Free choline levels in the rat brain

C holine levels in nervous tissue have been investigated by a number of workers in recent years. The values have varied widely from 39.3 nmol/g (E wetz , S parf and S öbo , 1970) to 700 nmol/g (S mith and S aelens , 1967). Many of the values published may have been too high for one of the following reasons: (1) post mortem formation of choline, (2) hydrolysis of phospholipids (PL) by extractants and (3) inadequate assay systems. In the past we too have obtained values which we can now with confidence say were too high due to the post mortem formation of choline. In a method which employed bioassay as an end-step after extraction of choline by acid-ethanol the values we obtained were 138 ±27 nmol/g. Despite criticism of this method by E wetz et al. (1970) and S chuberth , S parf and S undwall (1970a) we were reasonably sure that the assay system was both sensitive and specific, and that extraction with acid-ethanol did not lead to liberation of choline from PL, especially since values for plasma choline by this method were in a number of trial extractions as low as 8 nmol/ml. In view of these results we decided to re-investigate free choline levels in the brain using a method similar to that of E wetz et al. (1970) in that the living animal (in this case anaesthetized) was frozen in liquid nitrogen before removing the brain, and comparing the results of three different methods of analysis applied to brain extracts prepared in this way.     

Immunological properties of rat brain choline acetyltransferase.

Abstract— Four antisera active against choline acetyltransferase (ChAc) were obtained by injecting 22 rabbits with rat brain ChAc. The ChAc preparations used for immunization (specific activity from 015 to 2 μmol/min/mg of protein) were not pure and the antisera produced were not monospecific. The antisera inhibited and precipitated ChAc, but the precipitated enzyme-antibody complexes still retain ChAc activity. One millilitre of the most active serum precipitates 0–5 μmol/min of rat brain ChAc at the equivalence point. Its titre expressed in mg/ml of immunoglobulins precipitated with the antigen and the equivalence point was calculated at about 0.08 mg/ml of serum. This relatively low titre explains the lack of any visible ChAc immunoprecipitate in an immunodiffusion test. Cross-reactivity studies revealed that ChAc has undergone few changes during evolution, since antisera produced against rat brain ChAc still precipitate ChAc from fish (Torpedo).     

Biophysical properties of rat brain choline acetyltransferase.

Abstract— The molecular weight of choline acetyltransferase (ChAc) was determined by using a variety of methods. A molecular weight of 68,000 daltons was found. ChAc is a globular protein with an apparent radius of 3.39 nm (value of the Stokes radius). The existence of a dimeric form and higher aggregates is discussed     

Monoclonal antibodies to choline acetyltransferase of rat brain

Monoclonal antibodies to rat brain choline acetyltransferase were produced by the hybridoma technique. Two stable cell lines, Ab-57 and Ab-60, secreted immunoglobulin of subclass IgG1. The monoclonal antibodies bound to choline acetyltransferase without blocking catalytic activity. Affinity of Ab-57 was 100-times higher than that of Ab-60. Both antibodies bound to the rat enzyme in a mutually exclusive fashion. The antibodies showed cross-species reactivity with choline acetyltransferase from several mammalian brains.     

Increase in rat brain acetylcholine induced by choline or deanol.

Total catecholamines, norepinephrine and small amounts of dopamine were measured in the spinal cord of rats by sensitive radiometric enzymatic assays. There was a considerable disparity between levels of NE and those of total CA suggesting the presence of significant amounts of another catecholamine in spinal cord. The demonstration of the presence of specific phenylethanolamine-N-methyl transferase activity in spinal cord suggested that the catecholamine could be epinephrine. The presence of epinephrine in spinal cord was confirmed by quantitative mass fragmentography. Pretreatment with intracisternal and intracerebroventricular 6-hydroxydopamine markedly reduced the contents of total catecholamines, no norepinephrine and dopamine-β-hydroxylase activity when compared to vehicle treated controls. In contrast, the levels of dopamine only fell to 50% of controls after 6-hydroxydopamine. Thus, in addition to descending noradrenergic tracts, adrenergic neurons appear to be present in the spinal cord.     

Detection of choline kinase in purified rat brain myelin

Choline kinase, an enzyme involved in the Kennedy pathway conversion of diacylglycerol to phosphatidylcholine, was detected in highly purified rat brain myelin at a level equal to 20% that of whole brain homogenate. This was an order of magnitude higher than the specific activity of lactate dehydrogenase, marker for cytosol. Choline kinase was also detected in the P1, P2, P3, and cytosolic fractions with highest relative specific activity in the latter. Myelin washed with buffered sodium chloride or taurocholate retained most of its kinase, indicating that adsorption of the soluble enzyme was unlikely. The results of mixing experiments and repeated purification further indicated that the enzyme is intrinsic to myelin. This finding in concert with previous studies supports the concept that myelin has all the enzymes needed to convert diacylglycerol to phosphatidylcholine.     

Inhibition of the rat blood–brain barrier choline transporter by manganese chloride

Choline transport has been characterized by multiple mechanisms including the blood-brain barrier (BBB), and high- and low-affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd(2+) (73 +/- 2%) and Mn(2+) (44 +/- 6%), whereas no inhibition was observed with Cu(2+) and Al(3+). Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 +/- 3%, 68 +/- 18%, 58 +/- 9% and 46 +/- 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.     

Arterio-venous differences of choline and choline lipids across the brain of rat and rabbit.

The concentration of unesterified choline in the plasma in the jugular vein of the rat (0.85 nmol/ml) was found to be three times that of the arterial supply to the brain (0.25 nmol/ml), indicating a higher efflux than uptake of unesterified choline by the brain. No such difference was found for the rabbit and no arterio-venous difference for phosphatidylcholine or lysophosphatidylcholine was observed in either species. No arterio-venous difference was found for choline in blood cells. The infusion of [Me-3H]choline into the circulation of the rat or rabbit indicated an uptake of radioactive choline by the brain and an efflux of non-radioactive choline. In the rabbit such an infusion produced a steady rise in the labelling of phosphatidylcholine and lysophosphatidylcholine in the plasma. When [14C2]ethanolamine was injected intraperitoneally into the rat there was a labelling of phosphatidylcholine, lysophosphatidylcholine and sphingomyelin in the plasma and cells of blood from the jugular vein and the arterial supply, as well as in the brain tissue. However, no labelling of unesterified choline in these tissues could be detected. Unesterified choline was shown to be liberated into the plasma when whole blood from the rat or man, but not the rabbit, was incubated for short periods at 30 degrees C.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Seizures increase acetylcholine and choline concentrations in rat brain regions

Seizures induced by three convulsant treatment produced differential effects on the concentration of acetylcholine in rat brain. Status epilepticus induced by (i) coadministration of lithium and pilocarpine caused massive increases in the concentration of acetylcholine in the cerebral cortex and hippocampus, (ii) a high dose of pilocarpine did not cause an increase of acetylcholine, and (iii) kainate increased acetylcholine, but the magnitude was lower than with the lithium/pilocarpine model. The finding that the acetylcholine concentration increases in two models of status epilepticus in the cortex and hippocampus is in direct contrast with manyin vitro reports in which excessive stimulation causes depletion of acetylcholine. The concentration of choline increased during seizures with all three models. This is likely to be due to calcium- and agonist-induced activation of phospholipase C and/or D activity causing cleavage of choline-containing lipids. The excessive acetylcholine present during status epilepticus induced by lithium and pilocarpine was responsive to pharmacological manipulation. Atropine tended to decrease acetylcholine, similar to its effects in controls. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, reduced the excessive concentration of acetylcholine, especially in the cortex. Inhibition of choline uptake by hemicholinium-3 (HC-3) administered icv reduced the acetylcholine concentration in controls and when given to rats during status epilepticus. These results demonstrate that the rat brain concentrations of acetylcholine and choline can increase during status epilepticus. The accumulated acetylcholine was not in a static, inactive compartment, but was actively turning-over and was responsive to drug treatments. Excessive concentrations of acetylcholine and/or choline may play a role in seizure maintenance and in the neuronal damage and lethality associated with status epilepticus.     

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.     

Formation and metabolism of hepoxilin A3 by the rat brain

Incubation of homogenates of the rat cerebral cortex with arachidonic acid led to the appearance of hepoxilin A3, analysed as its stable trihydroxy derivative, trioxilin A3, by high resolution gas chromatography/electron impact mass spectrometry. Using the stable deuterium isotope dilution technique, it is estimated that the cerebral cortex generates 5.0 +/- 0.2 ng/mg protein of hepoxilin A3. The formation of this product was stimulated by the addition of exogenous arachidonic acid (12.9 +/- 1.5 ng/mg protein) and blocked by boiling of the tissue. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C at a concentration of 75 microM did not result in a blockade of hepoxilin formation. Three other regions were also tested for their ability to form hepoxilin A3 upon stimulation with exogenous arachidonic acid, i.e. median eminence, 11.7 +/- 1.6 ng/mg protein, pituitary, 12.3 +/- 0.7 ng/mg protein; pons, 26.6 +/- 0.2 ng/mg protein. In a separate study, 14C-labelled hepoxilin A3 was transformed into 14C-labelled trioxilin A3 by homogenates of the rat whole brain, demonstrating the presence of epoxide hydrolases in the CNS which utilise the hepoxilins as substrates. This is the first demonstration of the occurrence of the hepoxilin pathway in the central nervous system.