Rate Of Acetylcholine Synthesis Research Articles

Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei

The degree of lesion produced by 192 IgG-saporin relative to controls was compared using three independent methods. Microdialyzed acetylcholine (ACh), choline acetyltransferase (ChAT) activity, and the rate of ACh synthesis were compared in the frontal cortex and hippocampus. Microdialysis of rats was performed 1 and 15 weeks post-lesion. In week 16, the rats were sacrificed after an injection of deuterated choline (Ch) for determination of the rate of ACh synthesis. ChAT activity was determined at the same timepoints in a separate set of rats. At 1 week, ChAT activity and microdialyzed ACh showed similar degrees of depletion. At 15 weeks, microdialyzed ACh was significantly lower than the synthesis rate in cortex, but not in hippocampus. A small increase in ChAT activity between 1 and 15 weeks was found in the cortex, but not hippocampus. In the hippocampus, however, the rate of ACh synthesis was significantly greater than ChAT activity. This was true for two doses of immunotoxin; the greater compensation occurring with the lesser lesion. Microdialyzed ACh levels were not different from the other measures in hippocampus. Residual cholinergic terminals in the hippocampus, but not frontal cortex, compensate for a selective cholinergic lesion by increasing the rate of synthesis and may thereby alleviate hippocampus-dependent behavioral deficits.

Effects of subchronic administration of metrifonate on cholinergic neurotransmission in rats.

The effects of subchronic oral administration of metrifonate, a long-acting cholinesterase (ChE) inhibitor, on cholinergic neurotransmission were assessed in young adult male Wistar rats. Animals were treated twice daily with metrifonate. In a pilot study testing a 100 mg/kg dose of metrifonate for up to 14 days, ChE activity was found to steadily decrease to reach maximum inhibition levels of about 55%, 80% and 35% in brain, erythrocytes and plasma. Steady-state inhibition levels were attained by the 10th day of treatment. When metrifonate-treatment was discontinued, ChE activity in plasma returned to control levels within another day, while erythrocyte and brain ChE activity took more than 2 weeks to recover. In subsequent dose-response studies, metrifonate treatment was given for 3 and 4.5 weeks at doses of 0, 12.5, 25, 50, and 100 mg/kg, to different groups of animals, respectively. Correlation analysis indicted that brain ChE inhibition was more accurately reflected by erythrocyte than by plasma ChE inhibition, although all effects were highly correlated. The changes in ChE activity were not paralleled by changes in other parameters of the cholinergic neurotransmission, such as acetylcholine synthesis rate or acetylcholine receptor binding. It is therefore concluded that repeated administration of metrifonate to rats induces a long-lasting inhibition of ChE activity in a dose-related and predictable manner, which is neither subject to desensitization nor paralleled by counterregulatory downregulation of muscarinic or nicotinic receptor binding sites in brain.

Intoxications with anticholinesterases: effect of different combinations of antidotes on the dynamics of acetylcholine in mouse brain.

Intoxications with organophosphorus compounds are normally treated with a mixture of atropine and an enzyme regenerating oxime. The addition of diazepam to the conventional drug therapy is reported to greatly improve the antidotal effect. The implication of the cholinergic system in such intoxications prompted us to study the effect of different combinations of antidotes on the acetylcholine (ACh) synthesizing system in mouse brain in vivo. The antidotes studied in this paper are diazepam, HI-6 and 1-hyoscyamine, the active enantiomer of atropine. Diazepam decreases the synthesis rate of ACh both when administered separately and in combination with 1-hyoscyamine and HI-6. This is in contrast to 1-hyoscyamine which, in addition to blocking muscarinic receptors, also increases the release and rate of synthesis of ACh, which probably is an unfavourable effect of the antidote. This might at least partly explain the advantage of combining 1-hyoscyamine and an oxime with diazepam in intoxications with anticholinesterases. Mice administered soman (0.75 x LD50), after pretreatment with the three-drug combination of antidotes, show no cholinergic symptoms despite a 50% increase in endogenous ACh. The rate of synthesis of ACh in these mice is in the same range as in animals administered diazepam alone. Mice administered the same dose of soman with no antidotal pretreatment suffer from severe tremor and salivation, and have a strongly reduced synthesis rate of ACh.

Choline and acetylcholine metabolism in rat neostriatal slices.

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).

Mode of action of gamma-butyrolactone on the central cholinergic system.

Gamma-butylactone (GBL), a drug depressing the central nervous system, produced marked increases in acetylcholine contents in rat brain hemispheric regions (striatum, hippocampus, cortex) and in striatal choline content without modifying choline acetyltransferase or acetylcholinesterase activities. In the hippocampus GBL also strongly decreased the acetylcholine turnover rate and inhibited the high affinity uptake of choline. Its increase in acetylcholine content was prevented by an acute electrolytic lesion of the medial septum but not by a wide array of drug treatments designed to interfere with neurotransmission in various pathways. The results are taken to indicate that GBL directly depresses the cholinergic septal-hippocampal afferents by interrupting impulse flow. In the striatum, too, GBL markedly depressed the acetylcholine synthesis rate but had no effect on the high affinity choline uptake process. Such dissociation of the two phenomena had previously been observed using other drugs and may denote that acetylcholine synthesis in this region is regulated differently from that in the hippocampus. By comparison, gamma-hydroxybutyric acid (GHBA), an active metabolite which shares with GBL the capacity to produce a somnolent state and depress impulse flow in the dopaminergic nigroneostriatal pathway, had no effect on either striatal acetylcholine content or on hippocampal high affinity choline uptake. The results suggest that GBL can be distinguished from GHBA in its neuropharmacological central cholinergic effects.

On the turnover of acetylcholine in mouse brain: Influence of dose size of deuterium labelled choline given as precursor

The influence of the dose size of precursor choline (Ch) on the turnover rate of acetylcholine (ACh) has been studied in whole brain and striatum of mice. Different doses of deuterium labelled Ch (d 6-Ch) were injected i.v. and concentrations of endogenous and d 6-labelled Ch and ACh were estimated at various times by mass fragmentography using deuterium labelled (d 9 − ) Ch and ACh as internal standards. At doses of 1.25–20 μmole/kg of d 6-Ch the endogenous concentration of Ch in blood wastransiently elevated for 5 min. However, the endogenous concentrations of Ch and ACh in whole brain and striatum were not affected. The fractional rate constant of ACh synthesis and efflux, K a a measure of the turnover rate of ACh, was influenced by the dose size of precursor d 6-Ch. In whole brain, K a was ca. 0.7 at doses of 5–20 μmole/kg of d 6-Ch. At lower doses K a increased to ca. 1.0. In striatum, K a was ca. 0.7 at doses of 20–40 μmole/kg of the precursor. At lower doses K a became smaller and decreased to 0.10 at a dose of 1.25 μmole/kg of d 6-Ch. When the calculations of K a were corrected for the part of d 6-Ch concentration in the brain contained in blood the figures were invariably increased by 20–40%. However, the increase was not correlated with the precursor dose size. It was found that the ratio between S ACh and S Ch decreased with increasing dose of d 6-Ch until it reached plateau values at doses of 2.5 μmole/kg and higher in whole brain and at 10–20 μmole/kg and higher in striatum. It is proposed that the decreased ratio at low concentrations of d 6-Ch depends on saturation of the high affinity (HA) uptake system for Ch coupled to ACh synthesis. At higher doses of d 6-Ch, ACh synthesis by the low affinity (LA) uptake system for Ch became increasingly important and was not yet saturated at the highest dose given (40, μmole/kg). It is suggested that the synthesis rate of ACh can be studied with either very low doses of labelled Ch that do not saturate the HA uptake system or with sufficiently high doses at which the influence of the LA uptake system on the synthesis rate of ACh is dominant.

Increase in exogenous choline fails to elevate the content or turnover rate of cortical, striatal, or hippocampal acetylcholine.

The present experiments were designed to test whether increasing the availability of choline to rat brain increases the rate of acetylcholine synthesis in that organ. The content of choline and acetylcholine and the turnover rate of acetylcholine in striatum, hippocampus, and cerebral cortex were measured following changes in dietary choline, intraperitoneal choline, or intravenous infusion of choline. Increasing plasma choline caused some increase in tissue choline but did not increase acetylcholine levels nor acetylcholine turnover rate in any of the areas of brain studied. Indeed, in hippocampus, choline decreased the turnover rate of acetylcholine.

Acetylcholine metabolism in the rat hippocampus and striatum following one-trial passive training

The steady state levels of acetylcholine and choline and the synthesis of acetylcholine in the hippocampus and striatum of rat brain have been determined after one-trial passive avoidance training. Steady state levels of acetylcholine and choline were not altered, but the rate of acetylcholine synthesis was significantly elevated in the striatum but not significantly elevated in the hippocampus at one hour after training.

Acetylcholine synthesis by displaced amacrine cells.

The ganglion cell layer of the rabbit retina contains neurons that synthesize acetylcholine. To identify these neurons, the ganglion cells were labeled by retrograde transport of a fluorescent dye, and the acetylcholine-synthesizing cells of the same retinas were labeled by exposing the tissue to tritiated choline. Autoradiographs inspected by fluorescence microscopy showed that tritiated acetylcholine and the dye accumulated in different cells. Optic nerves of other animals were sectioned, causing degeneration of many neurons of the ganglion cell layer. This loss affected neither the retina's overall rate of acetylcholine synthesis nor the number of acetylcholine-containing cells in the ganglion cell layer. The acetylcholine-synthesizing neurons thus appear to be displaced amacrine cells.

Lesion-induced plasticity of high affinity choline uptake in the developing rat fascia dentata

After removal of the perforant path input to the rat fascia dentata at the age of 11 days, cholinergic septohippocampal fibers invade the denervated area. We have examined the effect of this lesion on hemicholinium-sensitive, high affinity choline uptake and its coupling to acetylcholine synthesis, specific properties of the septohippocampal input. Removal of the ipsilateral perforant path fibers increased the velocity of high affinity choline uptake by dentate particulate preparations, usually within 1 day. Studies conducted 5–104 days after operation showed a consistent 50–65% elevation in the molecular (denervated) layer. In contrast, the choline uptake rate in the granular layer eventually decreased slightly. Calculation of choline uptake rates independently of protein (per whole region) revealed that fasciae dentatae from operated and control sides accumulated choline at approximately equal rates, but on the operated side a greater percentage was transported by structures from the molecular layer and a lesser percentage by those from the granular layer. The rate of acetylcholine synthesis from exogenous choline increased to the same extent as high affinity choline uptake from 3 days after operation onward. The changes in high affinity choline uptake and acetylcholine synthesis coincided spatially and temporally with the reactive growth of septohippocampal fibers. Our results support the view that a perforant path lesion during development permanently alters the distribution of functional septohippocampal boutons in the fascia dentata. Acetylcholine synthesis is regulated to the same extent by high affinity choline uptake in the anomalous boutons as in normally located boutons.

Transformation and action of metrifonate.

Chemical formation of dichlorvos (2, 2-dichlorovinyl dimethyl phosphate) was found to occur in mouse brain after i.p. injection of metrifonate (2, 2, 2-trichloro-1-hydroxyethyl dimethyl phosphonate). A mass fragmentographic technique was used. Different isotopic variants were used both as internal standards and to compensate for dichlorvos formed during the workup procedure. The dichlorvos formed in vivo was found to have its maximal concentration a few minutes after the maximum of the metrifonate itself. The effect of metrifonate and dichlorvos on acetylcholine levels, acetylcholinesterase activity and synthesis rate of acetylcholine in mouse brain was also studied. In all three cases the effect of metrifonate was found to be prolonged and delayed as compared to the effect of dichlorvos. It is concluded that metrifonate acts as a slow release formulation in the body giving rise to dichlorvos nonenzymatically. This circumstance at least partly explains its efficacy in the treatment of schistosomiasis.

Der Einflu� von Hemicholinium Nr. 3 auf die Acetylcholin-Bildung in Lactobacillus arabinosus

The effect of HC-3 on the rate of acetyl choline synthesis in growing Lactobacillus arabinosus 17-5 and on the activity of the isolated bacterial choline acetylase has been investigated.

Choline Acetylase in the Human Placenta at Different Stages of Development

IT has long been known that there is acetylcholine in the human placenta1 and Comline's2 experiments established that choline acetylase, the enzyme which synthesizes acetylcholine, is in the placenta as well. The rate of acetylcholine synthesis, which he observed in extracts of acetone powders, made from mature placentae, was between 0.6 and 0.75 mgm./gm. acetone-dried tissue/hr. Later in this laboratory, when a coupled enzyme system was used to provide an independent supply of the substrate acetyl-co-enzyme A, rates of 5–6 mgm./gm./hr. were obtained3.

Vitamin B1 and Acetylcholine Formation in Isolated Brain

WE have shown in earlier papers1 that in the presence of glucose or of pyruvate and under aerobic conditions brain tissue, whether sliced or minced, synthesizes acetylcholine. In the absence of such substrates, little or no synthesis takes place. Since it is well known from the work of Peters and his colleagues that the presence of vitamin B1 (as cocarboxylase) is necessary for the oxidation of pyruvic acid in brain in vitro, it becomes of interest to discover whether the presence of vitamin B1 has any influence on the rate of acetylcholine synthesis in isolated brain tissue. MacIntosh2 investigated the acetylcholine contents of brains of polyneuritic pigeons and found these to be of the same order as those in the brains of normal pigeons. We have examined the abilities of intact brain slices, from normal and polyneuritic pigeons, to synthesize acetylcholine under suitable aerobic conditions in bicarbonate-Locke-pyruvate media containing eserine. (The eserine is added to prevent the decomposition of the acetylcholine by the choline esterase in the tissue.) We have found no appreciable difference between the rates of acetylcholine formation in normal or polyneuritic brain tissue examined under these conditions; nor have we found as a general rule that the addition of vitamin B1 has any significant effect upon these rates. (On one occasion we found that the addition of vitamin B1 definitely increased the rate of acetylcholine formation by polyneuritic pigeon brain in a phosphate-pyruvate medium.)