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.     

Alterations in hepatic metabolism of sulfur-containing amino acids induced by ethanol in rats

Summary.  Alterations in hepatic metabolism of S-amino acids were monitored over one week in male rats treated with a single dose of ethanol (3 g/kg, ip). Methionine and S-adenosylhomocysteine concentrations were increased rapidly, but S-adenosylmethionine, cysteine, and glutathione (GSH) decreased following ethanol administration. Activities of methionine adenosyltransferase, cystathionine γ-lyase and cystathionine β-synthase were all inhibited. γ-Glutamylcysteine synthetase activity was increased from t = 8 hr, but GSH level did not return to control for 24 hr. Hepatic hypotaurine and taurine levels were elevated immediately, but reduced below control in 18 hr. Changes in serum and urinary taurine levels were consistent with results observed in liver. Cysteine dioxygenase activity was increased rapidly, but declined from t = 24 hr. The results show that a single dose of ethanol induces profound changes in hepatic S-amino acid metabolism, some of which persist for several days. Ethanol not only inhibits the cysteine synthesis but suppresses the cysteine availability further by enhancing its irreversible catabolism to taurine, which would play a significant role in the depletion of hepatic GSH. Received April 26, 2002 Accepted June 12, 2002 Published online October 14, 2002 Authors' address: Young C. Kim, Ph.D., Professor of Toxicology, College of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Ku, Seoul, Korea, Fax: +82-2-872-1795, E-mail: youckim@snu.ac.kr Abbreviations: CβS, cystathionine β-synthase; CDC, cysteine sulfinate decarboxylase; CDO, cysteine dioxygenase; CγL, cystathionine γ-lyase; GCS, γ-Glutamylcysteine synthetase; GSH, glutathione; MAT, methionine adenosyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.     

Hypotaurine disproportionation reaction: Identification of products

Summary Hypotaurine, concentrated under reduced pressure in HCl solution, partially (30–40%) degrades into taurine (about 30%), 2-aminoethyl-2-aminoethanethiolsulfonate (about 10%) and ethanolamine. The degradation products were identified using LC/APCI-MS, NMR, amino acid analysis and various chromatographics. The identities were confirmed by comparing the HPLS, MS and NMR characteristics of authentic compounds. One of the degradation processes during concentration of HCl solution of hypotaurine is therefore a disproportionation reaction which can interfere with the experimental results, when studying hypotaurine in biological systems.Abbreviations LC/APCI-MS liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry - TLC thin layer chromatography - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

In Vitro Studies on the Putative Function of N-acetylaspartate as an Osmoregulator

Efflux and tissue content of N-acetylaspartate (NAA) and amino acids were evaluated from cultured and acutely prepared hippocampal slices in response to changes in osmolarity. The osmoregulator taurine, but not NAA, was lost from both types of slices after moderate reductions in extracellular osmolarity (−60 mOsm) for 10–48 h. Hypoosmotic shock (−166 mOsm) for 5 min resulted in unselective efflux of several amino acids from acutely prepared slices. Notably, the efflux of taurine, but not NAA, was prominent also after the shock. Efflux of NAA was markedly enhanced by NMDA and high K⁺, in particular after the stimulation period. The high K⁺-mediated efflux was decreased by high extracellular osmolarity and a NMDA-receptor antagonist. The results indicate that NAA efflux can be induced by a sudden non-physiological decrease in extracellular osmolarity but not by prolonged more moderate changes in osmolarity. The mechanisms behind the efflux of NAA by high K⁺ are complex and may involve both swelling and activation of NMDA-receptors.     

Simple high-performance liquid chromatographic method for assaying cysteinesulfinic acid decarboxylase activity in rat tissue

A simple method is described for determining cysteinesulfinic acid decarboxylase activity in rat tissue. Enzyme preparations from the liver, kidney and brain were incubated with cysteinesulfinic acid substrate in the presence of pyridoxal 5-phosphate. The enzyme product, hypotaurine, was derivatized with o-phthalaldehyde and separated by reversed-phase high-performance liquid chromatography (Capsel Pack AG 120A C₁₈ column) using a mobile phase of acetonitrile–water (20:80, v/v) containing 50 mM sodium phosphate buffer (pH 6.0) and detected using a fluorometer (excitation at 360 nm and emission at 455 nm). The method described is reproducible and sensitive enough to determine the activity of cysteinesulfinic acid decarboxylase activity in the liver, kidney and brain. This assay was subsequently used to evaluate the effect of dietary proteins whose sulfur amino acid contents differ. Consistent with reported data, compared to casein and whole egg protein, a dietary protein low in sulfur amino acid (soybean protein) increased cysteinesulfinic acid decarboxylase activity in the liver and kidney. This method is therefore applicable to studies on the dietary regulation of cysteinesulfinic acid decarboxylase in rat tissue.     

Antioxidant Properties of Sulfinates: Protective Effect of Hypotaurine on Peroxynitrite-Dependent Damage

It has been proposed that hypotaurine may function as an antioxidant in vivo. We investigated whether this compound can act as protective agent able to prevent damage from peroxynitrite, a strong oxidizing and nitrating agent that reacts with several biomolecules. The results showed that the compound efficiently protects tyrosine against nitration, alpha1-antiproteinase against inactivation, and human low-density lipoprotein against modification by peroxynitrite. Hypotaurine is also highly effective in inhibiting peroxynitrite-mediated nitration of tyrosine in the presence of added bicarbonate. This result suggests that hypotaurine could play an important role as protective agent under physiological conditions. Moreover, it was found that cysteine sulfinic acid, but not taurine, possesses protective properties against peroxynitrite-dependent damage similar to hypotaurine. These findings indicate that the protective effects exerted by these compounds may be attributable to the presence of the sulfinic group oxidizable into sulfonate by scavenging peroxynitrite and/or its derived species.     

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1O2) we found that azide, usually used as 1O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed.     

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by ¹H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-¹³C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with ¹³C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-¹³C]cysteine. The presence of [1-¹³C]hypotaurine and [1-¹³C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-¹³C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-¹³C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.     

Precursors of taurine in female genital tract: Effects on developmental capacity of bovine embryo producedin vitro

Summary Two precursors of taurine have been studied: cysteamine and hypotaurine. Cysteamine has been quantified in genital secretions and found in follicular fluids of all species tested. On the contrary cysteamine was not detected (or traces) in tubal fluids of the same species. Addition of 50, 100 or 250M of cysteamine to the maturation medium used in the culturing of bovine oocytes did not improve the cleavage rate nor the embryo's developmental potentialin vitro. Furthermore, at 250M, cysteamine seems to be toxic to the embryo. Addition of 0.5–1 mM hypotaurine to the bovine embryo culture medium improved significantly blastocyst production and quality. The respective roles of these 2 taurine precursors on maturation and embryo development are discussed.     

Production of hypotaurine, taurine and sulfate in rats and mice injected with L-cysteinesulfinate

Summary. We studied in vivo production of taurine, hypotaurine and sulfate following subcutaneous administration of L-cysteinesulfinate (CSA) to rats and mice. When 5.0 mmol/kg of body weight of CSA was injected to rats, increased urinary excretions of taurine, hypotaurine and sulfate in 24 h urine were 617, 52 and 1,767 μmol/kg, respectively. From these results together with our previous data, sulfate production was calculated to be 1.6 times greater than taurine production. Increased contents (μmol/g of wet tissue) over the control of taurine and hypotaurine in mouse tissues at 60 min after the injection of 5.0 mmol/kg body weight of CSA were: liver, 3.5 and 9.9; kidney, 0.3 and 5.2; heart, 3.7 and 0.2; blood plasma, 0.4 and 0.2, respectively. Upon loading of hypotaurine or taurine, tissue contents of these amino acids in liver and kidney increased greatly. Our results indicate that liver is the most active tissue for taurine production, followed by kidney, and that external CSA, hypotaurine and taurine are easily taken up by these tissues.