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Notes: Abstract: Uptake of L-glutamine (2 mM) by rat brain cortex slices against a concentration gradient is markedly inhibited (40%) by branched-chain Lamino acids (1 mM), L-phenylalanine (1 mM), or L-methionine (1 mM); that of L-asparagine (2 mM) is much less affected by these amino acids. Other amino acids investigated have little or no effect on cerebral L-glutamine uptake. The suppressions of L-glutamine uptake by the inhibitory amino acids are apparently blocked by high [K+], which itself has little or no effect on glutamine uptake. This abolition of suppression is partly explained by high [K+] retention of endogenous glutamine; in the absence of Ca2+ such retention disappears. The inhibitory amino acids (1 mM) also enhance the release of endogenous glutamine, exogenous glutamine with which slices have been loaded, or glutamine synthesized in the slices from exogenous glutamate. The enhanced release of endogenous glutamine is diminished by high [K+]. The suppression of glutamine uptake by the branched-chain amino acids is independent of the concentration of glutamine at low concentrations (0.25–0.5 mM), indicating non-competition, but is reduced with high concentration of glutamine. The inhibition by L-phenylalanine is noncompetitive. L-Glutamine (2 mM) exerts no inhibition of the cerebral uptakes of the branched-chain L-amino acids or Lphenylalanine (0.25–2 mM). The inhibitory amino acids are as active in suppressing L-glutamine uptake with immature rat brain slices as with adult, although the uptake, against a gradient, of L-glutamine in the infant rat brain is about one-half that in the adult. They are also just as inhibitory on the concentrative uptake of L-glutamine by a crude synaptosomal preparation derived from rat brain cortex. Such a nerve ending preparation takes up L-glutamine (0.25 mM), against a gradient, at about ninefold the rate at which it is taken up by cortex slices (for equal amounts of protein), and the uptake process is markedly suppressed by high [K+] in contrast to the effects of high [K+] with slices. The possible physiological and pathological consequences of the suppression of glutamine uptake are discussed.

Notes: Abstract— Radioactive acetylcholine ([14C]ACh) that is taken up by rat cerebral cortex slices, incubated aerobically in a physiological saline-glucose paraoxon-[14C]ACh medium, apparently by a passive diffusion process at concentrations 〉 1 mm consists essentially of two forms, a readily exchangeable and releaseable or mobile form, and a bound or retained form, poorly (or not) exchangeable. The quantity of retained ACh consists of a considerable fraction of that taken up amounting to 54% with external 0.1 mm-[14C]ACh and about constant, 27%, for the range 5-50mm-[14C]ACh. All its ACh is released on homogenization with 0.1 n-perchloric acid or on tissue disintegration in distilled water. The cerebral uptake of ACh differs basically from that of urea as there is no retention of the latter following its uptake. Cerebral cortex slices are superior to those of cerebellar cortex, subcortical white matter, kidney cortex, liver and spleen in taking up and retaining [14C]ACh. Deprivation in the incubation media of glucose or Na+ or Ca2+. or the presence of dinitrophenol, whilst causing little change in ACh uptake, induces considerable changes in swelling and ACh retention; the greater the amount of swelling the smaller is that of retention. It seems that the latter is segregated in compartments characterized by a low permeability to exogenous ACh. About half of it is independent of changes in incubation conditions whilst the other half enters the compartment by an Na+, Ca2+ and energy-dependent process. At least part of the retention is neuronal as it is diminished by protovera-trine, the diminution being blocked by tetrodotoxin. Mobile ACh (i.e. total uptake minus retained ACh) is largely unaffected by protoveratrine, ouabain, etc. It seems that the retained ACh is directly proportional to the amount of mobile ACh minus the amount that enters with swelling. If the latter is largely glial in location, then the retained ACh is simply proportional to the mobile neuronal ACh. Suggestions are made as to the location of the retained ACh in the brain cells and to the processes involved in its segregation there. Release of retained ACh occurs on change of the Na+ gradient. Atropine and d-tubocurarine also diminish the amount of retained ACh but the percentage diminution falls with increase of the concentration of exogenous ACh.

Notes: The effects of ammonium ions on the frequency of spontaneous action potentials in guinea-pig cerebellar slices, recorded with an extracellular microelectrode, and on the contents of sodium, potassium and chloride ions in incubated guinea-pig cerebellar, and rat brain cortex, slices have been investigated. The frequencies of the spontaneous action potentials are partially suppressed by concentrations of NH4Cl less than 2 mm and completely abolished by concentrations exceeding 2 mm. The amplitudes of the spike discharges are unaffected. A lag period of at least 15 s precedes the inhibition. The suppressing action of NH on the spike frequency is reversible, as shown by complete recovery on removal of NH after short time intervals. Deficiency of Cl− in the superfusion medium causes conversion of inhibition by NH to excitation. Reduction of [K+], or of [Na+], causes increase of inhibition by NH in a normal [Cl1], and reduction of excitation in a low [Cl1], medium. The inhibitory effects of NH on spike frequency are unaffected by picrotoxin or strychnine. NH4Cl, even at 1 or 2 mm, causes a significant increase of aerobic glycolysis. It is suggested that the lag period preceding the suppression of the frequency of spike discharges by NH is partly due to a metabolic change induced by NH, perhaps a transient lowering of pH in the responsible neurons, causing changed permeability to Cl− and possibly to K+ and Na+, NH promotes, in guinea-pig cerebellar slices, an inward flow of Na+ and an outward flow of K+, the latter being greater than that due to exchange of K+ for NH. NH4Cl at 1 or 2 mm causes an outward flow of K+ and an inward flow of Cl− in rat brain cortex slices. The movement of Cl− is biphasic, the first phase, seen with low [NH], consisting of an increase of tissue content of Cl− with little or no fluid uptake and a second phase, seen with high (〉 5 mm) concentrations of NH, in which the uptake of Cl− is directly proportional to the fluid uptake. It is suggested that the first phase is largely neuronal in location whilst the second is largely glial. In infant rat brain cortex slices, there seems to be predominantly an equal exchange of NH for K+. There is little evidence of energy assisted concentrative uptake of NH by brain slices and this is thought to be due largely to the rapid diffusion of undissociated NH3 across cell membranes. It is suggested that some NH (amounting to about 2 mequiv/1) may be bound in the brain. It is concluded that changes in ionic permeabilities, particularly that of Cl−, partly due to a metabolic action, may be responsible for some of the acute cerebral effects of NH administration.

Notes: Abstract— (1) The sum of the values of total (tissue + medium) amino acid-N of glutamate, glutamine, γ-aminobutyrate, and aspartate (referred to as the glutamate system) and of ammonia-N of incubated rat brain cortex slices is approximately constant under a variety of metabolic conditions (presence or absence of glucose or of oxygen or in the presence of metabolic inhibitors such as aminooxyacetate, malonate, methionine sulfoximine, fluoroacetate, ouabain, 2:4 dinitrophenol, or Amytal). Fluctuations in the value of one constituent are compensated by fluctuations in the values of other constituents. The same applies to infant rat brain cortex slices and to rat brain synaptosome preparations. It is suggested that the constancy of the glutamate-ammonia system implies a coupling of neurons and glia in such a manner that glutamate released from the neurons during excitation is taken up by the glia and there converted to glutamine. The glutamine is returned to the neurons where it is hydrolysed to glutamate and ammonia. The glia, on this view, exercise an important buffering effect on the extracellular content of the excitatory amino acid, glutamate, and possibly on that of other functionally active amino acids emanating from the neurons. (2) The magnitude of the glutamate-ammonia system in the infant rat brain cortex is about 43% of that in the adult. It is suggested that, with maturity, the development of the glutamate-ammonia system is linked with the development of the citric acid cycle of operations. (3) The ammonia in the system is tightly linked to the activity of the ATP-controlled glutamine synthetase. (4) Proteolytic ammonia and amino acids are formed, during the incubation, to values that seem to be independent of a wide variety of metabolic conditions. The total value is approximately 10 μmol/g in the first h of incubation. (5) As the ammonium ion is necessary for the return of glutamate to the neuron in the form of glutamine, it is inferred that the ion plays a functional role in the nervous system by helping to maintain the steady state of glutamate in the neuron.

Notes: Abstract: Estimates have been made of the amounts and rates of uptake of radioactive branched-chain i-amino acids, L-phenylalanine, and L-glutamine into incubated rat brain cortex slices. Estimates have also been made of the binding of these amino acids to brain cell fragments. It is shown that such binding, as well as the process of passive diffusion, is not affected by the presence of ouabain (0.2 mM), which suppresses the energy-dependent concentrative uptakes of the amino acids investigated. The maximum specific binding of L-glutamine is about three times that of the other amino acids and amounts to about 11% of the total uptake of the amino acid by rat brain cortex slices in 12 min from a medium containing 0.25 mM-glutamine. The sodium-ion concentration of the medium appears not to play a significant role in determining the rate of L-glutamine uptake in brain slices except at relatively low concentrations (〈20 mequiv./l). The presence of Na+, however, is essential for the attainment of a tissue-to-medium concentration ratio greater than 2.0 for L-glutamine. At relatively low concentrations (0.25 mM) the rapidity of uptake of L-glutamine into a suspension of nerve terminals exceeds that into brain cortex slices. The uptakes of L-glutamine (Km's = 0.66 mM and 2.25 mM) and of the branched chain L-amino acids (Km's approx. 0.3 mM and 2 mM) by rat brain cortex slices are characterized by a double affinity system, but that of L-phenylalanine has only one affinity system (Km= 0.23 mM). The Km's have been calculated after subtracting the ouabain-insensitive passive uptakes of the amino acids from the total observed uptakes.

Notes: Abstract— 1. Whereas exogenous l-glutamate enters rat brain cortex slices incubated in a glucose-physiological saline medium by both low affinity (Km= 0.7 mm) and high affinity (Km= 27−30 μM) processes, the uptake of d-glutamate occurs only by a low affinity (Km= 2mm) system. 2. d-glutamate appears to release l-glutamate from incubated rat brain cortex slices only to a very small extent, whether the tissue l-glutamate is of endogenous or exogenous origin. 3. Competitive inhibition takes place between l- and d-glutamates at the low affinity carrier. This indicates that a common carrier exists for l- and d-glutamates for the low affinity uptake process. 4. Apparently non-competitive inhibition by d-glutamate of l-glutamate uptake occurs at the high affinity carrier, but the affinity of d-glutamate for this carrier is about 0.4% of that of l-glutamate. 5. Both d-, and l-glutamate exchange freely with labelled d-glutamate taken up by preliminary incubation of the brain slices with this amino acid. Whereas l-glutamate exchanges freely with labelled l-glutamate taken up by preliminary incubation, d-glutamate shows little or no exchange. 6. The uptake of labelled d-glutamate by exchange diffusion into brain slices previously loaded with unlabelled d-glutamate proceeds by a low affinity system. Therefore, the process of exchange diffusion does not necessarily involve a high affinity uptake component. 7. Whereas ouabain suppresses both high and low affinity concentrative uptakes of l- and d-glutamate it has little apparent effect on the exchange diffusion process. 8. Sensitivity to tetrodotoxin of evoked release of l- and d-glutamates, taken up by brain slices by preliminary incubation with these amino acids, indicates that the major proportion of the uptake of exogenous l- or d-glutamate proceeds into non-neuronal structures (presumably the glia). 9. At 0°C non-carrier mediated (passive) diffusion of labelled d- and l-glutamate takes place in brain slices.

Notes: Abstract— It is shown, using aminooxyacetate as metabolic inhibitor, that the process of oxidation of endogenous glutamate in incubated rat brain cortex slices follows a different course from that of exogenous l-glutamate. Whereas endogenous glutamate is largely oxidized by an initial reaction with glutamate dehydrogenase with release of ammonia, exogenous l-glutamate undergoes initial transamination to aspartate and α-oxoglutarate before oxidation occurs. In the presence of 2·5 mm l-glutamate, it is found that, of the total exogenous glutamate utilized, 49 per cent is converted to aspartate, 37 per cent is converted to glutamine and the rest is f uily oxidized through glutamate dehydrogenase. It is suggested that endogenous glutamate is normally oxidized in the neurons, and that glutamate released from neurons during excitation, and acting therefore as exogenous glutamate, is taken up by the glia where, besides conversion to glutamine, it largely undergoes initial transamination before oxidation takes place.