Hypotaurine Uptake Research Articles

Role of Hypotaurine in Protection against UVA-Induced Damage in Keratinocytes.

Photoageing and skin cancer are major causes of morbidity and are a high cost to society. Interest in the development of photoprotective agents for inclusion in topical cosmetic and sunscreen products is profound. Recently, amino acids with a sulfinic group, notably hypotaurine, have been included as ingredients in cosmetic preparations. However, the mechanism of action of hypotaurine as a possible anti-aging agent is unknown, despite its use as a free radical scavenger. To address this issue, we investigated hypotaurine uptake in a human keratinocyte model and examined its effect on UVR-induced cytotoxicity. Hypotaurine was taken up by keratinocytes in a time- and concentration-dependent manner, with levels remaining significantly above baseline 48h after washout. A cytoprotective effect of pre-incubation with 2.5-5mMhypotaurine was shown as indicated by increased cell viability when keratinocytes were irradiated with UVA at 5 or 10 Jcm-2 , with the level of hypotaurine also significantly reduced. These findings indicate a potential cytoprotective effect of hypotaurine against the deleterious effects of UVA irradiation. This provides support for further studies to evaluate the potential photoprotective benefits of hypotaurine supplementation of topical cosmetic and sunscreen products.

Hypotaurine Is a Substrate of GABA Transporter Family Members GAT2/Slc6a13 and TAUT/Slc6a6.

Hypotaurine is a precursor of taurine and a physiological antioxidant that circulates in adult and fetal plasma. The purpose of the present study was to clarify whether hypotaurine is a substrate of Slc6a/gamma-aminobutyric acid (GABA) transporter family members. Radiolabeled hypotaurine was synthesized from radiolabeled cysteamine and 2-aminoethanethiol dioxygenase. The uptakes of [3H]GABA, [3H]taurine, and [14C]hypotaurine by HEK293 cells expressing mouse GAT1/Slc6a1, TAUT/Slc6a6, GAT3/Slc6a11, BGT1/Slc6a12, and GAT2/Slc6a13 were measured. TAUT and GAT2 showed strong [14C]hypotaurine uptake activity, while BGT1 showed moderate activity, and GAT1 and GAT3 showed slight but significant activity. Mouse TAUT and GAT2 both showed Michaelis constants of 11 µM for hypotaurine uptake. GAT2-expressing cells pretreated with hypotaurine showed resistance to H2O2-induced oxidative stress. These results suggest that under physiological conditions, TAUT and GAT2 would be major contributors to hypotaurine transfer across the plasma membrane, and that uptake of hypotaurine via GAT2 contributes to the cellular resistance to oxidative stress.

Fetal growth retardation and lack of hypotaurine in ezrin knockout mice.

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez−/−) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis–time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez−/− mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

Taurine and Hypotaurine Content of Human Leukocytes

Taurine (T) was reported to be at high concentrations in human leukocytes. It was proposed that T is a scavenger for chlorinated oxidants produced by the myeloperoxidase system of monocytes and neutrophils. Hypotaurine (HT) would be a more effective scavenger, and HT could also detoxify products of bromide or iodide oxidation produced by the eosinophil peroxidase system. Methods previously used to measure T in leukocytes might oxidize HT to T or fail to separate T and HT. Therefore, we examined T and HT content, uptake, and biosynthesis in isolated blood cells and cultured tumor cells derived from hematopoietic/lymphoid cells. Platelets and all leukocytes including monocytes, lymphocytes, neutrophils, and eosinophils had high T levels (10-20 mM), and all except eosinophils had substantial HT levels (0.3-1 mM). Intracellular levels were 500-times higher than in plasma. Erythrocytes were the only blood cells with low levels of both T and HT. Tumor cells from lymphoid (CCRF-CEM) and myeloid (HL-60, K-562, RWLeu4, HEL) lineages took up and concentrated T and HT from the bovine calf serum in the culture medium, and intracellular levels were similar to those in leukocytes. When cells were cultured in HT-supplemented media, HT almost completely replaced T, and HT was not converted to T. Levels of T were not raised by culturing cells with possible precursors, but HT levels were raised when cysteine sulfinic acid was present. Washed tumor cells took up T and HT by way of a beta-amino acid transport system, but uptake by leukocytes was very low. Therefore, leukocytes may acquire T and HT by active uptake rather than biosynthesis, and uptake may be completed during differentiation in the bone marrow. Though HT is low relative to T, HT levels may be sufficient to protect leukocytes from toxic oxidants.

Taurine and hypotaurine transport by a single system in cultured neuroblastoma cells.

The kinetics of mutual inhibition of taurine and hypotaurine uptake were studied using neuroblastoma C1300 cells as neuronal model. Hypotaurine and GABA inhibited taurine uptake competitively, increasing the apparent Km. High-affinity uptake of hypotaurine was completely abolished and the low-affinity component competitively inhibited by taurine. GABA affected noncompetitively low-affinity hypotaurine uptake, whereas the effect on high-affinity uptake was competitive, with an increase in the apparent Km. All structural analogues tested inhibited taurine and hypotaurine uptakes similarly. The most potent inhibitors were beta-alanine and 2-guanidinoethanesulphonic acid. The mutual inhibition and similar specificity profiles of taurine and hypotaurine uptakes showed that these amino acids employ a single transport system in neuroblastoma cells. Competitive inhibition by GABA of the high-affinity uptake of taurine and hypotaurine further suggests that also GABA uses the same carrier system.

Modification of taurine and hypotaurine uptake systems in cultured primary astrocytes by serum-free medium and dibutyryl cyclic AMP treatment

The characteristics of taurine and hypotaurine uptake were studied during astrocyte maturation in cultures grown in normal, serum-containing medium or in serum-free medium in the presence of 0.1 mmol/l dibutyryl cyclic AMP (dBcAMP). The uptake of both amino acids consisted of one saturable high-affinity component in both control and treated cultures. The dBcAMP treatment produced no marked modification of the transport systems. In the treated cultures the kinetic parameters of taurine and hypotaurine uptake remained unaltered during maturation. In the control cells the transport constant and maximal velocity of taurine uptake were greater in 21-day-old than in 16-day-old cultures, while the changes in hypotaurine transport were the opposite. The uptakes were strictly sodium-dependent and also considerably decreased when potassium ions were omitted from incubation medium. The uptake of both amino acids was affected more by potassium omission in the dBcAMP-treated than in the control cultures. Also the results with metabolic poisons suggest that physiological ion gradients sustained by an active Na+, K+-pump are essential for the normal uptake of taurine and hypotaurine.

Taurine and hypotaurine transport in neuroblastoma cells: effects of cations

The cation requirements of [(3)H]taurine and [(35)S]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 Ca(2+) or Mg(2+) 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.

Comparison of taurine, hypotaurine and β-alanine uptake in brain synaptosomal preparations from developing and adult mouse

The properties of taurine, hypotaurine and β-alanine uptakes were compared in brain synaptosomal preparations from 6-day-old and adult mice. The uptakes of these structurally related amino acids resembled each other, being concentrative, sodium-dependent and inhibited by the same analogues. The absolute sodium requirement of uptake was already evident in developing brain. The affinity of the lowaffinity uptake for taurine was higher in immature than in adult brain. Both affinity and maximal velocity increased in hypotaurine uptake during development, whereas in β-alanine uptake only the maximal velocity did so. The efficient synaptosomal taurine and hypotaurine transport systems in immature brain could contribute to the high taurine level in developing brain.

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.

Hypotaurine transport in mouse brain synaptosomal preparations.

The transport of hypotaurine, the precursor of the probable neuromodulator taurine, was investigated using mouse brain synaptosomal preparations. Hypotaurine uptake was concentrative, energy- and sodium-dependent and strongly inhibited by GABA, L-DABA (L-2,4-diaminobutyric acid) and beta-alanine, suggesting interactions with GABA uptake systems. The uptake consisted of only one saturable transport component both in adult and in 6-day-old brain, being more efficient in the latter.

Effects of cations on taurine, hypotaurine, and GABA uptake in mouse brain slices.

The effects of cations on taurine, hypotaurine and GABA uptake were studied in mouse brain slices under identical experimental conditions. The uptakes were all strictly sodium-dependent. The omission or excess of K+ inhibited similarly taurine, hypotaurine and GABA uptake. The effects of omission of Ca2+ or Mg2+ were less pronounced. In both normal-sodium and low-sodium media all uptakes were saturable, consisting of both low- and high-affinity transport components. The Km constants for both low- and high-affinity transport components of hypotaurine and GABA increased in low-sodium medium, suggesting that sodium ions are necessary for their attachment to possible carrier sites in plasma membranes. In the case of taurine, however, the translation rate rather than the affinity of carrier sites was affected in Na+-free media. More than two sodium ions may be involved in the transport of one hypotaurine and one GABA molecule, whereas the coupling ratio between sodium and taurine was at least three. In its cation dependence hypotaurine uptake thus resembled more GABA uptake than taurine uptake.

Hypotaurine uptake in mouse brain slices.

Hypotaurine, 2-aminoethanesulfinic acid, an immediate precursor in taurine biosynthesis (3), is converted to taurine also in brain tissue (10). Hypotaurine concentration in the rat brain is 0.07 mmole/kg fresh weight, i.e. higher than that of the biogenic amine precursors, phenylalanine and tryptophan (21). Taurine transport has been studied in different nervous tissue preparations (16,19) and its penetration into brain tissue has been found to be very slow both in vivo (18) and in vitro (8) when compared to the putative amino acid transmitters. Now we have characterized the properties of hypotaurine by mouse brain slices in vitro. The uptake was found to be strikingly fast and efficient, generating exceptionally high concentration gradients and consisting of two sodium-dependent high-affinity and low-affinity transport systems.

Cation dependence of hypotaurine uptake in mouse brain slices.

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 Km 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.

Hypotaurine uptake by brain slices from adult and 8-day-old mice.

Uptake of [35S]hypotaurine by brain slices prepared from adult and 8-day-old mice was studied at varying temperatures, under O2 and N2 atmospheres, and in the presence of metabolic inhibitors and varying concentrations of hypotaurine in the incubation medium. The tissue/medium concentration gradients generated were exceptionally high for an amino acid. Hypotaurine uptake was energy- and temperature-dependent, more strictly in adult mice. Uptake was saturable, containing a high-affinity and a low-affinity component. The estimated transport constants for the high-affinity uptake of hypotaurine (8-day-old mice, 17.2 mumol/liter; adults, 35.3 mumol/liter) were of the same order of magnitude as the reported transport constants of putative amino acid transmitters, but the total transport capacity appears to be greatest for hypotaurine.