MEASUREMENT OF ACETYLCHOLINE TURNOVER WITH GLUCOSE USED AS PRECURSOR: EVIDENCE FOR COMPARTMENTATION OF GLUCOSE METABOLISM IN BRAIN

The turnover of acetylcholine in whole mouse brain in vivo has been determined using [U-¹⁴C]glucose as a precursor of the acetyl moiety. The standard requirements for the measurement of turnover were met: the injection did not change the concentrations of precursor or product, the amount of radioactivity in the brain was proportional to the amount injected, and the relationship between the specific activity of glucose and that of acetylcholine was typical of a precursor and a product. The value for acetylcholine turnover was 64 pmol/min per mg protein, approx 6.4 nmol/min per g brain. Treatment with amobarbital (0.16 mmol/kg) decreased the incorporation of glucose into acetylcholine by 73 × 7%, and treatment with atropine increased it by 18 × 6%. These values agree with those using choline as a precursor, supporting the validity of the values for turnover obtained with either labelled precursor. The specific activity of acetylcholine was higher than that of pyruvate at all times in mouse brain in vivo and in rat brain slices in vitro. These observations demonstrate compartmentation of glucose metabolism with respect to acetylcholine synthesis in the brain. They agree with observations by others of compartmentation of acetyl metabolism. They provide an explanation for the close linkage which has been observed between carbohydrate catabolism and acetylcholine synthesis in the CNS.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

ON THE ORIGIN OF THE ACETYL MOIETY OF ACETYLCHOLINE IN BRAIN STUDIED WITH A DIFFERENTIAL LABELLING TECHNIQUE USING 3H-14C-MIXED LABELLED GLUCOSE AND ACETATE

—Cortex slices of rat brain were incubated with glucose mixed-labelled with ³H and ¹⁴C in the 6-position and the ³H/¹⁴C ratios of lactate, acetate, citrate and acetylcholine were determined. The values obtained were: lactate 0·95, acetate 0·85, citrate 0·65 and acetylcholine 0·67 when expressed in relation to a glucose ³H/¹⁴C ratio of 1·00. When brain slices were incubated with [2-¹⁴C, 2-³H]acetate in the presence of unlabelled glucose, labelled acetylcholine was formed with a ³H/¹⁴C ratio not significantly different from the labelled substrate. The results indicate that citrate is a precursor to the acetyl moiety of acetylcholine.     

CONTROL OF ACETYLCHOLINE SYNTHESIS—THE INHIBITION OF CHOLINE ACETYLTRANSFERASE BY ACETYLCHOLINE

Abstract— The inhibition of choline acetyltransferase by acetylcholine in vitro occurs at a concentration of 10 m m and increases progressively to 45 per cent at a concentration of 100 m m . The inhibition is competitive for choline and noncompetitive for acetyl-CoA. It is suggested that the synthesis of acetylcholine may be controlled by its accumulation in synaptic vesicles.     

THE SYNTHESIS OF ACETYLCHOLINE BY THE OLIVOCOCHLEAR BUNDLE

Abstract— Choline acetyltransferase (ChAc; EC 2.3.1.6) was assayed in the membranous cochlea and in the eighth cranial nerve (both the vestibular and cochlear components) from the point where it leaves the brain stem to the internal auditory meatus of the cat. To determine the contribution of the efferent innervation of the cochlea to this enzymatic activity both eighth nerves and both membranous cochleae were assayed at 17–29 days following section of the right, crossed and uncrossed olivo-cochlear bundles (OCB) in the cat. The lesion was produced along the right sulcus limitans on the floor of the fourth ventricle. The left eighth nerves and cochleae served as controls in the ChAc assay. There was a significant decrease in ChAc activity in the right cochlea and eighth nerve after OCB section and degeneration. The mean activity of ChAc in the right cochleae of the 6 operated cats was 15 ± 7 μg of ACh formed. h⁻¹ (g wet wt. of tissue) ⁻¹ in comparison to the rate of all the intact cochleae of 156 ± 38 μg of ACh formed. h⁻¹. (g of tissue)⁻¹, a statistically significant difference ( P < 0005). The mean activity of ChAc in the right eighth nerves of the cats with OCB lesions was 30 ± 8 n-g of ACh formed. h⁻¹. (g of tissue)⁻¹in comparison to 91 ± 19 fig of ACh formed . h⁻¹. (g of tissue)⁻¹ found for intact eighth nerves. This difference was also significant ( P < 0005). Thus, section and degeneration of the crossed and uncrossed OCB reduce the activity of ChAc in the eighth nerve and membranous cochlea. This finding provides support for the hypothesis suggesting the cholinergic nature of olivo-cochlear transmission.     

METABOLISM OF THE ASPARTYL MOIETY OF N-ACETYL-l-ASPARTIC ACID IN THE RAT BRAIN

The metabolism of N-acetyl-l -aspartic acid (NAA) was studied in rat brain. [Aspartyl-U-¹⁴C]NAA was metabolized predominantly by deacylation. Studies of NAA biosynthesis from l -[U-¹⁴C]aspartic acid have confirmed previous reports that NAA turns over slowly in rat brain. However, intracerebrally-injected N-acetyl-l -[U-¹⁴C]asparticacid was rapidly metabolized. Exogenous NAA appears to be taken up rapidly into a small, metabolically-active pool. This pool serves as substrate for a tricarboxylic acid cycle associated with the production of glutamate for the biosynthesis of glutamine. The bulk of the NAA content in brain appears to be relatively inactive metabolically.     

THE ORIGIN OF THE ACETYLCHOLINE RELEASED FROM THE SURFACE OF THE CORTEX

—The specific radioactivity of acetylcholine liberated from the surface of the rabbit occipital cortex has been compared with that of the underlying cortical synaptosomal and vesicular acetylcholine at varying times after the administration of [N-Me-³H]choline. Choline was administered by diffusion from solutions placed in cups formed by Perspex cylinders applied to the surface of the cortex. Acetylcholine was collected by diffusion into these cups. The specific radioactivity of the acetylcholine declined progressively. The effect of stimulation of afferent cholinergic pathways was to cause a fall in the specific radioactivity of the released acetylcholine. However this was always higher than that of the synaptosomal or vesicular acetylcholine as represented by fractions P₂ and D of the authors’fractionation scheme. It is concluded that acetylcholine released from the cortex must come from a store or stores more recently synthesized than the endogenous acetylcholine of these subcellular fractions.     

GLYCOPROTEIN MOIETY IN THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA) AS THE BIORECOGNITION SITE FOR THE CRYPTHECODINIUM COHNII-LIKE DINOFLAGELLATE

The Crypthecodinium cohnii -like heterotrophic dinoflagellate preys on the cells of the red microalga Porphyridium sp. UTEX 637, and not on other microalgae. The dinoflagellate contains enzymes that degrade the cell wall complex of this species of alga and not that of other red microalgae. The cells of the red microalgae are encapsulated within a cell wall complex composed of about 10 sugars, sulfate, and proteins. We previously hypothesized that the dinoflagellate recognizes the cell wall of this alga. In this study, we have shown that the biorecognition site is the 66-kDa glycoprotein in the algal cell wall complex. The methodology used in this study was based on changing the algal cell wall composition and examining the prey and chemosensory response of the dinoflagellate. The dinoflagellate was not attracted to the cell wall of other red microalgae, which are similar to that of Porphyridium sp., or to sugars composing its cell wall. However, the dinoflagellate preyed on and was attracted to Porphyridium sp. mutants (DCB resistant) having modified cell wall polysaccharide composition, probably because the 66-kDa cell wall glycoprotein was not changed. The dinoflagellate did not respond chemotactically to enzymatically degraded cell wall complex. Treatment of the cell wall complex with antiserum to the 66-kDa glycoprotein or with the lectin concanavalin A (con A), which binds specifically to α-d-mannosyl and α-d-glucosyl residues, did not affect the chemotactic attraction. However, prey by the dinoflagellate was prevented when the algal cells were blocked with antiserum specific to the 66-kDa glycoprotein or with con A. These latter results provide direct proof that the 66-kDa cell wall glycoprotein isthe recognition site and prey-prevention results from the blocking of this site on the cell wall.     

Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves : I. DETERMINATION OF THE ROLE OF METHIONINE AS THE PRECURSOR OF ETHYLENE

The hypersensitive reaction of Samsun NN tobacco leaves to tobacco mosaic virus (TMV) was accompanied by a large increase in ethylene production, just before necrotic local lesions became visible. Normal and virus-induced ethylene production were both largely inhibited by 0.1 millimolar aminoethoxyvinylglycine indicating that methionine is a main ethylene precursor.     

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.     

CHANGES IN SUBCELLULAR COMPONENTS OF YEAST AS RELATED TO NUCLEOTIDE RELEASE FROM THE CELLS INCUBATED IN CITRATE BUFFER

It was found that when intact cells of a yeast, Saccharomycescerevisiae ‘Yebisu’, were incubated in 0.08 M citratebuffer (pH 6.0) containing 2 per cent glucose, nucleotides werereleased in the medium. In this connection, experiments havebeen carried out to elucidate biochemical changes in subcellularstructure of such cells. Microscopic observation showed that the longer the durationof incubation of the cells in the citrate buffer, the more markedbecomes the granulous appearance of the cytoplasm. Among various subcellular fractions of freshly disrupted cells,the highest content in nucleic acid was found in the cell membranefraction and in the small granule fraction. The nucleic acidcontent in the former fraction decreased markedly, even aftera short period of incubation with citrate, accompanied by anabundant release of nucleotides. In contrast, the nucleic acidcontent in the small granule fraction scarcely changed. Continuedincubation with citrate, however, caused a decrease of nucleicacid content also in this fraction. In this case, also the extracellularrelease of amino acids increased and a partial loss of viabilityof the cells was observed. Ultracentrifugal analysis showedthat the sedimentation pattern of the small granule fraction,consisting of an 80 S (major) and a 40 S (minor) component,did not change on incubation with citrate. ¹Present address. Department of Biochemistry, School of Medicine,Tohoku University, Sendai. (Received May 18, 1962; )