Acetylcholine In Tissue Research Articles

Radio-enzymatic assay of acetylcholine in tissues

A method for the determination of ACh in tissues and subcellular fractions is described. The ACh is extracted by an ion pair exchange method, purified by paper chromatography and determined by enzymatic phosphorylation with choline kinase after enzymatic hydrolysis with AChE.

Simultaneous analysis of choline and acetylcholine levels in rat brain by pyrolysis gas chromatography

Gas chromatographic analysis of the tertiary amines resulting from either chemical (1,2) or heat-catalyzed (3,4) removal of a quarternary methyl group from choline esters has provided a sensitive chemical assay for acetylcholine (ACh) in various tissues. In order to study ACh turnover using precursor labeling techniques it is also necessary to measure the level of free choline in tissue. Recent publications on the level of choline in the central nervous system (5,6) and on the role its uptake plays in the regulation of ACh synthesis in cholinergic neurons have also stimulated interest in the measurement of choline. Methods for simultaneous analysis of choline and ACh employing chemical demethylation have previously been published (7). The present paper describes the modification of a previous method (4) which is necessary for simultaneous analysis of choline and ACh by pyrolysis gas chromatography. These modifications are required because endogenously occurring amounts of choline are not reproducibly precipitated as the eneiodide salt from aqueous solutions and choline cannot be quantitatively converted to its tertiary amine analog by pyrolysis. It is therefore quantitatively isolated and converted to a choline ester prior to gas chromatographic analysis.

Triethylcholine as a precursor to a cholinergic false transmitter

A FALSE transmitter is a substance that is stored and released by a nerve terminal instead of its normal transmitter. Several false transmitters have been demonstrated at adrenergic synapses1, but there has been no report of the synthesis, storage and release of a false transmitter at cholinergic synapses. It has been suggested that the triethyl analogue of choline (2-hydroxyethyl-triethylammonium iodide, TEC) might form a cholinergic false transmitter; in vitro studies have shown that it can be accumulated by tissues that take up choline2, and that choline acetyltransferase can acetylate TEC (refs 3 and 4). Nothing is known, however, about the specificity of the processes associated with storage and release of acetylcholine in intact tissue. The possibility that TEC can form a false transmitter was studied in the present experiments. The results demonstrate the uptake of TEC, its acetylation, and the release of acetyl-TEC by the superior cervical ganglion of the cat.

Heart tissue acetylcholine in chronically exercised rats.

Le taux de l'acetylcholine du myocarde a ete dose par chromatographie en phase gazeuse chez le rat. Chez l'animal exerce le taux de l'acetylcholine etait plus eleve que chez son homologue sedentaire.

Changes of Tissue Acetylcholine in the Distended Intestine

The effects of distension of the intestinal wall on the liberation and the synthesis of acetylcholine (ACh) were studied in the isolated guinea-pig ileum measuring the changes in the amount of free and bound ACh. In the presence of eserine longitudinal distension increased the amount of bound ACh in the intestinal wall without increasing the free ACh, while circumferential distension or mucosal rubbing increased free but not bound ACh. These effects were evident within 30 sec. In the eserine-free medium the amount of bound ACh was increased in cither direction of distension. Addition of more than 5×10−8 g/ml ACh to the bathing medium completely blocked the distension-induced increase. It is concluded that the release and the synthesis of ACh may be both augmented by the circumferential and the longitudinal distension in the physiological conditions. In the presence of eserine, however, the presence of unphysiologically high extracellular ACh levels presumably inhibits the distension-induced augmentation of ACh synthesis in the tissue.

Estimation of choline and acetylcholine in tissue by pyrolysis gas chromatography.

Estimation of choline and acetylcholine in tissue by pyrolysis gas chromatography.

Quantitative Measurements of Endogenous Levels of Acetylcholine and Choline in Tetrathyridia of Mesocestoides corti (Cestoda) by Means of Combined Gas Chromatograph-Mass Spectrometry

Endogenous levels of choline and acetylcholine in tetrathyridia of Mesocestoides corti were determined under different environmental conditions. The highest values of choline and ACh were obtained when tetrathyridia were washed in media NCTC 135 and Eagle's Basal Medium. The lowest values were obtained when organisms were washed in 0.85% NaCl, or BME without choline chloride. Incubation of organisms in different concentrations of physostigmine showed an increase in the ACh level. The levels of choline and ACh decreased with decreasing drug concentration. DFP also showed a similar effect on choline and ACh. Oxotremorine increased choline and ACh levels but the increase was considerably lower than in physostigmine or DFP-treated organisms. While the histochemical association of acetylcholinesterase (AChE) with the nervous system has been the primary means of claiming the presence of acetylcholine (ACh) in helminthic tissue, a few workers have demonstrated ACh by biochemical isolation. Bueding (1952) showed that extracts of Schistosoma mansoni contain high levels of AChE and choline acetylase activities. Barker et al. (1966) reported the level of ACh in S. mansoni to be 5.3 jug/g wet weight. Chance and Mansour (1953) demonstrated AChE and choline acetylase activities in the liver fluke, Fasciola hepatica. Furthermore, they reported that this parasite contains an ACh-like substance. The presence of ACh was demonstrated in Ascaris by Mellanby (1955) who showed that the head region contains a much higher level than the body. Several biochemical methods are routinely used for determining ACh-like activity in extremely small amounts, but their reliability has been questioned (Hosein and Orzek, 1966; Stockley, 1969) in view of the fact that their response is not specific to one compound alone. Definitive identification of ACh and choline Received for publication 2 November 1973. * This investigation was aided in part by research grant USPHS AI-11107-01 from the NIH, Bethesda, Maryland. t Recipient of USPHS-NIAID Training Grant No. TOI-A1-00249. + Grand Island Biological Company, 519 Aldo Avenue, Santa Clara, California 95050. was made in tetrathyridia of Mesocestoides corti by means of a combined gas chromatograph-mass spectrometry (GC-MS) system. The functional aspects of ACh in this parasite will be reported elsewhere. MATERIALS AND METHODS Tetrathyridia of Mesocestoides, originally isolated from lizards (Specht and Voge, 1965), were maintained in Swiss albino mice. The animals were killed by cervical dislocation and tetrathyridia were transferred from the body cavity into petri dishes containing GIBCOt NCTC 135, "Eagle's Basal Medium" (BME) with or without choline chloride, or physiological saline solution (0.85% NaCl). Worms were preincubated in BME without choline chloride for a maximum of 1/2 hr before exposure to drug. The number of worms never exceeded 20 per ml of medium. The maximum period of incubation was 1 hr. For each drug concentration, the motility of worms was observed under the dissecting microscope at 15, 30, 45, and 60 min. The effect on choline and ACh levels by 3 different concentrations of physostigmine salicylate and 2 concentrations of oxotremorine and diisopropylfluorophosphate (DFP) was tested. The effect of each concentration was tested 3 times and organisms for each concentration were obtained from a different mouse. After incubation at 37 C, parasites were washed and dried by use of aspiration of fluids or by means of a Millipore filter. Organisms were then removed with a frozen spatula and immediately immersed in liquid nitrogen. The entire process of washing and drying lasted 6 to 8 min. Organisms were pulverized by the method of Campbell et al. (1970). The finely powdered tissue, 0.066 to 1.11 g, was transferred to a tared, chilled glass homogenizer to prevent thawing, and weighed. With few exceptions, the

Fluorophotometric method for estimation of tissue acetylcholine contents

Fluorophotometric method for estimation of tissue acetylcholine contents

An enzymatic method for measuring picomole quantities of acetylcholine and choline in CNS tissue

A rapid and sensitive enzymatic assay for measuring picomole quantities of both acetylcholine (ACh) and choline (Ch) in tissue extracts has been developed. After ACh and Ch were extracted into 15% 1 n formic acid/85% acetone by the procedure of Toru and Aprison, lipids were removed by a heptane-chloroform extraction. All quaternary ammonium compounds were isolated by precipitation with periodide. After the precipitate (including ACh and Ch) was dissolved in a known volume of water, aliquots were taken for both assays. In the ACh assay, endogenous Ch was removed after conversion to choline phosphate by choline kinase, whereas ACh was subsequently hydrolyzed by base. In the presence of [ 14C]acetyl-CoA and choline acetyltransferase, the choline moiety was converted into [ 14C]ACh. The labeled ACh was extracted into sodium tetraphenylboron/butenenitrile and then counted in a scintillation counter. In the Ch assay, the first enzyme reaction step is omitted and only the second is used. The lower limit of sensitivity in both assays is 20 pmoles. Once the tissue has been carried through the extraction step, over eighty determinations can be made in one day. In vivo levels of ACh and Ch in the cerebrum of rats are reported for totally frozen rats and for rats sacrificed by the near-freezing procedure of Takahashi and Aprison. Mean ACh values in the two groups statistically were the same (26.5 ± 2.2 and 25.3 ± 1.7 nmoles/g, respectively) whereas the mean Ch values were significantly different (25.7 ± 0.9 and 64.0 ± 3.6 nmoles/g, respectively). The difference in the Ch levels as well as the importance of specifying the conditions that effect the measurement of ACh and Ch are discussed.

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).

Autonomic nervous control of cardiac frequency in the exercise-trained rat.

Autonomic nervous control of cardiac frequency in the exercise-trained rat.

Biosynthesis of acetylcholine in nervous tissue

articleBiosynthesis of acetylcholine in nervous tissueC. HebbC. HebbPublished Online:01 Oct 1972https://doi.org/10.1152/physrev.1972.52.4.918MoreSectionsPDF (6 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByEffects of unilateral vestibular ganglionectomy on glutaminase activity in the vestibular nerve root and vestibular nuclear complex of the rat1 January 2004 | Journal of Neuroscience Research, Vol. 77, No. 4Simultaneous measurement of oxygen and dopamine: Coupling of oxygen consumption and neurotransmissionNeuroscience, Vol. 47, No. 3The regulation of quantal sizeProgress in Neurobiology, Vol. 36, No. 2Effects of soman and sarin on high affinity choline uptake by rat brain synaptosomesBrain Research Bulletin, Vol. 22, No. 5Impairment of cerebellar blood flow autoregulation during cerebral ischemia in spontaneously hypertensive rats.Stroke, Vol. 19, No. 5Distinct influences of nerve growth factor and a central cholinergic trophic factor on medial septal explantsBrain Research, Vol. 422, No. 1Inputs to motoneurones in the hypoglossal nucleus of the rat: Light and electron microscopic immunocytochemistry for choline acetyltransferase, substance P and enkephalins using monoclonal antibodiesNeuroscience, Vol. 17, No. 1Evidence for the Presence of Cholinergic Nerves in Cerebral Arteries: An Immunohistochemical Demonstration of Choline Acetyltransferase29 June 2016 | Journal of Cerebral Blood Flow & Metabolism, Vol. 5, No. 2Choline acetyltransferase: Purification and immunohistochemical localizationLife Sciences, Vol. 34, No. 24Problems in the organization and control of acetylcholine synthesis in brain neuronsProgress in Biophysics and Molecular Biology, Vol. 44, No. 1Evidence for high affinity choline transport in synaptosomes prepared from hippocampus and neocortex of patients with Alzheimer's diseaseBrain Research, Vol. 289, No. 1-2Muscle reinnervation—I. Restoration of transmitter release mechanismsNeuroscience, Vol. 8, No. 3Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylaseNeurochemistry International, Vol. 5, No. 6Differential sensitivity of soluble and membrane-bound forms of choline O-acetyltransferase to inhibition by Coenzyme ABiochemical Pharmacology, Vol. 30, No. 17Innervation of intrapulmonary airway smooth muscle of the dog, monkey and baboonJournal of the Autonomic Nervous System, Vol. 3, No. 1Purification of chicken brain choline acetyltransferaseNeurochemistry International, Vol. 3, No. 6Increase in activity of choline acetyltransferase in the dorsal raphe nucleus following habenular deafferentationBrain Research, Vol. 194, No. 2Choline uptake and permanent memory storagePharmacology Biochemistry and Behavior, Vol. 13, No. 1Cholinergic synaptic vesicles isolated from Torpedo marmorata: Demonstration of acetylcholine and choline uptake in an in vitro systemNeuroscience, Vol. 5, No. 6A comparison of solubilized and membrane bound forms of choline-O-acetyltransferase (EC 2.3.1.6) in mouse brain nerve endingsBrain Research, Vol. 185, No. 2Axonal transport of norepinephrine and choline acetyltransferase in regenerating sciatic nerve of the ratExperimental Neurology, Vol. 62, No. 1Effect of hemicholinium-3 on the release and net synthesis of acetylcholine in Auerbach's plexus of guinea pig ileumNeuroscience, Vol. 2, No. 5Regulation of acetylcholine synthesis in nervous tissueLife Sciences, Vol. 20, No. 9Combined pharmacological and fine-structural studies of choline O-acetyltransferase at the myoneural junctionBrain Research, Vol. 119, No. 1The measurement of acetylcholine turnover rate in brain structuresNeuropharmacology, Vol. 15, No. 12Preparation and characterization of two isozymes of choline acetyltransferase from squid head gangliaBiochimica et Biophysica Acta (BBA) - Enzymology, Vol. 445, No. 1Histochemistry of putative transmitter substances in the insect brainProgress in Neurobiology, Vol. 7Accumulation in vitro of [methyl-14C]choline and the synthesis of radioactive acetylcholine in the nervous system of the prawn Palaemonetes varians (leach)Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, Vol. 54, No. 1Choline acetyltransferase of rat retina a comparison of its properties with the enzyme from brainComparative Biochemistry and Physiology Part C: Comparative Pharmacology, Vol. 54, No. 2Choline acetyltransferase activity and mass fragmentographic measurement of acetylcholine in specific nuclei and tracts of rat brainNeuropharmacology, Vol. 14, No. 11Increase in rat brain acetylcholine induced by choline or deanolLife Sciences, Vol. 17, No. 6Brain tissue and fluorocitrate synthesisBiochemical Pharmacology, Vol. 24, No. 11-12Comparative studies on synaptosomes: high-affinity uptake and acetylation of N-[Me-3H]choline and N-[Me-3H]N-hydroxyethylpyrrolidiniumBrain Research, Vol. 86, No. 2Two forms of guanylate cyclase in mammalian tissues and possible mechanisms for their regulationMetabolism, Vol. 24, No. 3Factors influencing transmission at adrenergic synapsesProgress in Neurobiology, Vol. 4Special chemistry of the basal ganglia 2. distribution of acetylcholine, choline acetyltransferase and acetylcholinesterasePharmacology & Therapeutics. Part B: General and Systematic Pharmacology, Vol. 1, No. 1Injection of radioactive materials into an identified axon of AplysiaBrain Research, Vol. 68, No. 2Histochemistry of choline acetyltransferase: A critical analysisBrain Research, Vol. 62, No. 2Neurotransmitter synthetic enzymesProgress in Neurobiology, Vol. 2 More from this issue > Volume 52Issue 4October 1972Pages 918-957 Copyright & PermissionsCopyright © 1972 the American Physiological Societyhttps://doi.org/10.1152/physrev.1972.52.4.918PubMed4345069History Published online 1 October 1972 Published in print 1 October 1972 Metrics

A method for differential measurement of free and bound acetylcholine in the small intestine.

Abstract It is well known that almost all acetylcholine (ACh) in tissues is present in a bound form and is released during excitation. In order to study the physiological role of ACh, the free form should be distinguishable from that of the bound. In previous papers (1-4) the amount of ACh diffused into the bathing solution of the intestine was considered to exhibit the entire amount of ACh released from tissue, however, there remains the possibility of retainment. Extraction of free ACh would be more satisfactory if the tissue were sliced before extraction. In this report, an attempt has been made to determine a method which would demonstrate the total picture of the tissue bound and free ACh in one operation.

A Method for Differential Measurement of Free and Bound Acetylcholine in the Small Intestine

It is well known that almost all acetylcholine (ACh) in tissues is present in a bound form and is released during excitation. In order to study the physiological role of ACh, the free form should be distinguishable from that of the bound. In previous papers (1-4) the amount of ACh diffused into the bathing solution of the intestine was considered to exhibit the entire amount of ACh released from tissue, however, there remains the possibility of retainment. Extraction of free ACh would be more satisfactory if the tissue were sliced before extraction. In this report, an attempt has been made to determine a method which would demonstrate the total picture of the tissue bound and free ACh in one operation.

Effect of triperidol on processes involving acetylcholine in rat brain in vitro

The effect of triperidol on the synthesis of free acetylcholine in fresh tissue, on the activity of the choline acetylase system in extracts of acetone powders, and on the activity of acetylcholinesterase in homogenates was studied in various regions of rat brain. Triperidol at a concentration 0·2 mM decreases the free acetylcholine synthesis in brain cortex slices by about 80 per cent. This decrease, although considerably lower, is statistically significant even at a concentration of the drug of 0·002 mM. Triperidol is without effect on the activity of the choline acetylase system in cerebral cortex at concentrations varying from 0·001 mM to 0·2 mM. Triperidol is without effect on the activity of acetylcholine esterase in all regions studied at concentrations ranging from 0·001 mM to 0·2 mM.

Synthesis of radioactive acetylcholine from ( 3 H)choline and its release from cerebral cortex slices in vitro.

Abstract— Guinea pig cerebral cortex slices were incubated for 60 min in a medium containing [3H]choline with or without the addition of 33 mM‐KCl for the last 30 min. KC1 caused the release into the medium of large amounts of both bioassayable and radioactive ACh, while at the same time their concentrations in the tissue decreased. The specific activity (d.p.m./pmol) of the ACh released by KC1 was greater than that released in control incubations, indicating that it comes from a newly synthesized, more radioactive store. The amounts of [3H]choline, [3H]ACh and the specific activity of tissue acetylcholine reached a plateau in the tissue 30 min after the addition of isotope. However isotopic equilibrium was not achieved because the specific activity of the ACh released, with or without KC1 in the subsequent 30 min, was less than the specific activity of the ACh remaining in the tissue. This implies the existence of a pool of ACh in the tissue which is turning over very slowly or is being synthesized from a less radioactive pool of choline. This pool of ACh does not contribute substantially to that released by KC1. Levorphanol at 10−3 M, as well as the analgesically inactive stereoisomer, dextrorphan, blocked the KCl‐stimulated release of both bioassayable and radioactive ACh. These drugs demonstrate the coupling of synthesis and release of ACh in cerebral cortex slices.

Status of the Microcirculation during Acute Choline Deficiency

Weanling 21-day-old male rats of the Fischer strain were fed a choline-deficient diet, some with and some without choline supplementation. Five days later, none had any signs of nephropathy by blood urea nitrogen concentration or by gross and microscopic examination of the kidneys. The mesoappendiceal circulation of the nonsupplemented rats appeared ischemic when compared with that of the choline-supplemented rats. There was a striking alteration in the characteristic reaction of the microvessels to topical epinephrine. In choline-supplemented rats, an average of 0.48 μg/ml epinephrine caused maximal responses of the precapillary sphincters, lesser responses of the precapillary arterioles, and no reaction of the supplying artery. In marked contrast, less than 10% this concentration, 0.03 μg/ml epinephrine, resulted in pronounced vasoconstriction of the supplying artery with minimal or no reactions of the smaller vessels in the rats not supplemented with choline. These data are consistent with our view that the feeding of diets low in choline results in an imbalance in vasoactive mediators due to a decrease in tissue acetylcholine which leads to vasospasm and ischemia of the kidneys resulting in the characteristic nephropathy of acute choline deficiency.

Tissue Levels of Acetyl Choline and Acetyl Cholinesterase in Weanling Rats Subjected to Acute Choline Deficiency

The concentrations of acetyl choline and acetyl cholinesterase were assayed in the brain, small intestine and kidneys of choline-deficient and supplemented weanling male Fischer rats. These measurements were made to explore our thesis that acute choline deficiency induces a deficiency in acetyl choline which makes the renal vasculature hyperresponsive to vasopressor amines, resulting in vasospasm, ischemia, vascular rupture, hemorrhage and renal tubular necrosis. We found that 5 days of choline deficiency resulted in a 30–35% decrease in the concentration of acetyl choline in the brain and small intestine and a 50–75% decrease in the kidney. The level of acetyl cholinesterase of the brain was unchanged; kidney acetyl cholinesterase was 18% lower in choline-deficient rats. These changes in tissue acetyl choline and acetyl cholinesterase levels occurred before there were changes in kidney weight and gross appearance or blood urea concentration. These experiments support our thesis that the nephropathy of acute choline deficiency results from vascular disturbances caused by a deficiency of acetyl choline.

The subcellular distribution of acetylcholine, choline acetyltransferase and acetyl cholinesterase in nerve tissue.

The subcellular distribution of acetylcholine, choline acetyltransferase and acetyl cholinesterase in nerve tissue.

Effect of cysteine on acetylcholine synthesis.

CHOLINE acetyltransferase (choline acetylase; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) (ChA) is a sulphydryl enzyme responsible for the synthesis of acetylcholine (ACh) in nervous and other tissues. Early attempts to purify ChA from rat brain and calf caudate nucleus showed that this enzyme is unstable and attempts were made to protect it by addition of cysteine to the medium. It was observed, however, that high concentrations of cysteine decreased the synthesis of ACh. Similar findings were recently reported by Morris, Hebb and Bull1 in Torpedo Marmorata and rabbit brain extracts. They attributed the decreased synthesis of ACh to the inhibition of ChA by cysteine. Subsequently, Morris2 studied the effect of a number of sulphydryl compounds on ChA activity. He demonstrated that their mode of action was not directly on ChA but that they acted by depletion of the acetyl-CoA in the system. We have been able to produce evidence to support Morris's suggestion that the reaction involved is a spontaneous transacetylation between acetyl-CoA and cysteine. This mechanism presumably applies to the other sulphydryl compounds which he studied.