Application to Mammalian Tissues of the Chemiluminescent Method for Detecting Acetylcholine

Abstract: It is now possible to extend to mammalian tissues the chemilumi-nescent acetylcholine assay. Mammalian tissue extracts must be treated with oxidants (which is not necessary for electric organ extracts). The assay can then be performed as previously described (acetylcholinesterase hydrolyses acetylcholine; choline oxidase converts choline to betaine and H₂O₂, which gives off light in the presence of luminol and peroxidase). It is also shown that release experiments can be performed on mammalian tissue slices (mouse caudate nucleus) after the slice is washed in oxygenated saline solutions.     

A Simple, Sensitive, and Economic Assay for Choline and Acetylcholine Using HPLC, an Enzyme Reactor, and an Electrochemical Detector

A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.     

Simple and rapid measurement of acetylcholine and choline by HPLC and enzymatic-electrochemical detection

A simple, rapid and sensitive method for the detection of acetylcholine and choline in tissue extracts is reported. Acetylcholine and choline are first separated by HPLC then react in a mini-column with acetylcholinesterase and choline oxidase immobilized on Sepharose. The resulting H₂O₂ produced by choline oxidase is then detected electrochemically. The assay is more sensitive than existing methods. We believe that the principle involved in this method namely the combination of immobilized enzymes and the high sensitivity of electrochemical detection may be applied to other substances that can be converted by immobilized enzymes into an electrochemically detectable compound.     

High-performance liquid chromatography followed by peroxyoxalate chemiluminescence detection of acetylcholine and choline utilizing immobilized enzymes

A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.     

Chemiluminescent determination of acetylcholine,and continuous detection of its release from torpedo electric organ synapses and synaptosomes

A chemiluminescent procedure to determine acetylcholine is described. The enzyme choline oxidase recently purified, oxidises choline to betaine, the H₂O₂ generated is continuously measured with the luminol-peroxidase chemiluminescent reaction for H₂O₂. Other chemi or bioluminescent detectors for H₂O₂ would probably work as well. The chemiluminescent step provides great sensitivity to the method which is slightly less sensitive than the leech bio-assay but much more sensitive than the frog rectus preparation. The specificity of the chemiluminescent method depends on the fact that choline oxidase receives its substrate only when acetylcholine is hydrolysed by acetylcholinesterase. The acetylcholine content of tissue extracts was determined with the chemiluminescent method, and with the frog rectus assay, the values found were very comparable. The chemiluminescent procedure was used to follow the release of acetylcholine from tissues. When a slice of electric organ is incubated with choline oxidase, luminol and peroxidase, KCl depolarization or electrical stimulation in critical experimental conditions triggered an important light emission, which was blocked in high Mg²⁺. The venom of Glycera convoluta, known to induce a substantial transmitter release, was also found to trigger the light emission from tissue slices. Suspensions of synaptosomes release relatively large amounts of acetylcholine following Glycera venom action; this was confirmed with the chemiluminescent reaction. The demonstration that the light emission reflects the release of acetylcholine is supported by several observations. First, when the tissue is omitted no light emission is triggered after KCl or venom addition to the reagents. Second, the time course of the light emission record is very similar to the time course previously found for ACh release with radioactive methods. Third, if choline oxidase is omitted, or if acetylcholinesterase is inhibited by phospholine, the light emission is blocked, showing that the substance released has to be hydrolyzed by acetylcholinesterase and oxidised by choline oxidase to generate chemiluminescence.The procedure described has important potential applications since other transmitters can similarly be measured upon changing the oxidase.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.     

Metabolism of acetylcholine in the nervous system of Aplysia californica. III. Studies of an indentified cholinergic neuron

[3H] choline and [3H] acetyl CoA were injected into the cell body of an identified cholinergic neuron, the giant R2 of the Aplysia abdominal ganglion, and the fate and distribution of the radioactivity studied. Direct eveidence was obtained that the availabliity of choline to the enzymatic machinery limits synthesis. [3H] choline injected intrasomatically was converted to acetylcholine far more efficiently than choline taken up into the cell body from the bath. Synthesis from injected [3H] acety CoA was increased more than an order of magnitude when the cosubstrate was injected together with a saturating amount of unlabeled choline. In order to study the kinetics of acetylcholine synthesis in the living neuron, we injected [3H] choline in amounts resulting in a range of intracellular concentrations of about four orders of magnitude. The maximal velocity was 300 pmol of acetylcholine/cell/h and the Michaelis constant was 5.9 mM [3H] choline; these values agreed well with those previously reported for choline acetyltransferase assayed in extracts of Aplysia nervous tissue. [3H] acetylcholine turned over within the injected neuron with a half-life of about 9 h. The ultimate product formed was betaine. Subcellular distribution of [3H] acetylcholine was studied using differential and gradient centrifuagtion, gel filtration, and passage through cellulose acetate filters. A small portion of acetylcholine was contained in particulates the size and density expected of cholinergic vesicles.     

Microanalysis of Endogenous Acetylcholine Released from the Hemidiaphragm of the Rat

A chemiluminescence method for the determination of endogenous acetylcholine was adapted to transmitter released from the hemidiaphragm of the rat. Released choline and acetylcholine were isolated and concentrated using KI3 precipitation. Between 1 and 20 pmol of acetylcholine may be measured without sample dilution. The detection limit for acetylcholine luminescence is 1.0 pmol; thus, with an observed precipitation recovery of approximately 30%, the lowest detectable released amount is 3.5 pmol. The limitations of the method are discussed.     

Acetylcholine and Choline in Neuronal Tissue Measured by HPLC with Electrochemical Detection

Abstract: A simple, rapid method is presented for the determination of acetylcholine (ACh) and choline (Ch) in neuronal tissue using HPLC with electrochemical detection. The method is based on the separation of ACh and Ch by reverse-phase HPLC and mixing the effluent as it emerges from the column with acetylcholinesterase and Ch oxidase, which converts endogenous Ch and Ch produced by the hydrolysis of ACh to betaine and hydrogen peroxide. Production of hydrogen peroxide is continuously monitored electrochemically. The sensitivity of the procedure is 1 pmol for Ch and 2 pmol for ACh. Specificity of the method is based on HPLC, two specific enzymatic reactions, and the detection of hydrogen peroxide.     

Determination of picomole quantities of acetylcholine and choline in physiologic salt solutions

An assay capable of detecting tens-of-picomole quantities of choline and acetylcholine in milliliter volumes of a physiological salt solution has been developed. Silica column chromatography was used to bind and separate 10–3000 pmol [¹⁴C]choline and [¹⁴C]acetylcholine standards made up in 3 ml of a bicarbonate-buffered Krebs-Ringer solution. The silica columns bound 95–98% of both choline and acetylcholine. Of the bound choline 84–87% was eluted in 1.5 ml of 0.075 n HCl, whereas 95–98% of the bound acetylcholine was eluted in a subsequent wash with 1.5 ml of 0.030 n HCl in 10% 2-butanone. Vacuum centrifugation of the eluants yielded small white pellets with losses of choline and acetylcholine of only 1%. Dried pellets of unlabeled choline and acetylcholine standards were assayed radioenzymatically using [γ-³²P]ATP, choline kinase, and acetylcholinesterase. The net disintegrations per minute of choline[³²P]phosphate product was proportional to both the acetylcholine (10–3000 pmol) and choline (30–3000 pmol) standards. The “limit sensitivity” was 8.5 pmol for acetylcholine and 11.4 pmol for choline. Cross-contamination of the choline assay by acetylcholine averaged 1.3%, whereas contamination of the acetylcholine assay by choline averaged 3.1%.     

Determination of acetylcholine by on-line microdialysis coupled with pre- and post-microbore column enzyme reactors with electrochemical detection

A sensitive procedure consisting of a pre- and post-microbore column reactor sequence of a LC-electrochemical detection system coupled with on-line microdialysis system is described in the present study to measure endogenous acetylcholine concentration in freely moving rats. The pre-column packed, with immobilized choline oxidase and catalase, was used to remove choline, whereas the post-column, packed with immobilized acetylcholine oxidase and choline oxidase, was used to measure acetylcholine selectively. The detection limit of acetylcholine was found to be 5 fmol/μl (50 fmol/10 μl). The usefulness of the described methodology was evaluated by examining the change in the striatal acetylcholine concentration of freely moving rats after physostigmine (0.5 mg/kg, s.c.) administration.     

Use of monoclonal anti-enzyme antibodies for analytical purposes.

This review discusses the analytical applications of monoclonal antibodies specific for enzymes. One important, but not well-studied, application of these monoclonal antibodies is their use in immobilizing enzymes on solid supports. This method is based on binding the enzymes to an immobilized antibody through the antigen binding site of the antibody. Enzymes immobilized this way retain much of their activity. The utility of immobilized enzyme reactors prepared by immobilizing the enzymes through antibodies is demonstrated by using them in the determination of acetylcholine and choline in brain tissue extracts. Currently available methods for immobilizing antibodies and enzymes are reviewed. Other issues discussed in this review include the problems and advantages of immobilized enzyme reactors, especially when used in conjunction with HPLC. In addition, the applications of monoclonal antibodies for the detection and measurement of enzymes and their isoforms are summarized.     

Measurement of acetylcholine and choline in brain by HPLC with electrochemical detection

A simple and rapid method for measuring acetylcholine and choline using high performance liquid chromatography (HPLC) with electrochemical detection is presented. Acetylcholine and choline were first separated using reverse-phase chromatography; acetylcholine was then hydrolyzed post-column to choline by acetylcholinesterase. Choline was oxidized enzymatically by choline oxidase to betaine and hydrogen peroxide, and the peroxide was detected electrochemically. Changes in methodology from previous procedures include a different mobile phase, controlled heating of chromatography column and post-column reaction coil, and a different extraction method for quaternary amines. The changes resulted in less inhibition of derivatizing enzymes by mobile phase, narrow and consistent elution of peaks, and a rapid and efficient extraction of quaternary amines. Measurement of acetylcholine and choline in brain tissue was found to be replicable, and the levels agreed with literature values.     

Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity.

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.     

Enzymic radioassay for acetylcholine and choline in brain

This assay for acetylcholine (ACh) or choline in extracts of rat brain involves the isolation of the choline ester by high-voltage paper electrophoresis, alkaline hydrolysis of ACh to choline, and the quantitative enzymic conversion of choline to a radioactive derivative, P³²-phosphorylcholine. The method is specific, is applicable to large numbers of tissue samples, and has a blank value of about 3 nanograms of choline.     

The subcellular distribution of [N-Me-3H]-acetylcholine synthesized by brain in vivo

1. Three forms of acetylcholine occur in subcellular fractions of brain tissue: free acetylcholine, present in the high-speed supernatant from eserinized sucrose homogenates; stable bound acetylcholine, present in synaptic vesicles; and labile bound acetylcholine, present in the cytoplasm of synaptosomes (detached presynaptic nerve terminals). 2. The relationship between these forms has been investigated by isolating the subcellular fractions from the cortical tissue of cats and guinea pigs excised 1hr. after infiltration of [N-Me-(3)H]choline into the cortex in vivo. 3. Since choline is a ubiquitous metabolite, means were devised for isolating the radioactive acetylcholine on columns of the weak acid ion-exchange resin IRF-97; control experiments with samples of extracts treated with acetylcholinesterase showed that the radioactivity attributed to acetylcholine migrated to the choline peak after cholinesterase treatment. 4. The specific radioactivities of the various forms of acetylcholine were different: labile bound (synaptosomal cytoplasmic) acetylcholine had the highest, stable bound (vesicular) acetylcholine the next highest, and the high-speed-supernatant form the lowest. 5. It is concluded that the various forms of acetylcholine could not have arisen during fractionation from a single pre-existing pool of acetylcholine.     

Effect of Increasing Choline, In Vivo and In Vitro, on the Synthesis of Acetylcholine in a Sympathetic Ganglion

Abstract: The experiments described in this paper were designed to test whether increasing choline availability over normal physiological levels increases acetylcholine synthesis in the cat's superior cervical ganglion. When ganglia were perfused with Krebs solution, an increase in the medium's choline concentration over physiological (10⁻³M) levels increased tissue choline but did not increase tissue acetylcholine or the release of acetylcholine from stimulated ganglia. However, increasing plasma choline in the whole animal increased ganglionic acetylcholine levels. The basis for this difference in the effects of in vivo and in Vitro exposure to elevated choline levels on the tissue acetylcholine content was found to involve plasma factor(s), rather than indirect actions of choline, and the acetylcholine content of isolated ganglia was increased when the tissue was perfused with plasma, instead of Krebs solution, containing 10⁻³M-choline. The extra acetylcholine generated by this procedure was associated with a subsequent transient increase in transmitter release during short intervals of stimulation, but most of the extra acetylcholine was not readily available for release from stimulated ganglia. It is concluded that increasing choline available to sympathetic ganglia over physiological concentration does not have a sustained effect on the turnover of releasable transmitter under the conditions of these experiments.     

Some components of the cholinergic system in the prawn Palaemonetes varians (Leach)

1. An enzyme similar to mammalian acetylcholinesterase is found in high activity in the nervous tissue of Palaemonetes varians, i.e. eyes plus stalks, brain, suboesophageal ganglion and ventral cord. Acetylcholinesterase is also found associated with the abdominal muscles. Multiple enzyme forms are found in extracts of nervous tissues and muscles by electrophoresis and isoelectric focusing. 2. Cholinesterase is present in high activity in the stomatogastric system of P. varians. Three electrophoretically separable forms are found, having isoelectric points at pH4.2, 4.5 and 5.4. 3. Approx. 50% of the total acetylcholinesterase activity, approx. 80% of the choline acetyltransferase activity and 100% of the acetylcholine equivalents are found associated with the nervous tissue. Among the tissues examined, eyes plus stalks were the richest source of both choline acetyltransferase and acetylcholine equivalents. Suboesophageal ganglion and brain also contained large amounts of these components. 4. The distribution of these components could support the function of acetylcholine as a central and/or sensory transmitter in P. varians.     

The use of choline acetyltransferase for measuring the synthesis of acteyl-coenzyme A and its release from brain mitochondria

1. A method for measuring small amounts of acetyl-CoA synthesized in subcellular fractions of the brain from pyruvate and released from particles into the incubation medium has been developed by using placental choline acetyltransferase and choline in the incubation medium to transform acetyl-CoA into acetylcholine. Acetylcholine is measured by biological assay. Optimum conditions of incubation are described. 2. With fresh mitochondria, a decrease of acetyl-CoA output into the medium is observed in the presence of ATP or ADP, and an increase in the presence of calcium chloride or 2,4-dinitrophenol. Fluorocitrate and malonate have little or no effect. 3. After the mitochondria had been treated with ether, the release of acetyl-CoA into the medium is much larger; presumably, nearly all acetyl-CoA synthesized is then released and transformed into acetylcholine under the conditions used. The release of acetyl-CoA is diminished in the presence of Krebs-cycle intermediates and ADP. 4. Of all subcellular fractions, the highest acetyl-CoA production from pyruvate is found in the crude mitochondria; rates up to 51 mumoles of acetyl-CoA/g. of original tissue/hr. are observed in ether-treated samples. 5. The activities of acetyl-CoA synthetase and ATP citrate lyase found in homogenates and nerve-ending fractions of brain tissue are considerably lower than those of pyruvate oxidase complex and choline acetyltransferase. 6. The bearing of some of the findings on the question of the source of acetyl radicals for the synthesis of acetylcholine in vivo is discussed.