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.     

Hypotaurine transport in brain slices

Hypotaurine uptake was compared to taurine and GABA uptakes in brain slices under identical experimental conditions. The slices effectively concentrated both hypotaurine and GABA from the medium, whereas taurine was taken up more slowly. The uptakes of these three structurally related amino acids were all saturable, consisting of one low-and one high-affinity transport component. The kinetic parameters of hypotaurine uptake were of the same order of magnitude as those of GABA uptake. All uptake systems were sensitive to temperature, metabolic poisons, and sodium omission. Hypotaurine uptake was inhibited by GABA,l-2,4-diaminobutyric acid (l-DABA), cysteic acid, and -alanine, but not by taurine. Taurine uptake was strongly reduced by hypotaurine, -alanine, andl-DABA, as well as by GABA, whereas GABA uptake was affected only by cystamine,l-DABA, and nipecotic acid.The uptake processes of hypotaurine, taurine, and GABA were thus fairly similar and showed properties characteristic for neurotransmitter uptake. Hypotaurine uptake resembled more GABA than taurine uptake. The present inhibition studies suggest that there may exist only one common two-component transport system for these three amino acids.     

Release of preloaded taurine and hypotaurine from astrocytes in primary culture: Stimulation by calcium-free media

The spontaneous and stimulated release of taurine and hypotaurine from astrocytes in primary cultures were investigated. Spontaneous efflux was slow, less than one half of preloaded labeled taurine and hypotaurine still remaining in the cells after a 60-min efflux period. The release processes of both amino acids were in principle similar. No homo- or heteroexchange with extracellularly added taurine, hypotaurine or GABA could be detected, and depolarizing potassium concentrations failed to stimulate taurine or hypotaurine release. On the other hand, omission of calcium ions from medium increased efflux of taurine and hypotaurine about three- and twofold, respectively, in both high-K⁺ and normal-K⁺ media.     

Glial uptake system of GABA distinct from that of taurine in the bullfrog sympathetic ganglia

The kinetics and specificity of GABA and taurine uptake were studied in the bullfrog sympathetic ganglia. GABA uptake system consisted of simple saturable component and taurine uptake system consisted of two saturable components exclusive of non-saturable influx. Taurine unaffected GABA uptake while GABA inhibited taurine uptake competitively with theK _i/K_m ratio of 38. GABA (5.14 M) uptake was inhibited by -aminovaleric acid and slightly by 2,4-diaminobutyric acid (5 mM, each) among ten structural analogs. Taurine uptake under high-affinity conditions was most strongly suppressed by hypotaurine and -alanine competitively with theK _i/K_m ratio of 1.0 and 1.9, respectively. Autoradiography showed that glial cells were heavily labeled by both [³H]GABA and [³H]taurine. These results suggest that GABA is transported by a highly specific carrier system distinct from the taurine carrier and that taurine, hypotaurine, and -alanine may share the same high-affinity carrier system in the glial cells of the bullfrog sympathetic ganglia.     

Cation Dependence of Hypotaurine Uptake in Mouse Brain Slices

Abstract: Mouse brain slices take up hypotaurine (2-aminoethanesulphinic acid) from medium by means of two concentrative low- and high-affinity transport systems. [³⁵S]Hypotaurine uptake by the slices was significantly reduced in the absence of external potassium, calcium, or magnesium ions. An excess of potassium ions also inhibited hypotaurine uptake by one-half. Uptake was almost completely abolished on removal of sodium ions. The K _m constants for both low- and high-affinity transport components increased in a low-sodium medium, suggesting that sodium ions are required when hypotaurine is attached to its possible carrier sites in plasma membranes. Sodium ions also mimicked allosteric effectors of hypotaurine transport, showing positive cooperativity. More than two sodium ions may be involved in the transport of one hypotaurine molecule across the cell membrane. The calculated activation energies of transport were fairly similar in normal and sodium-deficient media and thus sodium ions may not participate in the activation mechanisms of the transport. With respect to cation dependence, hypotaurine transport in brain slices exhibits features characteristic of neurotransmitter amino acids.     

Taurine and β-alanine uptake in primary astrocytes differentiating in culture: Effects of ions

The effects of ions on taurine and -alanine uptake were studied in astrocytes during cellular differentiation in primary cultures. The uptakes were strictly Na⁺-dependent and also inhibited by the omission of K⁺ and in the presence of ouabain suggesting that their transport is fuelled mainly by these cation gradients. Two sodium ions were associated in the transport of one taurine and -alanine molecule across cell membranes. A reduction in Cl^– concentration also markedly inhibited the uptake of both amino acids, indicating that this anion is of importance in the transport processes. The similar ion dependency profiles of taurine and -alanine uptake corroborate the assumption that the uptake of these amino acids in astrocytes is mediated by the same carrier. In Na⁺- and K⁺-free media both taurine and -alanine uptakes were reduced significantly more in 14-day-old or older than in 7-day-old cultures. No significant changes occurred in the coupling ratio between Na⁺ and taurine or -alanine as a function of spontaneous cellular differentiation or upon dBcAMP treatment. These results suggest that the uptake systems of these structurally related amino acids in astrocytes have reached a relatively high degree of functional maturity by two weeks in culture.     

Mutual interactions in the transport of taurine,hypotaurine, and GABA in brain slices

The mutual interactions and the effects of GABA on the saturable transport components of taurine and hypotaurine were investigated with mouse brain slices. The low-affinity taurine transport was competitively inhibited by both hypotaurine and GABA. Hypotaurine did not alter the kinetic parameters of high-affinity taurine uptake, whereas there occurred some stimulation with GABA, possibly by heteroexchange. Taurine had no significant effects on high-affinity hypotaurine uptake, whereas the low-affinity component was reduced by both taurine and GABA, GABA strongly interfered with the high-affinity hypotaurine uptake, being the preferred substrate in simultaneous uptake experiments. The results confirm that taurine, hypotaurine, and GABA are transported into brain slices by only one two-component system with affinities highest for GABA and lowest for taurine.     

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.     

High-affinity uptake of taurine and β-alanine in primary cultures of rat astrocytes

The kinetics and specificity of taurine and -alanine uptake were studied in primary cultures of rat astrocytes under identical experimental conditions. The uptake consisted of nonsaturable penetration and saturable high-affinity transport that was strictly sodium dependent. The cells accumulated taurine more effectively than -alanine, both the affinity and uptake capacity being greater for taurine. Taurine uptake was competitively inhibited by -alanine and GABA, the former being more potent. Also, hypotaurine and 2-guanidinoethanesulphonic acid strongly reduced taurine uptake, but L-2,4-diaminobutyric acid had no significant effect. -Alanine uptake was also competitively inhibited by GABA, but the most potent inhibitors were hypotaurine and 2-guanidinoethanesulphonic acid.l-2,4-Diaminobutyric acid was moderately active. The uptake systems for taurine and -alanine were thus in principle similar, and they exhibited certain characteristics typical for a neurotransmitter amino acid. The inhibition studies further suggest the existence of only one common transport system for taurine, -alanine, and GABA in cultured primary astrocytes. The same uptake system may also be used for hypotaurine.     

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.     

Molecular Characterization and In Situ Localization of a Mouse Retinal Taurine Transporter

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na⁺- and Cl^?-dependent [³H]taurine transport activity in Xenopus laevis oocytes that saturated with an average K_m of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl^? was varied, a single saturation process with an average apparent equilibrium constant (K_Cl?) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na⁺ was varied (apparent equilibrium constant, K_Na+～54.8 mM). Analyses of the Na⁺- and Cl^?-concentration dependence data suggest that at least two Na⁺ and one Cl^? are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β-alanine > l -diaminopropionic acid > guanidinoethane sulfonate > β-guanidinopropionic acid > chloroquine > γ-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.     

l-Carnitine uptake by mouse brain synaptosomal preparations: Competitive inhibition by GABA

The uptake ofl-carnitine was characterized in mouse brain synaptosomal preparations, with an emphasis on mutual interactions with GABA uptake systems. The uptake consisted of nonsaturable diffusion and one saturable energy- and sodium-dependent component. GABA,l-DABA and nipecotate were strong and hypotaurine and homotaurine moderate inhibitors of the uptake. The inhibition by GABA was shown to be competitive. GABA uptake contained two saturable transport components, high- and low-affinity. It was most strongly inhibited by nipecotate andl-DABA, but also by carnitine and hypotaurine. The high-affinity uptake of GABA was competitively inhibited by carnitine, but the inhibition of the low-affinity uptake of GABA was of the mixed type. The results suggest that GABA and carnitine share the same carrier system at synaptosomal membranes. However, GABA is the preferred substrate and the carnitine concentrations which significantly inhibited GABA uptake exceed the physiological carnitine levels in vivo.     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

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.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.     

Transport of valproate and its effects on GABA uptake in astroglial primary culture

The antiepileptic drug Na⁺-valproate (VPA) is a broadspectrum anticonvulsant. It has been proposed to be involved in the inhibitory mechanisms of GABA-ergic systems. In this study, transport of the drug and possible influence on the GABA uptake were investigated in primary astroglial cell cultures from newborn rat cerebral cortex. The results show a Na⁺ and K⁺ independent high affinity uptake for VPA, withK _m andV _max not significantly different from those observed for the GABA uptake. In the presence of the drug, the K_m-value of the GABA uptake increased. The GABA uptake inhibitors guvacine, (RS)-Cis-4-OH-nipecotic acid and 4,5,6,7-tetrahydroisoxazolo (4,5-c) pyridin-3-ol (THPO) did not influence upon the uptake of VPA, suggesting a transport mechanism for the drug, separated from the GABA uptake carrier.     

Sodium-dependent uptake of taurine in encapsulated Staphylococcus aureus strain M

A novel uptake system for the unusual sulfonated amino acid taurine was discovered in the prokaryote, encapsulated Staphylococcus aureus strain M. This strain has been shown previously to contain taurine in its capsular polysaccharide. Taurine uptake by whole cells incubated in buffer showed a saturable dependency upon Na⁺ and taurine uptake was itself a saturable process, stimulated by glucose, and markedly affected by temperature. No evidence was found for the inducibility of taurine uptake. In the presence of 10 mM NaCl Lineweaver-Burk plots revealed a K_m of 42 μM and V_max of 4.6 nmol/min per mg dry weight for taurine uptake at 37°C. Increasing concentrations of Na⁺ decreased the K_m of the system and appeared to increase the V_max. Of various other cations tested only Li⁺ supported marked taurine uptake. Excess unlabelled taurine did not cause efflux of radioactivity taken up. Taurine was taken up into cold trichloroacetic acid-soluble material and did not chromatograph as taurine, indicating rapid metabolism during or closely following uptake. Taurine uptake appeared to occur via a highly specific system because amino acids representing the major known groups of amino acid transport systems in S. aureus did not inhibit taurine uptake, and uptake was only slightly diminished by the structurally closely related compounds hypotaurine and 3-amino-1-propane sulfonic acid. Sulfhydryl group reagents, electron transport inhibitors, an uncoupler and inhibitors of Na⁺-linked transport processes inhibited taurine uptake. A variety of other metabolic inhibitors had little effect on taurine uptake.     

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the β-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na⁺ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K⁺ or Li⁺ do not increase taurine accumulation more than Na⁺-free mannitol, except that the combination of external K⁺ and Na¹ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN⁻ and NO₃⁻) or less permeant (SO₄²⁻), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl⁻ stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO₃ and LiSO₄. Internal LiCl, regardless of the final Na⁺ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO₃ or LiSO₄ with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl⁻ in taurine uptake in addition to Na⁺ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H⁺ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na⁺/H⁺ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na⁺ concentrations. Taurine uptake is blocked only by other β-amino acids and in a competitive fashion. d-glucose and p-aminohippurate at high concentrations (> 10⁻³ M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of d-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal β-amino acid transport system in brush-border vesicles and indicate a role for external Cl in this uptake system.     

CHARACTERIZATION OF TAURINE UPTAKE BY NEURONAL AND GLIAL CELLS IN CULTURE

Uptake of [³⁵S]taurine was studied in parallel on glial and neuronal cells maintained in continuous culture, including transformed neuronal cells. Both glial and neuronal taurine uptake systems were concentrative, highly sodium-dependent and inhibited by closely related structural analogues such as hypotaurine, β-alanine and GABA. Strychnine was found to be a potent inhibitor of taurine uptake, especially in the glial cells, while parachloromercuriphenylsulphonate was more efficient on the neuronal clones. In contrast with uptake by neuroblastoma cells, the glial transport was dependent on the presence of calcium in the incubation medium.