ENZYMIC AND CEREBRAL METABOLIC EFFECTS OF 2-DEOXY-d-GLUCOSE

—The time course of effects of 2-deoxy-d -glucose on cerebral glucose metabolism has been studied in vivo and the inhibitory actions of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate on cerebral glycolytic enzymes in vitro. Mice were given 2-deoxy-d -glucose 3 g/kg intraperitoneally. Blood 2-deoxy-d -glucose/glucose ratio was 2–3 from 5 to 30 min after injection, the hyperglycaemic response to 2-deoxy-d -glucose having been suppressed with propranolol. Maximal cerebral 2-deoxy-d -glucose uptake observed was 1μ11 μmol/g/min between 5 and 10 min after injection. At 10 min brain concentrations of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate were 5·82 and 3·12 μmol/g. Analysis of the fate of d -[U-¹⁴C] glucose given subcutaneously 5 min before death showed that glucose uptake was reduced to 40–60 per cent of control from 5 to 30 min after 2-deoxy-d -glucose. However brain glucose concentration rose three to five-fold 20–30 min after 2-deoxy-d -glucose. The majority of glucose entering the brain after 10 min of 2-deoxy-d -glucose treatment was recovered as glucose. Conversion of brain glucose to other acid soluble components was reduced to 1/3 at 10 min and 1/5 at 20–30 min. Glucose-6-phosphate concentration rose from 5 min onwards and was maintained at twice control concentration from 10–30 min. However, because of the rapid entry of 2-deoxy-d -glucose and its conversion to 2-deoxy-d -glucose-6-phosphate, the 2-deoxy-d -glucose 6-P/glucose 6-P ratio was between 19 and 32. Brain adenosine triphosphate concentration did not change, creatine phosphate concentration fell after 25 min. Measurement of enzyme activities in cerebral homogenates (using 1 mivs substrate concentration) showed that hexokinase (EC 2.7.1.1) was 40 per cent inhibited by 5 mm -deoxy-d -glucose (but not by 2-deoxy-d -glucose 6-P). Glucose 6-P dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.43) and phosphoglucomutase (EC 2.7.5.1) were not affected by either 2-deoxy-d -glucose (5 mm ) or 2-deoxy-d -glucose 6-P (5 or 20 mm ). Hexose-phosphate isomerase (EC 5.3.1.9) was 70 per cent inhibited by 20 mm -d -deoxy-d -glucose 6-P. Phosphofructokinase (EC 2.7.1.11) was inhibited by 17 per cent by 2-deoxy-d -glucose 6-P (20 mm ). During the initial impairment of cerebral function by 2-deoxy-d -glucose there is competitive inhibition of glucose transport into the brain; later, glycolysis is more powerfully depressed by the inhibition of isomerase produced by the high intracerebral concentration of 2-deoxyglucose-6-phosphate.     

The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

1. Cerebral-cortex slices prelabelled with gamma-amino[1-(14)C]butyrate (GABA) were incubated in a glucose-saline medium. After the initial rapid uptake there was no appreciable re-entry of (14)C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03mumol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of (14)C into amino acids associated with the tricarboxylic acid cycle was followed with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of (14)C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-(14)C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by alpha-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [(14)C]GABA into gamma-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-(14)C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of (14)C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-(14)C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-(14)C]GABA were different from those labelled by the metabolism of [(14)C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-(14)C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ;transmitter pool' was estimated to be 25-30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (aw) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the aw of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 aw. The maintenance coefficients for glucose uptake were similar at both aw values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 aw.     

Glucose metabolism in the developing rat. Studies in vivo

1. The specific radioactivity of plasma d-glucose and the incorporation of (14)C into plasma l-lactate, liver glycogen and skeletal-muscle glycogen was measured as a function of time after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into newborn, 2-, 10- and 30-day-old rats. 2. The log of the specific radioactivity of both plasma d-[6-(14)C]- and d-[6-(3)H]-glucose of the 2-, 10- and 30-day-old rats decreased linearly with time for at least 60min after injection of labelled glucose. The specific radioactivity of both plasma d-[6-(14)C]- and d-[6-(3)H]-glucose of the newborn rat remained constant for at least 75min after injection. 3. The glucose turnover rate of the 30-day-old rat was significantly greater than (approximately twice) that of the 2- and 10-day-old rats. The relative size of both the glucose pool and the glucose space decreased with age. Less than 10% of the glucose utilized in the 2-, 10- and 30-day-old rats was recycled via the Cori cycle. 4. The results are discussed in relationship to the availability of dietary glucose and other factors that may influence glucose metabolism in the developing rat.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (a_w) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the a_w of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 a_w. The maintenance coefficients for glucose uptake were similar at both a_w values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 a_w.     

Evidence that activation of 2-deoxy-D-glucose transport in rat thymocyte suspensions results from enhanced coupling between transport and hexokinase activity.

1. Suspensions of rat thymocytes accumulate free 2-deoxy-D-glucose (2-dGlc) within the cytosol to a concentration approx. 25-fold above the external concentration. This active accumulation was enhanced by 40 nM-phorbol 12-myristate 13-acetate (phorbol). 2. The Km for zero-trans uptake in control cells was 2.3 +/- 0.14 mM and Vmax. was 0.41 +/- 0.08 mumol/min per 10(10) cells (n = 6). In cells treated with phorbol (40 nM) the Km for zero-trans uptake was 1.2 +/- 0.13 mM and Vmax. 0.46 +/- 0.03 mumol/min per 10(10) cells (n = 6). The Km was decreased significantly by phorbol (P less than 0.01). 3. Phorbol-dependent activation of thymocytes delayed exit of free 2-dGlc into sugar-free solution and prevented exchange exit. Activation had no effect on 3-O-methyl D-glucoside (3-OMG) exit. 4. Coupling of 2-dGlc transport to hexokinase activity was determined by observing the effects of various concentrations of unlabelled cytosolic 2-dGlc on influx of labelled 2-dGlc into the hexose phosphate pool. In control cells this coupling was 0.81 +/- 0.02 and in phorbol-activated cells it was 0.92 +/- 0.01 (P less than 0.01). 5. The high-affinity inhibitor of hexokinase, mannoheptulose, inhibited uptake of 2-dGlc in both control and phorbol-treated cells. These data are consistent with a model for activation of sugar transport in which hexokinase activity is integrated with the sugar transporter at the endofacial surface. The results suggest that phorbol increases the degree of coupling transport with hexokinase activity, thereby leading to an increase in the rate of uptake of 2-dGlc, a decrease in exit of free 2-dGlc from the cytosol and an increase in free 2-dGlc accumulation.     

The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

1. The metabolism of gamma-aminobutyrate (GABA) was investigated in cerebral-cortex slices incubated in glucose-saline medium with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. 2. A rapid release of GABA from the tissue, amounting to 25-30% of the total, was observed on addition of 66m-equiv. of K(+)/1 to the medium; the liberation of other amino acids was relatively small. The effect was apparently specific for K(+); GABA was not released on addition of equivalent amounts of Na(+) or on increasing the respiration rate with 10mm-ammonium chloride. The results show that GABA behaves like the transmitter compounds (acetylcholine, catecholamines) on K(+) stimulation, and therefore now satisfies certain of the criteria required for a transmitter in mammalian brain. 3. The release of GABA from the tissue on addition of K(+) was followed by a slow re-uptake. The rate of uptake of GABA in a medium containing 5.9m-equiv. of K(+)/1 was more than four times that in a medium containing 66m-equiv. of K(+)/1. 4. The concentration of GABA in brain tissue incubated for 1h in a medium containing 66m-equiv. of K(+)/1 was about 50% higher than that observed under normal conditions. 5. There was evidence that exogenous [(14)C]GABA mixed with the endogenous pool(s), since the proportion of the total GABA released on K(+) stimulation was the same, and the specific radioactivity of the liberated GABA was close to that remaining in the tissue, whether the GABA was labelled by [1-(14)C]GABA from the medium or generated in the tissue from [(14)C]glucose. 6. On the basis of these findings and the observations outlined in the preceding papers it was possible to calculate the kinetic constants of GABA metabolism by computer simulation of the results. K(+) stimulation led to a 2.5-fold increase in the flux through the tricarboxylic acid cycle, whereas the flux in the GABA bypath was little affected; as a result the flux through the GABA bypath, which under normal conditions was 8% of that through the tricarboxylic acid cycle, decreased to 3-5%. 7. The metabolism of glutamine was greatly affected by K(+)-stimulation. The ratio of the concentration of glutamine in the slices to that in the medium, which under normal conditions was the smallest among the amino acids investigated, increased from about 17 to 63 in 1h. This effect was attributable partly to an uptake of glutamine from the medium (1.8mumol/h per g) and partly to a net increase in the total amount of glutamine (2.6mumol/h per g). At 1h after the addition of K(+) the net gain of glutamine could be accounted for by the decrease of glutamate. 8. Metabolic compartmentation was evident when brain-cortex slices were incubated in glucose-saline medium and the labelled substrate was [(14)C]GABA, since the specific radioactivity of glutamine exceeded that of glutamate. On addition of K(+) the signs of metabolic compartmentation promptly disappeared: this effect was apparently associated with an increase in the permeability of the compartments containing labelled metabolites derived from [(14)C]GABA. The change in the permeability, however, did not affect all the compartments; when the labelled substrate was [(14)C]glucose the equilibration of labelled amino acids between tissue and medium was similar under normal conditions and in the presence of high concentrations of K(+). 9. The metabolism of [(14)C]glucose was followed by measuring oxygen uptake, respiratory (14)CO(2), and incorporation of (14)C into amino acids. The results showed that K(+) stimulation increased the flux of glucose carbon, both in the glycolytic pathway and in the tricarboxylic acid cycle.     

Pretreatment with 3-O-methyl-d-glucose or 2-deoxy-d-glucose attenuates the post-mortem rise in rat brain lactate

The present investigation examined the effects of pretreatment with 3-O-methyl-d-glucose (3OMG) or 2-deoxy-d-glucose (2DOG) on post-mortem rise in rat brain lactate to evaluate their potential use for minimizing ischemia-induced rise in brain lactate. The results showed that iv administration of either glucose analogue (2 g/kg) at 2.5 min prior to sacrifice significantly attenuated (to 0.61 of control levels) post-mortem brain lactate rise. Pretreating rats with 2-deoxy-d-glucose (2 g/kg) 15 min prior to sacrifice resulted in a greater inhibition (to 0.52 of control) of the post-mortem lactate rise. The effects of these two analogues (3OMG and 2DOG) can be accounted for by their inhibition of brain glucose transport and inhibition of brain glucose metabolism by 2DOG. The present results suggest that intervention with either of these glucose analogues under the proper experimental procedures may minimize the cytopathological consequences of ischemia related to the rise in brain lactate.     

Sugar transport and metabolism in Schistosoma mansoni.

The absorption kinetics of some 14-C-labeled simple sugards in adults of Schistosoma mansoni are described. The influx of fructose and 3-0-methylglucose was by diffusion alone, while glucose, 2-deoxyglucose (2DOG), galactose, glucosamine, and mannose were absorbed by mediated transport as well as by diffusion. Although absorbed glucose was rapidly metabolized, uptake rates of radio-glucose in 2-min incubations corresponded with the amount of glucose (determined chemically) removed from the incubation medium. In 30-min incubations 2DOG was slowly metabolized and accumulated against an apparent concentration difference. The mediated transport of glucose and 2DOG was inhibited in Na+-free media, and by the presence of ouabain, phlorizin, phloretin, and other sugars. Accordingly, influxes of glucose of 2DOG and 22-Na+ were coupled. On a per mg protein basis, female worms transported more 2DOG and glucose, but less glycine, than did males. However, the rate of glucose metabolism by male and female worms incubated together was greater than that of either males or females incubated separately. The nature of sugar transport in schistosomes and other flatworms is similar to that in vertebrates.     

Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta

Summary Glucose uptake into plasma membrane vesicles from the maternal surface of the human placenta was measured with the Millipore filtration technique. Uptake ofd-glucose was dependent on the osmolarity of the incubation medium surrounding the vesicles. Uptake ofd-glucose exceeded that ofl-glucose. The uptake ofd-glucose was not enhanced by placing 100mm NaCl or NaSCN in the medium outside the vesicles (none inside) at the onset of uptake determinations.d-glucose transport was inhibited by cytochalasin B; phloretin, phlorizin, and 1-fluoro-2,4-dinitrobenzene.d-glucose uptake was inhibited by 2-deoxy-d-glucose, 3-O-methyl-d-glucose and to a lesser extent byd-galactose. It was not inhibited by -methyl-d-glucoside. Cytochalasin B binding to the vesicles was 30% inhibited in the presence of 80mm d-glucose. The results indicate that the system for facilitated transport ofd-glucose at the maternal face of the placenta is distinctly different from that on the brush-border membrane of intestine or renal tubule and more closely resembles that of human erythrocyte.     

Fluxes and accumulation of ethirimol in haustoria of Erysiphe pisi and protoplasts of Pisum sativum

Isolated purified fractions containing haustorial complexes and mesophyll protoplasts were used to investigate the relative affinities in vitro of the host-parasite interface and host for the mildew specific fungicide, ethirimol. Compounds labelled with [¹⁴C] were used throughout this study. Isolated haustorial complexes accumulated fungicide against a concentration gradient and, in 15 min, to much higher concentrations than they accumulated glucose, sucrose, uracil, glycine, ethanolamine hydrochloride, inulin carboxylic acid, thiocyanate ions or uridine diphosphate glucose. Accumulation of ethirimol ceased after 30 min when the internal concentration was over twenty times greater than in the ambient medium. Similar quantities were absorbed in 15 min at pH 4.2 and 6–2. The quantities absorbed during 1 h incubation were directly related to the ambient concentration (4–400 μm). Ethirimol introduced into haustorial complexes was easily removed by washing; one third was removed in the first 5 min and only one sixth of the original remained after 3 h. Analysis of the kinetics of ethirimol efflux showed that it was located in two compartments within the complexes; thus it most probably entered the fungal cytoplasam. Ethirimol entered pea mesophyll protoplasts showing biphasic kinetics with considerable uptake in the first 30 min and a more gradual influx during the following 18 h. It was not accumulated against a concentration gradient until 6 h and after 18 h the internal concentration exceeded that of the ambient by only 1.74 fold. Extraction and chromatography showed that only small proportions of the ethirimol were degraded by haustorial complexes and protoplasts during the experimental periods. These in vitro experiments indicate competitive accumulation of ethirimol by the host-parasite interface in vivo.     

d-glucose uptake in human liver cell cultures

Summary The kinetic parameters ford-glucose uptake were studied in human liver cell cultures under strictly defined experimental conditions. Using a wide concentration range (0.005 to 30 mmol/l), the kinetic data obtained suggested strongly thatd-glucose in human liver cell cultures can be transported by two separate systems. For the high-affinity system, the apparentK _m was 0.645±0.21 mmol/l and the V_max, 12.49±3.74 nmol/mg protein per min. For the low-affinity system, the apparentK _m was 6.91±0.58 mmol/l and the V_max, 79.90±5.27 nmol/mg protein per min. At a concentration of 2.1×10⁻⁷ mol/l, cytochalasin B preferentially inhibited the high-affinityd-glucose site or transport system. The time course ofd-glucose uptake, studied in two cell lines from patients with hereditary fructose intolerance, was significantly higher than for the control lines. This work was supported by Grant I.N.S.E.R.M. CRL 77-5-210-4.     

Biphasic stimulation of cellular calcium concentration by 3,5,3'-triiodothyronine in rat thymocytes

3,5,3'-Triiodothyronine (T3) produced a rapid and transient increase in 45Ca uptake and cytoplasmic free calcium concentration in rat thymocytes, which is the most rapid effect of T3 in this system. This effect was manifested in cells suspended in medium containing 1 mM calcium. The T3 effect on 45Ca uptake was evident at 15-30 s, reached maximum at 30-60 s, and returned to control values at 5 min. The T3 effect on cytoplasmic free calcium concentration was seen after 30 s, reached maximum at 7 min, and returned to control values after 24 min. In cells suspended in Ca2+-free medium, T3 produced a similar rapid increase in 45Ca uptake, which was sustained for at least 60 min, but T3 failed to change cytoplasmic free calcium concentration. Alprenolol (10 microM) blocked the stimulatory effects of T3 on these two functions in a similar fashion. From these results, I suggest that in rat thymocytes T3 influences cellular calcium economy through a biphasic mechanism in which T3 first increases calcium uptake which, in turn, is followed by a release of calcium from intracellular pool(s), resulting in a further increase in cytoplasmic free calcium concentration and the activation of Ca2+ -regulated systems. Moreover, the present study provides further support for the postulate that in the rat thymocyte calcium serves as the first messenger for the plasma membrane-mediated stimulatory effects of T3 on several metabolic functions.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of excess K+ on two different tricarboxylate cycles in rat brain cortex slices

The authors compared, in rat brain cortex slices, the oxidation of labelled glucose and acetate and the conversion of these precursors into amino acids during incubation in control salt-glucose medium and in medium with 47 mM K+, with the aim of determining with which of the two determinable tricarboxylate cycles raised oxygen consumption is associated in the presence of excess K+. Under the experimental conditions it was found that from U-[14C]-glucose more than double the amount of [14C]-CO2 was formed and that the rate of [14C] incorportation into the amino acids was likewise roughly doubled. This is indicative of activation of processes in the tricarboxylate cycle associated with the large glutamate pool. Incorporation from 1-[14C]-acetate into the total amino acids was not affected. Specific activity in glutamate and asparate was more than doubled, while glutamine specific activity fell to less than half. [14C]-CO2 production fell to 65%. This shows that the tricarboxylate cycle associated with the small glutamate pool, which is probably localized in the glia cells, did not participate in raised oxygen consumption in the presence of excess K+.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

Thymidine uptake by developing sea urchin embryos

Unfertilized Paracentrotus lividus eggs accumulate very little thymidine. Upon fertilization, however, uptake increases sharply. The pool for thymidine and/or its metabolic products is saturated after 40 min of exposure. Its size is expandable and proportional to the initial concentration of thymidine in the medium. The uptake rate is low shortly after fertilization, increases until 40 min after fertilization and remains constant thereafter. Of the radioactivity taken up in the form of [³H]thymidine during a 30 min exposure beginning at 60 min after fertilization, about 1% is associated with the acid-insoluble fraction and 99% with the acid-soluble fraction.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.