Juda Hirsch Quastel

EFFECTS OF AMMONIUM IONS ON SPONTANEOUS ACTION POTENTIALS AND ON CONTENTS OF SODIUM, POTASSIUM, AMMONIUM AND CHLORIDE IONS IN BRAIN IN VITRO

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.

Spontaneous action potentials in isolated guinea-pig cerebellar slices: effects of amino acids and conditions affecting sodium and water uptake.

The effects of incubation conditions on the frequency of spontaneous action potentials exhibited by guinea-pig cerebellar slices, and recorded with an extracellular microelectrode, have been investigated. Various incubation conditions that lead to tetrodotoxin-sensitive uptakes of water and of sodium ions by the incubated cerebellar slices lead to enhanced frequencies of the spontaneous action potentials, e. g. the presence of protoveratrine or of ouabain, the absence of glucose or the onset of anoxia. The frequency of the spikes is also enhanced by acetylcholine (in presence of neostigmine) or by the presence of excitatory amino acids, such as L-glutamate, D-glutamate or L-aspartate. It is suppressed by tetrodotoxin, or by the inhibitory amino acids, e. g. γ-aminobutyrate, glycine or taurine, or by ammonium ions or by pentobarbital. It is concluded that guinea-pig cerebellar slices, incubated under specified conditions, may provide a suitable means for quantitative correlation of neurochemical data with data obtained by electrophysiological techniques in tissue incubated under similar conditions and also for quantitative assessment of the effects of amino acids on cerebellar electrical activity.

Effects of drugs on the uptake of acetylcholine in rat brain cortex slices

Abstract The following drugs inhibit competitively uptake of acetylcholine against a concentration gradient into rat brain cortex slices incubated aerobically at 37° in a physiological saline-glucose medium containing 20 μM paraoxon: eserine (Ki = 0.9 × 10−5M), tetramethylammonium chloride (Ki = 0.4 × 10−5M), tetraethylammonium chloride (Ki = 0.5 × 10−5M), atropine (Ki = 1.8 × 10−5M), cocaine (Ki = 0.33 × 10−5M), procaine (Ki = 0.23 × 10−5M), lidocaine (Ki = 0.09 × 10−5M), chlorprocaine (Ki = 0.5 × 10−5M), methacholine (Ki = 0.4 × 10−5M), succinylcholine (Ki = 0.6 × 10−5M), d-tubocurarine (Ki = 0.6 × 10−5M), hexamethonium (Ki = 1.2 × 10−5M), pilocarpine (Ki = 0.5 × 10−5M), nicotine (Ki = 0.6 × 10−5M), strychnine (Ki = 0.6 × 10−5M), and hemicholinium (Ki = 0.6 × 10−5M). The following drugs inhibit noncompetitively: chlorpromazine and amphetamine. A comparison of the inhibitor constants of a number of these drugs, which act competitively on acetylcholine transport, with the inhibitor constants towards acetylcholine esterase or certain neurophysiological acetylcholine receptor sites leads to the conclusion that the transport carrier site for acetylcholine is not identical in chemical structure with the anionie site of acetylcholine esterase or with the acetylcholine receptor sites. The noncompetitive effects of certain drugs suggest the presence of a site on the transport carrier, with an affinity for these drugs, which affects the reactions of acetylcholine at the brain cell membrane.

Uptake of acetylcholine in rat brain cortex slices

Abstract Acetylcholine is taken up against a concentration gradient by rat brain cortex slices, incubated aerobically in a physiological saline medium, in the presence of paraoxon. Optimal rates of acetylcholine uptake are obtained with slices 0.2–0.4 mm thick, in the presence of glucose (> 3 mM), in oxygen at 37° and in the presence of paraoxon (> 5 μ M) which completely inactivates the choline esterase. Little or no uptake apart from that due to passive diffusion occurs in the presence of eserine. It is evident that the uptake is carrier-mediated. The concentration ratio (tissue:medium) of acetylcholine varies from approximately unity with relatively high external concentrations of acetylcholine (e.g. 5 mM) to 21 with low concentrations (0.01 μM) of acetylcholine. The optimal pH for acetylcholine uptake is 8.3 and the amount of uptake in 1 hr, corrected for passive diffusion, is doubled on increasing the temperature of incubation from 17° to 27°. The rate of acetylcholine uptake, corrected for the passive diffusion rate, is approximately proportional to the respiratory rate (in the presence of oxygen or air or in the absence of glucose). It is inhibited by 2:4 dinitrophenol (10 μM) and by ouabain (10 μM), their inhibitions being independent of the acetylcholine concentration. The rate of uptake declines (from its optimal value) in the absence of potassium and calcium ions, or in the presence of relatively high concentrations of potassium chloride (> 50 mM) or calcium chloride (8 mM) or magnesium sulfate (6 mM). It is also diminished, but not abolished, by the omission of sodium ions from the medium. It is concluded that the rate of uptake of acetylcholine (apart from passive diffusion) in the brain slices is partly dependent on the operation of the sodium pump. The possible involvement of exchange diffusion in the process of uptake is discussed.

Kinetics of Cerebral Uptake Processes In Vitro of L-Glutamine, Branched-Chain L-Amino Acids, and L-Phenylalanine: Effects of Ouabain

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 (Kms = 0.66 mM and 2.25 mM) and of the branched chain L‐amino acids (Kms 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 Kms have been calculated after subtracting the ouabain‐insensitive passive uptakes of the amino acids from the total observed uptakes.

Ribonucleic acid biosynthesis in adult and infant rat brain in vitro.

The rate of biosynthesis of ribonucleic acid (as judged by the rate of incorporation of uridine into ribonucleic acid) in infant and adullt rat brain cortex slices, incubated (aerobically in tile presenice of various substrates, is directly proportionial to the adenosine triphosphate concentration. This suggests that the adenosine triphosphate concentration is one of the factors involved in the control of ribonucleic acid biosynthesis in infant and adult rat brain. Acetoacetate or β-hydroxybutyrate is about 70 percent as effective as glucose, with both infant and adult brain, for the promotion of ribonucleic acid biosynthesis. but they are considerably mnore effective than succinate in infant brain than in adult brain.

Effects of neurotropic drugs on sodium influx into rat brain cortex in vitro

Abstract Two methods have been adopted for studying the effects of neurotropic drugs on sodium ion influx into rat brain cortex slices incubated aerobically in a physiological saline-glucose medium: (a) a direct method in which the influx of 22Na+ from the incubation medium is measured: (b) an indirect method in which the rate of [1-14C] acetate oxidation to 14CO2 is measured. The latter process is inhibited by sodium ions and stimulated by potassium ions. The results of both methods show that the local anesthetics (cocaine 0·2 mM lidocaine 0·5 mM and 1 mM and procaine 1 and 3 mM) block the increased influx of Na+ due to electrical stimulation without affecting the content of 22Na+ found in the brain tissue in the unstimulated state. Tetrodotoxin (1 μm), chlorpromazine (0·1 mM) and atropine (5 mM) behave similarly. Chlorpromazine at a higher concentration (0·5 mM) causes increased influx of 22Na+ into the brain slices in the unstimulated state and this is correlated with its marked inhibition, at this concentration, of the activity of membrane Na+-K+-ATPase (as occurs also with ouabain 10 μm). The local anesthetics at the concentrations quoted do not affect membrane Na+-K+-ATPase. The barbiturates investigated (amytal 0·25 and 0·5 mM and pentothal 0·1 and 0·2 mM) do not abolish the influx of Na+ following electrical stimulation and there is no evidence from the results of these experiments that the barbiturates, at the concentrations quoted, affect the movements of sodium or potassium ions across the brain cell membrane. In contrast, also, with the effects of local anesthetics, the barbiturates inhibit the oxidation of [1-14C]acetate to 14CO2 without markedly influencing the effects of potassium ions or of electrical stimulation on this process. The effects of barbiturates may be explained by their inhibitory action on brain cell energetics.

Effects of Branched-Chain L-Amino Acids, L-Phenylalanine, and L-Methionine on the Transport of L-Glutamine in Rat Brain Cortex In Vitro. Influence of Cations

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.

Effects of anesthetics on sodium uptake into rat brain cortex in vitro

Abstract Local anesthetics (cocaine, lidocaine) suppress the uptake of Na + into rat brain cortex slices incubated aerobically for 1 hr in the presence of protoveratrine or of ouabain, or in the absence of glucose, but not in the presence of L - or D -glutamate. Tetrodotoxin has similar effects. The barbiturates (amytal, pentothal), used at anesthetic concentrations that completely block potassium, or electrically stimulated brain respiration, have no such suppressing effect on Na + uptake. However, in calcium-free media and under conditions where brain respiration is being stimulated by the presence of protoveratrine or 30 mM KC1 or 0.03 mM 2,4-dinitrophenol, the barbiturates exert significant depressions on both respiration and the uptake of Na + . It is inferred that the effects of local anesthetics and of tetrodotoxin on the uptake of Na + into brain slices are due to their block of the generation of action potentials, whereas those of the barbiturates are due to their suppression of mitochondrial metabolism, causing release of mitochondrial Ca 2+ and thereby resultant changes in the permeability of the cell membrane to Na + and K + .

UPTAKE AND RELEASE OF EXOGENOUS [1-14C]ACETYLCHOLINE BY BRAIN CORTEX SLICES FROM THE RAT

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.