CONTROL OF AEROBIC GLYCOLYSIS IN GUINEA-PIG CEREBRAL CORTEX SLICES

—The effect of glutamate on aerobic glycolysis in guinea-pig cerebral cortex slices was analysed in comparison with that of high-potassium. In contrast to the increased glycolysis in 50 mm -potassium medium which was accompanied by increases of fructose diphosphate and triose phosphates in the slices, the addition of 5 mm -d -glutamate to the medium increased the rate of glycolysis without increasing these intermediates. When increasing the concentration of potassium in the medium up to 20 mm , the rate of aerobic glycolysis was not increased although fructose diphosphate and triose phosphates in the slices were increased. At this potassium concentration in the medium ATP in the slices was highest. At 30 mm -potassium the rate of glycolysis was increased significantly, but fructose diphosphate and triose phosphates were decreased. ATP was lower at 30 mm - than at 20 mm -potassium. By increasing potassium to 40 mm and above, the rate of glycolysis was further increased, and fructose diphosphate and triose phosphates were again increased. Between 5 and 20 mm -potassium in the medium the increasing effect of glutamate on glycolysis was very pronounced. d -Glutamate decreased the amounts of ATP, fructose diphosphate and triose phosphates at any concentration of potassium in the medium. When adding cyclic AMP and 5′AMP to the slices, fructose diphosphate and triose phosphates were increased, but the rate of glycolysis was not increased. On the basis of these observations mechanisms of the control over glycolysis in guinea-pig cerebral cortex slices are discussed. It is suggested that the glycolysis is controlled by the changes in ATP concentration through their action on the glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase system. The changed patterns of the glycolytic intermediate profile in the slices when adding ATP to the medium are consistent with this suggestion. The addition of l -phenylalanine to guinea-pig cerebral cortex slices did not inhibit the rate of glycolysis, although it inhibited the activity of pyruvate kinase.     

SODIUM AND POTASSIUM IONS AND ACCUMULATION OF LABELLED d-ASPARTATE AND GABA IN CRUDE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

The accumulation of labelled d -aspartate into crude synaptosomal fraction (P₂) prepared from the rat cerebral cortex proceeded by a ‘high affinity’ system (K_m= 15.1 μm The maximal velocity of d -aspartate uptake was higher than that of the ‘high affinity’ component of l -aspartate uptake and almost equal to that of l -glutamate under the same incubation conditions. Negligible metabolism of labelled d -aspartate was observed in the P₂ fraction. These findings are in accord with those which have been reported for rat cerebral cortical slices. The following observations were made on d -aspartate uptake into rat cerebral P₂ fraction. (1) The requirement of sodium is almost absolute and obligatory. (2) The affinity of the carrier for the substrate is increased by increasing sodium concentration in the medium, but the maximal velocity is not altered. (3) It is suggested that sodium ion is co-transported mole for mole with the substrate molecule. (4) Omission of potassium from the medium inhibits the uptake competitively. (5) Ouabain is a competitive inhibitor on the uptake. (6) Whereas thallium, rubidium and ammonium are efficient substitutes for potassium in exhibiting Na–K ATPase activity of the P₂ fraction, the uptake is activated only by rubidium in the absence of potassium. These observations were in common with the uptake of l -aspartate as well as of l - and d -glutamate, but not with GABA uptake. The requirement of sodium for the uptake of d -glutamate was indicated to be higher than that in the uptake of the other amino acids. Mutual inhibitions of the uptake among l - and d -isomers of glutamate and aspartate suggested that a common carrier is involved in the transport. Mechanisms of the transport of these amino acids in the crude synaptosomal fraction were discussed.     

THE EFFECT OF EXTRACELLULAR POTASSIUM CONCENTRATION ON PROTEIN SYNTHESIS IN GUINEA-PIG HIPPOCAMPAL SLICES1,2

Abstract— We have measured the effect of small variations in extracellular potassium concentrations ([K⁺]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K⁺ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K⁺ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K⁺] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K⁺] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.⁺] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

FLUID COMPARTMENTATION AND ELECTROLYTES OF CAT CEREBRAL CORTEX IN VITRO–II SODIUM, POTASSIUM AND CHLORIDE OF MATURE CEREBRAL CORTEX

The contents of K⁺, Na⁺ and Cl^? in various incubation media and in slices of adult cat cerebral cortex incubated in vitro under a variety of conditions have been determined in conjunction with studies on slice swelling and fluid compartmentation reported in the preceding paper (Bourke and Tower , 1966). Cortical slices incubated in media containing 16 Or 27 mm-K⁺ exhibit contents of K⁺ and Na⁺ most nearly comparable to those found in viuo. Substitution of isethionate^? For Cl^? or omission of Ca²⁺ in such media have little effect on slice cation composition. Rb⁺ can effectively substitute for K⁺, but substitution of Li⁺ or choline⁺ for most of the naf in incubation media is associated with accumulation of these cations in slices at the expense of both K⁺ and Na⁺. Compared to values in vivo for net contents and/or concentrations of electrolytes in the non-sucrose spaces of cortical slices, conditions yielding most favourable data in vitro appeared to be incubation of cortical slices in 16 mm -K⁺ medium or in 27 mm -K⁺ medium with either omission of Ca²⁺ or replacement of Cl^? by isethionate. Essentially complete inhibition of maintenance of K⁺ and extrusion of Na⁺ in slices of cat cerebral cortex occurs upon incubation with 10^?5 or 10^?4m -ouabain, with 50 per cent inhibition of cortical slice electrolyte metabolism occurring at about 8 × 10^?7m -ouabain. Cortical slices incubated in 27 mm -K⁺ medium in the presence of ⁴²K exhibited rates of exchange and turnover of slice K⁺ (in non-sucrose spaces) of 0·7 μequiv./min and 6.45 per cent respectively. In the presence of 10^?5m -ouabain, a maximal ratio of slice specific activity/medium specific activity is attained within about 5 min after ⁴²K addition, compared to >20 min for control slices. In neither case does the maximal specific activity ratio exceed about 0.85; this suggests that some 10-15 per cent of total cortical K⁺ comprises a “slowly exchangeable” fraction. In the presence of Ca²⁺ (1.3 mm ) slice oxygen consumption is markedly stimulated (39 per cent) and aerobic glycolysis is markedly depressed (54 per cent) in the presence of 10^?5m -ouabain; whereas on omission of Ca²⁺ from incubation media, both respiration and glycolysis are normally stimulated but, with 10^?5m -ouabain present, both are significantly depressed (20 per cent and 37 per cent respectively). Possible relevance of these effects to mobilization of tissue Ca²⁺ by ouabain and to effects of intracellular Ca²⁺ on mitochondrial respiratory metabolism is discussed.     

THE UPTAKE OF LOW CONCENTRATIONS OF LITHIUM IONS INTO RAT CEREBRAL CORTEX SLICES AND ITS DEPENDENCE ON CATIONS

—Rat cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline, and the uptake of Li⁺ was measured after periods of 15 s to 5 min. Saturation was not seen within the concentrations of Li⁺ employed (0·5-2·0 mm ). The half-time of the uptake was 7·9 min. At steady state, after 1 h incubation, the concentration of Li⁺ in the tissue was linearly related to that of the medium (0·5-1·5 mm Li⁺) with a concentration ratio of 1·29–1·66. The concentrations of K⁺ and Na⁺ in the slices incubated without Li⁺ were found to be (μmol/g incubated wt, mean ±s.d .) 63·8 ± 9·6 and 96·2 ± 7·8 respectively (n = 28). In the presence of media with 1·5 mm -Li⁺, the K⁺ and Na⁺ in the slices were 56·2 ± 8·8 and 101·0 ± 7·7 respectively (n = 37). The concentration of Li⁺ in the slices, after 1 h incubation, increased in a non linear way as the concentration of K⁺ in the medium was decreased within a range of 0·10 mm -K⁺. In the absence of K⁺ in the medium the uptake of Li⁺ was approx 50% higher than in the presence of 4·9 mm -K⁺. There was an inverse linear relationship between the concentration of Li⁺ in the slices and that of Ca²⁺ in the medium within the range of 0-5·2 mm (-0·13 mm -Li⁺/mm Ca²⁺). The concentration of Li⁺ in the slices increased by approx 10% when the Mg²⁺ in the medium was increased from 1·3 mm to 2·6 mm . Changes of the concentration of Na⁺ between 120 mm and 170 mm in the medium had no significant effect on the Li⁺ uptake.     

EFFECTS OF DIAMINOPROPIONATE, DEOXYADENOSINE, AND THEOPHYLLINE ON STIMULATED FORMATION OF CYCLIC AMP AND GMP BY DEPOLARIZING AGENTS IN SLICES OF GUINEA-PIG CEREBRAL CORTEX

In incubated slices of guinea-pig cerebral cortex depolarizing agents such as veratridine and high potassium ions caused 50 to 80-fold increases of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) levels and these responses were inhibited about 50% by 2, 3-diaminopropionate and 2'-deoxyadenosine: the former is a specific antagonist for glutamate-elicited accumulation of cyclic AMP and the latter selectively for adenosine-elicited accumulation. Methylxanthines were powerful ‘inhibitors’toward the responses not only to depolarizing agents but also to glutamate and adenosine. These findings are consistent with the hypothesis that releases of both glutamate and adenosine are involved in the depolarization-elicited increases of cyclic AMP levels. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP) levels in the slices were also elevated by veratridine as well as by glutamate, but always to a lesser extent (8 ～ 12 times the control value) than cyclic AMP levels were. The responses for cyclic GMP both to veratridine and glutamate were ‘augmented’by methylxanthines and were not inhibited by 2, 3-diaminopropionate. Thus, glutamate appears to cause the increase of cyclic GMP levels through a different mechanism or site of action from that for cyclic AMP.     

INFLUENCE OF POTASSIUM ON THE STEADY-STATE REDOX POTENTIAL OF THE ELECTRON TRANSPORT CHAIN IN SLICES OF RAT CEREBRAL CORTEX AND THE EFFECT OF OUABAIN

Abstract— The concentration dependence of the modifications by potassium of the respiratory intermediates in incubated slices of rat cerebral cortex has been examined in the presence and absence of calcium. In addition to the immediate increase in respiration and the concomitant oxidation of the respiratory intermediates, longer term increases in the steady-state redox potential were observed at higher potassium concentrations. Addition of calcium to the system did not appreciably alter the immediate effects of potassium, but shifted the redox state of the respiratory intermediates; these changes involved a decrease in reduced intermediate at low concentrations of potassium and a relatively higher level of reduced carriers at high concentrations of potassium. Ouabain (50 μ m ) inhibited both the initial responses to added potassium and modified the changes in steady-state levels of reduced intermediate in the absence of calcium. In the presence of calcium, ouabain (50 μ m ) inhibited the initial oxidation of NAD(P)H observed upon addition of potassium but had no effect on the respiratory response to the addition of low concentrations of potassium. The disassociation of these responses resulted in a large decrease in the steady-state levels of reduced cytochrome. At 30 m m potassium an oxidation of NAD(P)H was observed which accompanied by an increase in levels of reduced cytochromes. These changes in redox state of the respiratory carriers have been discussed in relation to previous reports dealing with the effects of potassium on aerobic glycolysis and oxygen consumption by brain slices.     

SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA-PIG CEREBRAL CORTEX AND SPLEEN

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea-pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non-occluded micro-particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [³H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non-occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6-0.8 m -sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub-fractions D to G (0.4-1.0 m -sucrose) and the other in sub-fraction J (1.4 m -sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.
Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4-0.6 m -sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m -sucrose).     

SODIUM AND THE FLUX OF CALCIUM IONS IN ELECTRICALLY-STIMULATED CEREBRAL TISSUE

Abstract— The rate of efflux of ⁴⁵Ca²⁺ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of ⁴⁵Ca²⁺ from the more stable pool and the time course for the redistribution of Na⁺ and K⁺ paralleled that for the increased efflux of Ca²⁺. This effect of stimulationwas dependent on the presence of Na⁺ in the incubation medium. Lack of Na⁺ in the medium during loading of the slices with ⁴⁵Ca²⁺ increased uptake but on subsequent transfer to a medium containing Na⁺, electrical pulses failed to increase the rate of efflux of ⁴⁵Ca²⁺. In unstimulated slices, the rate of efflux of ⁴⁵Ca²⁺ was dependent upon the concentration ratio of Na⁺ to Ca²⁺ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of ⁴⁵Ca²⁺ that occurred during electrical stimulation but exerted no effect on Ca²⁺-Ca²⁺ exchange. Our results suggest that there is a Na⁺-dependent turnover of Ca²⁺ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na⁺-Ca²⁺ interaction in the regulation of neurotransmitter function is discussed.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

THE UPTAKE OF SPERMIDINE AND SPERMINE BY SLICES OF MOUSE CEREBRAL HEMISPHERES

Abstract— Spermidine and spermine are taken up into mouse cerebral hemisphere slices by active transport and can be accumulated well above the medium concentration. The uptake process shows saturation kinetics and resembles that for amino acid uptake in that it is sensitive to temperature and inhibited by cyanide, 2,4-dinitrophenol or by the absence of glucose from the medium. However, at low initial medium concentrations spermine is taken up by a process which is insensitive to metabolic inhibitors or to temperature. It is suggested that either physical binding to a cellular constituent or exchange transport may account for this uptake. Ouabain does not inhibit polyamine uptake. Spermidine or spermine uptake is inhibited by cadaverine and putrescine. Spermine is the most potent inhibitor of spermidine uptake and vice-versa. Polyamine uptake differs from that of amino acids in that it is increased by a reduction in medium sodium or calcium content and decreased by an increase in medium potassium content. Recently taken up spermine undergoes heteroexchange with spermidine and homoexchange with recently entered spermine. Spermidine undergoes neither heteroexchange with spermine nor homoexchange.     

THE EFFECTS OF OXYGEN DEPRIVATION ON ELECTRICALLY STIMULATED CEREBRAL CORTEX SLICES

Abstract— (1) Thin slices were prepared from guinea pig cerebral cortex and allowed to incubate in oxygenated bicarbonate-buffered medium for 30 min. Subsequent to that time the slices were made hypoxic by passing 95% N₂-5% CO₂ through the medium. Hypoxic exposure caused the slices to gain Na⁺ and to lose K⁺ ions from the non-inulin space. These shifts were especially pronounced when slices were electrically stimulated during the hypoxic period. Thus, after 30 min of hypoxia plus stimulation, non-inulin Na⁺ had risen from 30 to 84, μequiv./g wet wt., and non-inulin K⁺ had fallen from 50·5 to 14·3 μequiv./g wet wt.
(2) The above shifts were in part reversible, but when reoxygenated slices were subsequently electrically stimulated in oxygenated media, they failed to lose K⁺ or to gain Na⁺.
(3) The induced inexcitable state could not be attributed to inability of the slices to replenish ATP and phosphocreatine and may indicate an alteration in membrane constituents necessary for preservation of membrane excitability.