Inhibition Of Nitric Oxide Generation Research Articles

GC–MS based antioxidants characterization in Saussurea heteromalla (D. Don) Hand-Mazz by inhibition of nitric oxide generation in macrophages

For centuries, medicinal plants have served as the cornerstone for traditional health care systems and same practice is still prevalent today. In the Himalayan region, Saussurea heteromalla holds a significant place in traditional medicine and is used to address various health issues. Despite its historical use, little exploration has focused on its potential for scavenging free radicals and reducing inflammation. Hence, our current study aims to investigate the free radical scavenging capabilities of S. heteromalla extracts. The n-hexane extract of entire plant revealed promising activity. This extract underwent extensive extraction on a larger scale. Subsequent purification, employing column chromatography, HPLC–DAD techniques, led to the identification of active compounds, confirmed via GC–MS and the NIST database as 1-O-butyl 2-O-octyl benzene-1,2-dicarboxylate and 2,4-ditert-butylphenol. Assessing the free radical scavenging properties involved utilizing RAW-264.7 macrophages activated by lipopolysaccharides. Notably, the compound 2,4-di-tert-butylphenol exhibited remarkable scavenging abilities, demonstrating over 80% inhibition of Nitric oxide. This study stands as the inaugural report on the isolation of these compounds from S. heteromalla.

Structure and anti-inflammatory activity of neo-clerodane diterpenoids from Scutellaria barbata

Herein, 13 previously undescribed neo-clerodane diterpenoids (1–13) and 27 known analogs (14–40) were isolated from the aerial parts of Scutellaria barbata. Absolute configurations of undescribed compounds were assigned based on single-crystal X-ray diffraction analysis and comparison of experimental and circular dichroism. All isolates were evaluated for the inhibition of nitric oxide generation induced by lipopolysaccharide in RAW 264.7 macrophages. Compound 36 was found to be the most active with an IC50 value of 10.6 μM. Structure–activity relations of these neo-clerodane diterpenoids revealed that the α, β-unsaturated-γ-lactone moiety with an exocyclic conjugated double bond was necessary for maintaining and increasing its activity. Further mechanistic studies show that compound 36 suppressed nitric oxide synthase enzymes (iNOS) expression without affecting iNOS activity. Additionally, compound 36 suppresses NF-κB signaling by inhibiting IκBα phosphorylation.

Ergostane-type sterols and sesquiterpenes with anti-neuroinflammatory activity from a Nigrograna species associated with Clematis shensiensis

Nigrograna sp. LY66, an endophytic fungus associated with the herbal medicinal plant Clematis shensiensis, produced four undescribed steroids, nigergostanes A−D (1−4), including an unusual ketal-containing nigergostane (1), and four undescribed sesquiterpenoids decorated with cyclohexanone motifs, nigbisabolanes A−D (7−10), along with three known compounds, 23R-hydroxy-(20Z,24R)-ergosta-4,6,8(14),20(22)-tetraen-3-one (5), ergosta-5,7,22-trien-3β-ol (6), and curculonone A (11). The structures and absolute configurations of these undescribed compounds were confirmed using spectroscopic data (NMR and HRESIMS), modified Mosher's method, and ECD experiments. Additionally, compounds 5 and 8 displayed significant inhibition of nitric oxide generation in lipopolysaccharide-induced BV-2 microglial cells with IC50 values of 2.8 and 2.7 μM, respectively, and is thus more potent than that of the positive control, quercetin (IC50 = 8.77 μM). A molecular docking study revealed that 23-OH of 5 binds to the Y347 residue of inducible nitric oxide synthase (iNOS), whereas the 2-OH and 9,10-diol moieties of 8 bind to R381 and W463 and haeme residues of iNOS, respectively, which has rarely been reported in previous studies. These findings provide a set of undescribed lead compounds that can be developed into anti-neuroinflammatory agents.

Transforming growth factor-β1/Thrombospondin-1/CD47 axis mediates dysfunction in CD34+ cells derived from diabetic older adults

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34+ cells. Transforming growth factor- β1 (TGF-β1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- β1 (TGF-β1) is upregulated in diabetic CD34+ cells and impairs NO generation via thrombospondin-1 (TSP-1)/CD47/NO pathway. CD34+ cells from nondiabetic (ND) (n=58) or diabetic older adults (DB) (both type 1 and type 2) (n=62) were isolated from peripheral blood. TGF-β1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-β1). Migration and proliferation in response to stromal-derived factor-1α (SDF-1α) were evaluated. NO generation and eNOS phosphorylation were determined by flow cytometry. CD34+ cells from older, but not younger, diabetics have higher expression of TGF-β1 compared to that observed in cells derived from healthy individuals (P<0.05, n=14). TSP-1 expression was higher (n=11) in DB compared to ND cells. TGFβ1-PMO decreased the secretion of TGF-β1, which was accompanied with decreased TSP-1 expression. Impaired proliferation, migration and NO generation in response to SDF-1α in DB cells were reversed by TGF-β1-PMO (n=6). TSP-1 inhibited migration and proliferation of nondiabetic CD34+ cells that was reversed by CD47-siRNA, which also restored these responses in diabetic CD34+ cells. TSP-1 opposed SDF-1α-induced eNOS phosphorylation at Ser1177 that was reversed by CD47-siRNA. These results infer that increased TGF-β1 expression in CD34+ cells induces dysfunction in CD34+ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation.

Effect of nifedipine, a calcium channel blocker, on the generation of nitric oxide and interleukin-1β by murine macrophages activated by lipopolysaccharide from Prevotella intermedia.

Nifedipine, a calcium channel blocker, has been reported to possess anti-inflammatory and immunosuppressive effects. The current study was undertaken to explore the influence of nifedipine on the generation of proinflammatory mediators by murine macrophages activated by lipopolysaccharide (LPS) prepared from Prevotella intermedia, a putative periodontal pathogen, and associated mechanisms of action as well. LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1β. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. NF-κB-dependent secreted embryonic alkaline phosphatase (SEAP) levels were estimated by reporter assay. Nifedipine markedly suppressed the generation of iNOS-derived NO and IL-1β together with their mRNA expressions in murine macrophages activated by P. intermedia LPS. LPS-stimulated cells exposed to nifedipine notably increased the mRNA levels of Arg-1, Ym-1, FIZZ1, and TGF-β, which are typical markers for M2 macrophage polarization. Nifedipine induced HO-1 at both gene and protein levels in cells challenged with P. intermedia LPS, and the nifedipine-mediated inhibition of NO generation was significantly abrogated by adding SnPP, an HO-1 inhibitor. Nifedipine inhibited LPS-evoked generation of NO and IL-1β in a PPAR-γ-independent manner. In addition, NF-κB activation as well as phosphorylation of STAT1/3 induced by P. intermedia LPS was suppressed by nifedipine. Nifedipine is an inhibitor of P. intermedia LPS-evoked production of NO and IL-1β in murine macrophages and encourages macrophage polarization toward the M2 phenotype. Nifedipine possibly has potential to be used for host modulation of periodontal disease and is worth being further researched.

Corticosterone rapidly improves the endurance of high-intensity exercise (swimming) via nongenomic mechanisms in mice.

Glucocorticoids (GCs) take a pivotal role during the stress response. Some clinical studies suggest short-term GCs intake improves exercise endurance. However, whether the rapid nongenomic effects of GCs are involved in acute exercise is still unknown. Here, we aimed to reveal the potential nongenomic effects of GCs in skeletal muscle of mice during exercise. Adrenalectomized mice subjected to a weight-loaded forced swim were used for detecting the changes of time to exhaustion. Corticosterone (CORT) and other drugs were injected via the coccygeal vein before swimming. After exhaustion, the injury of skeletal muscle, nitric oxide generation, blood glucose and lactic acid were determined. The results demonstrated that CORT rapidly extended the time to exhaustion within 30 min (~30%), which could not be abolished by glucocorticoid receptor antagonist RU486. Pretreatment with the nitric oxide synthesis inhibitor L-NAME prior to CORT administration further increased exercise tolerance compared to the increase caused by CORT alone. Moreover, CORT contributed to protecting skeletal muscle from injury and maintaining blood glucose. Considered together, our results suggest that GCs rapidly improve exercise tolerance via its nongenomic mechanism, which is associated with the inhibition of nitric oxide generation. Pretreatment of GCs may be helpful to enhance exercise tolerance during acute exercise.

Oxidative stress and inhibition of nitric oxide generation underlie methotrexate-induced senescence in human colon cancer cells

The response of human colon cancer C85 cells to methotrexate takes the form of reversible growth arrest of the type of stress-induced senescence. In the present study it is shown that during C85 cell progression into methotrexate-induced senescence, dihydrofolate reductase, the primary intracellular target for the drug, is stabilized at the protein level and its enzymatic activity, assayed in crude cellular extracts, decreases by 2-fold. Dihydrofolate reductase inhibition results in an increase in dihydrobiopterin level and an ultimate decrease in the tetrahydrobiopterin: dihydrobiopterin ratio in senescent cells. Endothelial nitric oxide synthase expression declines. Despite concomitant upregulation of inducible nitric oxide synthase expression, no nitric oxide generation in senescent cells is detected. Progressing oxidative stress accompanies establishment of the state of senescence. DNA damage, in the form of double strand-breaks, occurs at the highest level at the senescence initiation phase and decreases as cells progress into the senescence maintenance phase.

Structure-Antioxidative and Anti-Inflammatory Activity Relationships of Purpurin and Related Anthraquinones in Chemical and Cell Assays.

Anthraquinone (9,10-anthraquinone) and several hydroxy derivatives, including purpurin (1,2,4-trihydroxyanthraquinone), anthrarufin (1,5-dihydroxyanthraquinone), and chrysazin (1,8-dihydroxyanthraquinone), were evaluated for antioxidative and anti-inflammatory activities in chemical assays and mammalian cells (murine macrophage RAW 264.7 cells). Several tests were used to assess their activities: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical; ABTS radical cation; hydrogen peroxide scavenging; reduction of potassium ferricyanide; chelation of ferrous ions; inhibition of lipid peroxidation; inhibition of nitric oxide generation; scavenging of the intracellular hydroxyl radical; expression of NLRP3 polypeptide for inflammasome assembly; and quantitation of proinflammatory cytokine interleukin 1β (IL-1β) for inflammasome activation. The results show that purpurin, from the root of the madder plant (Rubia tinctorum L.), exhibited the highest antioxidative activity in both chemical and cultured cell antioxidant assays. The antioxidative activities of the other three anthraquinones were lower than that of purpurin. In addition, purpurin could down-regulate NLRP3 inflammasome assembly and activation, suggesting that it might protect foods against oxidative damage and prevent in vivo oxidative stress and inflammation. Structure-activity relationships and the significance of the results for food quality and human health are discussed.

Inwardly rectifying K+ channels are major contributors to flow-induced vasodilatation in resistance arteries.

Endothelial inwardly rectifying K+ (Kir2.1) channels regulate flow-induced vasodilatation via nitric oxide (NO) in mouse mesenteric resistance arteries. Deficiency of Kir2.1 channels results in elevated blood pressure and increased vascular resistance. Flow-induced vasodilatation in human resistance arteries is also regulated by inwardly rectifying K+ channels. This study presents the first direct evidence that Kir channels play a critical role in physiological endothelial responses to flow. Inwardly rectifying K+ (Kir) channels are known to be sensitive to flow, but their role in flow-induced endothelial responses is not known. The goal of this study is to establish the role of Kir channels in flow-induced vasodilatation and to provide first insights into the mechanisms responsible for Kir signalling in this process. First, we establish that primary endothelial cells isolated from murine mesenteric arteries express functional Kir2.1 channels sensitive to shear stress. Then, using the Kir2.1+/- heterozygous mouse model, we establish that downregulation of Kir2.1 results in significant decrease in shear-activated Kir currents and inhibition of endothelium-dependent flow-induced vasodilatation (FIV) assayed in pressurized mesenteric arteries pre-constricted with endothelin-1. Deficiency in Kir2.1 also results in the loss of flow-induced phosphorylation of eNOS and Akt, as well as inhibition of NO generation. All the effects are fully rescued by endothelial cell (EC)-specific overexpression of Kir2.1. A component of FIV that is Kir independent is abrogated by blocking Ca2+ -sensitive K+ channels. Kir2.1 has no effect on endothelium-independent and K+ -induced vasodilatation in denuded arteries. Kir2.1+/- mice also show increased mean blood pressure measured by carotid artery cannulation and increased microvascular resistance measured using a tail-cuff. Importantly, blocking Kir channels also inhibits flow-induced vasodilatation in human subcutaneous adipose microvessels. Endothelial Kir channels contribute to FIV of mouse mesenteric arteries via an NO-dependent mechanism, whereas Ca2+ -sensitive K+ channels mediate FIV via an NO-independent pathway. Kir2 channels also regulate vascular resistance and blood pressure. Finally, Kir channels also contribute to FIV in human subcutaneous microvessels.

Glucose-6-phosphate dehydrogenase plays critical role in artemisinin production of Artemisia annua under salt stress

Artemisinin, a natural sesquiterpenoid isolated from Artemisia annua L., is regarded as the most efficient drug against malaria in the world. Artemsinin production in NaCl-treated A. annua seedlings and its relationships with the glucose-6-phosphate dehydrogenase (G6PDH) activity and generation of H2O2 and nitric oxide (NO) were investigated. Results revealed that artemisinin content in the seedlings was increased by 79.3 % over the control after 1-month treatment with 68 mM NaCl. The G6PDH activity was enhanced in the presence of NaCl together with stimulated generation of H2O2 and NO. Application of 1.0 mM glucosamine (GlcN), an inhibitor of G6PDH, blocked the increase of NADPH oxidase and nitrate reductase (NR) activities, as well as H2O2 and NO production in A. annua seedlings under the salt stress. The induced H2O2 was found to be involved in the upgrading gene expression of two key enzymes in the later stage of artemisinin biosynthetic pathway: amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1). The released NO being attributed mainly to the increase of NR activity, negatively interacted with H2O2 production and enhanced gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Inhibition of NO generation partly blocked NaCl-induced artemisinin accumulation, and NO donor strongly rescued the decreased content of artemisinin caused by GlcN. These results suggest that G6PDH could play a critical role in NaCl-induced responses and artemisinin biosynthesis in A. annua.

Nitric oxide signals are interlinked with calcium signals in normal pancreatic stellate cells upon oxidative stress and inflammation.

The mammalian diffuse stellate cell system comprises retinoid-storing cells capable of remarkable transformations from a quiescent to an activated myofibroblast-like phenotype. Activated pancreatic stellate cells (PSCs) attract attention owing to the pivotal role they play in development of tissue fibrosis in chronic pancreatitis and pancreatic cancer. However, little is known about the actual role of PSCs in the normal pancreas. These enigmatic cells have recently been shown to respond to physiological stimuli in a manner that is markedly different from their neighbouring pancreatic acinar cells (PACs). Here, we demonstrate the capacity of PSCs to generate nitric oxide (NO), a free radical messenger mediating, for example, inflammation and vasodilatation. We show that production of cytosolic NO in PSCs is unambiguously related to cytosolic Ca2+ signals. Only stimuli that evoke Ca2+ signals in the PSCs elicit consequent NO generation. We provide fresh evidence for the striking difference between signalling pathways in PSCs and adjacent PACs, because PSCs, in contrast to PACs, generate substantial Ca2+-mediated and NOS-dependent NO signals. We also show that inhibition of NO generation protects both PSCs and PACs from necrosis. Our results highlight the interplay between Ca2+ and NO signalling pathways in cell–cell communication, and also identify a potential therapeutic target for anti-inflammatory therapies.

The Anti-inflammatory Effect of Gnaphalium affine Through Inhibition of NF-κB and MAPK in Lipopolysaccharide-Stimulated RAW264.7 Cells and Analysis of Its Phytochemical Components.

Gnaphalium affine is an annual herbaceous plant that is used as a traditional medicine in some Latin American and Asian countries. However, systematic studies on its anti-inflammatory activity and signaling pathways have not yet been reported. In this study, we investigated the anti-inflammatory effect of G. affine methanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells and fractioned the methanol extract into hexane, chloroform, ethyl acetate (EtOAc), butyl alcohol (BuOH), and distilled water (DW) by measuring the generation of nitric oxide (NO). G. affine inhibited the generation of NO and prostaglandin E2. The chloroform-soluble fraction most effectively inhibited LPS-stimulated NO production. We also examined the cytotoxicity of G. affine in three normal cell lines: RAW264.7, HEK293, and HaCaT. Cell viability assays showed that the methanol extract and chloroform-soluble fraction of G. affine had no cytotoxic effect on normal cell lines. The expression of pro-inflammatory mediators was also investigated. Western blotting and immunofluorescence showed that G. affine reduces the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in LPS-stimulated RAW264.7 cells. RT-PCR showed that G. affine also negatively regulates inflammatory cytokines at the gene expression level. Taken together, G. affine exerts its anti-inflammatory activity through inhibition of NO generation as a result of inhibiting NF-κB and MAPKs-related inflammatory signaling pathways. In addition, the result of GC-MS analysis revealed the presence of nineteen different types of constituents including guaiacol in the chloroform-soluble fraction of G. affine.

Anti-inflammatory activity of mycelial extracts from medicinal mushrooms.

Medicinal mushrooms have been essential components of traditional Chinese herbal medicines for thousands of years, and they protect against diverse health-related conditions. The components responsible for their anti-inflammatory activity have yet to be fully studied. This study investigates the anti-inflammatory activity of n-hexane, chloroform, ethyl acetate, and methanol extracts of mycelia in submerged culture from 5 commercially available medicinal mushrooms, namely Cephalosporium sinensis, Cordyceps mortierella, Hericium erinaceus, Ganoderma lucidum, and Armillaria mellea. MTT colorimetric assay was applied to measure the cytotoxic effects of different extracts. Their anti-inflammatory activities were evaluated via inhibition against production of lipopolysaccharide (LPS)-induced nitric oxide (NO) in murine macrophage-like cell line RAW264.7 cells. Of the 20 extracts, n-hexane, chloroform, ethyl acetate, and methanol extracts from C. sinensis, C. mortierella, and G. lucidum; chloroform extracts from H. erinaceus and A. mellea; and ethyl acetate extracts from A. mellea at nontoxic concentrations (<300 μg/mL) dose-dependently inhibited LPS-induced NO production. Among them, the chloroform extract from G. lucidum was the most effective inhibitor, with the lowest half maximal inhibitory concentration (64.09 ± 6.29 μg/mL) of the LPS-induced NO production. These results indicate that extracts from medicinal mushrooms exhibited anti-inflammatory activity that might be attributable to the inhibition of NO generation and can therefore be considered a useful therapeutic and preventive approach to various inflammation-related diseases.

Inhibition of Nitric Oxide Generation in Mononuclear Splenocytes from Multiple-Low-Dose-Streptozotocin Diabetic Mice

Inhibition of Nitric Oxide Generation in Mononuclear Splenocytes from Multiple-Low-Dose-Streptozotocin Diabetic Mice

Polycystic Ovarian Syndrome as a State of Premature Ageing of the Nitric Oxide System

Ageing per se represents an independent risk factor for ischaemic heart disease, but the mechanism(s) underlying this effect remains controversial. Polycystic ovarian syndrome (PCOS) appears to engender premature development of heart disease, irrespective of comorbidities. We have now evaluated the impact of ageing and menopause on vascular and platelet nitric oxide (NO) signalling in normal women aged 18–60 years, and compared these changes with an age-matched group of PCOS subjects. Methods: We evaluated platelet responsiveness to NO via whole blood aggregometry, vascular endothelial function and NO responsiveness using applanation tonometry, and utilised asymmetric dimethylarginine (ADMA) concentrations as an index of endogenous inhibition of NO generation. Results: In normal women (n = 132), platelet NO responsiveness (ANOVA, F = 9.9, p < 0.0001) and vascular endothelial function (F = 7.7, p < 0.0001) decreased whilst ADMA increased significantly (F = 6.4, p = 0.0005) with age. Detailed evaluation of 40 perimenopausal women revealed no correlation between plasma oestradiol levels and changes in NO response. In PCOS subjects (n = 109) there was no significant fluctuation of NO response (F = 1.9, p = 0.1), vascular endothelial function (F = 0.6, p = 0.6) or ADMA (F = 1.6, p = 0.2) with age. However, NO response and vascular endothelial function were impaired (2 way ANOVA, F = 4.2, p = 0.04 and F = 5.6, p = 0.02 respectively) and ADMA was elevated (F = 47.0, p < 0.0001) relative to normal women, with these anomalies already evident in women <40 years. Vascular NO responsiveness showed identical differences for both age and PCOS. Conclusions: (1) Normal female ageing is associated with progressive impairment of platelet/vascular NO signalling, (2) in PCOS subjects, similar changes are already present in young adults.

Effect of Aminoguanidine on Ischemia/Reperfusion Injury in Rat Small Intestine

Ischemia/reperfusion (I/R) injury is induced by reactive oxygen species (ROS). During intestinal I/R, the amount of nitric oxide (NO), which is a ROS, is increased. In this study, we examined the protection against I/R injury by inhibition of NO generation. Wistar/ST rats were exposed to 1 h of ischemia, followed by reperfusion for 4 h. The rats were intravenously injected with 100 mg/kg aminoguanidine (AG), which is a selective inducible NO synthase (iNOS) inhibitor, for 5 min before ischemia. The increase in NO(2)(-) by intestinal I/R was significantly inhibited by AG 1 h after reperfusion. Moreover, the increase in area under curve of 0 to 1 h after reperfusion (AUC(0-1)) of paracellular marker was inhibited. However, 3 h after reperfusion, the survival ratio of rats was significantly decreased in the intestinal I/R condition with AG. The amount of NO(2)(-) and AUC of 3 to 4 h after reperfusion (AUC(3-4)) of paracellular marker in intestinal I/R groups were increased by AG compared with those in the I/R condition without AG 3 h after reperfusion. These data indicated that AG, which was given by single pre-administration, can clearly inhibit intestinal I/R injury 1 h after reperfusion. However, the injury occurs again 3 h after reperfusion and grows worse.

Effects of nitric oxide on mucosal barrier dysfunction during early phase of intestinal ischemia/reperfusion

Ischemia/reperfusion (I/R) injury must be overcome for successful small intestinal transplantation. During intestinal I/R, the expression level of nitric oxide (NO) is increased, and vermiculation of the mucosal tract is induced by NO. Although NO has many beneficial effects on intestinal I/R injury, its role in intestinal I/R injury is controversial. Therefore, in the present study, we examined changes in the tight junctions (TJ) and P-glycoprotein (P-gp) by aminoguanidine (AG), which can be considered a selective inducible NO synthase inhibitor during intestinal I/R, to clarify the effect of NO on mucosal barrier dysfunction during intestinal I/R. A mucosal lesion was induced by intestinal I/R. The protein expression levels of the claudin family organizing TJ and P-gp, were decreased, and their functions were also decreased. Through the inhibition of NO generation by AG in the above mucosal lesion, TJ and P-gp dysfunction was significantly inhibited. NO participated in opening TJ and decreasing P-gp function and expression induced during intestinal I/R. Therefore, it is important to consider the level of NO generation in the ileal mucosa in drug therapy for intestinal I/R injury.

Mediatorwirkungen im trigeminovaskulären System als Grundlage für die nozizeptiven Vorgänge bei der Kopfschmerzentstehung

Nociceptive mechanisms in the trigeminovascular system are probably involved in the generation of different forms of hedaches, wherein the neuropeptide CGRP (calcitonin gene-related peptide), which is released from a subset of activated primary afferents, is regarded as an important mediator. CGRP is not only a potent vasodilator but is also involved in nociceptive transmission in the trigeminal nucleus. Inhibition of CGRP receptors by the high-affinity CGRP receptor antagonist BIBN4096BS, which had proved its therapeutic potency in migraine patients, led to a significant decrease in neuronal activity in the spinal trigeminal nucleus of the rat. Another important vasodilatory mediator in meningeal nociception seems to be nitric oxide (NO), which may facilitate CGRP release from peripheral and central trigeminal structures. Inhibition of NO generation was effective in lowering the neuronal activity in the spinal trigeminal nucleus as well, whereas infusion of NO donors led to an increase in activity. The activiation showed typically a biphasic pattern. The delayed response can only be explained by secondary processes such as the expression of pro-nociceptive substances in the trigeminal nucleus or ganglion, as indicated by preliminary experimental studies. The fact that clinical and basic experimental data complement each other highlights the interpretative value of basic experiments for the pathophysiological mechanisms underlying the generation of headaches.

Vascular endothelial growth factor and nitric oxide in rat liver regeneration

In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65 ± 10.70; PH-Control: 88.13 ± 1.60 ⁎; Sh-SNP: 90.35 ± 3.11 ⁎; PH-SNP: 119.5 ± 12.10 ⁎ #; Sh-AG: 33.27 ± 5.23, PH-AG: 36.80 ± 3.40 #) ( p < 0.05 vs Sh-Control; # p < 0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.

Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

Beta-thujaplicin Is a natural troponoid with strong antifungal, antiviral, and anticancer activities. Beta-thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O2*-) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O2*- induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O2*- shows that NO production and H2O2 production are interdependent, but NO and O2*- accumulation were negatively related because of coconsumption of NO and O2*-. NO- and O2*- -induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O2*- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.