Neurotoxicity of Ammonia.

Abnormal liver function has dramatic effects on brain functions. Hyperammonemia interferes profoundly with brain metabolism, astrocyte volume regulation, and in particular mitochondrial functions. Gene expression in the brain and excitatory and inhibitory neurotransmission circuits are also affected. Experiments with a number of pertinent animal models have revealed several potential mechanisms which could underlie the pathological phenomena occurring in hepatic encephalopathy.

Significance of Taurine in the Brain.

Two main functions of taurine in the brain are here discussed: the role of taurine in cell volume regulation and the neuromodulatory actions of taurine liberated by depolarization. Taurine takes part in cell volume regulation with other small-molecular compounds. Extracellular taurine inhibits neuronal firing through GABA and glycine receptors. However, the existence of specific taurine receptors is still not excluded.

Comparison of Toxicity of Taurine and GABA in Combination with Alcohol in 7-Day-Old Mice.

Previously, we described the combined toxicity of taurine and alcohol, and assumed hypoglycemia to be one reason of this toxicity. To understand whether taurine-ethanol combined toxicity is exclusively connected to taurine or whether other inhibitory amino acids may have similar effects when combined with ethanol, we tested different doses of gamma-aminobutyric acid (GABA) in combination with ethanol in 7-day-old mice. The minimal dose of GABA in combination with 5 g/kg ethanol which could kill a mouse was 2 g/kg. GABA combined with ethanol at doses of 3 g/kg, 4 g/kg, 6 g/kg induced lethality of 30%, 90% and 100%, correspondingly. Taurine at the doses of 4 and 6 g/kg combined with ethanol induced death in 60 and 100% of mice. Ethanol (5 g/kg), taurine (6 g/kg), GABA (4 g/kg) administered alone and the combination of ethanol (5 g/kg) with taurine (3 g/kg) have no lethal effects. GABA (6 g/kg) applied alone induced 90% lethality. Taurine or GABA alone decreased blood glucose in a dose-depending manner. Ethanol potentiated GABA- and taurine-induced decrease in blood glucose and in some animals it dropped from 8.8 (intact) to a hypoglycemic level 3.1-3.3 mmol/L (GABA 4 g/kg, taurine 6 g/kg), but this may not be considered a single reason of death. We conclude that the combination of GABA and ethanol has a lethal effect and this is stronger than the combined toxicity of ethanol and taurine.

Modulation of taurine release in glucose-free media by glutamate receptors in hippocampal slices from developing and adult mice.

Taurine has been thought to protect neural cells against cell-damaging conditions to which the hippocampus is particularly vulnerable. We studied now how the release of preloaded [(3)H]taurine is regulated by glutamate receptors in glucose-free media in slices prepared from the mouse hippocampus from developing (7 days old) and young adult (3 months old) mice, using a superfusion system. The lack of glucose enhanced taurine release more from slices from developing mice than from slices from adults. At both ages ionotropic glutamate agonists significantly increased the release in a receptor-mediated manner. Of the metabotropic glutamate receptors those belonging to the group III were effective. The release was enhanced in adult mice but attenuated in developing mice. Both effects were blocked by the receptor antagonists. The results show that glutamate receptors affect taurine release in the absence of glucose in which condition taurine should be neuroprotective.

Regulation of taurine release in the hippocampus of developing and adult mice.

Taurine release in mouse hippocampal slices is regulated by several neurotransmitter receptor systems. The ionotropic glutamate receptors and the adenosine receptor A(1) are the most effective. The effect of N-methyl-D-aspartate receptors is mediated via activation of the pathway involving nitric oxide and 3',5'-cyclic guanosine monophosphate. The activation of excitatory amino acid receptors causes at the same time an increase in taurine release. The activation of adenosine A(1) receptors also potentiates taurine release. The taurine released may counteract any excitotoxic effects of glutamate, particularly in the developing hippocampus.

Lethality of taurine and alcohol coadministration in mice.

Alcohol consumption by mothers during pregnancy causes a fetal alcohol syndrome associated with massive neuronal apoptosis. We have recently shown that taurine at a dose of 2 g/kg saves about 50% of dying cerebellar neurons from ethanol-induced apoptosis in 7-day-old mice. However, a further increase in the taurine dose to ethanol-treated mice had a toxic and in some cases lethal effect. In the present work we studied the toxic effects of taurine and ethanol coadministration in three age groups: 7-day-old, adult (5 to 6 months old), and old (12 to 13 months old) mice. Taurine and ethanol were injected in two half-doses: taurine at 0 and 4 h and ethanol at 1 and 3 h. The minimal 100% lethal doses in coadministration of taurine and ethanol were the following: 7-day-old mice-6 g/kg taurine + 5 g/kg ethanol, adult mice-10 g/kg of taurine + 8 g/kg of ethanol, and old mice-above 6 g/kg of taurine + 6 g/kg of ethanol. All mice treated with taurine or ethanol alone survived. The adult and old mice dying from the combined toxicity of taurine and ethanol showed a marked fall in blood glucose, which may be one reason for lethality. A comparison of the lethal doses of taurine and ethanol coadministration in different age groups allows us to conclude that the adverse effect of the combined toxicity of taurine and ethanol is age dependent.

Taurine protects cerebellar neurons of the external granular layer against ethanol-induced apoptosis in 7-day-old mice.

Acute alcohol administration is harmful especially for the developing nervous system, where it induces massive apoptotic neurodegeneration leading to alcohol-related disorders of newborn infants. Neuroprotection against ethanol-induced apoptosis may save neurons and reduce the consequences of maternal alcohol consumption. Previously we have shown that taurine protects immature cerebellar neurons in the internal granular layer of cerebellum from ethanol-induced apoptosis. Now we describe a similar protective action for taurine in the external layer of cerebellum of 7-day-old mice. The mice were divided into three groups: ethanol-treated, ethanol + taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 0 h and 2.5 g/kg at 2 h) to the ethanol and ethanol + taurine groups. The ethanol + taurine group also received subcutaneously two injections of taurine (1 g/kg each, 1 h before the first dose of ethanol and 1 h after the second dose of ethanol). To verify apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 8 h after the first ethanol injection. Ethanol induced apoptosis in the cerebellar external granular layer. Taurine treatment significantly reduced the number of activated caspase-3-immunoreactive and TUNEL-positive cells. Taurine has thus a neuroprotective antiapoptotic action in the external granular layer of the cerebellum, preserving a number of neurons from ethanol-induced apoptosis.

High activity of indoleamine 2,3-dioxygenase is associated with renal insufficiency in Puumala hantavirus induced nephropathia epidemica.

Nephropathia epidemica (NE) is a hemorrhagic fever with renal syndrome caused by Puumala hantavirus. The severity of NE varies greatly. The aim of the present study was to evaluate whether serum indoleamine 2,3-dioxygenase (IDO) activity is associated with the severity of NE. A prospectively collected cohort of 102 consecutive patients with acute serologically confirmed NE was examined. Serum kynurenine, tryptophan, creatinine, CRP, and blood cell count were measured for up to 5 consecutive days after admission. The kynurenine to tryptophan (kyn/trp) ratio reflecting IDO activity was calculated. A maximum kyn/trp ratio >202 µmol/mmol had a sensitivity of 85% and a specificity of 75% for detecting maximum serum creatinine values >250 µmol/L by receiver operating characteristic (ROC) analysis. A maximum kyn/trp ratio >202 µmol/mmol (high IDO level) was also associated with other parameters reflecting the severity of the disease and renal impairment. Patients with high IDO levels had higher maximum serum creatinine (379 vs. 102 µmol/L, P<0.001), plasma C-reactive protein (104.1 vs. 72.1 mg/L, P=0.029), and blood leukocyte values (11.9 vs. 9.0 × 10(9) /L, P<0.001) compared to patients with kyn/trp ratio ≤ 202 µmol/mmol. They also had lower minimum urinary output (1,100 vs. 1,900 ml/day, P<0.001) and longer hospital stays (8 vs. 5 days, P<0.001). In conclusion, high serum IDO activity was associated with increased disease severity and renal impairment in NE.

Release of endogenous amino acids from the striatum from developing and adult mice in ischemia.

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K(+)-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K(+) stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K(+) stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.

Neuroprotection by taurine in ethanol-induced apoptosis in the developing cerebellum.

BACKGROUND: Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse cerebellum during the postnatal period. METHODS: The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3 h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine (1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase high-performance liquid chromatography (HPLC). RESULTS: Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group diminished dramatically during the experiment, not being different at 13 h from that in the controls. CONCLUSIONS: We conclude that the neuroprotective action of taurine is not straightforward and seems to be different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in blood plasma.

Modulation of taurine release in ischemia by glutamate receptors in mouse brain stem slices.

Glutamate is the main excitatory transmitter in the brain stem, regulating many vital sensory and visceral processes. Taurine is inhibitory and functions as a neuromodulator and regulator of cell volumes in the brain, being especially important in the developing brain. Taurine release is markedly enhanced under ischemic conditions in many brain areas, providing protection against excitotoxicity. The involvement of glutamate receptors in the release of preloaded [(3)H]taurine was now characterized under ischemic conditions in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice. The ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate had no effect on ischemic taurine release in the immature brain stem, whereas in adults the release was enhanced in a receptor-mediated manner. The metabotropic receptor agonists of group I, (1+/-)-1-aminocyclopentane-trans-1,3-dicarboxylate and (S)-3,5-dihydroxyphenylglycine, potentiated both basal and K(+)-stimulated release in both age groups. The group III agonist L(+)-2-amino-4-phosphonobutyrate also enhanced the release. In both cases the effects were receptor-mediated, being reduced by the respective antagonists. The results show that activation of glutamate receptors in the ischemic brain stem generally enhances the release of taurine. This is beneficial to neurons in ischemia, offering protection against excitotoxicity and preventing neuronal damage.

Adenosine receptor agonists affect taurine release from mouse brain stem slices in ischemia.

The release of the inhibitory amino acid taurine is markedly enhanced under ischemic conditions in both adult and developing brain stem, together with a pronounced increase in the release of the neuromodulator adenosine. We now studied the effects of adenosine receptor agonists and antagonists on [(3)H]taurine release in the brain stem in normoxia and ischemia, using a superfusion system. Under standard conditions, the adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA) potentiated basal taurine release in adult mice, which response was blocked by the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CHA and the A(2a) receptor agonist 2-p-(2-carboxyethyl)phenylamino-5'-N-ethylcarboxaminoadenosinehydrochloride (CGS 21680) had no effect on the release in developing mice. In ischemia, CHA depressed both basal and K(+)-stimulated taurine release in developing mice in a receptor-mediated manner, blocked by DPCPX. The A(2a) receptor agonist CGS 21680 was also inhibitory. Taurine and adenosine may thus not cooperate in developing mice to prevent ischemic neuronal damage. On the other hand, CGS 21680 enhanced taurine release in the adult brain stem in ischemia, both basal and K(+)-stimulated release being affected. These effects were abolished by the antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), indicating a receptor-mediated process. In this case elevated levels of taurine could be beneficial, protecting against hyperexcitation and excitotoxicity.

Mechanisms of inhibitory amino acid release in the brain stem under normal and ischemic conditions.

This article deals with the release of GABA, glycine and taurine from the brain stem under normal conditions and in ischemia. The release mechanisms, the effects of glutamate and adenosine receptors, and the roles of nitric oxide and second messengers are reviewed.

Glutamine inhibits ammonia-induced accumulation of cGMP in rat striatum limiting arginine supply for NO synthesis.

Brain L-glutamine (Gln) accumulation and increased activity of the NO/cGMP pathway are immediate consequences of acute exposure to ammonia. This study tested whether excess Gln may influence NO and/or cGMP synthesis. Intrastriatal administration of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine or the system A-specific Gln uptake inhibitor methylaminoisobutyrate increased microdialysate Gln concentration and reduced basal and ammonia-induced NO and cGMP accumulation. Gln applied in vivo (via microdialysis) or in vitro (to rat brain cortical slices) reduced NO and cGMP accumulation in the presence and/or absence of ammonia, but not cGMP synthesis induced by the NO donor sodium nitroprusside. Attenuation of cGMP synthesis by Gln was prevented by administration of L-arginine (Arg). The L-arginine co-substrates of y(+)LAT2 transport system, L-leucine and cyclo-leucine, mimicked the effect of exogenous Gln, suggesting that Gln limits Arg supply for NO synthesis by interfering with y+LAT2-mediated Arg uptake across the cell membrane.

(R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action.

Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABA(B) receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABA(B) receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABA(B) receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABA(B) receptor antagonist and absent in GABA(B)-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concentrated in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABA(B) receptors and may offer a potential new approach to treatment of GERD.

Mechanisms of glycine release in mouse brain stem slices.

In the brain stem glycine is associated with multiple sensory and visceral regulations, being involved in, for instance, cardiovascular, respiratory and auditory functions. We here studied the mechanisms of the release of preloaded [(3)H]glycine from mouse brain stem slices in a superfusion system. A depolarizing concentration of K(+) ions (50 mM) evoked glycine release, but in the absence of Ca(2+) the effect was attenuated, indicating that a part of the evoked release represents Ca(2+)-dependent exocytosis. The Ca(2+)-independent release was enhanced by omission of Na(+) and Cl(-). The stimulatory effect of extracellular glycine confirmed the involvement of transporters functioning in a reverse direction. A part of the release is mediated by Na(+) and Cl(-) channels, since it was inhibited by the inhibitors of these, riluzole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate, respectively. Glycine release was potentiated by the activation of protein kinase C and diminished by increasing cyclic guanosine monophosphate levels with a phosphodiesterase inhibitor, zaprinast. The release was also modulated by the phospholipase inhibitor quinacrine and the tyrosine kinase inhibitor genistein. Adenosine A(1) receptors likewise regulate glycine release, since it was enhanced by their agonist R(-)N(6)-(2-phenylisopropyl)adenosine, which effect was blocked by the antagonist 8-cyclopentyl-1,3-dipropylxanthine. The ionotropic glutamate receptor agonists N-methyl-D: -aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate failed to have any effects contrary to their effects in higher brain regions, e.g., in the hippocampus. The group I and III metabotropic glutamate receptor agonists (S)-3,5-dihydroxyphenylglycine and O-phospho-L: -serine, respectively, increased the release in a receptor-mediated manner. Glycine release in the brain stem was also markedly enhanced by cell-damaging conditions, including hypoxia, hypoglycemia and ischemia.

Release of endogenous amino acids from the hippocampus and brain stem from developing and adult mice in ischemia.

The release of neurotransmitters and modulators has been studied mostly using labeled preloaded compounds. For several reasons, however, the estimated release may not reliably reflect the release of endogenous compounds. The basal and K(+)-evoked release of the neuroactive endogenous amino acids GABA, glycine, taurine, L-glutamate and L-aspartate was now studied in slices from the hippocampus and brain stem from 7-day-old and 3-month-old mice under control and ischemic conditions. The release of synaptically not active L-glutamine, L-alanine, L-threonine and L-serine was assessed for comparison. The estimates for the hippocampus and brainstem were markedly different and also different in developing and adult mice. GABA release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite, in the hippocampus in particular. K(+) stimulation enhanced glycine release more in the mature than immature brain stem while in the hippocampus the converse was observed. Ischemia enhanced the release of all neuroactive amino acids in both brain regions, the effects being relatively most pronounced in the case of GABA, aspartate and glutamate in the hippocampus in 3-month-old mice, and taurine in 7-day-old and glycine in 3-month-old mice in the brain stem. These results are qualitatively similar to those obtained on earlier experiments with labeled preloaded amino acids. However, the magnitudes of the release cannot be quite correctly estimated using radioactive labels. In developing mice only taurine release may counteract the harmful effects of excitatory amino acids in ischemia in both hippocampus and brain stem.

Taurine protects immature cerebellar granullar neurons against acute alcohol administration.

Acute ethanol administration causes extensive apoptosis throughout the nervous system. We studied the protective effect of taurine on alcohol-induced apoptosis in the cerebellum of developing mice. Taurine rescued a part of immature neurons by markedly reducing caspase-3 immunoreactivity and the number of TUNEL-positive cells in most cerebellar lobules.