Hydrogen peroxide potentiates volume-sensitive excitatory amino acid release via a mechanism involving Ca2+/calmodulin-dependent protein kinase II

Excessive excitatory amino acid (EAA) release in cerebral ischemia is a major mechanism responsible for neuronal damage and death. A substantial fraction of ischemic EAA release occurs via volume-regulated anion channels (VRACs). Hydrogen peroxide (H2O2), which is abundantly produced during ischemia and reperfusion, activates a number of protein kinases critical for VRAC functioning and has recently been reported to activate VRACs. In the present study, we explored the effects of H2O2 on volume-dependent EAA release in cultured astrocytes, measured as the release of preloaded D-[3H]aspartate. 100-1,000 microm H2O2 enhanced swelling-induced EAA release by approximately 2.5-3-fold (EC50 approximately 10 microM). The VRAC blockers ATP, phloretin, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) potently inhibited both control swelling-induced and the H2O2-potentiated release, suggesting a role for VRACs. The H2O2-induced component of EAA release was attenuated by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and completely eliminated by the calmodulin antagonists trifluoperazine and W-7 and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93. Inhibitors of tyrosine kinases, protein kinase C, and the myosin light chain kinase were ineffective in blocking the H2O2 response. H2O2 treatment of swollen astrocytes, but not swelling alone, resulted in CaMKII activation that was inhibited by KN-93, as determined by a phospho-Thr286 CaMKII antibody. These data demonstrate that H2O2 strongly up-regulates astrocytic volume-sensitive EAA release via a CaMKII-dependent mechanism and in this way may potently promote pathological EAA release and brain damage in ischemia.     

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

Volume-dependent ATP release andsubsequent activation of purinergic P2Y receptors have been implicatedas an autocrine mechanism triggering activation of volume-regulatedanion channels (VRACs) in hepatoma cells. In the brain ATP is releasedby both neurons and astrocytes and participates in intercellularcommunication. We explored whether ATP triggers or modulates therelease of excitatory amino acid (EAAs) via VRACs in astrocytes inprimary culture. Under basal conditions exogenous ATP (10 µM)activated a small EAA release in 70-80% of the cultures tested.In both moderately (5% reduction of medium osmolarity) andsubstantially (35% reduction of medium osmolarity) swollen astrocytes,exogenous ATP greatly potentiated EAA release. The effects of ATP weremimicked by P2Y agonists and eliminated by P2Y antagonists or the ATPscavenger apyrase. In contrast, the same pharmacological maneuvers didnot inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cellswas eliminated by a 5-10% increase in medium osmolarity or byanion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but actssynergistically with cell swelling. Moderate cell swelling and ATP mayserve as two cooperative signals in bidirectional neuron-astrocytecommunication in vivo.

     

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization. NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone. This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO. Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.     

Protective properties of neoechinulin A against SIN-1-induced neuronal cell death

Peroxynitrite (ONOO-) is thought to be involved in the neurodegenerative process. To screen for neuroprotective compounds against ONOO- -induced cell death, we developed 96-well based assay procedures for measuring surviving cell numbers under oxidative stress caused by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1), a generator of ONOO-, and sodium N,N-dietyldithiocarbamate trihydrate (DDC), an inhibitor of Cu/Zn superoxide (O2-) dismutase. Using these procedures, we obtained a microbial metabolite that rescued primary neuronal cells from SIN-1-induced damage, but not from DDC-induced damage. By NMR analysis, the compound was identified as neoechinulin A, an antioxidant compound that suppresses lipid oxidation. We found that the compound rescues neuronal cells such as primary neuronal cells and differentiated PC12 cells from damage induced by extracellular ONOO-. However, non-neuronal cells, undifferentiated PC12 cells and cells of the fibroblast cell line 3Y1 were not rescued. Neoechinulin A has scavenging, neurotrophic factor-like and anti-apoptotic activities. This compound specifically scavenges ONOO-, but not O2- or nitric oxide (NO). Similar to known neuroprotective substances such as nerve growth factor and extracts of Gingko biloba leaves, neoechinulin A inhibits the SIN-1-induced activation of caspase-3-like proteases and increases NADH-dehydrogenase activity. These results suggest that neoechinulin A might be useful for protecting against neuronal cell death in neurodegenerative diseases.     

Inhibition of Release of Taurine and Excitatory Amino Acids in Ischemia and Neuroprotection

Volume regulated anion channels (VRAC) have been extensively studied in purified single cell systems like cell cultures where they can be activated by cell swelling. This provides a convenient way of analyzing mechanisms and will likely lead to the holy grails of the field, namely the nature or natures of the volume sensor and the nature or natures of VRACs. Important reasons for such an understanding are that these channels are ubiquitous and have important physiological functions which under pathological conditions convert to deleterious effects. Here we summarize data showing the involvement of VRACs in ischemia-induced release of excitatory amino acids (EAAs) in a rat model of global ischemia. Using microdialysis studies we found that reversal of the astrocytic glutamate transporter and VRACs contribute about equally to the large initial release of EAAs and together account for around 80% of the total release. We used the very potent VRAC blocker, tamoxifen, to see if such inhibition of EAA release via VRACs led to significant neuroprotection. Treatment in the focal rat MCA occlusion model led to around 80% reduction in infarct size with an effective post initiation of ischemia therapeutic window of three hours. However, the common problem of other effects for even the most potent inhibitors pertains here, as tamoxifen has other, potentially neuroprotective, effects. Thus it inhibits nitrotyrosine formation, likely due to its inhibition of nNOS and reduction of peroxynitrite formation. Although tamoxifen cannot therefore be used as a test of the "VRAC-excitotxicity" hypothesis it may prove successful for translation of basic stroke research to the clinic because of its multiple targets.     

Two conventional protein kinase C isoforms, alpha and beta I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes

Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 μM ATP synergistically increased the release of excitatory amino acids, measured with a non-metabolized analog of l -glutamate, d -[³H]aspartate. Both Go6976, the selective inhibitor of Ca²⁺-sensitive PKCα, βI/II, and γ, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKCα and βI/II, reduced the effects of ATP on d -[³H]aspartate release by ∼45–55%. Similar results were obtained with a mixture of siRNAs targeting rat PKCα and βI. Surprisingly, down-regulation of individual α and βI PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKCα and βI.     

Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

Comparative vasodilation of peroxynitrite and 3-morpholinosydnonimine.

Vasorelaxation mediated by peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1) were investigated in isolated bovine intramammary arteries. Both ONOO- and SIN-1 relaxed U 46619-precontracted rings in a dose-dependent, endothelium-independent manner. Pretreatment with an adenylyl cyclase inhibitor, SQ 22536 [(9-tetrahydro-2-furyl)adenine], resulted in an enhanced ONOO--mediated relaxation, but did not modulate the response to SIN-1. ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a potent and selective inhibitor of soluble guanylyl cyclase (sGC), did not significantly affect relaxant actions of ONOO-, but ODQ markedly attenuated SIN-1-elicited relaxation with a rightward shift in the dose-response curve and an unaltered maximal response. In the presence of carboxy-PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide), a putative nitric oxide scavenger and ONOO- inactivator, the relaxant response to ONOO- was abolished, while relaxant actions of SIN-1 appeared to be unaffected. The results reveal a difference between ONOO- and SIN-1-mediated relaxation with regards to the role of the sGC and suggest that ONOO--evoked relaxation may not be associated with sGC activity, but rather depends on an sGC-independent mechanism triggered by ONOO- and/or NO itself. It also re-emphasizes that SIN-1 induces a vasorelaxant response, in part, via stimulation of sGC.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Rescue of volume-regulated anion current by bestrophin mutants with altered charge selectivity

Mutations in human bestrophin-1 are linked to various kinds of retinal degeneration. Although it has been proposed that bestrophins are Ca(2+)-activated Cl(-) channels, definitive proof is lacking partly because mice with the bestrophin-1 gene deleted have normal Ca(2+)-activated Cl(-) currents. Here, we provide compelling evidence to support the idea that bestrophin-1 is the pore-forming subunit of a cell volume-regulated anion channel (VRAC) in Drosophila S2 cells. VRAC was abolished by treatment with RNAi to Drosophila bestrophin-1. VRAC was rescued by overexpressing bestrophin-1 mutants with altered biophysical properties and responsiveness to sulfhydryl reagents. In particular, the ionic selectivity of the F81C mutant changed from anionic to cationic when the channel was treated with the sulfhydryl reagent, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) (P(Cs)/P(Cl) = 0.25 for native and 2.38 for F81C). The F81E mutant was 1.3 times more permeable to Cs(+) than Cl(-). The finding that VRAC was rescued by F81C and F81E mutants with different biophysical properties shows that bestrophin-1 is a VRAC in S2 cells and not simply a regulator or an auxiliary subunit. F81C overexpressed in HEK293 cells also exhibits a shift of ionic selectivity after MTSES(-) treatment, although the effect is quantitatively smaller than in S2 cells. To test whether bestrophins are VRACs in mammalian cells, we compared VRACs in peritoneal macrophages from wild-type mice and mice with both bestrophin-1 and bestrophin-2 disrupted (best1(-/-)/best2(-/-)). VRACs were identical in wild-type and best1(-/-)/best2(-/-) mice, showing that bestrophins are unlikely to be the classical VRAC in mammalian cells.     

Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1

Inducible nitric oxide synthetase plays an essential role in insulin resistance induced by a high-fat diet. The reaction of nitric oxide with superoxide leads to the formation of peroxynitrite (ONOO-), which can modify several proteins. In this study, we investigated whether peroxynitrite impairs insulin-signalling pathway. Our experiments showed that 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), a constitutive producer of peroxynitrite, dose-dependently inhibited insulin-stimulated glucose uptake. While SIN-1 did not affect the insulin receptor protein level and tyrosine phosphorylation, it reduced the insulin receptor substrate-1 (IRS-1) protein level, and IRS-1 associated phosphatidylinositol-3 kinase (PI-3 kinase) activity. Although SIN-1 did not induce Ser307 phosphorylation of IRS-1, tyrosine nitration of IRS-1 was detected in SIN-1-treated-Rat1 fibroblasts expressing human insulin receptors. Mass spectrometry showed that peroxynitrite induced at least four nitrated tyrosine residues in rat IRS-1, including Tyr939, which is critical for association of IRS-1 with the p85 subunit of PI-3 kinase. Our results suggest that peroxynitrite reduces the IRS-1 protein level and decreases phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death

3-Morpholinosyndnomine (SIN-1) has been reported to be a peroxynitrite (OONO(-)) donor because it produces both nitric oxide (NO) and superoxide (O(2)(-).) upon decomposition in aqueous solution. However, SIN-1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN-1 caused a concentration-dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3-nitrotyrosine into the culture medium. The increase in 3-nitrotyrosine immunoreactivity, a footprint of OONO(-) production, was specific for SIN-1 as exposure to neurotoxic concentrations of an NO donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN-1-induced injury and 3-nitrotyrosine staining were prevented by the addition of either 5,10,15,20-Tetrakis (4-sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO(-) decomposition catalyst, or uric acid, an OONO(-) scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO(-) from SIN-1 as well as its cytotoxicity. Removal of O(2)(-). and the subsequently formed H(2)O(2) by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein-bound tyrosine but actually enhanced the cytotoxicity of SIN-1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN-1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non-NMDA receptors contributed to the SIN-1-mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN-1 initiates neural cell death in an OONO(-)-dependent or -independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO-related species generated.     

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl^– currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[³H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[³H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (80% inhibition) by intracellular Ca²⁺ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[³H]aspartate release activated by the substantial hyposmotic swelling showed little (25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca²⁺-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca²⁺/calmodulin-independent signaling mechanism. Ca²⁺-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication     

Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 microM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/ SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO-scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1 -treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.     

NMDA-induced acetylcholine release in mouse striatum: role of NO synthase isoforms

Striatal cholinergic interneurons are stimulated by glutamatergic inputs from thalamus and cortex via NMDA receptors. The present microdialysis study was designed to characterize the role of nitric oxide (NO) in this process and to identify the NO synthase (NOS) isoform responsible for this effect. For this purpose, we studied the effects of NMDA and 3-morpholino sydnonimine (SIN-1) perfusions on the release of acetylcholine (ACh) in mouse striatum. In wild-type C57/Bl6 mice, perfusion of NMDA (100 micro m) induced a two-fold stimulation of ACh release. This effect was attenuated in mice lacking endothelial NOS but was completely absent in mice lacking neuronal NOS. Local perfusion of SIN-1 (300 micro m), an NO donor, increased ACh release by more than two-fold in all three mouse lines. We conclude that NO synthesized by neuronal NOS provides a nitrergic link in the glutamatergic stimulation of striatal cholinergic interneurons.     

Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO- may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO- on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO- or decomposed ONOO- in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2'-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO- treatment in vitro inhibited enterocyte proliferation. ONOO- treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO- blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO- may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.     

3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.     

一氧化氮抑制内皮素促血管平滑肌细胞增殖作用的信号转导途径

培养的家兔胸主动脉血管平滑肌细胞（ＶＳＭＣ）分别以内皮素（ＥＴ－１）、一氧化氮（ＮＯ）前体Ｌ－Ａｒｇ和ＮＯ供体ＳＩＮ－１刺激,或用ＥＴ－１＋Ｌ－Ａｒｇ、ＥＴ－１＋ＳＩＮ－１联合刺激,测ＶＳＭＣ＾３Ｈ－ＴｄＲ掺入、丝裂素活化蛋白激酶（ＭＡＰＫ）活性及蛋白激酶Ｃ（ＰＫＣ）活性的改变,以研究ＮＯ抑制ＥＴ－１促ＶＳＭＣ增殖作用的信号转导途径。结果表明：（１）ＥＴ－１ １０＾－８ｍｏｌ／Ｌ单独刺激,＾３Ｈ－