Excessive NO Generation Research Articles

Glutathione Depletion-Induced ROS/NO Generation for Cascade Breast Cancer Therapy and Enhanced Anti-Tumor Immune Response.

As an effective alternative choice to traditional mono-therapy, multifunctional nanoplatforms hold great promise for cancer therapy. Based on the strategies of Fenton-like reactions and reactive oxygen species (ROS)-mediated therapy, black phosphorus (BP) nanoplatform BP@Cu2O@L-Arg (BCL) co-assembly of cuprous oxide (Cu2O) and L-Arginine (L-Arg) nanoparticles was developed and evaluated for synergistic cascade breast cancer therapy. Cu2O particles were generated in situ on the surface of the BP nanosheets, followed by L-Arg incorporation through electrostatic interactions.In vitro ROS/nitric oxide (NO) generation and glutathione (GSH) depletion were evaluated. In vitro and in vivo anti-cancer activity were also assessed. Finally, immune response of BCL under ultrasound was investigated. Cu2O was incorporated into BP to exhaust the overexpressed intracellular GSH in cancer cells via the Fenton reaction, thereby decreasing ROS consumption. Apart from being used as biocompatible carriers, BP nanoparticles served as sonosensitizers to produce excessive ROS under ultrasound irradiation. The enhanced ROS accumulation accelerated the oxidation of L-Arg, which further promoted NO generation for gas therapy. In vitro experiments revealed the outstanding therapeutic killing effects of BCL under ultrasound via mechanisms involving GSH deletion and excessive ROS and NO generation. In vivo studies have illustrated that the nanocomplex modified the immune response by promoting macrophage and CD8+ cell infiltration and inhibiting MDSC infiltration. BCL nanoparticles exhibited multifunctional characteristics for GSH depletion-induced ROS/NO generation, making a new multitherapy strategy for cascade breast cancer therapy.

Role of LPS-elicited signaling in triggering gastric mucosal inflammatory responses to H. pylori: modulatory effect of ghrelin.

Infection with Helicobacter pylori is a primary culprit in the etiology of gastric disease, and its cell-wall lipopolysaccharide (LPS) is recognized as a potent endotoxin responsible for triggering a pattern of the mucosal inflammatory responses. The engagement by the LPS of gastric mucosal Toll-like receptor 4 (TLR4) leads to initiation of signal transduction events characterized by the activation of mitogen-activated protein kinase (MAPK) cascade, induction of phosphoinositide-specific phospholipase C (PLC)/protein kinase C (PKC)/phosphatidylinositol 3-kinase (PI3K) pathway, and up-regulation in Src/Akt. These signaling events in turn exert their influence over H. pylori-elicited excessive generation of NO and PGE2 caused by the disturbances in nitric oxide synthase and cyclooxygenase isozyme systems, increase in epidermal growth factor receptor transactivation, and the induction in matrix metalloproteinase-9 (MMP-9) release. Interestingly, the extent of gastric mucosal inflammatory response to H. pylori is influenced by a peptide hormone, ghrelin, the action of which relays on the growth hormone secretagogue receptor type 1a (GHS-R1a)-mediated mobilization of G-protein dependent transduction pathways. Yet, the signals triggered by TLR-4 activation as well as those arising through GHS-R1a stimulation converge at MAPK and PLC/PKC/PI3K pathways that form a key integration node for proinflammatory signals generated by H. pylori LPS as well as for those involved in modulation of inflammation by ghrelin. Hence, therapeutic targeting these signals' convergence and integration node could provide a novel and attractive opportunities for developing more effective treatments of H. pylori-related gastric disease.

Telmisartan attenuates myocardial apoptosis induced by chronic intermittent hypoxia in rats: modulation of nitric oxide metabolism and inflammatory mediators.

NO and NO synthase (NOS) are known to play key roles in the development of myocardial apoptosis induced by ischemia/hypoxia. Current evidence suggests that angiotensin II type 1 receptor blockers, such as telmisartan, lower blood pressure and produce beneficial regulatory effects on NO and NOS. Here, we examined the protective role of telmisartan in myocardial apoptosis induced by chronic intermittent hypoxia (CIH). Adult male Sprague-Dawley rats were subjected to 8h of intermittent hypoxia/day, with/without telmisartan for 8weeks. Myocardial apoptosis, NO and NOS activity, and levels of inflammatory mediators and radical oxygen species were determined. Treatment with telmisartan preserved endothelial NOS expression and inhibited inducible NOS and excessive NO generation, while reducing oxidation/nitration stress and inflammatory responses. Administration of telmisartan before CIH significantly ameliorated the CIH-induced myocardial apoptosis. This study show that pre-CIH telmisartan administration ameliorated myocardial injury following CIH by attenuating CIH-induced myocardial apoptosis via regulation of NOS activity and inhibition of excessive NO generation, oxidation/nitration stress, and inflammatory responses.

NitroDIGE analysis reveals inhibition of protein S-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells

BackgroundNitric oxide (NO) is a signaling molecule regulating numerous cellular functions in development and disease. In the brain, neuronal injury or neuroinflammation can lead to microglial activation, which induces NO production. NO can react with critical cysteine thiols of target proteins forming S-nitroso-proteins. This modification, known as S-nitrosylation, is an evolutionarily conserved redox-based post-translational modification (PTM) of specific proteins analogous to phosphorylation. In this study, we describe a protocol for analyzing S-nitrosylation of proteins using a gel-based proteomic approach and use it to investigate the modes of action of a botanical compound found in green tea, epigallocatechin-3-gallate (EGCG), on protein S-nitrosylation after microglial activation.Methods/ResultsTo globally and quantitatively analyze NO-induced protein S-nitrosylation, the sensitive gel-based proteomic method, termed NitroDIGE, was developed by combining two-dimensional differential in-gel electrophoresis (2-D DIGE) with the modified biotin switch technique (BST) using fluorescence-tagged CyDye™ thiol reactive agents to label S-nitrosothiols. The NitroDIGE method showed high specificity and sensitivity in detecting S-nitrosylated proteins (SNO-proteins). Using this approach, we identified a subset of SNO-proteins ex vivo by exposing immortalized murine BV-2 microglial cells to a physiological NO donor, or in vivo by exposing BV-2 cells to endotoxin lipopolysaccharides (LPS) to induce a proinflammatory response. Moreover, EGCG was shown to attenuate S-nitrosylation of proteins after LPS-induced activation of microglial cells primarily by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response.ConclusionsThese results demonstrate that NitroDIGE is an effective proteomic strategy for “top-down” quantitative analysis of protein S-nitrosylation in multi-group samples in response to nitrosative stress due to excessive generation of NO in cells. Using this approach, we have revealed the ability of EGCG to down-regulate protein S-nitrosylation in LPS-stimulated BV-2 microglial cells, consistent with its known antioxidant effects.

English

Introduction In this study, we report on the role of epidermal growth factor receptor (EGFR) transactivation in H. pylori lipopolysaccharide (LPS)-elicited induction in gastric mucosal expression of COX-2 and iNOS. Materials and methods We aimed to demonstrate that the LPS-induced p38 activation along with MMPs are of critical significance to EGFR transactivation that leads to up-regulation in the activation of ERK signalling cascade, amplification in iNOS and COX-2 induction, and consequently to the excessive increase in gastric mucosal PGE2 and NO generation. Results EGFR transactivation results in the amplification of the LPS-induced ERK phosphorylation and up-regulation in ERK-mediated IKK-β activation for the enhanced induction of NFκB-dependent expression of iNOS. The rise in iNOS-dependent NO generation, in turn, leads to an up-regulation in COX-2 activation through Snitrosylation and excessive PGE2 production. Conclusion Taken together, our data provide strong indications of the involvement of EGFR transactivation in the amplification of ERK signalling cascade associated with up-regulation in gastric mucosal induction of iNOS and COX-2, and excessive NO and PGE2 generation in response to H. pylor

The threshold of pentylenetetrazole-induced convulsive seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains

Alterations in the NO pathway play an important role in the development of convulsive seizures via the glutamatergic and GABAergic systems in acute pentylenetetrazole (PTZ) seizure animals. We previously reported that the background NO levels under physiological conditions negatively regulate convulsive seizures, while excess NO levels under pathologic conditions positively regulate PTZ-induced convulsive seizures. In this study, the NO content in various brain regions after a single dose injection of PTZ was quantitatively and directly measured using the ex vivo X-band electron paramagnetic resonance method with an NO-trapping agent. Experimental data demonstrated the effects of NO on the convulsive seizure threshold: a 1.5-fold increase in the NO level in all brain regions compared to that observed in the control state showed proconvulsive properties without any involvement with nonconvulsive seizures. The distribution of the background NO content in the normal animals was higher in the temporal region of the cerebral cortex, including the amygdala, than in the hippocampus, cerebellum and other regions of the cerebral cortex. However, the levels of NO after the occurrence of acute PTZ-induced convulsive seizures significantly increased by more than 50% in all brain regions, thus suggesting that the NO levels in all brain regions contribute to PTZ-induced convulsions as a seizure threshold. In a pharmacological study, the inhibitor of neuronal NO synthase and antagonists of ionotropic glutamate receptors prevented PTZ-induced convulsions and excessive NO generation. In addition, therapeutic drugs, such as valproate and ethosuximide used to treat generalized seizures not only inhibited the increase in NO generation induced by PTZ, but also prevented both convulsive and nonconvulsive seizures caused by PTZ. We herein provide novel insight into the involvement of NO in PTZ-seizure susceptibility at the whole-animal level.

SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI)

BackgroundUbiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration.MethodsTo study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining.ResultsOur data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured astrocytes following oxygen glucose deprivation and reperfusion was also suppressed by 1400W. Interestingly, these aggregates were colocalized with SOD1, which was found to co-immunoprecipitate with PDI.ConclusionsNO-mediated S-nitrosylation of PDI may be involved in the formation of the SOD1-linked ubiquitinated-protein aggregates in cerebral ischemia/reperfusion injury.

Baicalin protects human skin fibroblasts from ultraviolet A radiation-induced oxidative damage and apoptosis

Reactive oxygen species (ROS) are an important factor in the development of skin photodamage after ultraviolet A (UVA) radiation. A flavonoid antioxidant, baicalin, can selectively neutralize super-oxide anion (O2−) while having no significant effect on •OH. Fibroblasts are a key component of skin dermis. In the present study, we investigated the protective effects of baicalin on human skin fibroblasts (HSFs) under UVA induced oxidative stress. Fluorescence microscopy and flow cytometry were used to assay the intracellular O2−, NO, ROS concentrations and the mitochondrial membrane potential. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular MDA, SOD, GSH, T-AOC, and 8-oxo-dG were also measured. Cellular apoptosis was measured by flow cytometry and caspase-3 detection. The results revealed that UVA radiation could cause oxidative stress and apoptosis in HSFs. Interestingly, the use of baicalin after UVA radiation significantly reduced the level of intracellular O2−, NO, and ROS, stabilized the mitochondrial membrane potential, and attenuated production of MDA and 8-oxo-dG. These efficiently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage and apoptosis. In other words, baicalin decreased the excessive generation of intracellular ROS and NO, and elevated the cellular antioxidative defense, which eventually mitigate the UVA-induced apoptosis. Based on our results, baicalin may have applications in the treatment of skin photodamage caused by UVA irradiation.

Modulation of gastric mucosal inflammatory responses to <i>Helicobacter pylori</i> by ghrelin: Role of cNOS-dependent IKK-<i>β</i> S-nitrosylation in the regulation of COX-2 activation

Disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) isozyme systems, manifested by the excessive NO and prostaglandin (PGE2) generation, are well-recognized features of gastric mucosal inflammatory responses to H. pylori infection. In this study, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and COX-2 activation was accompanied by the impairment in constitutive (c) cNOS phosphorylation, up-regulation in the inhibitory κB kinase-β (IKKβ) activation and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that abrogation of cNOS control over NF-κB activation has lead to induction of iNOS expression and COX-2 activation through S-nitrosylation. Moreover, we demonstrate that the modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOS-mediated IKK-β protein S-nitrosylation. As a result, ghrelin exerted the inhibitory effect on NF-κB nuclear translocation, thus causing the repression of iNOS gene induction and the inhibition in COX-2 activation through iNOS-dependent S-nitrosylation. Our findings point to cNOS activation as a pivotal element in the signaling cascade by which ghrelin exerts modulatory control over proinflammatory events triggered in gastric mucosa by H. pylori infection.

S-Nitrosylation of Critical Protein Thiols Mediates Protein Misfolding and Mitochondrial Dysfunction in Neurodegenerative Diseases

Excessive nitrosative and oxidative stress is thought to trigger cellular signaling pathways leading to neurodegenerative conditions. Such redox dysregulation can result from many cellular events, including hyperactivation of the N-methyl-D-aspartate-type glutamate receptor, mitochondrial dysfunction, and cellular aging. Recently, we and our colleagues have shown that excessive generation of free radicals and related molecules, in particular nitric oxide species (NO), can trigger pathological production of misfolded proteins, abnormal mitochondrial dynamics (comprised of mitochondrial fission and fusion events), and apoptotic pathways in neuronal cells. Emerging evidence suggests that excessive NO production can contribute to these pathological processes, specifically by S-nitrosylation of specific target proteins. Here, we highlight examples of S-nitrosylated proteins that regulate misfolded protein accumulation and mitochondrial dynamics. For instance, in models of Parkinson's disease, these S-nitrosylation targets include parkin, a ubiquitin E3 ligase and neuroprotective molecule, and protein-disulfide isomerase, a chaperone enzyme for nascent protein folding. S-Nitrosylation of protein-disulfide isomerase may also be associated with mutant Cu/Zn superoxide dismutase toxicity in amyotrophic lateral sclerosis. Additionally, in models of Alzheimer's disease, excessive NO generation leads to the formation of S-nitrosylated dynamin-related protein 1 (forming SNO-Drp1), which contributes to abnormal mitochondrial fragmentation and resultant synaptic damage.

Role of constitutive nitric oxide synthase in regulation of <i>Helicobacter pylori</i>-induced gastric mucosal cyclooxygenase-2 ac-tivation through S-nitrosylation: mechanism of ghrelin action

Gastric mucosal inflammatory responses to H. pylori lipopolysaccharide (LPS), are characterized by the excessive NO and prostaglandin (PGE2) generation due to the disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) systems. Here, we report that the LPS-induced enhancement in gastric mucosal inducible (i) iNOS) activity and up-regulation in PGE2 production was associated with the suppression in Akt kinase activity and the impairment in constitutive (c) cNOS activation. The stimulatory effect of the LPS on PGE2 production, furthermore, was susceptible to suppression by COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400 W. Further, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in up-regu- lation in Akt activity and the increase in cNOS activation through phosphorylation, and accompanied by the suppression in iNOS expression and the reduction in COX-2 activity associated with the loss in COX-2 protein S-nitrosylation. Moreover, the effect of ghre-lin on the LPS-induced COX-2 S-nitrosylation was subject to repression by Akt inhibition. Our findings demonstrate that induction in iNOS with H. pylori in- fection leads to COX-2 activation through S-nitro- sylation and up-regulation in PGE2 generation, and that ghrelin counters these untoward consequences of the LPS through Akt-mediated up-regulation in cNO- S activation required for the iNOS gene repression.

Role of constitutive nitric oxide synthase S-nitrosylation in Helicobacter pylori-induced gastric mucosal cell apoptosis: effect of ghrelin

Infection with H. pylori is a primary factor in the etiology of gastric disease, and the excessive NO generation and a massive rise in apoptosis are well recognized features that characterize the mucosal inflammatory responses to the bacterium and its lipopolysaccharide (LPS). Here, we report that H. pylori LPS-induced enhancement in gastric mucosal cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked up-regulation in the activity of inducible nitric oxide synthase (iNOS). Further, we demonstrate that the detrimental effect of the LPS on cNOS was manifested in the enzyme protein S-nitrosylation, that was susceptible to suppression by iNOS inhibitor, 1400W. Moreover, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes in apoptosis and cNOS activity was reflected in the loss in cNOS S-nitrosylation and the increase in the enzyme phosphorylation. These findings demonstrate that the disturbances in gastric mucosal NO generation system caused by H. pylori result from the iNOS-derived NO suppression of cNOS activation through S-nitrosylation. We also report that ghrelin protection against H. pylori-induced gastric mucosal proapoptotic events involves cNOS activation manifested by the increase in enzyme protein phosphorylation and a decrease in its S-nitrosylation.

Correction: Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

Correction: Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

Correction: Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

Correction: Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

BackgroundA distinctive feature of type 2 diabetes is inability of insulin-secreting β-cells to properly respond to elevated glucose eventually leading to β-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of β-cell dysfunction.Principal FindingsWe show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon.ConclusionThe results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms.

Response of Asymmetric Dimethylarginine to Hemodialysis-Associated Hypotension in End-Stage Renal Disease Patients

Aims: To define the role of asymmetric dimethylarginine (ADMA) in the control of blood pressure (BP) during hemodialysis (HD). Methods:L-Arginine, ADMA and symmetric dimethylarginine (SDMA) levels of patients with (n = 18) or without (n = 13) hypotensive episodes during HD sessions were measured before and after HD treatment by liquid chromatography-mass spectrometry. Clinical variables, laboratory parameters and underlying pathologies of end-stage renal disease (ESRD) were comparable in the groups. BP was serially recorded. Results: In patients with ESRD, plasma dimethylarginines were markedly elevated and decreased significantly by the end of the HD sessions. ADMA levels in patients having hypotensive episodes during HD were significantly higher than in those maintaining their BP (before HD: 0.62 ± 0.11 µmol/l vs. 0.71 ± 0.13 µmol/l, p = 0.04; after HD: 0.31 ± 0.11 µmol/l vs. 0.43 ± 0.11 µmol/l, p = 0.01). There was a significant inverse relationship of the minimum systolic and diastolic BP during HD to the predialysis ADMA levels (for systolic BP r = –0.50, p < 0.01; for diastolic BP r = –0.59, p < 0.01) and to the postdialysis ADMA levels (for systolic BP r = –0.49, p < 0.01; for diastolic BP r = –0.51, p < 0.005), respectively. Conclusions: It is suggested that excessive NO generation is involved in the HD-associated hypotension and induces an increase in plasma ADMA levels to prevent further fall in BP.

Protective effects of burdock ( Arctium lappa Linne) on oxidation of low-density lipoprotein and oxidative stress in RAW 264.7 macrophages

The protective effects of burdock ( Arctium lappa Linne) on oxidation of low-density lipoprotein (LDL) and nitric oxide production were investigated. The results showed that methanolic extracts of burdock (MEB) and their major components, chlorogenic acid (CHA) and caffeic acid (CA), showed marked antioxidant activity against oxidative damage of liposome ( p < 0.05), deoxyribose ( p < 0.05) and protein ( p < 0.05). In addition, at a concentration of 500 μg/ml, the inhibitory effect of MEB on LDL oxidation was 66.9% compared to the control ( p < 0.05). MEB, at 200 μg/ml, not only enhanced GSH levels, but also increased activity of GSH reductase, GSH peroxidase, GSH transferase and catalase, which were 3.82-, 24.9-, 4.35- and 3.02-fold compared to the control ( p < 0.05), respectively. MEB directly scavenged nitric oxide in a concentration-dependent fashion ( p < 0.05). Moreover, MEB showed a reducing effect on nitric oxide production of lipopolysaccharide (LPS)-induced RAW 264.7 cells. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in activated RAW 264.7 cells were inhibited by MEB. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the expression of iNOS and COX-2 mRNA in activated macrophages were suppressed by a high concentration (500 μg/ml) of MEB. Furthermore, a downregulated degradation of IκB-α by MEB was found, indicating that MEB reduced iNOS enzyme expression as a result of preventing NF-κB activation. These results suggest that MEB displays an inhibitory action on biomolecules and has a bioactive action for attenuating excessive NO generation at inflammatory site as well as in cardiovascular disease.

Reciprocal changes in endothelium‐derived hyperpolarizing factor‐ and nitric oxide‐system in the mesenteric artery of adult female rats following ovariectomy

1. To explore the effects of estrogen on arterial functions, we examined endothelium-derived hyperpolarizing factor (EDHF)- and NO-mediated responses in isolated mesenteric arteries of female rats, 4 weeks after sham-operation (CON), ovariectomy (OVX) and OVX plus chronic estrogen treatment (OVX+E(2)). Tissue levels of connexins-40, 43 (major components of gap junction), inducible NOS (iNOS), endothelial NOS (eNOS) and eNOS regulator proteins such as calmodulin, heat shock protein 90 (hsp90) and caveolin-1 were also examined using Western blot. 2. In OVX, acetylcholine (ACh)-induced EDHF-mediated relaxation and membrane hyperpolarization of arterial smooth muscles were reduced, whereas ACh-induced NO-mediated relaxation was enhanced, leading to no change in ACh-induced relaxation. 3. In OVX, connexin-40 and 43 were decreased. Tissue levels of eNOS and its positive regulators (calmodulin and hsp90) were unchanged, but that of its negative regulator, caveolin-1, was decreased. The levels of iNOS in mesenteric artery and aorta and plasma levels of NO metabolites and cholesterol were elevated. 4. In OVX, contraction of the artery by phenylephrine was reduced, but augmented by nonspecific inhibitor of NOS to the comparable level as that in CON group. The contraction in OVX group unlike that in CON group was augmented by specific iNOS inhibitor, and the difference between contractions in the presence of nonspecific and specific inhibitor as an index of eNOS activity was increased. 5. In OVX+E(2), all these changes were recovered. 6. In all groups, EDHF-mediated relaxation was suppressed by 18beta-glycyrrhetinic acid, an inhibitor of gap junction. 7. These results indicate that estrogen deficiency does not change the diameter of mesenteric artery: it reduces EDHF-mediated relaxation by decreasing gap junction, whereas it augments NO-mediated relaxation via an increase in NO release. Increased NO result from increased activity of eNOS subsequent to a decrease in caveolin-1 and from induction of iNOS. However, excessive NO generation with elevated plasma cholesterol would raise a risk for atherosclerosis.

Calcium-activated NO production plays a role in neuronal death induced by beta-bungarotoxin in primary cultures of cerebellar granular neurons.

The aim of this study was to elucidate the mechanism underlying the neurotoxic effect of beta-bungarotoxin (beta-BuTX) on cultured cerebellar granular neurons (CGN). Beta-BuTX had a potent time- and concentration-dependent neurotoxic effect on mature CGN. Beta-BuTX appeared to destroy initially the neurites and then caused neuronal death by both apoptotic and necrotic processes. Inspection using Nomarski optics showed that these neurons displayed morphological features of necrotic cells, including cell swelling, loss of membrane integrity and eventual dissolution of the cell. Staining with the fluorescent dye Hoechst 33258 showed that beta-BuTX-treated neuron bodies stained more densely with smaller apoptotic bodies. Using microspectrofluorimetry and fura-2 to measure cytosolic [Ca(2+)] ([Ca(2+)](i)), beta-BuTX markedly increased [Ca(2+)](i). BAPTA-AM, EGTA, MK 801 and diltiazem not only attenuated the beta-BuTX-mediated rise in [Ca(2+)](i) but also attenuated beta-BuTX-mediated neurotoxicity. In addition, these Ca(2+) inhibitors prevented the beta-BuTX-induced generation of reactive nitrogen species. The NO synthase inhibitor N(G)-methyl- l-arginine) also exhibited neuroprotection. This is the first report showing that beta-BuTX-induced CGN death is mediated, at least in part, by excessive generation of NO triggered by [Ca(2+)](i) overloading. Activation of NMDA receptors and L-type calcium channels is apparently involved in the increase in [Ca(2+)](i) induced by this neurotoxin. This potent neurotoxin will be a useful tool for studying neurotoxic processes and using this model system will allow us to find neuroprotective agents.