Effects of sulfur containing amino acids on iron and nitric oxide stimulated catecholamine oxidation

Summary.  Taurine is a free amino acid found in high concentrations in tissues containing catecholamines. The ability of taurine and its metabolic precursors to inhibit or stimulate catecholamine oxidation and subsequent quinone formation was examined. Ferric chloride was used as the catalyzing agent to stimulate L-dopa or norepinephrine oxidation and NO donors were also examined for their actions to stimulate quinone formation. Taurine attenuated iron-stimulated quinone formation from catecholamines suggesting that it may function as an endogenous antioxidant. Several other sulfur-containing amino acids (homocysteic acid, cysteine sulfinic acid and SAM) were found to inhibit catecholamine oxidation. Among other amino acids tested, homocysteine had biphasic effects; attenuating L-dopa oxidation catalyzed by ferric chloride and potentiating norepinephrine's oxidation catalyzed by both ferric chloride and sodium nitroprusside (SNP). Homotaurine and homocysteine (1 or 10 mM) greatly stimulated SNP-induced norepinephrine oxidation. Homotaurine potentiated quinone formation in the presence of ferric iron and this effect was attenuated by desferroxamine. In order to exclude a possible NO/iron interaction in SNP's oxidizing action, SIN-1 chloride, a specific NO-donor, was tested as an oxidizing agent. The failure of desferroxamine or taurine to attenuate SIN-1 oxidation of norepinephrine suggests that peroxynitrite-mediated oxidation was likely the dominant mechanism. Our results show that endogenous sulfur containing amino acids, like taurine, could serve a protective role to reduce cellular damage associated with both NO and metal-stimulated catecholamine oxidation. Received August 20, 2001 Accepted October 10, 2001     

Ethyl Pyruvate Inhibits Peroxynitrite-induced DNA Damage and Hydroxyl Radical Generation: Implications for Neuroprotection

Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite elicited cytotoxicity. Incubation of φX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time- dependent manner. The presence of EP (0.5–10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent fashion. The consumption of oxygen induced by 250 μM SIN-1 was found to be decreased in the presence of EP (0.5–10 mM), indicating that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5–10 mM). EPR spectroscopy in combination with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite, and that EP at 0.5–10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation.     

Effects of zinc ex vivo and intracellular zinc chelator in vivo on taurine uptake in goldfish retina

Taurine and zinc exert neurotrophic effects. Zinc modulates Na⁺/Cl⁻-dependent transporters. This study examined the effect of zinc (ZnSO₄) ex vivo and zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) in vivo on [³H]taurine transport in goldfish retina. The effect of TPEN in vivo on taurine and zinc levels was determined. Isolated cells were incubated in Ringer with zinc (0.1–100 μM). Taurine transport was done with taurine (0.001–1 mM) and 50 nM [³H]taurine. Zinc (100 μM) noncompetitively inhibited taurine transport. TPEN was administered intraocularly and retinas extracted 3, 5 and 10 days later. Taurine was determined by HPLC (nmol/mg protein) and zinc by spectrophotometry ICP (mg/mg protein). Taurine and zinc levels decreased at 3 days and increased at 10 days after TPEN administration. At 10 days after intraocular TPEN, taurine transport affinity increased (K _s = 0.018 ± 0.006 vs. 0.028 ± 0.008 mM). Apparently, zinc deficiency affects the taurine–zinc complex and taurine availability. The increased taurine uptake affinity by TPEN was possibly associated with a response to maximize retinal taurine content at low zinc concentration.     

Taurine and Zinc Modulate Outgrowth from Goldfish Retinal Explants

Taurine and zinc, highly concentrated in the retina, possess similar properties in this structure, such as neuro-protection, membrane stabilization, influencing regeneration, and modulating development, maybe by acting in parallel or as interacting agents. We previously demonstrated that there are some correlations between taurine and zinc levels in hippocampus, dentate gyrus and retina of the developing rat. In the present study we evaluate the possible effects of taurine and zinc on outgrowth from goldfish retinal explants. The optic nerve was crushed 10 days before plating and culturing retinal explants in Leibovitz medium with 10% fetal calf serum and gentamicin. Neurites were measured with SigmaScanPro after 5 days in culture. Taurine (HPLC) and zinc (ICP) concentrations were determined in the retina between 1 and 180 days after crushing the optic nerve. Zinc sulfate (0.01–100 μM), N,N, N′,N′-tetrakis (pyridylmethyl) ethylenediamine (TPEN, 0.1–5 nM) and diethylenetriamine penta-acetic acid (DTPA, 10–300 μM), intracellular and extracellular zinc chelators, respectively, were added to the medium. TPEN was also injected intraocular (0.1 nM). Combinations of them were added with taurine (1–16 mM). Taurine concentrations were elevated in the retina 72 h after the crush, but were normalized by 180 days, those of zinc increased at 24 h, preceding the increase of taurine. The axonal transport of [³H]taurine from the optic tectum to the retina was not affected in fish with or without crush of the optic nerve at early periods after the injection, indicating an increase of it post-lesion. Zinc sulfate produced a bell-shaped concentration dependency on in vitro outgrowth, with stimulation at 0.05 μM, and inhibition at higher levels, also increased the effect of 4 mM taurine at 0.02 μM, but diminished it at higher concentrations in the medium. TPEN decreased outgrowth at 1 nM, but not at 0.5 nM, although the simultaneous presence of 4 mM taurine and 0.5 nM TPEN decreased outgrowth respecting the stimulation by taurine alone. The intraocular administration of TPEN decreased outgrowth in vitro, an effect counteracted by the addition of 4 mM taurine to the culture medium. DTPA decreased outgrowth from 10 μM in the medium. The present results indicate that an optimal zinc concentration is necessary for outgrowth of goldfish retinal explants and that, in zinc deficient retina, taurine could stimulate outgrowth. In addition, the observations of variations in tissue concentrations and of the effects of intraocular administration of TPEN indicate that these effects could occur in vivo. Special issue dedicated to Dr. Simo S. Oja     

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.     

Induction of SOS response in Salmonella typhimurium TA4107/pSK1002 by peroxynitrite-generating agent, N-morpholino sydnonimine

Salmonella typhimurium TA4107/pSK1002 strain was used to measure the SOS response induced by peroxynitrite. The parent strain TA4107 (oxydelta1[oxydelta(oxyR argH)1]) is sensitive to oxidative stress and the plasmid of pSK1002 carries a fused gene umuC'-'lacZ, in which umu and lacZ genes are involved in the induction of mutagenesis and beta-galactosidase activity, respectively. Therefore, the level of SOS response was monitored via beta-galactosidase activity. A bolus addition of authentic peroxynitrite (0.3-0.6 mM) increased about eight times the enzyme activity. In N-morpholino sydnonimine (SIN-1), which produces peroxynitrite from superoxide and nitric oxide generated through hydrolysis, addition of over 1mM SIN-1 induced four-five-fold activity. The SIN-1-induced SOS response was scarcely influenced by superoxide dismutase (SOD), catalase or a combination of both, removing the possibility of induction by superoxide, hydrogen peroxide and hydroxyl radical. Two types of peroxynitrite scavengers, mannitol (type I) and glutathione (type II), decreased the response. Mannitol showed a constant inhibition (70%) at a concentration up to 20 mM, exhibiting kinetics that are zero-order in mannitol and first-order in peroxynitrite. On the other hand, glutathione sharply reduced the response dependent on concentration up to 2 mM (90%), indicating second-order kinetics, first-order in both glutathione and peroxynitrite. Dihydrorhodamine (DHR)123, which traps peroxynitrite in a molar ratio of 1:1, efficiently inhibited the SOS response. These effects suggest that peroxynitrite, generated gradually from SIN-1, penetrates through the cell membrane, damages the DNA and induces the SOS response. This strain can thus, be used in screening of antioxidants against peroxynitrite-induced DNA damage in cells.     

Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions. Evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite

Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2).     

Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Peroxynitrite generated in arteries fromsuperoxide and nitric oxide (NO) may damage their function. Here, wecompare the effects of peroxynitrite and peroxynitrite/NO-generatingagents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP(S-nitroso-N-acetyl-penicillamine), SNP (sodiumnitroprusside), and NONOate (spermine NONOate) on pig coronary artery.Deendothelialized artery rings were pretreated with these agents andthen washed before examining their contractility. Pretreatment with allagents (200 µM) results in a decrease in the force of contraction inresponse to the sarco(endo)plasmic Ca²⁺ (SERCA) pumpinhibitor cyclopiazonic acid (CPA): SNAP > NONOate  peroxynitrite  SIN-1 > SNP. Pretreatment with SNAP,NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plussuperoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca²⁺transients produced in response to CPA. Pretreating isolated membranevesicles with peroxynitrite inhibits the Ca²⁺ uptake due tothe SERCA pump, with all the other agents being less effective. Thusperoxynitrite and NO both inhibit the CPA-induced contractions indeendothelialized artery rings, peroxynitrite by damage to the SERCApump and NO possibly by a step downstream from the increase incytosolic Ca²⁺.

     

The effect of taurine depletion on the contractile properties and fatigue in fast-twitch skeletal muscle of the mouse

Summary. Taurine as well as tauret (retinyliden taurine) levels were measured in locust Locusta migratoria compound eyes. HPLC measurements revealed relatively low taurine levels (1.9 ± 0.16 mM) in dark-adapted eyes. Glutamate, aspartate and glycine levels were 2.0 ± 0.2, 2.7 ± 0.4 and 3.0 ± 0.37 mM, respectively, while GABA was present only in trace amounts. After about 4 h of light adaptation at 1500–2000 lx, amino acid levels in the compound eye were as follows: taurine, 1.8 ± 0.17 mM; glutamate, no change at 2.1 ± 0.2 mM; aspartate sharply increased to 4.7 ± 0.7 mM; glycine slightly decreased to 2.8 ± 0.3 mM; and GABA trace levels. In the compound eye of locust Locusta migratoria, the existence of endogenous tauret in micro-molar range was established. In the dark, levels were several times higher compared with compound eye after light adaptation 1500 lx for 3 h, as estimated by TLC in combination with spectral measurements. Existence of tauret in compound eye is of special interest because in the compound eye, rhodopsin regeneration is based on photoregeneration.     

Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in φX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 μM SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.     

Sodium nitroprusside-induced seizure and taurine release from rat hippocampus

Summary. We have recently reported that the nitric oxide (NO) donor, sodium nitroprusside (SNP), induces seizures which are associated with an increase in the basal release of aspartate and glutamate from rat hippocampus (Kaku et al., 1998). In order to determine whether taurine release occurs with SNP-induced seizures, we examined the effects of NO-related compounds, i.e., the NO trapper, diethyldithiocarbamate (DETC), the superoxide radical scavenger, dithiothreitol (DTT), the xanthine oxidase inhibitor, oxypurinol and the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ), on SNP-induced seizures and in vivo taurine release from rat hippocampus using microdialysis. Perfusion with 0.5 mM SNP provoked seizures and significantly increased taurine release, with the increase in release occurring primarily during reperfusion with artificial cerebrospinal fluid lacking SNP. Perfusion with 5 mM DETC significantly abolished the SNP-induced seizures and reduced taurine release during and after perfusion with the drugs. Perfusion with 1 mM DTT significantly reduced both the frequency of the SNP-induced seizures and taurine release during and after perfusion with the drugs. Perfusion with 1 mM oxypurinol or 0.5 mM ODQ did not reduce the frequency of the SNP-induced seizures, but tended to decrease taurine release during and after perfusion with the drugs. These results demonstrate that SNP-induced seizures are triggered by an increase in both NO and peroxynitrite and are related to an increase in taurine release from rat hippocampus. Received January 25, 2000/Accepted January 31, 2000     

Nitric oxide is involved in taurine release in the mouse brain stem under normal and ischemic conditions

Summary. Nitric oxide (NO) has been shown to regulate neurotransmitter release in the brain; both inhibitory and excitatory effects have been seen. Taurine is essential for the development and survival of neural cells and protects them under cell-damaging conditions. In the brain stem, it regulates many vital functions such as cardiovascular control and arterial blood pressure. Now we studied the effects of the NO-generating compounds hydroxylamine (HA), S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) on the release of preloaded [³H]taurine under normal and ischemic conditions in slices prepared from the mouse brain stem from developing (7-day-old) to young adult (3-month-old) mice. In general, the effects of NO on the release were somewhat complex and difficult to explain, as expected from the multifunctional role of NO in the central nervous system. The basal initial release under normal conditions was enhanced by the NO donors 5 mM HA and 1.0 mM SNAP at both ages, but SNP was inhibitory in developing mice. The release was markedly enhanced by K⁺ stimulation. The effects of HA, SNAP and SNP on the basal release were not antagonized by the NO synthase inhibitor N^G-nitro-L-arginine (L-NNA, 1.0 mM), demonstrating that mechanisms other than NO synthesis are involved. Taurine release in developing mice in the presence of SNP was reduced by the inhibitor of soluble guanylate cyclase, 1H-(1,2,3)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), indicating the possible involvement of cGMP. In normoxia, N-methyl-D-aspartate (NMDA, 1.0 mM) enhanced the SNAP- and HA-evoked taurine release in developing mice and the HA-evoked release in adults. In ischemia, both K⁺ stimulation and NMDA potentiated the NO-induced release, particularly in the immature mice, probably without the involvement of the NO synthase or cGMP. The substantial release of taurine in the developing brain stem evoked by NO donors together with NMDA might represent signs of important mechanisms against excitotoxicity which protect the brain stem under cell-damaging conditions. Authors’ address: Prof. Pirjo Saransaari, Brain Research Center, Medical School University of Tampere, Tampere, FIN-3 3014, Finland     

Taurine stimulates proliferation and promotes neurogenesis of mouse adult cultured neural stem/progenitor cells

This study reports an effect of taurine (1-10 mM) increasing markedly (120%) the number of neural precursor cells (NPCs) from adult mouse subventricular zone, cultured as neurospheres. This effect is one of the highest reported for adult neural precursor cells. Taurine-containing cultures showed 73-120% more cells than controls, after 24 and 96 h in culture, respectively. Taurine effect is due to enhanced proliferation as assessed by BrdU incorporation assays. In taurine cultures BrdU incorporation was markedly higher than controls from 1.5 to 48 h, with the maximal difference found at 1.5 h. This effect of taurine reproduced at every passage with the same window time. Taurine effects are not mimicked by glycine, alanine or GABA. Clonal efficiency values of 3.6% for taurine cultures and 1.3% for control cultures suggest a taurine influence on both, progenitor and stem cells. Upon differentiation, the proportion of neurons in control and taurine cultures was 3.1% (±0.5) and 10.2% (±0.8), respectively. These results are relevant for taurine implication in brain development as well as in adult neurogenesis. Possible mechanisms underlying taurine effects on cell proliferation are discussed.     

Studies on the growth ofThiobacillus ferrooxidans

Summary Uranyl sulphate (0.2–0.9 mM) inhibited ferrous iron oxidation by growing cultures ofThiobacillus ferrooxidans. The addition of 5–100 mM uranium to the cultures caused immediate cessation of carbon dioxide fixation, rapid loss of viability and gradual depression of ferrous iron oxidation. Virtually no uranium was found in washed cells grown in the presence of subtoxic to toxic amounts of uranyl sulphate. Uranium-poisoned organisms appeared plasmolyzed in electron micrographs. Cultures tolerant to 5 mM UO₂ ²⁺ were develoepd by successive subculturing in increased uranium concentrations. The tolerance was maintained during subculturing in uranium-free medium. Frequency of mutants resistant to 1.0 and 1.5 mM UO₂ ²⁺ was 1 per 1.3×10⁶ and 1 per 9.0×10⁸, respectively. The frequency was increased in the presence of 15–150 mM nickel, zinc and manganese. In liquid cultures, bivalent cations and EDTA alleviated the toxicity of 2 mM uranyl sulphate.     

Protection against peroxynitrite-induced DNA damage by mesalamine: implications for anti-inflammation and anti-cancer activity

Mesalamine (5-aminosalicylic acid, 5-ASA) is known to be the first-line medication for treatment of patients with ulcerative colitis. Studies have demonstrated that ulcerative colitis patients treated with 5-ASA have an overall decrease in the risk of developing colorectal carcinoma. However, the mechanisms underlying 5-ASA-mediated anti-inflammatory and anti-cancer effects are yet to be elucidated. Because peroxynitrite has been critically involved in inflammatory stress and carcinogenesis, this study was undertaken to investigate the effects of 5-ASA in peroxynitrite-induced DNA strand breaks, an important event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA with the peroxynitrite generator 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration-dependent manner. The presence of 5-ASA at 0.1 and 1.0 mM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by SIN-1 was found to not be affected by 5-ASA at 0.1–50 mM, indicating that 5-ASA at these concentrations is not involved in the auto-oxidation of SIN-1 to form peroxynitrite. It is observed that 5-ASA at 0.1–1 mM showed considerable inhibition of peroxynitrite-mediated luminol chemiluminescence in a dose-dependent fashion, suggesting that 5-ASA is able to directly scavenge the peroxynitrite. Electron paramagnetic resonance (EPR) spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap resulting in the formation of DMPO-hydroxyl radical adduct from peroxynitrite, and 5-ASA only at higher concentration (1 mM) inhibited the hydroxyl radical adduct while shifting EPR spectra, indicating that 5-ASA at higher concentrations may generate a more stable free radical species rather than acting purely as a hydroxyl radical scavenger. Taken together, these studies demonstrate for the first time that 5-ASA can potently inhibit peroxynitrite-mediated DNA strand breakage, scavenge peroxynitrite, and affect peroxynitrite-mediated radical formation, which may be responsible, at least partially, for its anti-inflammatory and anti-cancer effects.     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000     

Neuroprotective Mechanism of Taurine due to Up-regulating Calpastatin and Down-regulating Calpain and Caspase-3 during Focal Cerebral Ischemia

Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover, taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.     

A new screening method to detect water-soluble antioxidants: acetaminophen (tylenol®) and other phenols react as antioxidants and destroy peroxynitrite-based luminol-dependent chemiluminescence

This study is based on a simple chemical interaction of peroxynitrite (ON O O⁻) and luminol, which produces blue light upon oxidation. Since peroxynitrite has a half-life of about 1 s, a drug known as linsidomine (SIN-1) is used as a peroxynitrite generator. Peroxynitrite can oxidize lipids, proteins and nucleic acids. Upon the stimulation of inflammation and/or infection, macrophages and neutrophils can be induced to produce large amounts of peroxynitrite, which can oxidize phenols and sulphhydryl-containing compounds. Therefore, phenols and sulphhydryls eliminate peroxynitrite. This is an example of the Yin–Yang hypothesis e.g. oxidation–reduction. Acetaminophen (Tylenol®) can inhibit fever and some types of pain without being a particularly effective anti-inflammatory. Since it is a phenol, it could act as a nitration target for peroxynitrite. Then peroxynitrite, the possible cause of pain and elevated temperature, might be destroyed in the reaction. Acetaminophen is a phenolic compound which produces a clear inhibitory dose–response curve with peroxynitrite in its range of clinical effectiveness. Whether acetaminophen actually works as we suggest is to be proven. Three different types of reaction could decrease the amount of peroxynitrite: (a) interference with base-catalysed opening of the SIN-1 molecule; (b) destruction of one or both substances needed to form it— superoxide and/or nitric oxide; when the SIN-1 degrades to superoxide and nitric oxide, the former may be destroyed by superoxide dismutase (SOD); (c) peroxynitrite may react directly with phenols (mono-, di-, tri- and tetraphenols), possibly by nitration. Nordihydroguaiaretic acid and 2-hydroxyestradiol (catechol estrogen) are potent inhibitors of luminol light emission. Epineprine, isoproterenol, pyrogallol, catechol and ascorbic acid (a classic antioxidant) are all inhibitors of luminol chemiluminescence. Isoproterenol, norepinephrine/and epinephrine first inhibit light but overall stimulate the light production. Initially, SIN-1 degrades to produce peroxynitrite, which reacts with luminol to produce blue light. If any of three catecholamines are present with the reaction that produces light, there is an initial inhibition of light production, and then a marked stimulation. A possible reason for this is that these catechols are oxidized and the metabolized phenol stimulates the production of light from luminol. Also, during oxidation of catecholamines superoxide is sometimes formed, which could stimulate production of peroxynitrite. This simple screening system is introduced to find useful antioxidants against peroxynitrite. © 1998 John Wiley & Sons, Ltd.     

Antioxidant enzyme inhibitors enhance peroxynitrite-induced cell death in U937 cells

Peroxynitrite, a potent physiological inorganic toxin, is known to play a critical role in cellular oxidative damage. The protective role of antioxidant enzymes against peroxynitrite-induced oxidative damage in U937 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole, and oxlalomalate, specific inhibitors of superoxide dismutase, catalase, and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to 1 mM 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion, to U937 cells, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage reflected by an increase in 8-hydroxy-2′-deoxyguanosine, were higher in the inhibitor-treated cells as compared to the control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2′7′-dichlorodihydrofluorescin as well as the significant decrease in the intracellular GSH level in the inhibitor-treated U937 cells upon exposure to SIN-1. These results suggest that antioxidant enzymes play an important role in cellular defense against peroxynitrite-induced cell death.     

Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency

To better understand the mechanism(s) underlying nitricoxide (· NO)-mediated toxicity, in the presence and absenceof concomitant oxidant exposure, postmitotic terminally differentiatedNT2N cells, which are incapable of producing · NO, wereexposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1).Exposure to SIN-1, which generated peroxynitrite in the range of25-750 nM/min, produced a concentration- and time-dependentdelayed cell death. In contrast, a critical threshold concentration(>440 nM/min) was required for · NO to produce significantcell injury. Examination of cells by electron microscopy shows alargely necrotic injury after peroxynitrite exposure but mainlyapoptotic-like morphology after · NO exposure. Cellularlevels of reduced thiols correlated with cell death, and pretreatmentwith N-acetylcysteine (NAC) fully protected from cell death ineither PAPA/NO or SIN-1 exposure. NAC given within the first 3 hposttreatment further delayed cell death and increased theintracellular thiol level in SIN-1 but not · NO-exposedcells. Cell injury from · NO was independent of cGMP,caspases, and superoxide or peroxynitrite formation. Overall, exposureof non-· NO-producing cells to · NO orperoxynitrite results in delayed cell death, which, although occurringby different mechanisms, appears to be mediated by the loss ofintracellular redox balance.