UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS

—Cortex slices, synaptosomes and C-6 glioma cells were used to study [³⁵S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle-bound taurine and 16% of the total in the tissue. The uptake of [¹⁴C]GABA by C-6 glioma cells was inhibited 3-fold more by β-alanine than by l -DABA, whilst synaptosome preparations showed the opposite pattern, l -DABA being 2 or 3 times more effective than β-alanine. [³⁵S]Taurine uptake inhibition by l -DABA was low for synaptosomes and C-6 glioma, whereas β-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β-alanine. When 30 min rather than 5 min incubations were employed, β-alanine depressed [³⁵S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.     

Taurine uptake in synaptosomal fractions of rat cerebral cortex.

Abstract— [³⁵S]Taurine was found to be accumulated in synaptosomal fractions of rat cerebral cortex. Kinetic analysis in the range of 1–800 μm -[³⁵S]taurine revealed at least two different uptake processes. A high affinity uptake with a K_m of 20 μM and a low affinity uptake with a K_m of about 450 μM. The high affinity component was dependent on temperature and energy, and virtually abolished in the absence of sodium. Examination of the influence of structural analogues and putative transmitter substances indicates that the high affinity uptake of taurine into synaptosomal fractions of rat cerebral cortex is unique and highly specific. No specific actions of several centrally acting drugs on taurine uptake could be observed.     

TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [³⁵S]taurine in the brain of the mother, the fetus and the neonate after [³⁵S]taurine was administered to pregnant rats. In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [³⁵S]taurine was soluble, S₃; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P₂). After further fractionation of P₂, most of the taurine and [³⁵S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions. In the neonatal brain, shortly after birth there was a decrease in taurine and [³⁵S]taurine recovered in the supernatant fraction, S₃, accompanied by an increase in the percentage of taurine and [³⁵S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [³⁵S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo-osmotic shock. The pattern of development in subcellular fractions of neonatal rat brain labelled with [³⁵S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.     

Evidence for specific synaptosomal localization of exogenous accumulated taurine

Abstract— Equilibrium or incomplete equilibrium density gradient centrifugation was used to characterize the subcellular localization of exogenous [^{3 5}S]taurine which was taken up by minces or homogenates of rat cerebral cortex. [^{3 5}S]Taurine is accumulated in synaptosomes, which sediment more slowly than l -[³H]norepinephrine-accumulating particles. When [^{3 5}S]taurine and [³H]GABA are accumulated by minces, a small difference in the sedimentation profile of taurine and GABA was observed, but no difference was found when taurine and intrasynaptosomal potassium were compared. However, potassium sedimented more slowly after incubation of homogenates than of minces. These data give evidence for the accumulation of [^{3 5}S]taurine by a specific synaptosomal population.     

Taurine deficiency in the kitten subcellular distribution of taurine and [35S]taurine in brain

Taurine concentration decreases rapidly in the tissues and physiological fluids of kittens fed a diet of partially purified casein which lacks taurine. We have studied the subcellular distribution in cerebrum of taurine and [³⁵S]taurine administered intravenously to these animals. The taurine concentration of all the fractions isolated from the cerebrum of taurine-deficient kittens was approximately sevenfold less than that observed in the fractions of cerebrum isolated from control kittens. The [³⁵S]taurine was approximately twofold greater in all the brain fractions isolated from the taurine-deficient kittens compared with those isolated from the control kittens. The percent distributions of taurine and [³⁵S]taurine in the fractions isolated from the cerebrum of control and deficient kittens were identical. Thus, in the face of a severe diet-induced deficiency of taurine in kitten brain, there appears to be no conservation of taurine by any particular subcellular pool of taurine. These studies provide no evidence for differences in compartmentation of taurine in cerebrum of taurine-deficient kittens compared with control kittens.     

Effects of the ionophores X537A and A23187 on GABA,glycine, and taurine release from chick retinal subcellular fractions

The ionophore X537A at concentrations of 5–20 M stimulated the release of [³H]GABA and [³⁵S]taurine, from retinal subcellular crude nuclear (P₁) and crude synaptosomal (P₂) fractions. The release of [³H]GABA increased 114% and 136% over control values in P₁ and P₂ fractions, respectively. The efflux of [³⁵S]taurine from P₁ was increased by 45% and that from P₂ by 21%. X537A increased⁴⁵Ca²⁺ uptake in the P₂ fraction but not in the P₁ fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [³H]GABA or [³⁵S]taurine uptake by the retinal fractions. A23187 enhanced [³H]GABA release from P₁ and P₂ by 52% and 105%, respectively. The ionophore also increased [¹⁴C]glycine liberation in both P₁ (35%) and P₂ (50%) but failed to stimulate [³⁵S]taurine release. A23187 produced a transient increase of⁴⁵Ca²⁺ uptake of 38% in P₁ and 30% in P₂. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consisent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.     

Properties of [3H]taurine release from crude synaptosomal fractions of rat cerebral cortex

The release of previously accumulated [³H]taurine and [¹⁴C]GABA from crude synaptosomal (P₂) fractions isolated from rat cerebral cortex was studied using a superfusion system. The spontaneous efflux of [³H]taurine and [¹⁴C]GABA was stimulated by elevated concentrations of K⁺ (15–133 mM) in a concentration-dependent manner. This K⁺-stimulated release of [¹⁴C]GABA but not of [³H]taurine was enhanced in the presence of Ca²⁺. However, addition of 3 mM Ca²⁺ to the superfusion medium in the presence of the ionophore A 23187 resulted in a stimulation of the release of both [³H]taurine and [¹⁴C]GABA. These results are discussed in connection with the cellular localization of tourine in the central nervous system.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Taurine in the developing cat: Uptake and release in different brain areas

Taurine is an important modulator of neuronal activity in the immature brain. In kittens, taurine deficiency causes serious dysfunction in the cerebellar and cerebral visual cortex. The processes of taurine transport in vitro were now studied for the first time in different brain areas in developing and adult cats. The uptake of taurine consisted initially of two saturable components, high- and low-affinity, in synaptosomal preparations from the developing cerebral cortex and cerebellum, but the high-affinity uptake component completely disappeared during maturation. The release of both endogenous and preloaded labeled taurine from brain slices measured in a superfusion system was severalfold stimulated with a slow onset by depolarizing K⁺ (50 mM) concentrations. K⁺ stimulation released markedly more taurine from the cerebral cortex, cerebellum and brain stem in kittens than in adult cats. The responses were largest in the cerebellum. Both uptake and release of taurine are thus highly efficient in the brain of kittens and may be of significance in view of the vulnerability of cats to taurine deficiency.     

Binding of Paroxetine to the Serotonin Transporter in Membranes from Different Cells,Subcellular Fractions and Species

The binding of [³H]-paroxetine to membrane serotonin transporter (SERT) has been studied in membranes from different sources and subcellular fractions. From rat were membranes from venous blood platelets, brain total cortex, brain microsomes, brain crude and purified synaptosomes. Membranes were obtained from venous blood platelets from human volunteers and from brain cortex tissue from neurosurgery (cerebral lobectomies following craniocerebral injuries). The main finding was that the K _D of paroxetine binding to the SERT was the same for platelet and nerve ending (synaptosomal) membranes. That parameter was significantly lower in membranes from brain microsomes and cortex total tissue. No species related difference was found, where comparison was possible, between human and rat tissue. The equality of K _D of paroxetine binding to blood platelet membranes and to membranes from nerve endings appears to encourage the use of such membranes as a model for brain SERT. Binding at two different temperatures for several of the fractions suggests that paroxetine–SERT interaction is entropy-driven.     

Sedimentation and release properties of P2 fractions derived from rat cerebral cortex slices incubated with radiolabeled GABA for a short or long time period

On homogenization of rat cerebral cortex slices previously incubated with [³H] GABA or [¹⁴C]GABA for 5 or 30 min, respectively, particles were recovered in P₂ fractions which exhibited similar buoyant density, but different sedimentation velocity on linear sucrose density gradient centrifugation. The K⁺-evoked release of [³H]GABA from particles isolated from slices previously incubated for 5 min with [³H]GABA was increased in the presence of exogenous Ca²⁺. In contrast, the K⁺-evoked release from particles isolated from slices previously incubated for 30 min with [³H]GABA, was not influenced by the presence of exogenous Ca²⁺.These results suggest that, depending on the incubation time of slices, exogenously applied GABA can be detected in differnnt pools. These pools not only seem to differ in their Ca²⁺ dependency of K⁺-evoked release but also in their subcellular localization.     

Subcellular distribution of cysteine oxidase activity in ox retina.

Cysteine oxidase activity has been determined in the primary and secondary subfractions of ox retina. About 30% of enzyme activity is found in the soluble fraction while about 70% is associated with particulate components.In the secondary subcellular fractions about 36% of enzyme activity, recovered from crude mitochondria, is present in the synaptosomal fraction.Enzymic activity is stimulated by Fe⁺⁺ and NAD⁺. The reason and significance of the cysteine oxidase activity in synaptosomal fraction are briefly discussed in relation with taurine function in retina.     

Chloride dependence of the K+-stimulated release of taurine from synaptosomes

Exposure of a crude synaptosomal fraction to K⁺ concentrations ranging from 25 to 100 mM evokes the release of [³H]taurine and [³H]GABA. These high concentrations of K⁺ induce, besides depolarization, a marked synaptosomal swelling, which is prevented by replacing chloride in the solutions with the largely impermeant anion gluconate. The depolarizing effect of K⁺ is unaffected by omission of chloride. The K⁺-evoked release of taurine seems related to K⁺-induced changes in synaptosomal volume rather than to a depolarizing effect, since it is totally calcium-independent but is abolished by reducing chloride and by making solutions hypertonic with mannitol. The release of [³H]GABA, in contrast is unaffected in chloride-free or hypertonic solutions.     

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids.

Abstract— Crude synaptosomal (P₂) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P₂ fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P₂ fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm -l -[³H]glutamate and l -[³H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm -[³H]GABA and [³H]taurine was unchanged. A more detailed kinetic analysis of l -[³H]glutamate uptake revealed there was a 20% decrease in the V_max value with X-irradiation treatment and no change in the apparent K_m value. In a second study, the uptake of l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was measured using P₂ fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm -l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was significantly (P < 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [³H]GABA uptake revealed no significant change in the apparent K_m and a 35% decrease in the V_max value. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.     

SUBCELLULAR DISTRIBUTION OF TAURINE AND CYSTEINE SULPHINATE DECARBOXYLASE ACTIVITY IN OX RETINA

Abstract– Taurine levels have been determined in primary and secondary subcellular fractions of ox retina and pigment epithelium.
About the 79.5% of recovered taurine is located in the soluble fraction (S₃), while the remainder is associated with the particulate components. In the secondary subcellular fractions, taurine is primarily associated with the synaptosomal fraction.
Cysteine sulphinate decarboxylase is predominantly associated with particulate components of retinal cells. About the 50% of the recovered enzyme activity of crude mitochondria is present in the synaptosomal fraction.     

Taurine and hypotaurine transport in neuroblastoma cells: effects of cations

The cation requirements of [³H]taurine and [³⁵S]hypotaurine uptake by cultured neuroblastoma C1300 cells were compared in Krebs-Ringer-Hepes-glucose medium. The uptakes were strictly sodium-dependent at both low and high taurine and hypotaurine concentrations. The omission of Ca²⁺ or Mg²⁺ ions affected uptakes only marginally. The optimal K⁺ concentration was equal to the physiological concentration, whereas abnormally high K⁺ levels inhibited similarly taurine and hypotaurine uptake. The sodium dependence curves of both uptakes were sigmoidal in character at low and high taurine and hypotaurine concentrations. Hill plots suggest that two Na⁺ ions are coupled with the transfer of one taurine or hypotaurine molecule into neuroblastoma cells. With respect to cation requirements taurine and hypotaurine transports are similar in cultured neuroblastoma cells and display features considered typical of the uptake of a neurotransmitter amino acid.     

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [³H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination of [³H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.Supported by the Research Service of the Veterans Administration and by Research Grant NS 13227 from NINCDS.     

Multiple dopamine binding sites: subcellular localization and biochemical characterization.

Abstract— The biochemical and pharmacological characteristics of dopamine agonist and antagonist binding to rat striatal subcellular fractions were studied and compared to the localization of dopamine–sensitive adenylate cyclase activity. The highest specific activity of adenylate cyclase sensitive to dopamine was associated almost exclusively with the crude synaptic membrane fraction (P₂). Using [³H]-haloperidol, [³H]apomorphine and [³H]spiroperidol as markers for the dopamine receptor, high affinity and stereoselective specific binding was observed for the crude synaptic fraction and the microsomal fraction (P₃). Analysis of the binding of [³H]haloperidol to the striatal microsomal preparation revealed a homogeneous receptor site with a K_d value of 3.0 nm . The data for [³H]haloperidol binding to the crude synaptosomal fraction showed two saturable binding sites with K_d values of 2.5 nm and 12.5 nm . A similar heterogeneous binding profile was observed in the P₂ fraction using [³H]apomorphine. The K_d values for [³H]apomorphine in this fraction were determined to be 1.2 nm and 7.2 nm . The effects of various biochemical parameters including ionic strength, salt concentration and pH on the binding of [³H]haloperidol to the P₂ fraction were also studied. Overall, these data show that the subcellular localization of multiple binding sites in the crude synaptosomal fraction and the identification of specific binding to purified synaptosomes correlate with the subcellular distribution of striatal dopamine-sensitive adenylate cyclase activity.     

The Effect of Anti-Synaptosomal Membrane Antibodies on Neurotransmitter Uptake

Antibodies raised against synaptosomal plasma membranes of rat hippocampus (anti-HPC IgG) caused inhibition of [3H]noradrenaline, [3H]5-hydroxytryptamine, [3H]GABA and [3H]aspartate uptake into S1 fractions and slices of hippocampus and cerebral cortex, but not those of caudate nucleus and hypothalamus. Similar inhibition was not observed on using antibodies against synaptosomal membranes of rat caudate nucleus. Anti-HPC IgG raised against synaptosomal membranes of hippocampus failed to alter both spontaneous and K+-evoked release of [3H]noradrenaline. They did not interfere with the binding of [3H]desipramine (the potent noradrenaline-uptake inhibitor) and with the binding of [3H]dihydroalprenolol, thus excluding any interaction of the antibodies with drug receptors which are located on either the pre- or postsynaptic membrane. The anti-HPC IgG inhibit the enzymatic activity of [Na+-K+-]ATPase by 30% upon incubation of the antibodies with crude membrane preparations. A comparison of their inhibitory effects with those of the neurotoxin 6-hydroxydopamine suggests that the corresponding hippocampal specific antigens are located at a presynaptic site.