KINETICS OF THE SODIUM-DEPENDENT TRANSPORT OF GAMMA-AMINOBUTYRIC ACID BY SYNAPTOSOMES

The kinetics of transport of gamma-aminobutyric acid [2,3-³H] by synaptosomes from rat brain was studied by means of a rapid filtration technique. The rate of uptake was proportional to the protein concentration over the range 0.05—0.2 mg of synaptosomal protein per ml. Although apparent allosteric kinetics were observed with sodium, transport followed simple saturation kinetics with respect to GABA and no heterotropic, cooperative effects of GABA on sodium on kinetics were observed. A minimum of three interacting sodium sites is suggested the basis of Hill plots of the sodium data. Both the apparent K_m and V_max for GABA were functions of the sodium ion concentration but the effect of sodium was considerably greater on V_max than on the apparent K_m The V_max for GABA was 1.1 ± 0.5 nmol.min^?1 mg^?1 of protein at 95 mm sodium and decreased to 12 per Cent of this value at 19 mm sodium. The apparent K^m for GABA increased from 4.0 ± 1.0 μm at 95 mm sodium to 8.4 ± 2.0 μm at 19 mm sodium. Potassium was a noncompetitive inhibitor with respect to GABA and did not affect the apparent cooperativity observed with sodium. These findings are discussed in terms of models of GABA transport.     

A MODEL OF HIGH AFFINITY GLUTAMIC ACID TRANSPORT BY CORTICAL SYNAPTOSOMES FROM THE LONG-EVANS RAT

Abstract— The initial velocity of uptake of L-glutamic acid by cortical synaptosomes from the Long-Evans rat has been measured as a function of sodium and glutamic acid concentration. These data were then fitted to the rate equation from each of several possible models, and the model giving minimum error identified. The major predictions from the best fit model are as follows: (1) The order of combination with the carrier should be sodium, sodium, glutamic acid. (2) Uptake should be 100% dependent on the presence of sodium in the incubation medium. (3) At a finite concentration of sodium, V_mx should be independent of the sodium concentration, which implies that translocation of the carrier across the membrane is independent of the sodium concentration. (4) Lineweaver-Burk plots should be linear with slopes depending on the sodium Concentration. (5) There should be co-transport of two sodium ions with each glutamic acid molecule. (6) The dependence of K _t, on the sodium concentration should have the following form: K _t= A/[Na]²+ 8/[Na] + C , where A, B , and C are constants. The results differ substantially from those previously reported for Sprague-Dawley rats. K _t, is 2–5 times less than that for Sprfague-Dawley rats, and the relation of sodium to K _t, is basically different. We also find a coupling ratio of two, whereas previous studies found a coupling ratio of one. Thus the results raise the possibility that there are fundamental differences between Sprague-Dawley and Long-Evans rats with regard to the mechanism by which sodium participates in amino acid transport.     

PROPERTIES OF THE UPTAKE AND RELEASE OF GLUTAMIC ACID BY SYNAPTOSOMES FROM RAT CEREBRAL CORTEX

Abstract– (1) The uptake and release of glutamic acid by guinea-pig cerebral cortex slices and rat synaptosomal fractions were studied, comparing the naturally occurring l - and non-natural d -isomers. Negligible metabolism of d -glutamic acid was observed in the slices. (2) Whereas in the cerebral slices the accumulation of glutamic acid was almost the same for the two isomers, d -glutamic acid was accumulated into the synaptosomal fraction at a markedly lower rate than was the L-isomer. (3) The uptake systems for d -isomer into the slices and synaptosomal fraction were found to be of single component, in contrast with the two component systems, high and low affinity components, for the uptake of l -glutamic acid. The apparent K_m values for the uptake of d -glutamic acid into the slices and synaptosomal fraction were comparable with those reported for the low affinity components for l -isomer. The uptake systems for d -glutamic acid were dependent on the presence of Na⁺ ions in the medium, like those for l -glutamic acid and GABA. (4) The evoked release of radioactive preloaded d -glutamic acid was observed both from the slices and synaptosomal fraction following stimulation by high K⁺ ions in the medium. From these observations, it is evident that the evoked release of an amino acid by depolarization in vitro is not necessarily accompanied by a high affinity uptake process. (5) The uptake of l -glutamic acid, expecially into the synaptosomal fraction, was highly resistant to ouabain. On the other hand, the uptake rate of d -glutamic acid and GABA into the synaptosomal fraction was inhibited by varying concentrations of ouabain in accordance with the inhibition for brain Na-K ATPase. (6) The uptake of l -glutamic acid into subfractions of the P₂ fraction was studied in relation to the distribution of the ‘synaptosomal marker enzymes’. An attempt to correlate the activities of enzymes of glutamic acid metabolism with the uptake of l -glutamic acid into the synaptosomal fraction from various parts of brain was unsuccessful. The high affinity uptake of l -glutamic acid was found to be very active in the synaptosomal fraction from any part of brain examined.     

THE EFFECT OF DELTA-AMINOLAEVULINIC ACID ON THE UPTAKE AND EFFLUX OF AMINO ACID NEUROTRANSMITTERS IN RAT BRAIN SYNAPTOSOMES

Abstract— δ-Aminolaevulinic acid (δ-ALA) is an omega amino acid structurally similar to γ-aminobutyric acid (GABA) and l -glutamate. We have examined the effect of δ-ALA on the uptake and efflux of radiolabelled GABA and l -glutamate in rat cortical synaptosomes and report: (1) low concentrations of δ-ALA reduced the potassium-stimulated release of [³H]GABA from the synaptosome preparation. This effect was reversed by the GABA receptor antagonist bicuculline. We postulate that GABA release is modulated by a feedback mechanism on presynaptic GABA receptors, and that δ-ALA has agonist activity at these receptors. (2) δ-ALA at high concentrations (0.75-5.0 m m ) stimulated the efflux of l -[¹⁴C]glutamate from preloaded synaptosomes. (3) δ-ALA had no effect on potassium-stimulated release of l -glutamate. (4) Uptake of labelled l -glutamate was inhibited by δ-ALA in a noncompetitive fashion. (5) Synaptosomes did not accumulate [¹⁴C]δ-ALA in the range 0.5-50 δ m . These results are discussed in relation to the control of GABA release from nerve endings, and the role of δ-ALA in the neuropsychiatric manifestations of the acute porphyric attack.     

D-GALACTOSE TRANSPORT BY SYNAPTOSOMES ISOLATED FROM RAT BRAIN

Abstract— Synaptosomes prepared by differential and Ficoll density gradient centrifugation took up d -galactose by two saturable transport systems: one. a high affinity system with a K _m of 0-25 mn and V_max of 075 nmol/mg protein 3 min, the other, a low affinity system with a K_m of 47 mM and a V_max of 135 nmol/mg protein/3 min. The high affinity system was inhibited by 1-5 mM phlorizin but was unaffected by the absence of sodium ion or the presence of 1 mM ouabain. The low affinity system was unaffected by phlorizin or ouabain. Both systems were inhibited by high concentrations of glucose. 2-deoxyga-lactose. and inositol, and by 2.4-dinitrophcnol. Galactose was rapidly converted in synaptosomes to phos-phorylatcd intermediates and was more slowly oxidized to ¹⁴CO₂     

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 .     

GABA IN THE OLFACTORY BULB AND OLFACTORY NUCLEUS OF THE RAT: THE DISTRIBUTION OF GAMMA-AMINOBUTYRIC ACID, GLUTAMIC ACID DECARBOXYLASE, GABA TRANSAMINASE AND SUCCINATE SEMIALDEHYDE DEHYDROGENASE

Abstract— The distribution of GABA and enzymes involved in its metabolism was investigated in the different regions of the olfactory bulb and olfactory nucleus. The highest levels of GABA in the olfactory bulb were found in the layers rich in nerve terminals (31 μmol/g dry wt.). A similar distribution was found in the olfactory nucleus although the overall level of GABA was only a quarter of that measured in the bulb. Glutamic acid decarboxylase (GAD) levels in the various layers of the olfactory nucleus were similar in distribution to those of GABA. However, the correlation between GAD and GABA did not hold for the olfactory bulb, particularly in the granule cell layer and the medulla. The activities of GAD and the levels of GABA are significantly higher in the bulb than in the nucleus but succinic acid scmialdehyde dehydrogenase and GABA aminotransaminase activities are almost identical in both regions.     

REGULATION OF ACETYLCHOLINE SYNTHESIS: CONTROL OF CHOLINE TRANSPORT AND ACETYLATION IN SYNAPTOSOMES

Abstract— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [²H₄]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr-uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [²H₄]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.     

ON THE UPTAKE OF INOSITOL BY RAT BRAIN SYNAPTOSOMES

The uptake of inositol by rat brain synaptosomes occurs via an unsaturable process that even at substrate concentrations as low as 1 μM is unable to achieve a concentration gradient indicative of active transport. Dinitrophenol, ouabain and cytochalasin B did not affect uptake of the cyclitol. The data indicate that inositol uptake by rat synaptosomes occurs by diffusion or by a system with an affinity so low it is difficult to discern. The low capacity, saturable inositol uptake system observed in rabbit brain slices may reflect a species difference or uptake by elements of the slice other than neuronal membranes.     

THE KINETIC PROPERTIES OF HEXOSE TRANSPORT INTO SYNAPTOSOMES FROM GUINEA PIG CEREBRAL CORTEX

Abstract— Hexose uptake into synaptosomes has been shown to occur by a saturable mechanism with a relatively small component due to passive diffusion. Competition between glucose and deoxyglucose for entry was demonstrated and the kinetic properties of the process were studied by using glucose as a competitive inhibitor of deoxyglucose entry. During kinetic analysis of the transport process higher affinities for both hexoses were indicated by the results from the synaptosome preparation compared with those from cerebral cortex slices. The kinetic properties of glucose inhibition of deoxyglucose uptake into synaptosomes could not be interpreted completely in terms of a unimolecular transport model. The results appear to follow predictions for 'doubly competitive inhibition', and some evidence for polyvalency of the uptake process was obtained.     

EFFECT OF ELECTRICAL STIMULATION AND CHLORPROMAZINE ON THE UPTAKE AND RELEASE OF TAURINE, γ-AMINOBUTYRIC ACID AND GLUTAMIC ACID IN MOUSE BRAIN SYNAPTOSOMES

—The concentrations of taurine and GABA were determined in isolated mouse brain synaptosomes incubated in Krebs-Ringer phosphate medium (pH 7·4). The concentration of GABA gradually decreased during incubation, but that of taurine remained approximately unchanged. In the presence of chlorpromazine the amount of GABA in the synaptosomes increased, but the efflux and influx of GABA were slightly reduced. The content and efflux of both taurine and GABA increased in electrically stimulated synaptosomes, and the influx of taurine, GABA and glutamate into the synaptosomes similarly increased. All three amino acids are taken up by the synaptosomes through at least two mechanisms: low-affinity and high-affinity. In the high-affinity system the K_m values were 33 μm for taurine, 24 μm for GABA and 68 μm for glutamate, and in the low-affinity one 1·1 mil, 0·9 mm and 1·2mm , respectively. The influx capacity (V_max) was highest for glutamate, second highest for GABA and lowest for taurine.     

THE EFFECTS OF ELECTROSHOCK SEIZURES ON POTASSIUM TRANSPORT WITHIN SYNAPTOSOMES FROM RAT BRAIN

—Potassium transport was assessed within synaptic terminals isolated from cerebral cortices of rats which experienced one or two maximal electroshock (ES) convulsions daily. No significant change in endogenous K content was present after 2 consecutive days of ES-seizures. However, K decreased 22 % and 41 % after 4 and 6 days of convulsions respectively. After 6 days, synaptosomes from ES rats were able to accumulate K to the same extent as controls and were inhibited by ouabain to an equivalent extent (50%). When these synaptosomes from ES rats were preloaded with high concentrations of K, K leaked out at an increased rate. When diphenylhydantoin (DPH) was administered intraperitoneally from the second to the sixth day of ES-convulsions, the decrease in endogenous K induced by chronic convulsions was corrected. In ion-free media or with 50 mM Na, DPH had no effects on the enhanced efflux of K observed in vitro after ES-seizures. However, with high Na (50–100 mm ) and low K (0.2–1 mm ) DPH stimulated K uptake but did not affect the ouabain inhibition. Under conditions optimal for K uptake (50 mm Na and 10 mm K), DPH did not affect K accumulation but it prevented in vitro ouabain inhibition. Our results indicate that repeated ES-convulsions decreased K content within isolated nerve terminals by enhancing its passive leakage. In vivo DPH prevented the effects of ES-seizures by stimulating the Na-K pump and not by directly blocking the K leak.     

DEVELOPMENTAL AND OTHER ASPECTS OF [35S]CYSTEINE TRANSPORT BY RAT BRAIN SYNAPTOSOMES

Abstract— Cysteine uptake by rat brain synaptosomes occurs by active transport. The uptake by synaptosomes isolated from newborn brain is slower and the concentration gradient achieved is lower than that observed in adult tissue. Synaptosomal fractions from both adult and newborn rat brains accumulate cysteine by two saturable systems. The calculated parameters show that the maximum rates of cysteine uptake in adult synaptosomes are approximately twice that observed in newborn synaptosomes for both the high and low affinity systems. The uptake by the high affinity system is sodium dependent and is inhibited by glycine and dibasic amino acids. Uptake by synaptosomes from 14-day-old animals is close to that observed in adult tissue. The uptake of cysteine differs greatly from that of cystine since the oxidized form, cystine, is taken up more slowly by systems with low affinities which are sodium independent, do not interact with dibasic amino acids and are independent of age.     

THE SULPHATION OF VANILYLMANDELIC ACID AND HOMOVANILLIC ACID BY LIVER, SMALL INTESTINE AND BRAIN

Abstract— The sulpho-conjugates of vanilylmandelic acid (VMA) and homovanillic acid (HVA) were biosynthesized in vitro from ATP and sodium sulphate and from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) obtained commercially or generated in vitro . Hydrolysis with sulphatase indicated the sulphate nature of these conjugates. In decreasing order of activity, the liver, small intestine and brain of various animals exhibited this sulpho-conjugatory ability. The K _m, values of VMA and HVA for the sulphotransferase of mouse liver were, respectively, 1740 and 93 μM. Competition studies suggested that the same enzyme may be involved in the sulphation of these two acids.     

DEVELOPMENTAL CHANGES IN THE KINETICS OF γ-AMINOBUTYRIC ACID TRANSPORT BY CHICK RETINA

—Isolated retinas from chick embryos and mature animals were incubated in [³H]GABA at 25°C for 10 min in order to investigate kinetic properties of the amino acid uptake system. Embryo retina accumulated [³H]GABA by two distinct kinetic systems with K_m values of the order 10⁻⁴m and 10⁻⁵m for the low- and high-affinity mechanisms respectively. However, as the retina matured, the high-affinity process disappeared and only the low-affinity system was detectable. No obvious explanation can be offered for this phenomenon although a similar observation has previously been made in chick brain by other workers.