Effect of acute cyanide intoxication on the active transport of amino acids in brain slices

(1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices. (2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used. (3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals. (4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals. (5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices. (6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.     

EFFECTS OF POTASSIUM ON INDICATOR SPACES AND FLUXES IN SLICES OF BRAIN CORTEX FROM ADULT AND NEW-BORN RATS

—Inulin, sucrose and chloride spaces were measured in slices of brain cortex from adult and from new-born rats incubated in‘balanced', potassium-rich and sodium-rich media. The efflux of the radioactive markers was followed in the two first media and the following results were obtained: (1) In brain slices from new-born rats inulin and sucrose spaces were of identical magnitude (35 per cent). The space magnitude was essentially unaffected by excess potassium. The chloride space was somewhat larger than the inulin (sucrose) space, and the difference increased continuously but relatively slightly with the external potassium concentration. By far the largest amount (i.e. about 90 per cent) of the efflux of radioactive inulin, sucrose and chloride occurred from a rapidly exchanging compartment during incubation in both ‘balanced’ and potassium-rich media. (2) In brain slices from adult rats the inulin space (35 per cent) was significantly smaller than that of sucrose (50 per cent) and of chloride (65 per cent); it seemed to represent the extracellular space relatively well although 10 per cent of the efflux occurred from a slowly exchanging (probably intracellular) compartment. High concentrations of potassium led to a reduction of the inulin space which was probably a result of the concomitant intracellular swelling. The hyperosmolarity per se did not affect the space magnitude, but an increase of the sodium concentration exerted a competitive inhibition of potassium effects on the inulin space. Of the sucrose efflux, 20 per cent occurred from a slowly exchanging compartment in both ‘balanced’ and potassium-rich media, and 30 per cent of the chloride exchanged with this compartment when the tissue was incubated in a ‘balanced’ medium. An increase of the external potassium concentration caused a drastic increase of the chloride space and a reduction of the slowly exhanging fraction of chloride efflux to less than 10 per cent.     

THE EFFECT OF NEONATAL THYROIDECTOMY ON THE DEVELOPMENT OF THE ADENOSINE 3'',5''-MONOPHOSPHATE SYSTEM IN THE RAT BRAIN

Abstract— The effect of neonatal thyroidectomy on the cyclic AMP system in the developing rat brain was examined. Administration of ¹³¹I at birth led to a 16 per cent reduction in brain weight and a 70 per cent reduction in body weight by 40 days of age. The level of cyclic AMP in the brain increased 5-fold between birth and 40 days of age and this increase was partially reduced by early thyroidectomy. A similar increase in the activity of adenyl cyclase and phosphodiesterase was observed during development, but thyroidectomy produced no detectable changes in the activity of either enzyme. The activity of the cyclic AMP-dependent protein kinase was already maximal at birth and also was unaffected by thyroidectomy.
Norepinephrine increased levels of cyclic AMP 4- to 5-fold in brain slices prepared from adult rats, but was without effect on slices prepared from newborn or 3-day-old rats. The response to norepinephrine in thyroidectomized rats did not differ from that in control rats at any of the ages examined. Our findings indicate that neonatal hypothyroidism does not deleteriously affect the development of the cyclic AMP system in the rat brain.     

The action of gamma-hydroxybutyric acid on cerebral glucose metabolism

Abstract— Experiments have been performed to study the effect of gamma-hydroxybutyric acid (GHB) on glucose metabolism in vivo and in vitro.

• 1 Administration of GHB (500 mg/kg) is followed by sleep in rats and mice.
• 2 GHB is shown to increase the 1-¹⁴C/6-¹⁴C ratio in expired CO² in mice in vivo by 300 per cent.
• 3 The same effect is obtained with slices of cerebral cortical grey matter from GHB-treated rats, where the 1-¹⁴C/6-¹⁴C ratio is increased from 1.72 to 3.63, but not with homogenates of cerebral cortex, nor with slices of kidney or diaphragm.
• 4 GHB (500 mg/kg, intrapentoneally) specifically increases the activity of glucosed-6-phosphate dehydrogenase in vivo in rat and mouse whole brain by 27 per cent. The time course of this effect correlates with the sleeping time in both species.
• 5 The activity of glucose-6-phosphate dehydrogenase is not altered in vitro by high concentrations of GHB.
• 6 GHB stimulates O₂ uptake by slices of cerebral cortical grey matter by 24 per cent, but it is not itself able to support respiration by the tissue.
• 7 It is proposed that GHB specifically increases the activity of the pentose phosphate pathway in brain, and that this effect is mediated by an increase in glucose-6-phosphate dehydrogenase activity.

     

Chromatin phospholipid changes during rat liver development

The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0.47-0.59 per cent of total chromatin in newborn animals and 0.22 per cent in 45-day-old animals. No such age-related differences are observed in the nuclei. The phospholipid composition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine.     

KINETIC ANALYSIS OF PHENYLALANINE-INDUCED INHIBITION IN THE SATURABLE INFLUX OF TYROSINE, TRYPTOPHAN, LEUCINE AND HISTIDINE INTO BRAIN CORTEX SLICES FROM ADULT AND 7-DAY-OLD RATS

—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7-day-old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7-day-old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7-day-old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7-day-old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

RATES OF CEREBRAL GLUCOSE UTILIZATION IN RATS ANAESTHETIZED WITH PHENOBARBITONE

Abstract—

• 1 Intraperitoneal injection of phenobarbitone (250 mg/kg body wt.) into rats caused increased brain concentrations of glucose (100 per cent), glucose 6-phosphate (16 per cent) and ATP (12 per cent) and decreased concentrations of lactate (33 per cent) and ADP (15 per cent). A 31 per cent decrease in glutamate content was not statistically significant. No significant change occurred in the cerebral contents of glycogen or creatine phosphate.
• 1 The rates of increase in the brain of specific activities, in the first few minutes after systemic injection of [U-¹⁴C]glucose, of glucose, lactate, glutamate and glycogen were all halved by phenobarbitone. Calculated flux rates of ¹⁴C from glucose into metabolic intermediates and from lactate to glutamate were also decreased by 27–47 per cent; the effects on rate constants showed inconsistencies. The rate constants for conversion of glucose to lactate and to glutamate were decreased by 60–70 per cent, but that from lactate to glutamate was virtually unchanged. The rate constant for the flux from glucose to glycogen was reduced by 39 per cent, but the accumulation of glucose meant that the actual flux into glycogen increased by 20 per cent.
• 1 The results are interpreted in terms of an effect of the barbiturate not only on glucose transport, but also at an enzymic stage in glycolysis, possibly hexokinase or phosphofructokinase.

     

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.

     

VERÄNDERUNGEN DER AMINOSÄUREKONZENTRATIONEN IM RATTENHIRN BEI VERRINGERUNG DER K+-ZUFUHR IN VIVO

Abstract—

• 1 Resonium A, a cation exchange resin, administered orally caused no decrease of the potassium content in the CNS of the rat, but it provoked a potassium depletion in the liver tissue. However a slight increase could be detected in the ‘cortex’ and ‘striatum’.
• 2 A rise of the concentration of the free amino acids was found in ‘cortex’, ‘striatum’, ‘thalamus’ and cerebellum. Glutamic acid showed an increase of 70–80 per cent. GABA and glycine showed a remarkable increase of 280–330 per cent.
• 3 Restitution of K⁺ by feeding a potassium-rich diet brought the amino acid concentrations in the ‘cortex’ and cerebellum within a normal range. In ‘striatum’ and ‘thalamus’ an overshoot could be observed.
• 4 The experimental procedure for the estimation of free amino acids in brain tissue is discussed.

     

Effect of deafferentation of the forelimb on the postnatal development of the synaptic plasticity in the hippocampus

The postnatal development of LTP in CA1 area of hippocampus was studied in hippocampal slices from 13-20-day-old intact rats, after unilateral resection of n. medianus on the 13th day, and sham-operated animals. In slices from the intact rats prepared on the 15th-16th-day of postnatal development, the LTP magnitude and duration were significantly larger than in adult animals. Partial deafferentation eliminated this overshoot. However, a less pronounced increase in synaptic plasticity was observed in operated animals on the 17th day. The LTP suppression in the experimental animals may be explained by a decrease in the NMDA receptor activity due to enhanced synaptic activity in the hippocampus. We think that the limited sensory inflow from the partially deafferented forelimb to the hippocampus via the entorhinal cortex may be compensated by activation of other inputs from specific or/and nonspecific pathways. In contrast, the LTP magnitude and duration were significantly increased in slices from the sham-operated rats. This increase may be explained by a decline of synaptic activation of the hippocampus under anesthesia.     

Fluid compartmentation and electrolytes of cat cerebral cotex in vitro. 3. Ontogenetic and comparative aspects

Studies on swelling and fluid compartmentation have been carried out in vitro on incubated slices of cerebral cortex from kittens 1.5-120 days post-natal age and on incubated sections of corpus callosum and slices of liver and kidney cortex from adult cats. The findings have been compared with analogous data for incubated slices of adult cat cerebral cortex, studied under identical conditions (Bourke and Tower , 1966a, b), in order to identify the probable morphological correlates of fluid and electrolyte distribution. Incubated cortical slices from neonatal (1.5-4-day-old) kittens exhibit none of the relevant characteristics of slices from adult cerebral cortex. By 1 month post-natal age, K⁺-dependent swelling of slices becomes demonstrable, and the K⁺ and Na⁺ contents of slices approximate adult levels. Both these developments coincide with the morphological and physiological maturation of cortical neurons. At 3 months post-natal age, slice swelling accessible to C1^? but not to sucrose becomes observable and the dependence of sucrose space size on time, during incubation, of solute addition becomes demonstrable. Both these developments follow completion of axonal myelination in the cortex but coincide with the period of cortical glial cell proliferation. Incubated sections of corpus callosum from adult cats exhibit none of the relevant characteristics observed for cortical slices under identical conditions. Tissue swelling is minimal and uninfluenced by K⁺ concentrations of incubation media. Tissue fluid spaces accessible to sucrose are approximately twice the size of spaces accessible to inulin. In general, qualitatively similar results have been obtained for incubated slices of cat liver or kidney cortex or for incubated sections of rat diaphragm under the same conditions. A behaviour for glial cells (? astrocytes) in cerebral cortex under such in vitro conditions distinctly different from behaviour of subcortical glial cells is suggested.     

THE MECHANISM OF THE EXCLUSION OF SODIUM IONS AND THE CONCENTRATION OF POTASSIUM IONS BY GUINEA-PIG CEREBRAL CORTEX SLICES

—Guinea pig cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline for periods of 1 s to 60 min, and their swelling and Na⁺ and K⁺ cone were measured. The swelling was at the rate of 8 per cent for the 1st min, and 0·8 per cent for the next 29 min; it fell significantly during the subsequent 30 min (P= 0·05). The Na⁺ and K⁺ concn in the tissue fluctuated during the 1st min of incubation, but the Na⁺ concn had risen to a mean of 108 mm after 1 min incubation and the K⁺ concn had fallen to a mean of 52 mm by 3 min. The concentrations of these cations did not change significantly after these times. Cerebral slices were also incubated for 30 min in isotonic media modified such that Na⁺, + K⁺, Na⁺+ choline⁺, or K⁺+ choline⁺ always added up to 150 mm . It was found that about half of the swelling (20-25 per cent) was independent of the Na⁺ or K⁺ concn and a further 20-25 per cent of the swelling varied with the cations only if Na⁺ and K⁺ were both present and was a function of the K⁺ concn in the medium (0·15 per cent m-mol). The Na⁺ concn in the tissue was a mean 8·4 mm after incubation in a Na⁺-free medium and 7·1 mm in K⁺ after incubation in a K⁺-free medium. Cerebral slices in the presence of Na⁺+ K⁺ excluded one molecule of Na⁺ for every four molecules in the incubating medium; they accumulated K⁺ from the medium until the concn in the medium exceeded 130 mm .     

THE EFFECT OF AGE, GROWTH RETARDATION AND ASPHYXIA ON ASCORBIC ACID CONCENTRATIONS IN DEVELOPING BRAIN

Abstract— The concentration of ascorbic acid in whole rat brain during the first week of postnatal life was up to 100 per cent higher than in adult animals. A progressive fall in concentration occurred between 4 and 30 days of age. Corresponding changes did not occur in liver and adrenal gland, two other organs rich in ascorbic acid. Rats subjected to growth retardation during the fetal and suckling periods had, at 25 days of age, levels of ascorbic acid in the cerebellum and brainstem significantly higher than those of control animals. A period of prolonged asphyxia in 5-day-old rats resulted in a significant 4 per cent reduction in whole brain ascorbic acid concentration.     

A COMPARATIVE STUDY OF BRAIN AND LIVER MITOCHONDRIA FROM NEW-BORN AND ADULT RATS

• 1 A method has been developed for the estimation of organelle number in subcellular fractions and applied to the estimation of the mitochondrial content of brain and liver from new-born and adult rats.
• 2 The respiratory enzyme content per mitochondrion of the adult brain was 3·5 times greater than that of the neonate. This increase in enzyme content was not correlated with an increase in the mean size of the organelle.
• 3 The succinate dehydrogenase activity per mitochondrion of the livers from neonatal and adult rats showed no obvious change. There was, however, an increase in the mean size of the organelle.
• 4 These findings are discussed with reference to the development of the brain and to the development of mitochondria.

     

Regulation of liver corticoid metabolism during early postnatal life: effects of surgical stress

Corticosterone metabolism by liver slices was investigated in suckling (10-day-old), weanling (21-day-old) and adult male rats. During the suckling period adrenalectomy as well as sham adrenalectomy increase the rate of steroid A ring reduction and also the rate of steroid side chain degradation by 20-40%. In older animals such changes were not detected. The results support an earlier assumption that liver steroid metabolism is regulated in an age specific manner.     

EXCHANGE OF TAURINE IN BRAIN SLICES OF ADULT and 7-DAY-OLD RATS

Abstract— The influx or efflux of taurine in brain slices prepared from adult and 7-day-old rats was studied in Krebs-Ringer bicarbonate-glucose medium with 0,2 and 10 m m -taurine. The exchange of taurine between the slices and the medium was slow, and no steady-state concentration was reached within the experimental period of 150 min. In both experimental groups there was a net influx of taurine into the slices from 10 m m -taurine and a slight net efflux from the slices into 2 m m -taurine. The rate of influx from 10 m m -taurine was about the same in the two groups after an initial period of faster influx into the slices of adult rats. There was some rapid initial efflux into 0 and 2 m m -taurine solutions from the slices from 7-day-old rats, but with prolonged incubation these slices were better able to maintain their intracellular taurine than the slices from adult rats. The reasons and significance of the high cerebral concentration of taurine in immature brain in vivo are briefly discussed in the light of the present and earlier studies.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Sex Steroids Inhibit Osmotic Swelling of Retinal Glial Cells

Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postischemic and diabetic retinas. Progesterone, testosterone, estriol, and 17ß-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 μM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y₁, and adenosine A₁ receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.     

Hydrolase activities in brain of neurological mutants: cerebroside galactosidase, nitrophenyl galactoside hydrolase, nitrophenyl glucoside hydrolase and sulphatase

Abstract—

• 1 The brains of 17-day-old quaking and jimpy mice were compared with those of the corresponding normal phenotypes. The concentrations of cerebroside and sulphatide were found to be markedly lower in the affected mutants, while the relative amounts of ceramide and free fatty acid appeared normal.
• 2 The concentration of cerebroside glactosidase was not significantly abnormal in the jimpy mice but was about 17 per cent lower in quaking mice. In contrast, the relative amount of the enzyme that could be dispersed by sonication was considerably higher in the jimpy animals. It is suggested that this increase is a causative factor in the aetiology of the latter disease.
• 3 The concentrations of other acid hydrolases were determined, as well as the relative amounts dispersible by sonication. No difference was seen between the phenotypes with NPGalH, NPGluH, and nitrocatechol sulphate hydrolase.
• 4 An improved solvent system for the TLC detection of ceramide in brain lipids is described.