaining Tyrode solution. The ACh release evoked by high K+ (45 or 108 μ) or by the nicotinic drug dimethylphenylpiperazinium (DMPP) (10 pM) was increased by atropine (0.1–10 μ) in a concentration-dependent fashion. Muscarinic agonists (oxotremorine; propargylester of arecaidine) prevented the facilitatory effect of atropine on ACh release. These results suggest that the ACh release from the myenteric plexus is regulated via presynaptic…

Functional relevance of presynaptic muscarinic autoreceptors.

Pre- and postsynaptic muscarinic receptors have been characterized in the isolated trachea and ileal circular muscle of the guinea pig. The muscarinic autoreceptors mediating inhibition of acetylcholine release in the circular muscle belong to the M1 subtype, whereas those inhibiting acetylcholine release in the trachea are M2 or M4 receptors. In both tissues the postsynaptic muscarinic receptors are M3 receptors. Blockade of the autoreceptors by selective M1 and M2/M4 receptor antagonists leads to facilitation of cholinergic neurotransmission.

Effects of Atropine on Acetylcholine Overflow from Perfused Chicken Hearts

Isolated chicken hearts were perfused (20 ml/min) with Tyrode’s solution. Release of acetylcholine (ACh) was evoked either by electrical stimulation (1 ms; 15 mA) of both preganglionic vagus nerves or by perfusion with dimethylphenylpiperazinium (DMPP). ACh was extracted from the perfusates by ion-pair extraction and determined by gas chromatography.

Muscarinic Modulation of Acetylcholine Release from the Myenteric Plexus of the Guinea Pig Small Intestine

The concept of a local feedback regulation of neurotransmitter release has been most intensively studied in the adrenergic system (See Ref. 13 for review). The feedback hypothesis of noradrenaline release is based on the findings that α-adrenolytic drugs enhance, and α-adrenoceptor agonists reduce neuronal noradrenaline release. Relatively few systematic studies have been made on the influence of cholinolytic and cholinomimetic drugs on the release of ACh. Szerb and Somogyi (14) reported that the potent muscarinic agonist oxotremorine inhibited ACh output from cerebral cortical slices. Further, the muscarinic antagonists atropine and hyoscine have been found to facilitate the release of ACh…

THE EFFECTS OF MUSCARINIC AGONISTS AND ANTAGONISTS ON ACETYLCHOLINE RELEASE FROM PERIPHERAL CHOLINERGIC NERVES IN THE ABSENCE AND PRESENCE OF A CHOLINESTERASE INHIBITOR

Chapter 21 Modulation of acetylcholine release by nitric oxide

Publisher Summary This chapter reviews the studies in which the effects of endogenous and exogenous nitric oxide (NO) on the release of acetylcholine were investigated. As tools for the involvement of endogenous NO, the effects of NO synthase inhibitors on release of acetylcholine were studied. The cholinergic neurons are tonically stimulated by endogenous NO. On the other hand, NO synthase inhibitors fail to change either basal or electrically evoked acetylcholine release from slices of rat hippocampus and striatum. Endogenous and exogenous NO increase basal acetylcholine release from central and peripheral cholinergic neurons. The effect is tetrodotoxin sensitive and calcium-dependent, th…

SUBTYPES OF MUSCARINE RECEPTORS IN THE GUINEA-PIG ILEUM

Storage and release of false transmitters after infusion of (+)- and (?)-?-methyldopamine

Rabbits were given an infusion of 10 mg/kg (−)- or 30 mg/kg (+)-α-methyldopamine and killed after 135 min. The noradrenaline content of the heart was decreased to 26±5 and 34±2%, respectively, of the control value. After infusion of the (+)-isomer the missing noradrenaline was replaced by (−)-α-methylnoradrenaline. Electrical stimulation of the sympathetic nerves or infusion of acetylcholine plus atropine caused an output of noradrenaline and (−)-α-methylnoradrenaline from the isolated heart. The two amines were released in the same proportion as they were stored in the heart and the total output of both amines equalled the output of noradrenaline from control hearts. Nerve stimulation caus…

Control of Acetylcholine Release and of Intestinal Motility by Subtypes of Muscarine Receptors

Two types of neuronal muscarine receptors have been investigated in the myenteric plexus preparation of the guinea-pig small intestine: 1. Presynaptic receptors activation of which inhibits the depolarization-evoked release of acetylcholine. Pirenzepine and dicyclomine have low affinities to the release-inhibitory receptors (pA2 values 6.9 and 7.6) which suggests that the presynaptic receptors (similar to the smooth muscle receptors) belong to the M2 subtype. The inhibition of the electrically-evoked acetylcholine release by muscarine (0.01 - 1 μmol/1) was not affected by forskolin (1μmol/l). This indicates that cyclic AMP is not crucially involved in the muscarinic inhibition of acetylchol…

GAS CHROMATOGRAPHIC ESTIMATION OF ACETYLCHOLINE IN THE RABBIT HEART USING A NITROGEN SELECTIVE DETECTOR

The distribution of ACh in the rabbit heart was investigated by a modified gas chromatographic estimation method. ACh was extracted with perchloric acid, precipitated as reineckate and demethylated with sodium benzenethiolate. The tertiary amines derived from ACh and other choline esters were concentrated by a microdistillation procedure. Gas chromatography was performed using a nitrogen selective detector. In the range of concentrations between 0.4 and 2.5 nmol ACh per tissue sample the coefficient of variation was 5.2 per cent. The recovery of ACh added to heart extracts was 101 per cent. Evidence for the identity of the choline ester isolated from rabbit hearts and authentic ACh was obta…