Metallothionein-III prevents glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons

Metallothionein (MT)-III, a member of the MT family of metal-binding proteins, is mainly expressed in the CNS and is abundant in glutamatergic neurons. Results in genetically altered mice indicate that MT-III may play neuroprotective roles in the brain, but the mechanisms through which this protein functions have not been elucidated. The aim of this work was to assess whether MT-III is able to prevent glutamate neurotoxicity and to identify the step of the neurotoxic process interfered with by MT-III. Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive activation of glutamate receptors, increased intracellular calcium, and increased nitric oxide. It is shown that MT-III prevented glutamate- and nitric oxide-induced neurotoxicity in a dose-dependent manner, with nearly complete protection at 0.3-1 microgram/ml. MT-III did not prevent the glutamate-induced rise of intracellular calcium level but reduced significantly the nitric oxide-induced formation of cyclic GMP. Circular dichroism analysis revealed that nitric oxide triggers the release of the metals coordinated to the cysteine residues of MT-III, indicative of the S(Cys)-nitrosylation of the protein. Therefore, the present results indicate that MT-III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.     

Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.     

Protective role of autotypic contacts under cerebellar neural net injury by toxic doses of NO-generative compounds

In the present work, cerebellar neural net injury was induced by toxic doses of NO-generative compound (NaNO2). A protective role of glial cells was revealed in such conditions. The present results were compared with those of the previous work concerning the action of high concentration glutamate on the frog cerebellum (Samosudova et al., 1996). In both cases we observed the appearance of spiral-like structures--"wrappers)"--involving several rows of transformed glial processes with smaller width and bridges connecting the inner sides of row (autotypic contact). A statistic analysis was made according to both previous and present data. We calculated the number and width of rows, and intervals between bridges depending on experimental conditions. As the injury increased (stimulation in the NO-presence), the row number in "wrappers" also increased, while the row width and intervals between bridges decreased. The presence of autotypic contacts in glial "wrappers" enables us to suppose the involvement of adhesive proteins--cadherins in its formation. The obtained data suggested that the formation of spiral structures--"wrappers" may be regarded as a compensative-adaptive reaction on the injury of cerebellar neural net glutamate and NO-generative compounds.     

Metabotropic glutamate receptors as targets of neuromodulatory influence of nitric oxide

A possible effect of nitric oxide (NO) on metabotropic glutamate receptor (mGluR) function in the amino acid afferent synapse was investigated in the isolated labyrinth of the frog Rana temporaria. The modification of the amplitude of responses of metabotropic glutamate receptor agonist trans-ACPD was analyzed during bath applied NO donor S-nitroso-N-acetyl-DL-penicillamine SNAP (0.1–100 μM) or nitric oxide synthase inhibitor L-NAME. It was shown that NO donor SNAP (1 μM) inhibited mGluR induced responses, and the inhibitor of NO-synthase L-NAME (100 μM) increased the amplitude of trans-ACPD evoked answers. The results suggest that NO can depress mGluR function due to modulation of functions of the endoplasmic reticulum channels.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

Developmental changes in the response of murine cerebellar granule cells to nitric oxide

Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.     

Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.     

Effect of the interaction of formaldehyde and nitric oxide metabolism pathways in mechanism of their toxic action. I. Exo- and endogenous sources of formaldehyde and nitric oxide. Toxic action of formaldehyde

Possible exogenous sources of formaldehyde and nitric oxide have been considered; the environment pollution conditions under which these compounds and their precursors have mutual effect on the organism; endogenous sources of FA and NO which are intermediates of the metabolism and key enzymes of their transformation (semicarbazide-sensitive amine oxidase and NO-synthase) the role of the C1 metabolic cycle pathways and methyl cycles in the FA formation and accumulation have been considered as well, various paths of FA toxic action have been characterized.     

Energy exchange and ultrastructure of plant cells under generation of nitric oxide

The effects of nitric oxide (NO) on oxygen consumption, heat generation, and cell ultrastructure were investigated in the seedlings of wheat (Triticum aestivum L.). The experiments were conducted with the excised roots of 5-day-old seedlings grown in the solution of CaCl₂ (2.5 × 10⁻⁴ M). The source of NO was NaNO₂ (5 × 10⁻³ M) where the roots were incubated. Production of NO was determined by means of EPR, respiration — gasometrically, heat generation — using a microcalorimeter. The results showed that NO was formed in the presence of NaNO₂. This was accompanied by a decrease in the respiration rate by about 30%, which lasted for 5–6 h. Apparently, NO inhibited mitochondrial oxidation because stimulation of oxygen consumption induced by 2,4-DNP was completely removed in the presence of NaNO₂. When the cells were affected by succinic acid in the presence of NaNO₂, respiration was strongly inhibited. The effects of succinic acid and NaNO₂ were negated by ascorbic acid. A decrease in the rate of respiration was accompanied by a reduction in heat generation. Moreover, the efflux of potassium ions to the root incubation medium was stimulated, which may point to changes in ionic membrane permeability. The observed changes in energy exchange were accompanied by disturbances in the cell ultrastructure. Nitric oxide induced a clarification of the mitochondrial matrix and a reduction in the number of cristae. It was concluded that NO excess in plant tissues brings about a deceleration of energy exchange, disturbance of the ultrastructural organization, and cell death.     

Synergistic action of rutin and orthovanadate on nitric oxide release from mouse macrophage cells

The cooperative action of sodium orthovanadate (a putative protein-phosphotyrosine phosphatase inhibitor) and rutin (an effective superoxide scavenger) on the nitric oxide (NO) production of J774A.1 mouse macrophage cells has been investigated. Orthovanadate alone caused a mild but significant increase in NO production of the cells at its highest concentration used (500 microM). Orthovanadate and rutin together caused a significant increase in the nitrite level of the supematants of the J774A.1 cells after a 24-hour incubation period, in a concentration dependent manner. The optimal doses for orthovanadate and rutin were 50 microM and 100 microM, respectively. This cooperative action of rutin and orthovanadate was totally inhibitable by catalase, reduced glutathion, N-acetylcystein, cycloheximide, pyrrolidine dithiocarbamate (a putative NF-kappaB inhibitor), genistein and tyrphostin-AG126 (two protein tyrosine-kinase inhibitors). Superoxide dismutase had no inhibitory effect. Orthovanadate and rutin (only together) could induce the oxidation of 2',7'-dichlorofluorescein-diacetate, a marker of hydrogen peroxide. This effect was inhibitable by reduced glutathion, a hydrogen peroxide specific scavenger. These findings suggest, that orthovanadate can induce the production of NO by J774A.1 macrophages not only by inhibition of protein tyrosine-phosphatases, but, using it with rutin, by increasing the level of hydrogen peroxide in the cells.     

A 27-bp region of the inducible nitric oxide synthase promoter regulates expression in glial cells

The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cAMP levels. We examined the molecular basis for this effect using a 2.2-kb fragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Promoter activation (up to six-fold) by lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp -130 and containing the proximal nuclear factor-kappa B (NF-kappaB) binding site was minimally activated by LPS and cytokines, but activated up to three-fold by NE. Deletion analysis identified a 27-bp region (bp -187 to -160) as critical for mediating this suppressive effect. This region also enhanced promoter activation by LPS and cytokines, and prevented activation by NE alone. Gel shift analysis revealed constitutive binding to this region, and induction by NE of additional complexes which could be blocked by an antibody against CREB. NE also increased levels of the IkappaBalpha protein which could contribute to its suppressive effects. These results identify a critical role for this 27-bp region in regulation of NOS2 promoter activation and suppression by cAMP.     

Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of alpha-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the alpha-amino group to glutamate. Measurements of synthesis of (14)C-glutamate and of (14)C-glutamine from H(14)CO(3) have shown that (14)C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The specific radioactivity of (14)C-glutamine indicated that approximately 30% of glutamine was derived from (14)CO(2) fixation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of (14)C-glutamate and (14)C-glutamine from H(14)CO(3)(-) was inhibited by 31%. These results suggest that transamination of alpha-ketoglutarate to glutamate in Müller cells is slow, the supply of branched-chain amino acids may limit flux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H(14)CO(3)(-). (14)C-metabolites in brain extracts were measured to determine net incorporation of (14)CO(2) and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from (14)CO(2) fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for approximately 60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.     

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transamination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.     

眶额叶区5-HT与Glu、NO在急性强迫游泳应激抑郁症模型中的关系

目的：探讨眶额叶区5-羟色胺（5-HT）与谷氨酸（Glu）、一氧化氮（N0）在急性强迫游泳应激抑郁症模型中的相互作用。方法：雄性SD大鼠随机分为对照组及各种药物注射组,强迫游泳制造大鼠应激性抑郁模型,眶额叶区微量注射各组药物,敞箱实验及游泳测试观察大鼠的抑郁样行为表现。结果：①与对照组比,注射Glu使大鼠强迫游泳不动时间显著增加;注射NMDA受体拮抗剂（MK-801）使大鼠强迫游泳不动时间减少;与Glu组比,MK-801预注射后Glu注射使大鼠强迫游泳不动时间减少;②与5-HT组比,MK-801预注射后5-HT注射使大鼠强迫游泳不动时间增加;③与对照组比,注射L-精氨酸（L-Ars）使大鼠强迫游泳不动时间显著增加;注射NOS抑制剂（L-NAME）（10μg/μl）使大鼠强迫游泳不动时间减少;L-NAME（20μg／μl）注射使大鼠强迫游泳不动时间增加;L-NAME（40μg/μl）注射使大鼠强迫游泳不动时间增加;④与L-NAME（10μg/μl）组比较,5-HT1A受体拮抗剂spipemne预注射后LNAME（10μg／μl）注射使大鼠强迫游泳不动时间增加。结论：眶额叶（OFC）区Glu含量的增加能够诱发抑郁,其作用可能主要是通过NMDA受体实现的,Glu经NMDA受体引发抑郁的同时还可能通过调节突触后膜上5-HT1A受体减弱5-HT的抗抑郁作用;OFC区NO可通过调节5-HT神经元进而参与抑郁的发生。