Fluorometric enzyme assay for choline and acetylcholine

A sensitive and specific assay for choline and ACh which may be applied directly to brain extracts is described. The method is based upon enzymic coupling to the oxidation of fluorescent NADH. The following enzymic sequence is utilized: acetylcholinesterase, choline phosphokinase, pyruvate kinase, and lactate dehydrogenase. The method detects as little as 0.1 mμmole of choline or ACh, which is the amount of metabolite present in 1 mg or 8 mg of whole rat brain, respectively. The specificity of the method is such that only choline and ACh of tissue samples react. Extraction of whole brain samples by either heating at pH 4 or by chloroform/methanol or perchloric acid were compared in order to find a single procedure which was useful for extraction of both ACh and free choline from brain samples. Perchloric acid extraction proved to be the most efficient of the three methods for extraction of the two constituents. By this procedure the ACh content of whole rat brain was found to be 11.5 mμmoles/gm and the choline content of the same samples was 107 mμmoles/gm. Both values are in agreement with other published results.     

Acetylcholine binding site in the vesicular acetylcholine transporter

This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily.     

Ouabain blocks some rapid concentration-induced clamp acetylcholine responses onHelix neurons

1. The effects of ouabain, a potent inhibitor of Na(+)-K+ ATPase, were determined on the transmembrane responses of internally dialyzed Helix neurons to rapid acetylcholine (ACh) application using the "concentration clamp" technique. 2. Ouabain selectively depressed "A"-type responses to ACh, which are due to a selective increase in membrane permeability to chloride. In contrast, the "B"-type responses, due primarily to an increase in monovalent cation permeability, was unaffected. 3. The blockade of the Cl- responses was not associated with a change of the reversal potential of the response. Ouabain depressed the maximal response without shifting the dose-response curve. 4. Ouabain caused an increase in the time constant of decay of the ACh current, but the value in the presence of ouabain was not different from that of a lower concentration of ACh determined so as to give a response of the same peak amplitude. Therefore, the effect of ouabain is not on the process of receptor desensitization directly.     

The source of choline for acetylcholine synthesis in brain

More is known about the synthesis and metabolism of acetylcholine (ACh) than other choline (Ch) containing compounds in the brain in spite of the fact that ACh represents only a small fraction of the total Ch esters. This review will attempt to summarize the evidence for the source of Ch in the brain and its relation to the turnover of ACh. Ch is a precursor not only for ACh but also for phosphoryl Ch and phospholipids. It appears that in the rat a bound form of Ch in the brain can produce free Ch which can leave the brain, be converted to ACh or be reutilized for phospholipid synthesis. There is evidence that one of the sources of free Ch that is utilized for ACh synthesis is outside the cholinergic nerve terminal.     

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals

The effects of hemicholinium-3 (HC-3) or 4-(1-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 μg HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP resulted in a further reduction in ACh. Pretreatment with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR) against soman was increased from 0.8 to 7.3. These data are consistent with the hypothesis that excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.     

The influence of aminopyridines on Ca2+-dependent evoked release of acetylcholine from rat cortex slices

The release of acetylcholine (ACh) elicited by electrical stimulation was investigated in rat brain cortical slices preloaded with³H-choline. Decreasing the [Ca²⁺]o from 2.5 to 0.3 mM caused a progressive reduction of the evoked release of ACh. 4-Aminopyridine (4AP) or LF14 [(1,1-dimethyl-3-(4-amino-3-pyridyl)], 4×10^–5 M doubled the evoked release of ACh when the [Ca²⁺]o was 2.5 mM and quadrupled it when it was 0.3 mM, to levels higher than those obtained with 2.5 mM [Ca²⁺]o alone. This indicates that both 4AP and LF14 decrease the Ca²⁺ requirements for the evoked release of ACh. The findings of this study indicate that LF14 may be suitable for the symptomatic treatment of senile dementia of Alzheimer's type, presumably caused by dysfuntion of cholinergic transmission in the brain.     

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [¹⁴C]ACh newly synthesized during incubations from [U-¹⁴C] glucose depends on the concentration of Ca²⁺ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K⁺, significant increases in the release of [¹⁴C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca²⁺. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca²⁺ that is required for transmitter liberation.     

Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks. Although the synthesis and release of ACh by hippocampal slices from diabetic animals tended to be less than controls, these alterations were not statistically significant. Investigations into the mechanism(s) mediating the deficit in ACh synthesis exhibited by striatal slices indicated that it did not involve alterations in precursor choline availability, nor could it be attributed to alterations in the activities of the synthetic or hydrolytic enzymes choline acetyltransferase or acetylcholinesterase; rather, the decreased turnover of ACh may be secondary to other STZ-induced, hyperglycemia-mediated neurochemical alterations.     

Measurement of acetylcholine turnover rate in discrete areas of rat brain

The turnover rate of ACh was estimated in brain stem, two cortical areas and striatum of rat brain. The turnover rate was highest in the striatum (1.3 μmoles/g/hr); lowest in brain stem (0.092 μmoles/g/hr); and intermediate values were observed in limbic and occipital cortex. The highest ACh concentrations were measured in striatum, those in brain stem were intermediate but in the two cortical areas the ACh concentrations were the lowest. The results of the turnover estimations with the finite difference method yielded values similar to those obtained with the procedure described in this paper. Moreover, once the baseline was established, this method could be reliably used to estimate turnover rate using a single infusion time. The latter simplication would be very useful to compare ACh turnover rate in drug studies.     

Fractional Ca(2+) current through human neuronal alpha7 nicotinic acetylcholine receptors

The neuronal alpha7 nicotinic acetylcholine (ACh) receptor is believed to be a highly Ca(2+) permeable ligand-gated receptor-channel. However, the contribution of Ca(2+) to cationic current generated by ACh has not yet been directly measured to date. Simultaneous fluorescence and whole-cell current measurements using the Ca(2+) indicator dye fura-2 were made in GH4C1 pituitary cells stably expressing human alpha7 receptors and the fractional Ca(2+) current (the proportion of whole-cell current carried by Ca(2+); P(f)) was determined. We report that the P(f) value was 11.4+/-1.3%. This value was significantly larger than P(f) of human L248Talpha7 receptor mutant (P(f)=6.3+/-1.0%) and of rat alpha7 receptor (P(f)=8.8+/-1.5%) both determined in transiently transfected GH4C1 cells. In our knowledge, the findings here reported indicate the human alpha7 receptors are the most Ca(2+) conductive homomeric ligand-gated receptor-channels expressed in a heterologous cell system.     

Effect of intracerebroventricularly administered insulin on brain monoamines and acetylcholine in euglycaemic and alloxan-induced hyperglycaemic rats.

There is now conclusive evidence for the presence of insulin and insulin receptors in the mammalian CNS and it has been postulated that they can modulate peripheral glucose homeostasis. Since a number of central neurotransmitters are also known to influence glucose levels and it is likely that CNS insulin receptors act through neurotransmitter mediation, the present study was conducted to investigate the effect of intracerebroventricularly (icv) administered insulin on rat brain dopamine (DA), noradrenaline (NA), serotonin and acetylcholine (ACh) activity in normal and alloxan-induced hyperglycaemic animals. Insulin was administered in doses (50 and 100 microU) which induced minimal hypoglycaemia, so as to obviate the likely effects of hypoglycaemia on neurotransmitter function. DA was estimated in midbrain-diencephalon (MD) and caudate nucleus (CN), NA and serotonin in MD and pons-medulla (PM), while ACh was estimated in all the three areas, namely, MD, CN and PM. The regional brain concentrations of DA, NA and serotonin were more in the hyperglycaemic rats as compared to their euglycaemic counterparts. However, the reverse was noted in case of ACh. Insulin induced a decrease in rat brain DA and NA levels, which was more marked in the hyperglycaemic animals. Conversely, insulin induced an increase in rat brain serotonin concentration which was not significantly different in normal and hyperglycaemic rats. Insulin induced marked increase in rat brain ACh levels, which was accentuated in hyperglycaemic animals. The present study reports for the first time the likely interaction between CNS insulin receptors and brain monoamines, and ACh, in euglycaemic and hyperglycaemic states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of brain regional physostigmine concentration to cholinesterase activity and acetylcholine and choline levels in rat

The relationship between physostigmine (Phy) concentration, acetylcholine (ACh), choline (Ch) and cholinesterase (ChE) activity was examined in whole rat brain after the administration of [³H]Phy (650 g/kg i.m.). Cholinesterase inhibition was found to be inversely related to Phy levels. Maximal inhibition (80%) was seen at 5 min and by 2 hrs ChE activity had returned to control levels. Acetylcholine levels in whole brain peaked at 30 min at a concentration (80 nmol/g) 2.3 times higher than controls (33 nmol/g). Choline levels were not significantly altered. The regional distribution of Phy concentration and ChE activity was studied in six areas of the brain following i.m. administration of three different dosages of [³H]Phy. Physostigmine concentration and ChE activity showed a dose dependency in each area examined except in SP (medial septum). Striatum (ST) showed the greatest relative increase of ACh up to 30 min, when compared to other areas. Choline levels were not changed in any area with the exception of ST at 5 min where a decrease was seen. There was a relationship between ChE activity, Phy concentration and ACh levels in all areas examined with exception of the medulla oblongata (MO). Our results indicate that even though ChE was inhibited practically uniformly in all brain areas, the percent increase with respect to control animals and the relative increase of ACh varied widely from area to area. This finding has clinical implications in cases in which cholinomimetic therapy is used to elevate ACh levels in specific brain areas which show a cholinergic deficit.Special issue dedicated to Prof. Eduardo De Robertis.     

A comparison of the effects of scorpion venom tityustoxin and ouabain on the release of acetylcholine from incubated slices of rat brain.

The increase in the release of acetylcholine (ACh) from cortical slices of rat brain elicited by the depolarizing agents, ouabain and tityustoxin (TsTX) was compared in the presence of tetrodotoxin, an inhibitor of Na⁺ transport and of ethyleneglycol tetraacetic acid, a specific chelator of Ca²⁺. TsTX stimulated the release of ACh independently of K¹ but required both Na⁺ and Ca²⁺. Unlike ouabain, TsTX failed to inhibit the Na⁺, K⁺ -ATPase of rat brain homogenates. The uptake of ²⁴Na⁺ and of ⁴⁵Ca²⁺ by the slices was significantly enhanced by TsTX over the entire incubation period during which this process was compared to TsTX-free controls. A hypothesis of TsTX action is proposed which differs from that necessary to explain the ACh releasing effect of ouabain.     

An enzyme-reactor for electrochemical monitoring of choline and acetylcholine: applications in high-performance liquid chromatography, brain tissue, microdialysis and cerebrospinal fluid.

A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the on-line real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.     

Evoked release of tissue-bound exogenous [1-14C]acetylcholine from rat brain cortex slices

Exogenous labeled acetylcholine ([¹⁴C]ACh) bound, in rat brain cortex slices, in a poorly (or non-) exchangeable form, by prior incubation of the slices in presence of 5 mM [¹⁴C]ACh, is partly released in an ACh-free physiological saline-glucose-paraoxon medium by a variety of conditions. Among these are high [K⁺], lack of Na⁺ or Ca²⁺, and the presence of protoveratrine or ouabain. The releasing effect of protoveratrine is completely abolished by tetrodotoxin which itself is without effect. Only about half of the retained or tissue-bound [¹⁴C]ACh is affected by these conditions. The whole of the bound ACh is released by treatment with acid or by dissolution of the cell membranes. The stimuli that release part of the bound exogenous [¹⁴C]ACh appear to be similar to those that release glucose-derived tissue-bound ACh formed during normal cerebral metabolism.     

Dynamics of hippocampal acetylcholine release during lithium‐pilocarpine‐induced status epilepticus in rats

The lithium‐pilocarpine model is a rat model of epilepsy that mimics status epilepticus in humans. Here, we report changes of acetylcholine (ACh) release in the hippocampus before, during and after status epilepticus as monitored by microdialysis in unanesthetized rats. Administration of pilocarpine (30 mg/kg s.c.) to rats pretreated with lithium chloride (127 mg/kg i.p.) caused a massive, six‐fold increase of hippocampal ACh release, paralleling the development of tonic seizures. When seizures were stopped by administration of diazepam (10 mg/kg i.p.) or ketamine (75 mg/kg i.p.), ACh levels returned to normal. Extracellular concentrations of glutamate remained unchanged during this procedure. Administration of atropine (1 mg/kg i.p.) 2 h after pilocarpine caused a further increase of ACh but did not affect seizures, whereas injection of mecamylamine (5 mg/kg i.p.) reduced ACh levels and seizures in a delayed fashion. Local infusion of tetrodotoxin, 1 μM locally) or hemicholinium (10 μM locally) strongly reduced ACh release and had delayed effects on seizures. Administration of glucose or inositol (250 mg/kg each i.p.) had no visible consequences. In parallel experiments, lithium‐pilocarpine‐induced status epilepticus also enhanced striatal ACh release, and hippocampal ACh levels equally increased when status epilepticus was induced by kainate (30 mg/kg i.p.). Taken together, our results demonstrate that seizure development in status epilepticus models is accompanied by massive increases of extracellular ACh, but not glutamate, levels. Treatments that reduce seizure activity also reliably reduce extracellular ACh levels.

     

The activation of the nicotinic acetylcholine receptor by the transmitter

Experimental evidence has been published from isolated guinea pig muscle in vitro, and from direct ligand binding to receptors from T. californica, indicating that two agonist ions react with the nicotinic receptor by exchanging for one magnesium ion. It is the basis of the ion exchange receptor pair model, in which two acetylcholine ions exchange for one magnesium ion in contact with and between a pair of negatively charged receptor groups about 4 A apart. In the resting state the electrostatic attraction between the negatively charged receptor groups and the Mg2+ ion exerts a binding force. This binding force is opposed by the quantum mechanical repulsions of the electron clouds of the charged groups and ions in contact, together with the mutual repulsion of the pair of receptor oxyanions. When the Mg2+ ion is replaced by two acetylcholine ions the quaternary heads of the latter are positioned so that they form two mutually repelling ACh+ receptor group dipoles. As the Mg2+ ion leaves, its rehydration energy contributes to the sum of the electron cloud repulsions and the ACh+ receptor group dipole repulsions, causing the receptor groups to be forced apart activating the receptor macromolecule. The subsequent decrease in ACh+ concentration results in the reestablishment of the resting state. The coulombic electrostatic energy, the Born repulsion energy, the London attraction energy and the oxyanion ACh+ dipole repulsion energies have been calculated and shown to be consistent with the model. The displacement of the Mg2+ by two ACh+ ions makes several hundred kcals of energy available for receptor group separation and receptor activation.     

Concomitant protein phosphorylation and endogenous acetylcholine release induced by nicotine: Dependency on neuronal nicotinic receptors and desensitization

Summary 1. Nicotine stimulated two Ca²⁺-dependent processes in rat frontal cortex synaptosomes: the phosphorylation of an 80-kDa protein band and the release of endogenous ACh.³ Both effects were mediated by neuronal nAChRs and coincided with depolarization of the synaptosomal plasma membrane induced by the drug. Changes in the state of phosphorylation of the 80-kDa band (presumed to contain synapsin I) were correlated with changes in the release of ACh as follows, from 2 to 4.2. Blockade of predominant, nerve terminal P-type Ca²⁺ channels with -agatoxin-IVA, did not prevent nicotine from stimulating ACh release. In contrast, exposure to the toxin partially inhibited the release promoted by the depolarizing agent veratridine and attenuated protein phosphorylation induced by either nicotine or veratridine. Taken together, these data suggest that, upon nicotine stimulation, Ca²⁺ enters nerve terminals through two distinct pathways. The first, via Ca²⁺ channels, is necessary (but not sufficient) for both nicotine-induced phosphorylation and ACh release. The second, both necessary and sufficient for nicotine-induced phosphorylation and release, is the neuronal nAChR itself.3. Preincubation of the synaptosomes with a subeffective concentration of nicotine inactivated both nicotine-induced ACh liberation and phosphorylation. This shows that diminished release is associated to decreased phosphorylation of the 80-kDa protein band, most likely as a consequence of nicotine-promoted nAChR desensitization.4. Augmented ACh release and phosphorylation of the 80-kDa protein band were achieved by using the protein phosphatase inhibitor okadaic acid. However, okadaic acid did not summate with either nicotine or veratridine to increase ACh release further. This is probably because okadaic acid, as in other neurons, increases intracellular Ca²⁺ (Cholewinskiet al., 1993), thus promoting desensitization of ACh release.     

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.     

Noradrenergic-mediated potentiation of acetylcholine release from the phrenic nerve: evidence for presynaptic alpha 1-adrenoceptor involvement

This study, conducted in the rat phrenic nerve-diaphragm preparation, was designed to establish more direct evidence that norepinephrine enhances acetylcholine (ACh) release from motor neurons and characterize the alpha-adrenoceptor type mediating this action. Norepinephrine (50 microM, alpha 1 + alpha 2 agonist) increased nerve-stimulated release by 183%, as determined by radioenzymatic assay. This effect was completely abolished by pretreatment with the alpha-adrenoceptor antagonists phentolamine (alpha 1 + alpha 2) and by WB 4101 (alpha 1) but only modestly reduced by yohimbine (alpha 2). Clonidine (alpha 2 agonist) did not enhance ACh release or nerve-stimulated muscle contractions, while phenylephrine (alpha 1 agonist) and norepinephrine increased muscle contractions up to 19.5-22.4%. These results support the hypothesis that norepinephrine increases ACh release from somatic motor nerves via a presynaptic alpha 1 interaction.