Acid In Slices Research Articles

Activation of D1 receptors stimulates accumulation of gamma-aminobutyric acid in slices of the pars reticulata of 6-hydroxydopamine-lesioned rats.

D1 dopamine receptors are present on terminals of striatal neurons to the pars reticulata of the substantia nigra in the rat. Here we have studied the effect of the activation of these receptors on the synthesis of gamma-aminobutyric acid (GABA) in slices of the pars reticulata of the substantia nigra isolated from 6-hydroxydopamine-lesioned rats. The synthesis was judged by the accumulation of GABA after inhibiting GABA transaminase with aminooxyacetic acid. Both dopamine and SCH 23390, a D1 agonist, stimulated the synthesis. The effect of both compounds was blocked by SCH 23390, a D1 antagonist, but not by sulpiride, a D2 antagonist. In the absence of receptor activation, the synthesis was very slow. The results suggest a trophic influence of dopamine upon the synthesis of GABA via D1 receptors.

Effects of Creatine on Synthesis and Release of γ‐[3H]Aminobutyric Acid

Creatine has been used previously to alter the energy balance of neurons in brain slices. In the present experiments, it was found to reduce the accumulation of gamma-[3H]aminobutyric acid ([3H]GABA) as synthesized from [3H]glutamine or [3H]glutamic acid in slices of rat neostriatum. The lowest effective concentration was 5 mM. Creatine (25 mM) was also effective when the degrading enzyme of GABA, i.e., GABA-alpha-oxoglutarate transaminase, was blocked by gabaculine. Creatine (25 mM) did not inhibit the uptake and subsequent accumulation of [3H]GABA. Thus, indirect evidence was obtained that creatine decreased the activity of the synthesizing enzyme of GABA, i.e., glutamate decarboxylase. When the direct effect of creatine (25 mM) on glutamate decarboxylase was studied in vitro, the agent indeed decreased the activity of the enzyme. Creatine (25 mM) also diminished the release of [3H]GABA (expressed as dpm/mg wet weight) from rat neostriatal slices, probably by reducing its synthesis and thus its readily releasable pool. These data are of importance for studies with creatine in complex neuronal systems, because they show that the agent changes not only neuronal energy balance, but also synthesis and release of the ubiquitous transmitter GABA.

Augmentation by glycine and blockade by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) of responses to excitatory amino acids in slices of rat neocortex

Responses of neocortical pyramidal cells to excitatory amino acids were recorded intracellularly. Agonists and antagonists were applied electrophoretically from a separate multibarrel pipette and care taken to ensure that the pipette was positioned to evoke optimal responses to N-methyl- d-aspartate (NMDA), or homocysteic acid, before control responses were recorded. Responses to NMDA, but not those to α-amino-3-hydroxy-5-methyl-4-isoxazdepropionic acid (AMPA) or quisqualate, were enhanced when glycine was co-applied. Responses to AMPA, quisqualate and NMDA were reduced by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) applied either electrophoretically, or in the bathing medium, with responses to quisqualate being the least and those to AMPA being the most sensitive to CNQX. The blockade of NMDA responses by CNQX was selectively reversed by additional glycine confirming that CNQX blocks NMDA receptor-channel complexes at the glycine, rather than at the NMDA site. Under control conditions, responses to glutamate resembled responses to quisqualate, and were relatively insensitive to CNQX, 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid and 2-amino-5-phosphonovalerate, while responses to homocysteic acid resembled responses to NMDA and were blocked by these antagonists. This suggested that homocysteic acid acted at NMDA receptors, while glutamate acted primarily at non-NMDA receptors. However, responses to both glutamate and homocysteic acid were augmented by additional glycine when these transmitter candidates were applied close to a “hot spot” for NMDA receptor activation. The glycine enhancement of responses to glutamate was sensitive to NMDA antagonists, indicating that glutamate can activate NMDA receptors in an intact preparation if glycine levels are high enough.

Formation of leukotriene B4 and monohydroxy acids in slices of porcine thyroid gland.

Slices of porcine thyroid gland were incubated with arachidonic acid and ionophore A 23187. This led to the formation of 5-hydroxy-eicosatetraenoic acid (5-HETE), 12-HETE, 15-HETE and leukotriene B4 (LTB4). Time course studies demonstrated that levels of detected LTB4 reached a plateau after 30 min and that in parallel with the synthesis of this compound, greater amounts of 5-HETE was formed. The present results demonstrate the formation of lipoxygenase products in the porcine thyroid gland, indicating possible physiological/pathophysiological roles for these compounds in this organ.

Effects of barbiturates on responses evoked by excitatory amino acids in slices of rat olfactory cortex

A study has been made of the effects of ranges of concentrations of phenobarbitone, pentobarbitone and thiopentone on responses evoked by γ-aminobutyric acid (GABA), l-glutamate, l-aspartate, N- methyl- D- aspartate , kainate and quisqualate in slices of olfactory cortex of the rat. All three barbiturates affected GABA-evoked depolarizations similarly in that responses were potentiated by small doses but markedly inhibited at greater concentrations. Responses to l-aspartate and l-glutamate were little affected except at the largest dose of phenobarbitone tested (10 mM). The responses evoked by the selective agonists of excitatory amino acid receptors were inhibited by the barbiturates, the relative sensitivities being quisqualate ⩾ N- methyl- D-aspartate ⩾ kainate with phenobarbitone, quisqualate ⩾ kainate > N- methyl- D-aspartate with pentobarbitone and quisqualate > kainate = N- methyl- D-aspartate with thiopentone. The possible significance of these findings is discussed.

Contrasting properties of K+ conductances induced by baclofen and γ‐aminobutyric acid in slices of the guinea pig hippocampus

Properties of membrane K+ conductances induced by baclofen and gamma-aminobutyric acid (GABA) in the hippocampus were investigated by using guinea-pig brain slices. Baclofen hyperpolarized the membrane and decreased the input resistance of pyramidal cells through the activation of a membrane K+ conductance. GABA caused a biphasic response in pyramidal cells, consisting of hyperpolarizing and depolarizing components. Combined application of picrotoxin and bicuculline eliminated the major part of the depolarizing component of the biphasic response and produced a relatively pure hyperpolarizing response which was also mediated by an increase in K+ conductance. The K+ conductance change induced by baclofen showed prominent inward rectification. However, the K+ conductance induced by GABA did not show an obvious rectifying property. The K+ conductance activated by baclofen was strongly antagonized by a low concentration (5 x 10(-6) M) of 4-aminopyridine (4-AP). In contrast, the K+ conductance activated by GABA was insensitive to 4-AP even at a high concentration of 10(-3) M. The slow inhibitory postsynaptic potential (slow i.p.s.p.) evoked by stimulation of the mossy fibres was totally suppressed by a low concentration of baclofen (5 x 10(-6) M). Whereas GABA (10(-3) M) decreased the amplitude of the slow i.p.s.p., the reduction of the amplitude was proportional to the decrease in the amplitude of the electrotonic potentials produced by constant inward current injections. These results suggest that the hyperpolarizations induced by GABA and baclofen may be generated by K+ conductances of different kinetic and pharmacologic properties.

A γ-aminobutyric acid-specific transport mechanism in mammalian kidney

We describe high-affinity, sodium-dependent transport of gamma-aminobutyric acid in slices exposing basal lateral membranes and brush-border membrane vesicles prepared from rat renal cortex. In the presence of aminooxyacetic acid, to block γ-aminobutyric acid oxidation, uptake into the intracellular space of slices was saturable (apparent K t , 26±4 μ M , mean and S.E.) and concentrative (steady-state distribution ratio at 50 μM γ-aminobutyric acid, 47.7±2.4, mean and S.E.). Brush-border membrane vesicles accumulated γ-aminobutyric acid in the presence of an inward-directed sodium chloride gradient, (apparent K t , 30–36 μ M ) with the peak of ‘overshoot’ at 10 min. Uptake by vesicles responded to manipulation of the transmembrane potential gradient with valinomycin or impermeant anion, β-Alanine inhibited γ-aminobutyric acid transport by slices and brush-border membrane vesicles; inhibitors of neuronal-type γ-aminobutyric acid transport (e.g., nipecotic and diaminobutyric acids) did not. An ‘ABC test’ indicated that γ-aminobutyric acid and β-alanine do not share a single carrier in either the brush-border or basal-lateral membrane of renal cortex. Influx of γ-aminobutyric acid into brush-border membrane vesicles, at transequilibrium NaCl, was stimulated by trans-γ-aminobutyric acid but not by trans-taurine. Ion gradient-driven γ-aminobutyric acid co-transport was unaffected in freeze-thawed brush-border membrane vesicles; this treatment abolished β-alanine and taurine co-transport. We conclude that rat kidney membranes (brush-border and basal-lateral) possess a γ-aminobutyric acid-preferring, high-affinity transport mechanism.

Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes

5-Keto-l-idionic acid ( identical with5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv ;Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.Label from dehydro-l-[1-(14)C]ascorbic acid, 2-keto-l-[1-(14)C]idonic acid (l-xylo-2-hexulosonic acid), l-[1-(14)C]idonic acid, or 5-keto-l-[1-(14)C] idonic acid was incorporated into l-(+)-tartaric acid in high yields as it was in the l-[1-(14)C]ascorbic acid experiment. In a double label experiment involving a mixture of l-[1-(14)C]idonic acid and l-[2-(3)H]idonic acid, the (3)H/(14)C ratios of 5-keto-l-idonic acid and l-(+)-tartaric acid synthesized in young grape leaves were almost the same as the value of the l-idonic acid fed. Label from 5-keto-l-[6-(14)C]idonic acid was incorporated into sugars and insoluble residue in the same way as l-[6-(14)C]ascorbic acid was metabolized in grapes.These results provide strong evidence that in grapes l-(+)-tartaric acid is synthesized from the C(4) fragment that corresponds to the C1 to C4 group of the 5-keto-l-idonic acid derived from l-ascorbic acid via 2-keto-l-idonic acid and l-idonic acid.

Kinetics of tryptophan influx into brain slices depleted of sodium and loaded with L-histidine.

The kinetics of tryptophan influx were studied with rat brain slices preloaded with L-histidine and/or depleted of sodium ions. The best fits of the data (velocity of influx versus tryptophan concentration) were computed by use of a model consisting of a saturable (Michaelis-Menten type) and an unsaturable (diffusional) component with an iterative nonlinear regression analysis. Sodium depletion of the slices reduced the maximal velocity of saturable influx. In histidine-preloaded slices, depleted or not depleted of sodium ions, the most marked alteration again occurred in the maximal velocity, which more than doubled. Slices preloaded with histidine contained greatly elevated levels of glutamine and histidine, which may have stimulated the influx by exchange with extracellular tryptophan even in the absence of sodium ions. The maximal velocity was higher with increasing concentration of large neutral amino acids in slices at the start of the influx measurements. The influx of tryptophan in brain cells is apparently modified by changes in the intracellular amino acid pool, which, when increased, also counteracts the effect of sodium depletion on the tryptophan influx.

Conversion of L-Ascorbic Acid to L-Idonic Acid, L-Idono-γ-lactone ane 2-Keto-L-idonic Acid in Slices of Immature Grapes

Conversion of L-Ascorbic Acid to L-Idonic Acid, L-Idono-γ-lactone ane 2-Keto-L-idonic Acid in Slices of Immature Grapes

Choline uptake into renal phospholipids following renal ischemia in rats.

Renal phospholipid metabolism was studied after ischemia was induced by occlusion of the left renal artery in the rat. There was no change in the rate of cellular [14C]choline uptake after 25 or 60 minutes of ischemia. However, [14C]choline incorporation into phospholipid was two to three times greater in slices from the ischemic kidney than in slices from the contralateral control kidney. The increase occurred after 25 minutes of ischemia plus 15 minutes of reflow, and after 60 minutes of ischemia with or without reflow. When [14C]choline was injected into rats after a 60-minute period of renal ischemia, the rate of incorporation into phospholipid in the ischemic kidney was almost twice that of the control kidney. These results were similar to those of the in vitro experiments. Since virtually all of the cellular phospholipids of the kidney are present in cellular membranes, renal ischemia affects membrane metabolism. The mean distribution ratio of alpha-aminoisobutyric acid in slices of kidneys ischemic for 60 minutes was similar to that of control slices: 4.11 +/- 0.2 (SEM) vs. 4.30 +/- 0.30. The normal uptake of alpha-aminoisobutyric acid indicates that the increased incorporation of choline is associated with functional integrity of the membrane.

Perinatal changes of transport systems for amino acids in slices of mouse brain

Perinatal changes in the uptake of amino acids were measured in slices of fetal (15- and 19-day) and newborn (4-, 24-, and 48-hr-old) mouse brain. Uptake increased with age; smaller changes occurred with basic and neutral amino acid transport systems, and the largest changes occurred in fetal brain with amino acids of putative neurotransmitter function (taurine, glycine, GABA, and the acidic amino acids). The pattern of increase in uptake was similar at high and at low external amino acid concentrations. Developmental changes in tissue content of Na(+), K(+), or ATP were small during this period, and so are unlikely to be responsible for the observed changes in uptake. It appears that by the 15th day of fetal life, the transport systems for essential amino acids are fairly well developed in the brain, and the transport systems for neurotransmitter amino acids are not so well developed, but undergo a rapid increase in the 15-19-day period. From birth to adulthood, the concentrative capacity of slices of mouse brain for nonessential (putative neurotransmitter) amino acids is much greater than for essential amino acids.

Detection of the feedback control of pyrimidine biosynthesis in slices of several rat tissues

Measurements of the rate of incorporation of labeled precursors into orotic acid in slices of several rat tissues reveal that the rate of incorporation of bicarbonate, but not carbamoylphosphate or carbamoylaspartate, is significantly inhibited by the addition of uridine to the reaction mixture. These experiments demonstrate the operation of a feedback control mechanism governing the rate of pyrimidine biosynthesis in the intact cell and provide evidence that the reaction catalyzed by carbamoylphosphate synthetase is the site of end-product inhibition.

High affinity uptake of transmitters: studies on the uptake of L-aspartate, GABA, L-glutamate and glycine in cat spinal cord.

Abstract— The uptake of l‐aspartate, l‐glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent Km's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically‐sensitive particles of equilibrium density equivalent to that of 1.0 m‐sucrose were at least in part responsible for the uptake of these amino acids.Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids: specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord; specific for GABA—not inhibited by glycine, taurine, l‐aspartate or l‐glutamate and (3) specific for l‐aspartate and l‐glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l‐cysteate and l‐cysteine sulphinate. The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l‐glutamate diethyl ester (l‐aspartate and l‐glutamate). p‐Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l‐aspartate/l‐glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l‐aspartate or l‐glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.

Studies on the subcellular distribution of γ-aminobutyric acid in slices of rat cerebral cortex

Studies on the subcellular distribution of γ-aminobutyric acid in slices of rat cerebral cortex

Control of pyrimidine biosynthesis in rat liver by feedback inhibition of aspartate carbamoyltransferase

Summary Studies on the incorporation of labeled precursors into orotic acid in slices of rat liver reveal aspartate carbamoyltransferase to be a site for the feedback control of hepatic pyrimidine biosynthesis through end-product inhibition.

Amino acid transport in the rat renal papilla

The uptake of amino acids in slices of renal papilla of rats has been investigated in vitro under environmental conditions of high sodium and non-electrolyte concentrations and low O 2 tensions, and compared with uptake under similar conditions in slices of renal cortex. l-Lysine, cycloleucine and α-aminoisobutyric acid were actively concentrated in Krebs—Ringer-bicarbonate buffer up to 8 times the concentration of the media. The accumulation was 1.7 times greater in the papilla than in the cortex and behaved as a saturable transport system. The papilla actively accumulated the above amino acids in media of increasing sodium concentrations up to 439 mequiv/l and in media made hyperosmolal with sucrose, whereas active accumulation of the amino acids in the renal cortex was absent or greatly diminished under these same experimental conditions. Slices of renal papilla also concentrated α aminoisobutyric acid and lysine in oxygen-deficient media. Aldosterone and vasopressin did not influence the amino acid uptake in the renal papilla or cortex. The results of this study indicate that the papilla is able to accumulate amino acids actively in vitro under conditions which simulate the environment of the papilla in vivo and which are detrimental to the accumulation of amino acids in the renal cortex.

Effect of methimazole on amino acid utilization by calf thyroid slices.

The effects of methimazole on the formation of 14CO2 and 14C-protein from carboxyl-labeled leucine and tyrosine were investigated in calf thyroid slices. Decarboxylation was inhibited to a moderate but significant degree by 0.6 × 10−2 − 0.6 × 10−5m methimazole, and a similar effect on protein synthesis occurred with 0.6 × 10−4 − 0.6 × 10−6m methimazole. The greatest inhibition of either route of amino acid utilization occurred with 0.6 × 10−5m methimazole. Methimazole had no inhibitory effect on CO2 formation or protein synthesis from amino acids in slices of calf liver. The mechanism is unknown. (Endocrinology 81: 1169, 1967)

Biosynthesis of Δ9 monounsaturated fatty acids in slices of the ice-plant, Carpobrotus chilense

Biosynthesis of Δ9 monounsaturated fatty acids in slices of the ice-plant, Carpobrotus chilense

The metabolism of glutamic acid in rat retina

Investigation of the metabolism of L-glutamic acid in slices of adult rat retina under aerobic conditions using the technique of quantitative automatic paper radio-chromatography is described, L-glutamic acid was converted into aspartate, γ -aminobutyrate, glutamine and carbon dioxide. In the presence of added glucose, the formation of glutamine and γ aminobutyrate from glutamate was markedly increased, whereas the formation of glutamine was not appreciably affected by the addition of pyruvate to the medium. The addition of either glucose or pyruvate caused a marked decrease in the production of aspartate from glutamate. The production of carbon dioxide from glutamate, or from glutamate in the presence of glucose or pyruvate, appears to be quantitatively the same.