Replacement Of Glucose Research Articles

Effects of ketosis on glucose flux in children and adults.

Sequential glucose flux studies were carried out in five normal and six epileptic children and ten adult volunteers using [6,6-2H2]glucose to determine the effect of ketosis on carbohydrate homeostasis in children and adults. All subjects were studied after 14 and 30-38 h of fasting while consuming a normal diet and the epileptic children under 14 h of fasting while consuming an isocaloric ketogenic diet (75% fat wt/wt). Glucose flux, when expressed per kilogram body weight, was inversely correlated with the degree of ketosis in children (P less than 0.001) and in adults (P less than 0.01), but not when both children and adults were considered together (r = 0.078). When glucose flux was corrected for estimated brain weight, the relationship between glucose flux and ketonemia was linearly related in children (P less than 0.001), in adults (P less than 0.02), and when all subjects were considered together (P less than 0.001). The inverse relationship between ketonemia and glucose flux corrected for estimated brain mass is consistent with a partial replacement of glucose by ketone bodies for cerebral metabolism and may provide a more rational means of expressing glucose flux data to take into account the higher brain-to-body ratio in children.

Electrical activities of the frog spinal cord are resistant to perfusion with hypoxic medium

Synaptic functions of the isolated and intra-arterially perfused frog spinal cord were investigated in terms of dorsal root-dorsal root potential (DR-DRP) and dorsal root-ventral root reflexes (DR-VRR), and under the influences of hypoxic Ringer solution, replacement of medium glucose with 2-deoxyglucose (‘hypoglycemic Ringer’), and suspension of perfusion. The synaptic functions were markedly degraded during the suspension of perfusion, but they were remarkably resistant to hypoxic or hypoglycemic Ringer for more than 6 h, if the preparation was continuously perfused. The presynaptic inhibition of the first spike potential of the DR-VRR induced by a conditioning antidromic stimulation, however, was markedly reduced during prolonged exposure to hypoxic conditions. In contrast to earlier concepts that the spinal cord is vulnerable to hypoxia, the present results demonstrate that the frog spinal cord is resistant to prolonged perfusion with hypoxic and hypoglycemic media, although they suggest a latent deterioration of some processes which are believed to be more energy dependent.

Purines as 'hyper-repressors' of glucose transport. A role for phosphoribosyl diphosphate.

Under selected conditions the rate of glucose transport and the intracellular phosphoribosyl diphosphate (PPRibP) concentrations of chick-embryo fibroblasts are inversely correlated. This relationship holds when cells are incubated with mannose, fructose, xylose or various concentrations of glucose. The metabolic inhibitors 2,4-dinitrophenol, rotenone and Methylene Blue increased glucose transport and decreased PPRibP. The addition of any pyrimidine or purine base or ribonucleoside dramatically depleted PPRibP pools, regardless of the carbon source. Addition of guanine (10 microM) or hypoxanthine (100 microM) decreased transport in glucose-grown chick cells to barely detectable values, but did not affect increases observed in cells depressed by substitution of xylose for glucose. Guanosine, inosine and the purine analogues 6-thioguanine, 6-thioguanosine, 8-azaguanine and 6-methylmercaptopurine riboside sharply decreased transport in glucose-grown cells and blocked the increase in transport resulting from the replacement of glucose by fructose or xylose in the culture medium.

Glucose homeostasis during the early stages of liver regeneration in fasted rats.

Kinetic studies with [2-3H]glucose in vivo and gluconeogenic activity measurements in vivo and in vitro were performed in 70% hepatectomized rats submitted to fasting, which represents an extra burden for glucose synthesis but does not impair liver regeneration. Rates of glucose replacement, under steady-state conditions, 14 and 24 h postoperatively, did not differ in partially hepatectomized fasted rats and sham-operated controls. Phosphoenolpyruvate carboxykinase activities increased more rapidly during fasting in remnant livers than in intact livers from controls. Rates of incorporation of 14C from alanine into circulating glucose in hepatectomized rats were already maximal 14 h after surgery, whereas in controls they continued to augment. The maximal rates after partial hepatectomy could not be surpassed by performing the operation in diabetic animals. It is concluded that the relatively high blood sugar levels during fasting in hepatectomized rats do not depend on a reduced peripheral utilization of glucose, but only on a rapid increase in the gluconeogenic activity. The data suggest that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities.

Glucose and lactate kinetics and interrelations in an antarctic bird (emperor penguin)

The isotope single-injection method was used to investigate the glucose and lactate kinetics and the interrelationships between the glucose and lactate pools in fasting emperor penguins. In these remarkably fast-resistant birds, mean lactate concentration, replacement rate, pool, space, and transit time were 1.5 mmol.1-1,53 mumol.min-1.kg-1, 900 mumol.kg-1, 60% of body mass, and 17 min, respectively. Mean glucose concentration, replacement rate, pool, space, and transit time were 20 mmol.1-1, 23 mumol.min-1.kg-1, 4,300 mumol.kg-1, 24% of body mass, and 196 min, respectively. Maximum conversions of lactate into glucose and of glucose into lactate were 29 +/- 2.9% and 75.5 +/- 4.2%, respectively, which indicates that lactate is an effective gluconeogenic precursor and a major fate of glucose metabolism in fasting penguins. The lactate replacement rate and incorporation into glucose were related to the plasma lactate concentration, which suggests that the rate of formation of glucose from lactate is dependent on the availability of lactate. The glucose replacement rate and reduction into lactate were related with the plasma glucose concentration, suggesting that the rate of lactate formation from glucose is dependent on the plasma glucose concentration. These data suggest that in the fasting emperor penguin glucose and lactate availability is capable of regulating the rate at which these substrates are utilized and interconverted. To our knowledge, this is the first evidence for such regulatory capacities in birds.

Development of Extender and Techniques for Frozen Turkey Semen: 1. Development

An extender for turkey semen with a frozen-thawed recovery of ≥50% motile spermatozoa and a vigorous swirl was developed. The effect of glucose, a comparison of 10 different sugars in the extender, the use of sodium acetate and potassium acetate, the ratio of dimethylsulfoxide to ethylene glycol, the percent total cryoproteetant, equilibration time, and osmotic pressure were all tested.Replacement of approximately half of the extender with an iso-osmolar glucose solution yielded higher percentages of motile spermatozoa with both fresh and frozen-thawed semen samples. Replacement of glucose with other carbohydrates did not enhance recovery of frozen-thawed semen. The proportion of sodium and potassium acetates to TESNaK2 in the extender had no effect on motility. Approximately equal proportions of ethylene glycol to dimethylsulfoxide, with a final concentration of 11.2% after dilution, produced at or near optimal recovery of spermatozoal motility after freezing. Twenty to 90 min equilibration times before freezing yielded significantly higher recovery of motile sperm postthaw than shorter periods. A wide range of extender osmotic pressures were compatible with recovery of spermatozoal motility postthaw. The final extender consisted of 4.70 g sodium acetate, 3.39 g potassium acetate, 9.23 g TES,2 .353 g sodium hydroxide, .492 g potassium hydroxide, 32.00 g glucose, H2O q.s. 1000 ml, 370 mOsm/kg, pH 7.2. Ethylene glycol and dimethylsulfoxide were added (1:1 ratio) for a final concentration of 11.2% cryoprotectant after dilution. Semen was held undiluted for 6 min, extended 1:4 at 22 C, and equilibrated for 30 min at 0 C before pellet freezing.

Energy linked uptake of demethylphalloin by isolated rat liver cells

Isolated hepatocytes accumulate demethylphalloin (DMP) under aerobic conditions. In the absence of oxygen the initial rate of the DMP uptake is reduced to less than 20%, while reoxygenation restores the transport. Liver cells release previously accumulated phallotoxin when the oxygen supply is interrupted. DMP uptake is blocked by oligomycin, antimycin A, carbonylcyano-chlorophenylhydrazon (CCCP) or dinitrophenol and is partially inhibited by carboxyatractyloside. Depletion of ATP in hepatocytes by replacement of glucose by fructose reduces the accumulation of toxin too. Below 22 degrees C no uptake was measurable. Between 22 degrees and 37 degrees C an apparent activation energy of 76.6 kJ/mol toxin and a Q10 of 2.6 was calculated for the carrier mediated uptake of DMP. The results suggest that the uptake of demethylphalloin is an energy dependent substrate transfer very similar to that of cholate.

Glucose turnover in fast-growing, lean and in slow-growing, obese swine.

Glucose turnover and associated measurements were compared in genetically obese, slow-growing feral pigs (Ossabaw) and domestic lean, fast-growing (Yorkshire) pigs. Five Ossabaw and five Yorkshire pigs 8 wk of age were prepared with indwelling arterial catheters to facilitate injection of tracer and serial sampling of blood. After a 14-h fast, pigs were administered 100 muCi of glucose-6-3H in a single injection; 12 blood samples were obtained over the subsequent 4-h period to obtain tracer dilution curves. Plasma glucose concentrations were the same in both strains (88 mg/100 ml) prior to tracer injection and remained constant for the duration of the 4-h sampling period. Ossabaw pigs exhibited a smaller minimal glucose mass (144 vs 179 mg/kg body weight, P less than .01) and space (16 vs 20%, P less than .01) when compared with Yorkshire pigs. Glucose replacement rate was greater for Ossabaw pigs than for Yorkshire pigs (3.96 vs 2.97 mg.min-1.kg-1 body weight, P less than .001). Minimal transit time was less in Ossabaw pigs than Yorkshire pigs (36 vs 60 min, P less than .001), which reflected the greater rate of irreversible disposal of tracer from the glucose pool of Ossabaw pigs. In conclusion, under these experimental conditions and at similar fasting glucose concentrations, glucose turnover and metabolic clearance rates were greater in Ossabaw than Yorkshire pigs. The results suggest a greater rate of fasting liver gluconeogenesis during short-term fasting in the young Ossabaw than the Yorkshire pig.

A defined growth medium for the production of chloroperoxidase by Caldariomyces fumago.

Ten strains of Caldariomyces fumago and related fungi were found to produce extracellular chloroperoxidase when grown on a glucose - malt extract medium. High enzyme levels and pigment production were observed for C. fumago ATCC 16373 and C. fumago CMI 89362. Removal of malt extract from the medium and the replacement of glucose by fructose as the carbon source provided a defined medium which, by comparison with the complex medium, produced the following results with both fungal strains. Chloroperoxidase was produced to similar levels, with maximum production after 6 days rather than 12 days of growth; pigmentation of the medium was reduced by 90% and the pH of the medium remained constant, thus stabilizing enzyme activity. Addition or urea or proline as a nitrogen supplement to nitrate enhanced enzyme production by strain CMI 89362. Comparison of the two strains indicated that CMI 89352 produced higher levels of chloroperoxidase than ATCC 16373.

Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.

Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.

Amino acid gluconeogenesis and glucose turnover in kelp bass (Paralabrax sp.).

The glucose replacement rate, plasma glucose concentration, glucose body mass, and amino acid gluconeogenesis were determined in vivo in fed and fasted kelp bass (Paralabrax clathratus) using [6-3H]glucose administered with [U-14C]glutamate, [U-14C]aspartate, or [U-14C]alanine. Fasting (14 days) and prolonged starvation (72 days) do not produce changes in the replacement rate, body mass, or plasma concentration of glucose. The removal of amino acids from the circulation is rapid in both fed and fasting states with nearly 50% of the administered 14Ctracer disappearing by 5 min. The incorporation of [14C]amino acid carbon into the body glucose mass is also rapid with significant amounts of tracer appearing within 15 min after administration. Gluconeogenesis from alanine and glutamate is increased by fasting whereas that from aspartate is diminished. The gluconeogenic rate is comparable to that previously observed in rats (Dunn, A., M. Chenoweth, and J. G. Hemington. The relationship of adrenal glucocorticoids to transaminase activity and gluconeogenesis in the intact rat. Biochim. Biophys. Acta 237: 192-202, 1971), although the glucose replacement rate is significantly lower. We propose that the paradoxically high rate of gluconeogenesis in fish may serve to provide carbohydrate precursors for mucus synthesis in these carnivorous animals with limited carbohydrate intake.

Effect of substrates on the malonate inhibitory pattern and brefeldin A formation in Curvularia lunata.

The extracellular level of brefeldin A fluctuates with the length of malonate inhibition. Following treatment with malonate, mycelial multiplication as opposed to brefeldin A formation, was preferentially increased in the maleate, fumarate, succinate, citrate, methyl palmitate and glucose replacement cultures. Competitive maleate--malonate, fumarate--malonate, succinate--malonate and citrate--malonate-inhibited replacement cultures gave significantly higher mycelial and brefeldin A yields than the sole malonate-inhibited replacement cultures.

Glucose kinetics and respiratory metabolism in fed and fasted chickens

1. 1. Glucose replacement rate, recycling and oxidation rates, as well as respiratory metabolism, were studied in unanaesthetized chickens which were fed or fasted, 0, 24 and 72 hr. (U[ 14C], 6[ 3H]) glucose was used for glucose kinetics study while oxygen consumption ( VO 2) and CO 2 production ( VCO 2) were determined simultaneously in an open circuit system. 2. 2. Glucose apparent replacement rate decreased during fasting from 17 to 9 mg/min/kg while glucose recycling did not change significantly. 3. 3. The net conversion of glucose carbon to expired CO 2 decreased from 2.1 to 0.89 mg/min/kg during the first 24 hr of fasting. Glucose oxidation contributed 31 and 12% of the total heat production ( M) in fed and fasted chickens respectively. 4. 4. The relations between glucose homeostasis and heat production in the chicken are discussed.

Glucose homeostasis in a carnivorous animal (cat) and in rats fed a high-protein diet.

Glucose homeostasis in a carnivorous (cat) and in an omnivorous (rat) animal fed a high-protein, low-carbohydrate (HP) diet followed a pattern similar to that previously found in carnivorous birds [Am. J. Physiol. 234 (Regulatory Integrative Comp. Physiol. 3): R115-R121, 1978]. Thus, compared to controls given carbohydrate-rich (HC) diets. HP-fed animals had, in the fed state, lower levels of blood sugar, lower concentrations of liver glycogen, and a tendency to reduced body glucose mass. During fasting, their liver glycogen was little mobilized and their glycemia remained remarkably constant. Accordingly, their body glucose mass remained unchanged. Gluconeogenic capacity, already high in the fed state, was not further activated by fasting. Phosphoenolpyruvate carboxykinase activity actually decreased in rat liver cytosol, remaining unchanged in cat liver cytosol and mitochondria. [3H]glucose turnover studies revealed that in HP-fed cats and rats glucose replacement rates in the fed state were as high as in HC-fed animals and that the resistance of their glycemia to fasting depended on the high gluconeogenic capacity and not on a more efficient reduction of glucose utilization.

Questionnaire

Baker's yeast, a unicellular eukaryote, has been a model organism for biochemists, geneticists and most recently for molecular biologists. Pioneering biochemical studies were conducted on yeast, such as the study of glucose fermentation and amino acid metabolism. The powerful tools of yeast genetics have allowed a comprehensive study of important issues such as the cell cycle and meiosis. In recent years, it has been established that Saccharomyces cerevisiae, the most extensively characterized of the yeasts, shares key molecules and biochemical pathways with higher eukaryotes. For example, actin, tubulin, ubiquitin, calmodulin, GTP regulatory proteins, different protein kinases including protein tyrosine kinases, were all found to play central roles in yeast [1,2]. Furthermore, structurally homologous proteins [3,4,5], as well as transcription regulating elements [6,7], of yeast and higher eukaryotes, including mammals, were shown to be structurally and functionally interchangeable. It has also been found that yeast can express human genes [8].Technically, yeasts are simple to handle, inexpensive to grow, complete a cell cycle within 90 min, and therefore can yield relatively quick results. These qualities are useful in biotechnological applications.Saccharomyces cerevisiae, can be genetically manipulated fairly easily, and has been tinkered with more than any other system. A cloned, in vitro mutated gene, can be transformed into wild type yeast and by homologous recombination, can replace the native gene and generate the desired mutant [9]. Such manipulations, not possible yet in other eukaryotic cells, allow the precise definition of the role played by different genes and their domains.These unique features of Saccharomyces cerevisiae, together with rapidly evolving techniques of molecular biology, have made it a successful model organism for the study of numerous questions. It is a key cell in the study of major issues in biology since it provides an in vivo model where specific systems can be dissected and analysed. Combining genetic manipulations, molecular biology techniques and biochemical tools, in ways as yet not possible in other eukaryotes, has allowed the more precise treatment of problems such as the cell cycle, meiosis and secretion.Like other unicellular organisms, yeast cells respond individually to the environment. Changes in temperature, pH, cell density, oxygen pressure and especially changes in the availability of nutrients, have dramatic and rapid effects on yeast metabolism and, as a result, on the yeast cell cycle. For example, replacement of glucose by ethanol as the carbon source results in enhanced gluconeogenesis and suppression of glycolysis within seconds [10]. Depletion of glucose from the yeast culture blocks all biochemical pathways involved in the ‘start’ of cell cycle and the cells arrest at the G1 stage within 2 h [reviewed in 11]. Diploid cells undergo meiosis in response to glucose and nitrogen starvation.The cell cycle can also be controlled by mating pheromones secreted into the medium by haploid cells. MATa and MATα cells secrete a-factor and α-factor, respectively, which bind to specific receptors on the opposite mating type cell membrane. Pheromone binding causes cell cycle arrest in G1. This device allows the two mating type cells (MATa and MATα) to synchronize, mate and exchange genetic material [12, 13].These biochemical events, triggered in response to environment, are similar to those encountered in higher eukaryotic cells. This analogy attracted many investigators of transmembrane signalling, since in yeast, one can exploit in a more definitive fashion genetic manipulations and the tools of molecular biology to elucidate the mechanism of transmembrane signalling. It is likely that understanding the details of transmembrane signalling in yeast can advance the research tools as well as the thinking on transmembrane signalling in more complex eukaryotic cells.

Glucose metabolism and recycling of radioactively labelled glucose in the Zucker genetically obese rat (fa/fa).

1. The glucose metabolism of conscious lean and obese rats of the Zucker strain was studied by using doubly labelled glucose ([6-3H,U-14C]glucose) given by intravenous injection as a single dose. Fed animals were used, allowing the study to be made in conditions favouring active lipogenesis. 2. At any given prior food intake (consumption during preceding 24 h), the irreversible glucose replacement rate, R0, was considerably higher in the growing obese rat (4-6 months old) when both of these variables were scaled in terms of the total body water of the animals. 3. When scaled in a similar way, the minimal mass of glucose (Mmin.) was also larger in the obese rats. The mean transit time, t, through the pool did not differ significantly between the two groups, but there was a tendency for this to be shorter in obese rats. 4. There was no difference in the proportion of 14C (derived from metabolized labelled glucose) that recycled as [14C]-glucose after passing through the pyruvate pool in the two groups of rats if the rate of recycling of radioactivity (Rc) was expressed as a percentage of R0.

Influence of Carbon Source, Bile Salts and Incubation Temperature on Recovery of Enterobacteriaceae from Foods Using MacConkey-type Agars

The plea made many years ago (27) to replace the ill-defined coli-aerogenes (‘coliform’) bacteria as indicator organisms in foods processed for safety with the Enterobacteriaceae which are taxonomically accurately defined and as a rule more abundant has of late been more generally accepted. This called for development of a rigorously standardized formula for violet red bile glucose agar and for assessment of the optimal incubation temperature. Four reference strains of Enterobacteriaceae. 120 samples of minced meat and 100 samples of frozen broiler chickens were used in these studies. Considerable differences in the performance of commercially available dried formulae, when used as poured plates were observed. These applied both to productivity and to the type of colony produced by a given pure culture. As expected, replacement of lactose plus glucose by an equimolar amount of glucose did not influence the performance of the medium. Intrinsic toxicity of some batches of medium to non-stressed Enterobacteriaceae appeared to be mainly responsible for substandard performance. It could be overcome by careful selection of the preparations of crystal violet and particularly the bile salts (39) used in the formulae. Incubation at 30 C led to higher confirmed colony counts in minced meat than at 37 C. However, incubation temperature did not greatly influence similar counts in broiler drip. This observation could be substantiated by identification of the types of Enterobacteriaceae isolated from the two commodities. Psychrotrophic species predominated in minced meats, which are often made from raw materials stored for some time under refrigeration, whereas mesophilic species were in the majority on frozen broilers, which are generally frozen shortly after slaughter.

The effect of dietary fat on longevity of Drosophila melanogaster

The effects on the lifespan of adult male Drosophila melanogaster of replacing dietary glucose by fructose, glycerol or one of a variety of fats has been studied. Isocaloric replacement of glucose by fructose or glycerol has no significant effect on lifespan but substituting lard, butter, margarine, dripping or palmitic acid for glucose led to a significant reduction in lifespan. A detailed study of the nature of the life shortening produced by one of the fats, palmitic acid, suggested that most of the life shortening effects are due to rapidly reversible processes, but approximately 20% of the life shortening effects persist irreversibly when transferred back to a low fat medium. This suggests that a high fat medium may accelerate the ageing process. In contrast, a short exposure to palmitic acid early in the life of the flies produces an irreversible increase in the life expectancy which is very variable. This may be due to an action on endogeneous microflora.

Ouabain and regulation of cellular volume in freshly prepared slices of rabbit renal cortex.

1. Changes in the water and ion contents of rabbit renal cortical slices, which had been bathed, immediately after slicing, in air-equilibrated media at room temperature ('freshly prepared slices'), were followed during subsequent incubation at 25 degrees C in oxygenated media of the same composition as the initial bathing medium. 2. In comparison with conventional 'equilibrated' slices (slices incubated at 25 degrees C in oxygenated ordinary medium immediately after slicing) these 'freshly prepared' slices had increased tissue water, sodium and chloride contents and low tissue potassium contents. 3. Control freshly prepared slices incubated at 25 degrees C in oxygenated ordinary medium recovered within 4 min to the tissue water content that is usual for rabbit renal cortical slices incubated in oxygenated ordinary medium at 25 degrees C. Freshly prepared slices incubated at 25 degrees C in oxygenated media containing 1 mM-oubain took 75 min or more to recover to this usual tissue water content. Thus the presence of 1 mM-oubain in both bathing and incubation media produced a marked inhibition of the volume recovery observed when freshly prepared slices are incubated in oxygenated media at 25 degrees C. 4. Reduction of the ouabain concentration reduced the inhibition of cell volume recovery. 5. Replacement of medium glucose by 3-O-methylglucose did not inhibit cell volume recovery in the absence of ouabain. 6. The oxygen consumptions of slices that were bathed and incubated in 1 mM-ouabain media were similar to those of slices initially bathed and incubated in ouabain-free media and then incubated in ouabain media. Thus the effect of ouabain in inhibiting cell volume recovery was unlikely to be secondary to inhibition of cellular energy production. 7. The tissue potassium content of slices incubated aerobically in 1 or 10 mM ouabin fell to an apparently stable value of approximately 100 m-mole/kg dry wt., which corresponds to a calculated concentration ratio of 10:1 across the cellular membrane, suggesting that some residual potassium uptake may still have been occurring. 8. These results indicate that in freshly prepared rabbit renal cortical slices ouabain-sensitive mechanisms play a major role in cell volume recovery. They are not in accord with the postulate that renal cortical cells possess a separate ouabain-insensitive mechanism regulating cell volume.

Regulation of acetylcholine synthesis: control of choline transport and acetylation in synaptosomes.

Abstract— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [2H4]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr‐uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [2H4]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.