Regulation Of Choline Acetyltransferase Activity Research Articles

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis.

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.

Ethanol alone or with dexamethasone alters the kinetics of choline acetyltransferase

Choline acetyltransferase activity was measured in rats treated with daily injections of ethanol (0.1 g/kg body wt) and or dexamethasone (1 mg/kg body wt) for 5 consecutive days. Ethanol produced a biphasic reduction of choline acetyltransferase activity in rat cerebral cortex, which at most time points was further decreased by simultaneous injection of dexamethasone. Kinetic studies of cortex choline acetyltransferase activity in rats that had received 5 daily injections of ethanol or ethanol and dexamethasone indicated that the observed reduction in enzyme activity was due to an apparent reduction in affinity ( K m) of the enzyme for acetyl coenzyme A with no significant change in the total amount of enzyme present ( V max). This finding has implications with respect to the use of choline acetyltransferase as a marker for cholinergic neurons, and for the understanding of the regulation of choline acetyltransferase activity in the brain.

Mechanism of activation of choline acetyltransferase in a human neuroblastoma cell line

In our previous report we have shown that the enzyme choline acetyltransferase (ChAT), responsible for the synthesis of the neurotransmitter acetylcholine, can be regulated in response to treatment by either retinoic acid or sodium butyrate. These responses were dose and time dependent, but the mechanism by which these agents were acting was not understood. We now report the results of studies aimed at elucidating the level at which both sodium butyrate and retinoic acid are able to increase ChAT activity. The effects of these agents on macromolecular synthesis appeared to be limited to small but statistically significant increases in the rate of RNA synthesis. However, inhibition of DNA, RNA and protein synthesis in these cells had no effect on the stimulation of ChAT activity by either sodium butyrate or retinoic acid. Several experiments appeared to rule out a role for cyclic AMP or protein kinase C in the regulation of ChAT activity, even though retinoic acid treatment could increase endogenous levels of cyclic AMP 3- to 4-fold over the time course of ChAT activity stimulation. Experiments performed to determine kinetic parameters of this enzyme demonstrated changes only in the V max, but not the K m of ChAT, suggesting that the affinity of enzyme for either of its substrates was not responsible for the increase in specific activity. Taken together, this evidence suggests that the activation of choline acetyltransferase in this human neuroblastoma cell line occurs at the post-translational level.

Regulation of Choline Acetyltransferase Activity by Cell Density in a Cultured Human Neuroblastoma Cell Line

Choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine metabolism, is expressed in a human cholinergic neuroblastoma cell line; MC-IXC. We demonstrate that ChAT activity is regulated in this cell line by cell density. It is believed that the mechanism of stimulation of enzyme activity involves cellular contact, as medium conditioned by cells of high density failed to mimic the effect of density alone, and trypsinization reversed this effect. Density did not increase acetylcholinesterase activity, another marker for the cholinergic phenotype, in MC-IXC cultures, demonstrating the independent regulation of these two cholinergic enzymes. Since increased density slows the rate of cell division, we used a DNA synthesis inhibitor to uncouple DNA replication from cell density. This had no effect on the specific activity of ChAT, suggesting that a cell-cell contact was the mediating factor. Other neuroblastoma cell lines were tested, and only cell lines which already contain ChAT activity were sensitive to its regulation by cell-cell contact, suggesting that cell-cell contact is permissive rather than instructive in this process. The effect of cell passage on ChAT activity is also discussed.

An interaction between thyroid hormone and nerve growth factor in the regulation of choline acetyltransferase activity in neuronal cultures, derived from the septal-diagonal band region of the embryonic rat brain

Culture conditions have been established for growing neurons from the medial frontal part of the forebrain, containing the septum and the diagonal band of Broca, of 17-day-old rat embryos in a chemically defined medium. At 10 days in vitro, the cultures contained more than 96% nerve cells of which about 18% were cholinergic neurons, while the proportion of astrocytes was less than 1%. The majority of the cells that stained for acetylcholinesterase were bipolar but with different sizes and shapes. During development both the specific activity of choline acetyltransferase (ChAT) and the amount of protein increased markedly in the cholinergic cultures, ChAT activity rising much more than the protein content. Exposure of the cultures to nerve growth factor (NGF) or 3,3′,5-triiodo- l-thyronine (T 3) enhanced the expression of ChAT activity in a dose-dependent manner. The elevation of ChAT activity was due to an increase in the amount of enzyme per cholinergic cell, since, during the experimental period studied, neither treatment with NGF nor with T 3 had significant effects on the total protein content of the cultures or on the number of cells, including the cholinergic neurons. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT activity was much greater than the sum of the individual effects. The observations indicate that subcortical cholinergic neurons, which are affected in Alzheimer's disease and in Down's syndrome, are subject to regulation by an interaction between thyroid hormone and local humoral factors such as NGF.

An endogeneous inhibitory factor for choline acetyltransferase

Chromatography of partially purified choline acetyltransferase (CAT) over carboxymethyl cellulose may result in the loss of up to 95% of the enzyme activity. This loss of activity can be prevented by running the chromatographs at low protein concentration with a large gradient volume suggesting that interactions between CAT and other endogenous proteins are involved in the mechanism of inactivation. Further experiments showed that CM-cellulose chromatography separates an endogenous inhibitory factor(s) and an endogenous activating factor(s) which protects the enzyme from the action of the former. The inhibitory factor elutes with CAT and produces almost complete inactivation unless the protein concentration is maintained below 0.05 mg/ml. Mixing experiments demonstrated that the activating factor is capable of blocking the effect of the inhibitory factor. The low degree of temperature dependence of the inhibitory factor essentially rules out the possibility that the inhibitor is a proteolytic enzyme. The I50 was estimated to be 10(-7) M or less suggesting a possible physiological role of these factors in the regulation of CAT activity.

Interactions between afferents to the dentate gyrus after entorhinal lesion during development: long-term regulation of choline acetyl-transferase activity

Interactions between afferents to the dentate gyrus after entorhinal lesion during development: long-term regulation of choline acetyl-transferase activity

The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function.

1. The role of muscle mass and function in the regulation of choline acetyltransferase activity at the neuromuscular junction has been investigated in the rat.2. Choline acetyltransferase (ChAc) is located in presynaptic nerve terminals and is a specific enzymatic marker of cholinergic innervation in muscle.3. ChAc activity increased co-ordinately with developmental growth of the soleus muscle. However, another form of muscle growth, work hypertrophy, did not produce an increase in ChAc.4. Growth arrest of muscle by hypophysectomy did not alter the normal development of ChAc activity, and cortisone-induced muscle atrophy did not reduce ChAc activity in the soleus or plantaris.5. Tenotomy-induced muscle atrophy provoked a significant fall in ChAc in the soleus and plantaris.6. The tonic soleus had significantly greater ChAc activity than the phasic plantaris.7. These observations suggest that muscle mass per se does not influence the development and regulation of ChAc in muscle but that the quality of muscle contraction may modulate enzyme activity.