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     

Effects of endogenous glutamate on extracellular concentrations of taurine in striatum and nucleus accumbens of the awake rat: Involvement of NMDA and AMPA/kainate receptors

Summary. Using microdialysis, the effects of endogenous glutamate on extracellular concentrations of taurine in striatum and nucleus accumbens of the awake rat were investigated. The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was used to increase the extracellular concentration of glutamate. PDC (1, 2 and 4 mM) produced a dose-related increase of extracellular concentrations of glutamate and taurine in striatum and nucleus accumbens. Increases of extracellular taurine were significantly correlated with increases of extracellular glutamate, but not with PDC doses, which suggests that endogenous glutamate produced the observed increases of extracellular taurine in striatum and nucleus accumbens. The role of ionotropic glutamate receptors on the increases of taurine was also studied. In striatum, perfusion of the antagonists of NMDA and AMPA/kainate glutamate receptors attenuated the increases of extracellular taurine. AMPA/kainate, but not NMDA receptors, also reduced the increases of extracellular taurine in nucleus accumbens. These results suggest that glutamate-taurine interactions exist in striatum and nucleus accumbens of the awake rat. Received March 5, 1999/Accepted September 22, 1999     

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

Activation of adenosine A2 receptors enhances high K+-evoked taurine release from rat hippocampus: A microdialysis study

Summary The present study was designed to examine which type of adenosine receptors was involved in enhancement of high K⁺-evoked taurine release fromin vivo rat hippocampus using microdialysis. Perfusion with 0.5 or 5.0 mM adenosine enhanced high K⁺-evoked taurine release. Perfusion with 2M R(–)-N⁶-2-phenylisopropyladenosine (PIA), a selective adenosine A₁ receptor agonist, did not modulate taurine release. Perfusion with 1M 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective adenosine A₁ receptor antagonist, increased taurine release. On the other hand, perfusion with 20M 2-[4-(2-carboxyethyl)phenethylamino]-5-N-ethyl-carboxamideadenosine (CGS21680), a selective adenosine A_2A receptor agonist, enhanced taurine release, while perfusion with 1 mM 3,7-dimethyl-propagylxanthine (DMPX), an adenosine A₂ receptor antagonist, did not affect taurine release. These results demonstrate that adenosine enhances high K⁺-evoked taurine release via activation of adenosine A_2A receptors from both neurons and glial cells ofin vivo rat hippocampus.     

Alterations of taurine in the brain of chronic kainic acid epilepsy model

Summary. The aim of the study was to investigate the changes of taurine in the kainic acid (KA, 10 mg/kg, s.c.) chronic model of epilepsy, six months after KA application. The KA-rats used were divided into a group of animals showing weak behavioural response to KA (WDS, rare focal convulsion; rating scale <2 up to 3 h after KA injection) and a group of strong response to KA (WDS, seizures; rating >3 up to 3 h after KA injection). The brain regions investigated were caudate nucleus, substantia nigra, septum, hippocampus, amygdala/piriform cortex, and frontal, parietal, temporal and occipital cortices. KA-rats with rating <2 developed spontaneous WDS which occurred chronically and six months after KA injection increased taurine levels were found in the hippocampus (125.4% of control). KA-rats with rating >3 developed spontaneous recurrent seizures and six months after injection increased taurine levels were found in the caudate nucleus (162.5% of control) and hippocampus (126.6% of control), while reduced taurine levels were seen in the septum (78.2% of control). In summary, increased taurine levels in the hippocampus may involve processes for membrane stabilisation, thus favouring recovery after neuronal hyperactivity. The increased taurine levels in the caudate nucleus could be involved in the modulation of spontaneous recurrent seizure activity.     

Taurine and neural cell damage

Summary. The inhibitory amino acid taurine is an osmoregulator and neuromodulator, also exerting neuroprotective actions in neural tissue. We review now the involvement of taurine in neuron-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals, metabolic poisons and an excess of ammonia. The brain concentration of taurine is increased in several models of ischemic injury in vivo. Cell-damaging conditions which perturb the oxidative metabolism needed for active transport across cell membranes generally reduce taurine uptake in vitro, immature brain tissue being more tolerant to the lack of oxygen. In ischemia nonsaturable diffusion increases considerably. Both basal and K⁺-stimulated release of taurine in the hippocampus in vitro is markedly enhanced under cell-damaging conditions, ischemia, free radicals and metabolic poisons being the most potent. Hypoxia, hypoglycemia, ischemia, free radicals and oxidative stress also increase the initial basal release of taurine in cerebellar granule neurons, while the release is only moderately enhanced in hypoxia and ischemia in cerebral cortical astrocytes. The taurine release induced by ischemia is for the most part Ca²⁺-independent, a Ca²⁺-dependent mechanism being discernible only in hippocampal slices from developing mice. Moreover, a considerable portion of hippocampal taurine release in ischemia is mediated by the reversal of Na⁺-dependent transporters. The enhanced release in adults may comprise a swelling-induced component through Cl⁻ channels, which is not discernible in developing mice. Excitotoxic concentrations of glutamate also potentiate taurine release in mouse hippocampal slices. The ability of ionotropic glutamate receptor agonists to evoke taurine release varies under different cell-damaging conditions, the N-methyl-D-aspartate-evoked release being clearly receptor-mediated in ischemia. Neurotoxic ammonia has been shown to provoke taurine release from different brain preparations, indicating that the ammonia-induced release may modify neuronal excitability in hyperammonic conditions. Taurine released simultaneously with an excess of excitatory amino acids in the hippocampus under ischemic and other neuron-damaging conditions may constitute an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death. The release of taurine may prevent excitation from reaching neurotoxic levels. Received January 25, 2000/Accepted January 31, 2000     

Evidence that nitroprusside stimulates glucose uptake in isolated rat cardiomyocytes via mitogen-activated protein kinase

Sodium nitroprusside (SNP), a nitric oxide (NO.) donor, stimulates glucose uptake in skeletal muscle. We investigated the stimulatory effect of SNP on glucose uptake in cardiomyocytes and the possible role of soluble guanylate cyclase, phosphatidylinositol-3-kinase (PI-3-kinase) and the mitogen-activated protein kinases (MAPKs). Cardiomyocytes were isolated from adult male Wistar rats by trypsin/collagenase perfusion and glucose uptake determined from the accumulation of 3H-2-deoxyglucose. SNP caused a dose-dependent increase in glucose uptake with 200-300% increase at 30 mM. Cytochalasin B completely prevented the SNP-induced increase in glucose uptake. 8-Br-cGMP (100 microM) and the NO. donor spermineNONOate (100 microM) were without effect on basal glucose uptake. SNP-stimulated glucose uptake was not inhibited by the guanylate cyclase inhibitor ODQ (10 microM). Sodium ferrocyanide (Na4Fe(CN)6), a compound structurally related to SNP, but without any NO. group, also stimulated glucose uptake in cardiomyocytes suggesting that the effect of SNP could be unrelated to liberation of NO. Wortmannin, an inhibitor of PI-3-kinase, inhibited insulin-stimulated glucose uptake completely but did not affect SNP-stimulated glucose uptake. SNP-stimulated glucose uptake was inhibited by 50 microM PD 098059 (inhibitor of the MAPK-kinases that activate external regulated kinase [ERK1/2]) and by 50 microM SB203580 (inhibitor of p38MAPK). In conclusion, high SNP concentrations dose-dependently stimulate glucose uptake in cardiomyocytes and our data suggest a role for MAPK signalling, but not PI-3-kinase and soluble guanylate cyclase, in stimulation of glucose uptake.     

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     

Characteristics of nitric oxide-evoked [H]taurine release from cerebral cortical neurons

Pharmacological characteristics of [³H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. NO generators such as S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) dose-dependently increased [³H]taurine release from neurons. Such stimulatory effects of NO generators were completely abolished by hemoglobin, a NO radical scavenger, indicating that these [³H]taurine releases might be due to NO liberated from SNAP and SNP. Sodium withdrawal from incubation buffer significantly inhibited the SNAP- and SNP-induced [³H]taurine releases, whereas the removal of calcium showed no alterations in the [³H]taurine release evoked by NO generators. β-Alanine and guanidinoethane sulfonate, inhibitors of carrier-mediated taurine transport system, inhibited the SNAP- and SNP-evoked releases of [³H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [³H]taurine release from cerebral cortical neurons is mediated by the reverse process of sodium-dependent carrier-mediated taurine transport system.     

Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice

Summary. In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K⁺-stimulated release of [³H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA_B and GABA_C receptors. The involvement of GABA_A receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K⁺-evoked taurine release. Furthermore, several GABA_B receptor effectors were able to inhibit K⁺-stimulated taurine release in the adults, while the GABA_C receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds. Received June 28, 1999, Accepted August 31, 1999     

Sodium nitroprusside activates p38 mitogen activated protein kinase through a cGMP/PKG independent mechanism

The objective of this study was to understand the mechanism of action of nitric oxide (NO) in the heart by determining whether nitric oxide (NO) released from sodium nitroprusside (SNP) induces p38 mitogen activated protein kinase (p38 MAPK) phosphorylation and whether this is mediated through a cyclic GMP (cGMP)/protein kinase G (PKG) pathway. p38 MAPK activation was examined by Western blotting of whole cell lysates of embryonic chick cardiomyocytes with antibodies specific to the native or phosphorylated forms of p38 MAPK. SNP, 1 mM, which released significant amounts of NO as determined by Griess reaction, induced p38 MAPK phosphorylation that was apparent within 10 min, was significantly (p<0.05) greater than control at 60 min and remained higher than initial levels up to the 4 h end point of the experiment. This could not be attributed to hydrogen peroxide release from SNP as catalase did not affect SNP-induced p38 MAPK phosphorylation. SB202190, a relatively selective inhibitor of p38 MAPK, mainly p38alpha MAPK, inhibited SNP-induced p38 MAPK phosphorylation. SNP-induced p38 MAPK phosphorylation was not altered by pre-treatment with the PKG inhibitor KT 5823 or by ODQ a potent and selective inhibitor of NO-sensitive guanylyl cyclase. p38 MAPK phosphorylation was not induced by the cell permeable cGMP analogue, 8-Br-cGMP. In summary, considering that new therapeutic strategies aimed at NO and p38 MAPK are being considered for myocardial injury and heart failure, these data demonstrate that SNP induces p38 MAPK phosphorylation through a pathway that is independent of NO-induced activation of cGMP/PKG pathways and suggest that non cGMP/PKG regulatory proteins leading to p38 MAPK phosphorylation merit further investigation to address this therapeutic target.     

Which mechanisms are involved in taurine-dependent granulocytic immune response or amino- and α-keto acid homeostasis?

We examined the effects of beta-alanine (taurine analogue and taurine transport antagonist), taurine (regarding its role in neutrophil (PMN) immunonutrition) and taurine combined either with L-NAME (inhibitor of *NO-synthase), SNAP (*NO donor), DON (glutamine-analogue and inhibitor of glutamine-requiring enzymes), DFMO (inhibitor of ornithine-decarboxylase) and beta-alanine on neutrophil amino- and alpha-keto acid profiles or important PMN immune functions in order to establish whether taurine transport-, nitric oxide-, glutamine- or ornithine-dependent mechanisms are involved in any of the taurine-induced effects. According to the present findings, the taurine-mediated effect appears to be based primarily on a modulation of important transmembraneous transport mechanisms and only secondarily on directly or indirectly induced modifications in intragranulocytic amino- and alpha-keto acid homoeostasis or metabolism. Although a direct relation to the parallel observed immunological modifications can only be presumed, these results show very clearly that compositional modifications in the free intragranulocytic amino- and alpha keto-acid pools coinciding with changes in intragranulocytic taurine levels are relevant metabolic determinants that can significantly influence the magnitude and quality of the granulocytic immune response.     

Nitric oxide relaxes rat tail artery smooth muscle by cyclic GMP-independent decrease in calcium sensitivity of myofilaments

The effects of authentic nitric oxide (NO, 10(-6) M) and NO-donors such as sodium nitroprusside (SNP, 10(-5) M) and glyceryl trinitrate (GTN, 10(-4) M) on contractile force and free intracellular calcium level ([Ca2+]i) were studied on precontracted with high potassium chloride (KCl, 70 mM) isolated rings of rat tail artery. The sensitivity of contractile myofilaments to Ca2+ was measured using chemically permeabilized (alpha-toxin, beta-escin, Triton X-100) vascular rings. [Ca2+]i and contractile activity were measured simultaneously. The relationship of [Ca2+]i and tension developed was studied in endothelium-denuded rings and controlled calcium response was evaluated in both endothelium-denuded and permeabilized vascular rings. Both authentic NO and NO-donors decreased [Ca2+]i and high potassium-induced tension with a different time course. Inhibitor of soluble guanylyl cyclase (sGC) LY83583 (10(-5) M) did not affect SNP-induced relaxation whereas the other sGC inhibitor ODQ (10(-6) M) attenuated SNP-induced relaxation. Both inhibitors had no effect on NO- and SNP-induced reduction in [Ca2+]i. On the contrary, GTN induced neither relaxation nor decrease in [Ca2+]i on application of both LY83583 and ODQ. Tail artery rings permeabilized with alpha-toxin, beta-escin, but not with Triton X-100 were relaxed by authentic NO and NO-donors, but to a less extent than non-permeabilized rings. Dithioerythritol (DTE, 5 x 10(-3) M) that maintains sulfhydryl (SH) groups in reduced state preventing their nitrosylation attenuated NO-induced relaxation in both non-permeabilized and permeabilized tail artery rings. The cyclic heptapeptide mycrocystin-LR (MC-LR) (10(-5) M), an inhibitor of type 1 and 2A phosphatases, induced sustained increase in tension of beta-escin permeabilized rings in low Ca2+ (10(-8) M) solution. The tension was not affected by authentic NO and SNP. We conclude that authentic NO and SNP relax rat tail artery smooth muscle (SM) in the presence of inhibitors of sGC via cyclic guanosine monophosphate (cGMP)-independent pathway, whereas relaxation induced by GTN is inhibited. The data demonstrate that cGMP-dependent pathway in vascular smooth muscle is ubiquitous, but not the only way of relaxation induced by NO. NO can modulate vascular tone directly by reducing sensitivity of contractile myofilaments to [Ca2+]i and may involve activation of protein phosphatase(s).     

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     

Nitric oxide decreases pacemaker activity in lymphatic vessels of guinea pig mesentery

Intracellular microelectrode recordings were used to determine whether nitric oxide (NO), affects the pacemaker events that initiate vasomotion in lymphatic vessels of the guinea pig mesentery. This pacemaker activity is recorded as spontaneous transient depolarizations (STDs) and is likely to arise through synchronized Ca2+ release from intracellular stores. We show here that acetylcholine-induced endothelium-derived NO and exogenous NO released by sodium nitroprusside (SNP; 100 microM) and DEA-NONOate (500 microM) reduced the frequency and amplitude of STDs. This inhibition of STD frequency and amplitude was independent of the NO-induced hyperpolarization of the smooth muscle. The SNP-induced inhibition of STD frequency and amplitude was abolished during superfusion with the soluble guanylyl cyclase inhibitor ODQ (10 microM) and was diminished in the presence of cGMP and cAMP-dependent protein kinase inhibitors. The data are consistent with the hypothesis that NO inhibits vasomotion primarily by production of cGMP and activation of both cGMP- and cAMP-dependent protein kinases, which reduce the size and frequency of STDs, probably by acting on the underlying synchronized Ca2+ release from intracellular stores.     

Mechanisms underlying the nitric oxide inhibitory effects in mouse ileal longitudinal muscle

We investigated the mechanisms involved in the nitric oxide (NO)-induced inhibitory effects on longitudinal smooth muscle of mouse ileum, using organ bath technique. Exogenously applied NO, delivered as sodium nitroprusside (SNP; 0.1-100 micromol/L) induced a concentration-dependent reduction of the ileal spontaneous contractions. 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (ODQ; 1 micromol/L), a guanilyl cyclase inhibitor, reduced the SNP-induced effects. Tetraethylammonium chloride (20 mmol/L), a non-selective K+ channel blocker, and charybdotoxin (0.1 micromol/L), blocker of large conductance Ca2+-dependent K+ channels, significantly reduced SNP-induced inhibitory effects. In contrast, apamin (0.1 micromol/L), blocker of small conductance Ca2+-dependent K+ channels, was not able to affect the response to SNP. Ciclopiazonic acid (10 micromol/L) or thapsigargin (0.1 micromol/L), sarcoplasmatic reticulum Ca2+-ATPase inhibitors, decreased the SNP-inhibitory effects. Ryanodine (10 micromol/L), inhibitor of Ca2+ release from ryanodine-sensitive intracellular stores, significantly reduced the SNP inhibitory effects. The membrane permeable analogue of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (100 micromol/L), also reduced spontaneous mechanical activity, and its effect was antagonized by ryanodine. The present study suggests that NO causes inhibitory effects on longitudinal smooth muscle of mouse ileum through cGMP which in turn would activate the large conductance Ca2+-dependent K+ channels, via localized ryanodine-sensitive Ca2+ release.     

Characterization of N-methyl-D-aspartate-evoked taurine release in the developing and adult mouse hippocampus

Taurine is an inhibitory amino acid acting as an osmoregulator and neuroromodulator in the brain, with neuroprotective properties. The ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) greatly potentiates taurine release from brain preparations in both normal and ischemic conditions, the effect being particularly marked in the developing hippocampus. We now characterized the regulation of NMDA-stimulated taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mouse using a superfusion system. The NMDA-stimulated taurine release was receptor-mediated in both adult and developing mouse hippocampus. In adults, only NO-generating compounds, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and hydroxylamine reduced the release, as did also NO synthase inhibitors, 7-nitroindazole and nitroarginine, indicating that the release is mediated by the NO/cGMP pathway. On the other hand, the regulation of the NMDA-evoked taurine release proved to be somewhat complex in the immature hippocampus. It was not affected by the NOergic compounds, but enhanced by the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate and adenosine receptor A(1) agonists, N(6)-cyclohexyladenosine and R(-)N(6)-(2-phenylisopropyl)adenosine in a receptor-mediated manner. The activation of both ionotropic 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and metabotropic glutamate group I receptors also enhanced the evoked release. The NMDA-receptor-stimulated taurine release could be a part of the neuroprotective properties of taurine, being important particularly under cell-damaging conditions in the developing hippocampus and hence preventing excitotoxicity.     

Observations on the relationship between the extracellular changes of taurine and glutamate during cortical spreading depression, during ischemia, and within the area surrounding a thrombotic infarct

Summary. Taurine and glutamate were monitored by microdialysis technique during various cerebral insults: a. Application of K⁺ triggered a cortical spreading depression (CSD). Taurine and glutamate increased concomitantly but recovery of glutamate was faster than that of taurine. b. Application of NMDA induced also CSD but only taurine increased. c. Induction of an infarct triggered repetitive CSDs. Taurine increased rapidly whereas glutamate rose slowly starting with some delay. d. After induction of ischemia, taurine and glutamate increased after onset of depolarisation. The increase of glutamate occurred late after a small, transient increase in parallel with the depolarisation. These data suggest a close functional relationship between the changes of both amino acids. Therefore, they should be monitored together especially in clinical settings: during excitation, only taurine will increase; during overexcitation, taurine will also increase but to a higher maximum followed by a moderate rise of glutamate; after energy failure, taurine will accumulate to its highest level followed by a continuous rise of glutamate. Received January 25, 2000/Accepted January 31, 2000     

Taurine Release in the Developing and Adult Mouse Hippocampus: Involvement of Cyclic Guanosine Monophosphate

The inhibitory neuromodulator taurine is involved in osmoregulation and cell volume adjustments in the central nervous system. In addition, taurine protects neural cells from excitotoxicity and prevents harmful metabolic events evoked by cell-damaging conditions. The release of taurine in nervous cell preparations is greatly enhanced by glutamate receptor agonists and various cell-damaging conditions. NO-generating compounds also increase taurine release in the mouse hippocampus. The further involvement of the NO/cGMP pathway and protein kinases in preloaded [³H]taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mice in normoxia and in ischemia was now studied using a superfusion system. The release was enhanced by 8-Br-cGMP and the phosphodiesterase inhibitor 2-(2-propyloxyphenyl)-8-azapurin-6-one (zaprinast), particularly in the immature hippocampus, indicating that increased cGMP levels induce taurine release. The release was also increased by the inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo-(4,3a)quinoxalin-1-one (ODQ) and the protein kinase C activator 4-phorbol 12-myristate 13-acetate (PMA), but only in the adult hippocampus. The ischemia-induced release was also enhanced by increased cGMP levels in both adult and developing mice, whereas protein kinase inhibitors had no effects in any conditions. The results demonstrate that cGMP is able to modulate hippocampal taurine release in both adult and developing mice, the rise in cGMP levels evoking taurine release in normoxia and in ischemia. This could be part of the neuroprotective properties of taurine, being thus important particularly in cell-damaging conditions and in preventing excitotoxicity.     

Effects of Microdialysis-Perfusion with Anisoosmotic Media on Extracellular Amino Acids in the Rat Hippocampus and Skeletal Muscle

Changes in the levels of amino acids have been implicated as being important in osmoregulation both within and outside the CNS. The present study addressed the question of whether changes in osmolarity affect the extracellular concentration of amino acids in the rat hippocampus and femoral biceps muscle (FBM). Microdialysis probes were implanted in these tissues and perfused with standard physiological saline. Amino acid concentrations in the dialysate were determined with HPLC separation of o-phthaldialdehyde derivatives and fluorescence detection. The osmolarity of the perfusion buffer was gradually decreased by reduction of the concentration of NaCl from 122 to 61 to 0 mM. In other experiments, the osmolarity was increased by elevation of the NaCl level from 122 to 183 to 244 mM or by addition of mannitol. Glutamate, aspartate, gamma-aminobutyrate, and alanine levels in dialysate from the hippocampus increased when the concentration of NaCl was decreased by 61 mM, and they were further elevated when NaCl was omitted. Taurine and phosphoethanolamine (PEA) levels were maximally elevated at the intermediary decrease of NaCl concentration, and glutamine in particular but also methionine and leucine were suppressed by perfusion with hypoosmolar medium. The amino acid response of the FBM differed substantially from that of the hippocampus. The aspartate content increased slightly, and there was a marginal transient increase in PEA level. Perfusion with media containing high concentrations of NaCl induced diminished dialysate levels of taurine, PEA, and glutamate, whereas levels of other amino acids were either unaffected or increased. Mannitol administration via the perfusion fluid led to reduced levels of taurine, PEA, glutamate, and aspartate. In contrast to the effects of high NaCl levels, hyperosmotic mannitol did not induce increases in level of any of the amino acids detected. The results suggest that taurine and PEA are involved in osmoregulation in the mammalian brain. From a quantitative viewpoint, taurine seems to be most important. Transmitter amino acids may also be involved in the maintenance of the volume of neural cells subjected to severe disturbances in osmotic equilibrium.