Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

The relationship between sodium and high-affinity taurine uptake in hypothalamic crude P2 synaptosomal preparations

Two uptake systems for taurine transport in a rat hypothalamic crude synaptosomal preparation were identified. The true transport constants were, for the high-affinity uptake system,K _m=240 M andV (maximum velocity)=400 nmol/g protein/min, and for the low-affinity uptake system.K _m=5290 M and V=1640 nmol/g protein/min. The initial velocity of high-affinity taurine uptake by the crude synaptosomal preparation was studied as a function of sodium and taurine concentration. Hill plots were constructed from these data. The requirement of high-affinity taurine uptake on a sodium gradient was examined by utilizing monensin, and the metabolic poisons, 2,4-dinitrophenol and ouabain. The major findings are as follows: 1) One sodium ion is co-transported with each taurine molecule; 2) the high-affinity uptake process is driven by the sodium concentration gradient across the membrane; 3) sodium increases the maximal velocity rather than the affinity of the high-affinity taurine carrier for the taurine molecule; 4) one taurine molecule is transported per carrier for both the high- and low-affinity taurine uptake systems; and 5) high-affinity taurine uptake is an energy-dependent process.     

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.     

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.     

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP-Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.     

Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.     

Effect of taurine on passive ion transport in rat brain synaptosomes.

Taurine, in concentrations greater than 10 mM, was found to have an inhibitory effect on passive calcium uptake and release in rat brain synaptosomal preparations. Amino acids similar to that of taurine in chemical structure, β-alanine, hypotaurine, homotaurine and γ-amino-butyric acid were also shown to inhibit calcium uptake in this preparation. Taurine, though, did not alter the permeability of these preparations to sodium or potassium. It thus appears that taurine and chemically related amino acids can alter calcium movements in these preparations. It is postulated that this effect is due to binding to specific taurine sites in the synaptosomal membranes.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Mechanism of the inhibiting effect of atypical antidepressants and psychostimulants on the synaptosomal uptake of monoamines

The effects of viloxazine and zimelidine on reverse monoamine neurotransmitter uptake by crude synaptosomal fraction of the rat brain were studied and compared to those of imipramine, amphetamine and cocaine. Imipramine noncompetitively inhibited the uptake of all the monoamines under study, with a greater specificity as regards serotonin. Viloxazine and zimelidine strongly inhibited the transport of noradrenaline, dopamine and serotonine, the transport of the latter being inhibited to a greater degree. Kinetic analysis showed the active centers of noradrenaline and dopamine carriers to be very much alike, those of catecholamine and serotonin carriers to be less alike. The data obtained made it possible to describe (at least partly) the structure of the active centers of monoamine carriers and to specify the action modes of antidepressants and psychostimulants.     

Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.     

Analysis of dual transport systems by means of Hill plots

A method is proposed for calculating the parameters of dual solute transport systems utilizing Hill plots of isotope uptake data. The use of the method is illustrated with synaptosomal uptakes of phenylalanine and leucine and with data that have been reported in the literature for synaptosomal uptakes of taurine and glutamate via dual systems.     

HIGH AFFINITY UPTAKE SYSTEMS FOR TAURINE IN TISSUE SLICES AND SYNAPTOSOMAL FRACTIONS PREPARED FROM VARIOUS REGIONS OF THE RAT CENTRAL NERVOUS SYSTEM. CORRECTION OF TRANSPORT DATA BY DIFFERENT EXPERIMENTAL PROCEDURES

Abstract— —The uptake of taurine into tissue slices of specific regions of the rat central nervous system (CNS) was compared with the uptake of taurine into synaptosomal fractions prepared from the corresponding regions. Two different techniques for performing control experiments were also compared: procedure I, correction for the uptake of taurine obtained from duplicate incubations but at 2°c and procedure II, correction of taurine uptake into extracellular or extrasynaptosomal space measured by inulin uptake experiments plus correction for diffusion (non-saturable) processes.
Kinetic analyses of the uptake data in tissue slices utilizing the procedure I correction technique indicate that six regions of the rat CNS (spinal cord, diencephalon, cortex, striatum, hippocampus, and midbrain) possess high affinity uptake systems (K_m values approx 60 μM or less). The K_m value for the cerebellum (105.4 ± 15.7 μM) is intermediate between a high and low affinity uptake system while the K_m value for the pons-medulla (210.0 12.4 μM) is considered to be low affinity. When procedure II techniques were utilized for correcting the uptake data all eight regions demonstrated high affinity uptake systems (11.8–73.2μM).
Synaptosomal fractions prepared from the spinal cord, pons-medulla, diencephalon, and midbrain demonstrate high affinity uptake systems (procedure I) for taurine (10.3–47.2 μM) while the hippocampus, cortex, striatum, and cerebellum have intermediate (but still high affinity) values (59.4–96.4 μM). High affinity uptake systems (8.2–79.8 μM) were obtained for all eight regions of the rat CNS when procedure II was utilized for correction of the data.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles

Glutathione (GSH) transport was studied in synaptosomal membrane vesicles (SMV) of rat cerebral cortex. The present study shows that GSH uptake into SMV occurs very quickly in a time-dependent manner into an osmotically active intravesicular space. The initial rate of transport followed Michealis-Menten saturation kinetics with a K_m 4.5±0.8 μM that shows a high affinity of the transporter for GSH. Therefore GSH uptake in SMV occurs by a mediated transport system which can be activated by either an inward gradient of cations, like Na⁺ or K⁺, or membrane depolarization. These results, together with those obtained by valinomycin-induced K⁺ diffusion potential, indicate that GSH synaptosomal transport is electrogenic by a negative charge transfer. The increase of GSH uptake measured by trans-stimulation experiments confirms a GSH bidirectional mediated transport which seems susceptible of modulation by changes in ionic fluxes and in the membrane potential. These results may indicate a possible involvement of this transporter in the role suggested for GSH in synaptic neurotransmission; also considering that GSH precursor of neuroactive aminoacids (glyeine, glutamate), may contribute to regulate their level in synapses. Finally, a GSH transporter in synaptosomes may contribute to maintaining the GSH homeostasis in cerebral cortex, where decreases of GSH levels have been related to susceptibility to neuropathologies.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

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.     

Selective inhibition of synaptosomal proline uptake by leucine and methionine enkephalins

The high affinity, sodium-dependent uptake of proline by rat brain synaptosomes was inhibited by the opioid pentapeptides, Leu-enkephalin and Met-enkephalin. The synaptosomal uptake of other putative neurotransmitter amino acids including glutamic acid, aspartic acid, gamma-aminobutyric acid, and taurine was not altered in the presence of enkephalins. The uptake of a neuroinactive amino acid, leucine, was also unaffected by enkephalins. The extent of proline uptake was half-maximal at a Leu-enkephalin concentration of 1 microM. Both the initial rate of transport and the overall capacity for proline accumulation were reduced. The effect of the enkephalins was vectorial since carrier-mediated efflux of proline was not altered in the presence of enkephalins. Morphine and the opioid peptides, dynorphin and beta-endorphin, were without effect on proline uptake. The inhibition of proline uptake by enkephalins was not diminished by prior incubation of the synaptosomal preparation with naloxone; however, the inhibition was attenuated by 1-butanol. The des-tyrosyl fragments of the enkephalins were as inhibitory as the intact pentapeptides. A modified enkephalin ([D-Ser2]Leu-enkephalin-Thr) with selective affinity for the delta subclass of enkephalin receptor was effective in inhibiting proline uptake. On the basis of the selectivity of these effects, we propose that there is a specific population of nerve endings in the cerebral cortex that contains both a proline-transport system and binding sites for Leu- and Met-enkephalin and furthermore, that these binding sites may be related to the putative delta receptor.     

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.     

Regulation of Taurine Transport in Rat Skeletal Muscle

Taurine concentration of soleus muscle (SL, slow-twitch) was initially about twofold higher than that of extensor digitorum longus muscle (EDL, fast-twitch). Taurine concentration in gastrocnemius muscle (GC) was intermediate between that of EDL and SL. Four days after sciatic nerve section, taurine concentration in the EDL but not in the SL was increased by 2.5-fold. The increase was not due to the muscle atrophy and was observed 28 days after denervation. Tenotomy did not increase the total taurine content of the EDL. The increase in taurine concentration of the denervated EDL was prevented by simultaneous ingestion of guanidinoethane sulfonate, a competitive inhibitor of taurine transport. The initial and the maximal rates of [3H]taurine uptake were significantly higher in SL than in EDL. Denervation dramatically accelerated the initial and the maximal rates of the transport in EDL, whereas it significantly reduced those in SL. In contrast, the electrical stimulation of sciatic nerve accelerated the uptake of taurine by EDL and SL of the control but not of the curare-treated rats. These results suggest that transport of taurine into rat skeletal muscles is regulated differently by neural information and by muscular activity, and that the regulation is dependent on the muscle phenotype.