Effects Of Endogenous Nitric Oxide Research Articles

The influence of exogenous and endogenous nitric oxide on the human and animal body

It has been shown that the inhalation of gaseous NO (exogenous nitric oxide) leads to the formation of nitrosonium cations (NO+) in the circulating blood of humans and animals during the oxidation of NO, which can have a detrimental effect on pathogenic viruses and bacteria. When thiols enter the blood simultaneously with NO inhalation, they form S-nitrosothiols with NO+ and cause hypotensive effect in animals. The biological effect of endogenous NO, which is produced in cells and tissues with the participation of NO synthases in animals and humans, is mediated by the dinitrosyl iron complexes (DNIC) formed with thiol-containing ligands. As NO and NO+ donors, these complexes have a variety of regulatory and cytotoxic effects on the animal and human body. In particular, the NO+ released by DNIC was shown to suppress SARS-CoV-2 infection in Syrian hamsters.

Regulation of endogenous hormone and miRNA in leaves of alfalfa (Medicago sativa L.) seedlings under drought stress by endogenous nitric oxide

BackgroundAlfalfa (Medicago sativa. L) is one of the best leguminous herbage in China and even in the world, with high nutritional and ecological value. However, one of the drawbacks of alfalfa is its sensitivity to dry conditions, which is a global agricultural problem. The objective of this study was to investigate the regulatory effects of endogenous nitric oxide (NO) on endogenous hormones and related miRNAs in alfalfa seedling leaves under drought stress. The effects of endogenous NO on endogenous hormones such as ABA, GA3, SA, and IAA in alfalfa leaves under drought stress were studied. In addition, high-throughput sequencing technology was used to identify drought-related miRNAs and endogenous NO-responsive miRNAs in alfalfa seedling leaves under drought stress.ResultBy measuring the contents of four endogenous hormones in alfalfa leaves, it was found that endogenous NO could regulate plant growth and stress resistance by inducing the metabolism levels of IAA, ABA, GA3, and SA in alfalfa, especially ABA and SA in alfalfa. In addition, small RNA sequencing technology and bioinformatics methods were used to analyze endogenous NO-responsive miRNAs under drought stress. It was found that most miRNAs were enriched in biological pathways and molecular functions related to hormones (ABA, ETH, and JA), phenylpropane metabolism, and plant stress tolerance.ConclusionIn this study, the analysis of endogenous hormone signals and miRNAs in alfalfa leaves under PEG and PEG + cPTIO conditions provided an important basis for endogenous NO to improve the drought resistance of alfalfa at the physiological and molecular levels. It has important scientific value and practical significance for endogenous NO to improve plant drought resistance.

NO-IL-6/10-IL-1β axis: a new pathway in steatotic and non-steatotic liver grafts from brain-dead donor rats.

Brain death (BD) and steatosis are both risk factors for organ dysfunction or failure in liver transplantation (LT). Here, we examine the role of interleukin 6 (IL- 6) and IL-10 in LT of both non-steatotic and steatotic liver recovered from donors after brain death (DBDs), as well as the molecular signaling pathways underlying the effects of such cytokines. BD reduced IL-6 levels only in nonsteatotic grafts, and diminished IL-10 levels only in steatotic ones. In both graft types, BD increased IL-1β, which was associated with hepatic inflammation and damage. IL-6 administration reduced IL-1β only in non-steatotic grafts and protected them against damage and inflammation. Concordantly, IL-1β inhibition via treatment with an IL-1 receptor antagonist caused the same benefits in non-steatotic grafts. Treatment with IL-10 decreased IL-1β only in steatotic grafts and reduced injury and inflammation specifically in this graft type. Blockading the IL-1β effects also reduced damage and inflammation in steatotic grafts. Also, blockade of IL-1β action diminished hepatic cAMP in both types of livers, and this was associated with a reduction in liver injury and inflammation, then pointing to IL-1β regulating cAMP generation under LT and BD conditions. Additionally, the involvement of nitric oxide (NO) in the effects of interleukins was evaluated. Pharmacological inhibition of NO in LT from DBDs prompted even more evident reductions of IL-6 or IL-10 in non-steatotic and steatotic grafts, respectively. This exacerbated the already high levels of IL-1β seen in LT from DBDs, causing worse damage and inflammation in both graft types. The administration of NO donors to non-steatotic grafts potentiated the beneficial effects of endogenous NO, since it increased IL-6 levels, and reduced IL-1β, inflammation, and damage. However, treatment with NO donors in steatotic grafts did not modify IL-10 or IL-1β levels, but induced more injurious effects tan the induction of BD alone, characterized by increased nitrotyrosine, lipid peroxidation, inflammation, and hepatic damage. Our study thus highlights the specificity of new signaling pathways in LT from DBDs: NO-IL-6-IL-1β in non-steatotic livers and NO-IL-10-IL-1β in steatotic ones. This opens up new therapeutic targets that could be useful in clinical LT.

Role of Exogenous Nitric Oxide in Protecting Plants against Abiotic Stresses

The continuously changing environment has led to devastating effects on the normal growth and development of plants. This necessitates the understanding of different components that can be involved in alleviating these effects. In the last two decades, nitric oxide (NO) has been largely focused on as a molecule whose endogenous production and exogenous supply lead to several molecular and physiological changes in plants under stressed conditions. Although its role as a signaling molecule in endogenous production has been largely discussed, its function in dealing with contemporary abiotic stress conditions on exogenous supply remains comparatively less explored. There is growing evidence that NO plays a critical role in many physiological processes; however, there is debate about the exact mechanism(s) through which NO lessens abiotic stress on external supply. In this review, we discuss the studies that were focused on observing the effect of exogenous NO on different abiotic stresses including heavy metal stress, element deficiency or toxicity stress, salt stress, drought stress, ultraviolet radiation stress, waterlogging stress, and chilling stress. Though the positive effects of endogenous NO have been discussed in brief in different sections, the focus of the review is to discuss the effects of exogenous NO on plant grown under abiotic stresses. Deciphering the underlying mechanism of exogenous NO treatment may open up new ideas that can suggest the successful application of NO in agricultural regions to reduce the damaging influences of different abiotic stresses.

Endothelial Nitric Oxide Synthase (eNOS) and the Cardiovascular System: in Physiology and in Disease States

Endothelial nitric oxide synthase (eNOS) plays a critical role in regulating and maintaining a healthy cardiovascular system. The importance of eNOS can be emphasized from the genetic polymorphisms of the eNOS gene, uncoupling of eNOS dimerization, and its numerous signaling regulations. The activity of eNOS on the cardiac myocytes, vasculature, and the central nervous system are discussed. The effects of eNOS on the sympathetic autonomic nervous system (SANS) and the parasympathetic autonomic nervous system (PANS), both of which profoundly influence the cardiovascular system, will be elaborated. The relationship between the eNOS protein with cardiovascular autonomic reflexes such as the baroreflex and the Exercise Pressor Reflex will be discussed. For example, the effects of endogenous nitric oxide (NO) are shown to be mediated by the eNOS protein and that eNOS-derived endothelial NO is most effective in regulating blood pressure oscillations via modulating the baroreflex mechanisms. The protective action of eNOS on the CVS is emphasized here because dysfunction of the eNOS enzyme is intricately correlated with the pathogenesis of several cardiovascular diseases such as hypertension, arteriosclerosis, myocardial infarction, and stroke. Overall, our current understanding of the eNOS protein with a focus on its role in the modulation, regulation, and control of the cardiovascular system in a normal physiological state and in cardiovascular diseases are discussed.

Assessment of nitric oxide (NO) potential to mitigate COVID-19 severity.

Novel coronavirus disease by SARS-CoV-2 virus (also known as COVID-19) has emerged as major health concern worldwide. While, there is no specific drugs for treating this infection till date, SARS-CoV-2 had spread to most countries around the globe. Nitric oxide (NO) gas serves as an important signaling molecule having vasodilatory effects as well as anti-microbial properties. Previous studies from the 2004 SARS-CoV infection demonstrated that NO may also help to reduce respiratory tract infection by inactivating viruses and inhibiting their replication cycle and is an effective supportive measure for treating infection in patients with pulmonary complications. NO gas inhalation is being suggested as potential therapy for managing severe acute respiratory distress syndrome in COVID-19 patients. In view of COVID-19 pandemic, several clinical trials are underway to examine the effects of NO inhalation on infected patients. Previously published reports on beneficial effects of endogenous NO and NO inhalation therapy were thoroughly searched to assess the potential of NO therapy for treating COVID-19 patients. Present report summarized the therapeutic importance of NO to reverse pulmonary hypertension, restore normal endothelial activity and produce anti-thrombotic effects. In addition to this, NO also reduces viral infection by inhibiting its replication and entry into the host cell. In absence of vaccine and effective treatment strategies, we suggest that NO inhalation therapy and NO releasing foods/compounds could be considered as an alternative measure to combat COVID-19 infection.

Nitric oxide increases gain in the ventral cochlear nucleus of guinea pigs with tinnitus.

Previous work has led to the hypothesis that, during the production of noise-induced tinnitus, higher levels of nitric oxide (NO), in the ventral cochlear nucleus (VCN), increase the gain applied to a reduced input from the cochlea. To test this hypothesis, we noise-exposed 26 guinea pigs, identified evidence of tinnitus in 12 of them and then compared the effects of an iontophoretically applied NO donor or production inhibitor on VCN single unit activity. We confirmed that the mean driven firing rate for the tinnitus and control groups was the same while it had fallen in the non-tinnitus group. By contrast, the mean spontaneous rate had increased for the tinnitus group relative to the control group, while it remained the same for the non-tinnitus group. A greater proportion of units responded to exogenously applied NO in the tinnitus (56%) and non-tinnitus groups (71%) than a control population (24%). In the tinnitus group, endogenous NO facilitated the driven firing rate in 37% (7/19) of neurons and appeared to bring the mean driven rate back up to control levels by a mechanism involving N-methyl-D-aspartic acid (NMDA) receptors. By contrast, in the non-tinnitus group, endogenous NO only facilitated the driven firing rate in 5% (1/22) of neurons and there was no facilitation of driven rate in the control group. The effects of endogenous NO on spontaneous activity were unclear. These results suggest that NO is involved in increasing the gain applied to driven activity, but other factors are also involved in the increase in spontaneous activity.

Pulmonary vascular disease in swine with multiple comorbidities

IntroductionDiabetes mellitus (DM), chronic kidney disease (CKD) and hypercholesterolemia are common co‐morbidities associated with low‐grade inflammation and endothelial dysfunction leading to increased systemic vascular resistance, diastolic dysfunction and exercise intolerance. Diastolic dysfunction is a risk factor for development of post‐capillary pulmonary hypertension (PH), which, in a subgroup of patients, deteriorates into combined pre‐and post‐capillary PH. This process is characterized by an increase in pulmonary vascular resistance (PVR) due to pulmonary vascular dysfunction and remodeling. It is incompletely understood which factors predispose to the development of combined pre‐and post‐capillary PH. We hypothesize that comorbidities cause pulmonary endothelial dysfunction, which in turn initiates pulmonary vascular remodeling, increases PVR and further reduces exercise tolerance.ObjectiveTo investigate the contribution of endothelial dysfunction, characterized by perturbances in the nitric oxide (NO)‐cGMP‐phosphodiesterase 5 (PDE5) pathway, to changes in PVR at rest and during exercise in a swine model with multiple co‐morbidities.MethodsDM (streptozotocin 3×50mg/kg iv) and CKD (renal embolization) were induced in 6 female swine fed a high fat diet (DM+HFD+CKD), while 8 female healthy swine on normal diet served as controls (CON). Six months later, swine were chronically instrumented and hemodynamics were studied at rest and during exercise, in the absence and presence of eNOS‐inhibition (LNNA, 20 mg/kg iv) and PDE5‐inhibition (sildenafil, 100 mg iv).ResultsDM+HFD+CKD swine exhibited hyperglycemia (18.7±1.9 vs 7.5±0.6mmol/L), hypercholesterolemia (7.3±0.7 vs 1.7±0.1mmol/l), renal dysfunction (glomerular filtration rate: 123±12 vs 202±8 ml/min), and inflammation (TNF‐α 51.6±5.1 vs 24.6±5.5pg/ml, all P<0.05).DM+HFD+CKD swine had similar pulmonary arterial pressures at rest and during exercise compared to CON swine, while cardiac index was significantly reduced, reflecting the higher PVR in DM+HFD+CKD. DM+HFD+CKD swine showed a similar increase in PVR in response to eNOS‐inhibition compared to CON swine at rest and during exercise. However, PDE5 inhibition resulted in a significant reduction in PVR, reflecting pulmonary vasodilation in CON, but not in DM+HFD+CKD swine. Altogether, these data suggest that impaired PDE5‐activity acts to preserve the vasodilator effect of endogenous NO in DM+HFD+CKD.ConclusionsExposure to multiple co‐morbidities in a swine model causes an increase in inflammatory factors, which is accompanied by a reduced activity in the NO‐pathway, that is counterbalanced by reduction in PDE5‐activity in swine with DM+HFD+CKD. Hence, exposure to co‐morbidities causes pulmonary endothelial dysfunction and an increase in PVR, even in the absence of overt PH. Thus, pulmonary vascular disease might be an underestimated contributing factor in the disease progress in patients with multiple co‐morbidities and diastolic dysfunction.Support or Funding InformationFunding: CVON‐RECONNECTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of endogenous nitric oxide on adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide-containing nerve-mediated vasodilation in pithed rats

Vascular adrenergic nerves mainly regulate the tone of blood vessels. Calcitonin gene-related peptide-containing (CGRPergic) vasodilator nerves also participate in the regulation of vascular tone. Furthermore, there are nitric oxide (NO)-containing (nitrergic) nerves, which include NO in blood vessels as vasodilator nerves, but it remains unclear whether nitrergic nerves participate in vascular regulation. The present study investigated the role of nitrergic nerves in vascular responses to spinal cord stimulation (SCS) and vasoactive agents in pithed rats. Wistar rats were anesthetized and pithed, and vasopressor responses to SCS and injections of norepinephrine were observed. To evaluate vasorelaxant responses, the BP was increased by a continuous infusion of methoxamine with hexamethonium to block autonomic outflow. After the elevated BP stabilized, SCS and injections of acetylcholine (ACh), sodium nitroprusside (SNP), and CGRP were intravenously administered. We then evaluated the effects of the NO synthase (NOS) inhibitor, N-ω-nitro-L-arginine methylester hydrochloride (L-NAME), on these vascular responses. Pressor responses to SCS and norepinephrine in pithed rats were enhanced by L-NAME, while the combined infusion of L-NAME and L-arginine had no effect on these responses. L-NAME infusion significantly increased the release of norepinephrine evoked by SCS. In pithed rats with artificially increased BP and L-NAME infusion, depressor response to ACh (except for 0.05nmol/kg) was suppressed and SNP (only 2nmol/kg) was enhanced. However, depressor responses to SCS and CGRP were similar to control responses. The present results suggest endogenous NO regulates vascular tone through endothelium function and inhibition of adrenergic neurotransmission, but not through CGRPergic nerves.

Antagonistic Effects of Endogenous Nitric Oxide in a Glioblastoma Photodynamic Therapy Model.

Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In this study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA-PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased three- to fourfold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light-induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO-dependent increase in matrix metalloproteinase-9 activity, decrease in tissue inhibitor of metalloproteinase-1 expression and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT.

Reduction of PCN biosynthesis by NO in Pseudomonas aeruginosa

Pyocyanin (PCN), a virulence factor synthesized by Pseudomonas aeruginosa, plays an important role during clinical infections. There is no study of the effect of nitric oxide (NO) on PCN biosynthesis. Here, the effect of NO on PCN levels in Pseudomonas aeruginosa strain PAO1, a common reference strain, was tested. The results showed that the NO donor sodium nitroprusside (SNP) can significantly reduce PCN levels (82.5% reduction at 60μM SNP). Furthermore, the effect of endogenous NO on PCN was tested by constructing PAO1 nor (NO reductase gene) knockout mutants. Compared to the wild-type strain, the Δnor strain had a lower PCN (86% reduction in Δnor). To examine whether the results were universal with other P. aeruginosa strains, we collected 4 clinical strains from a hospital, tested their PCN levels after SNP treatment, and obtained similar results, i.e., PCN biosynthesis was inhibited by NO. These results suggest that NO treatment may be a new strategy to inhibit PCN biosynthesis and could provide novel insights into eliminating P. aeruginosa virulence as a clinical goal.

Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca2+

We determined the effects of endogenous nitric oxide and Ca2+ on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca2+ involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

Regulation of vascular tone in rabbit ophthalmic artery: Cross talk of endogenous and exogenous gas mediators

Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) modulate vascular tone. In view of their therapeutic potential for ocular diseases, we examined the effect of exogenous CO and H2S on tone of isolated rabbit ophthalmic artery and their interaction with endogenous and exogenous NO. Ophthalmic artery segments mounted on a wire myograph were challenged with cumulative concentrations of phenylephrine (PE) in the presence or absence of NG-nitro-l-arginine (LNNA) to inhibit production of NO, the CO-releasing molecules CORMs or the H2S-donor GYY4137. The maximal vasoconstriction elicited by PE reached 20–30% of that induced by KCl but was dramatically increased by incubation with LNNA. GYY4137 significantly raised PE-mediated vasoconstriction, but it did not change the response to PE in the presence of LNNA or the relaxation to sodium nitroprusside (SNP). CORMs concentration-dependently inhibited PE-induced constriction, an effect that was synergistic with endogenous NO (reduced by LNNA), but insensitive to blockade of guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-α]quinoxalin-1-one (ODQ). In vascular tissues cyclic GMP (cGMP) levels seemed reduced by GYY4137 (not significantly), but were not changed by CORM. These data indicate that CO is able per se to relax isolated ophthalmic artery and to synergize with NO, while H2S counteracts the effect of endogenous NO. CO does not stimulate cGMP production in our system, while H2S may reduce cGMP production stimulated by endogenous NO. These findings provide new insights into the complexities of gas interactions in the control of ophthalmic vascular tone, highlighting potential pharmacological targets for ocular diseases.

Role of soluble guanylate cyclase in renal hemodynamics and autoregulation in the rat.

We studied the influence of soluble guanylate (sGC) on renal blood flow (RBF), glomerular filtration rate (GFR), and RBF autoregulation and its role in mediating the hemodynamic effects of endogenous nitric oxide (NO). Arterial pressure (AP), heart rate (HR), RBF, GFR, urine flow (UV), and the efficiency and mechanisms of RBF autoregulation were studied in anesthetized rats during intravenous infusion of sGC activator cinaciguat before and (except GFR) also after inhibition of NO synthase (NOS) by Nω-nitro-L-arginine methyl ester. Cinaciguat (0.1, 0.3, 1, 3, 10 μg·kg(-1)·min(-1), n=7) reduced AP and increased HR, but did not significantly alter RBF. In clearance experiments (FITC-sinistrin, n=7) GFR was not significantly altered by cinaciguat (0.1 and 1 μg·kg(-1)·min(-1)), but RBF slightly rose (+12%) and filtration fraction (FF) fell (-23%). RBF autoregulatory efficiency (67 vs. 104%) and myogenic response (33 vs. 44 units) were slightly depressed (n=9). NOS inhibition (n=7) increased AP (+38 mmHg), reduced RBF (-53%), and greatly augmented the myogenic response in RBF autoregulation (97 vs. 35 units), attenuating the other regulatory mechanisms. These changes were reversed by 77, 78, and 90% by 1 μg·kg(-1)·min(-1) cinaciguat. In vehicle controls (n=3), in which cinaciguat-induced hypotension was mimicked by aortic compression, the NOS inhibition-induced changes were not affected. We conclude that sGC activation leaves RBF and GFR well maintained despite hypotension and only slightly impairs autoregulation. The ability to largely normalize AP, RBF, RBF autoregulation, and renovascular myogenic response after NOS inhibition indicates that these hemodynamic effects of NO are predominantly mediated via sGC.

Blockade of the purinergic P2Y 12 receptor greatly increases the platelet inhibitory actions of nitric oxide

Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y12 receptors, strongly amplifies aggregation. Platelet P2Y12 receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y12 receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y12 receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y12, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y12 receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.

261 P2Y12 INHIBITION GREATLY POTENTIATES THE ANTI-PLATELET EFFECTS OF PROSTACYCLIN AND NITRIC OXIDE

When P2Y₁₂ receptors on platelets are blocked by commonly used anti-platelet drugs such as clopidogrel and prasugrel, the inhibitory brake on adenylate cyclase (AC) activity is lifted and the anti-platelet effects of prostacyclin (PGI₂) and other agents that activate platelet AC are synergistically enhanced. We have recently demonstrated that blockade of the P2Y₁₂ receptor also enhances the anti-platelet effect of nitric oxide (NO). The aim of this study was to study the interaction between PGI₂, NO and P2Y₁₂ receptor inhibition on platelet aggregation and to determine pharmacologically, using isobolographic analysis, if this interaction constitutes a true synergy or simply an additive effect. <h3>Methods</h3> Blood was collected by venepuncture into 0.32% trisodium citrate. Platelet rich plasma (PRP) was isolated by centrifugation and either tested directly or further washed with Tyrode’s-Hepes buffer. The PRP or washed platelets (WP) were incubated with the P2Y₁₂ inhibitor prasugrel active metabolite (PAM; 3 µM) or vehicle (0.5% DMSO) for 30 min followed by 1 min incubation with the NO donor DEA/NONOate (10 nM–1 mM) and/or PGI₂ (0.2 nM–100 nM) and/or vehicle (0.01M NaOH). WP platelet aggregation to thrombin (1U/ml) was measured by 96-well aggregometry and PRP platelet aggregation to TRAP-6 (Thrombin Receptor Activating Peptide-6, 30 µM) or collagen (30 µg/ml) was measured by light transmission aggregometry (LTA). Isobolograms were constructed by plotting the IC₅₀ values for DEA-NONOate and PGI₂ in vehicle or PAM treated WP. Data represents mean±SEM % final platelet aggregation from 4–5 healthy volunteers. <h3>Results</h3> Thrombin (1U/ml), TRAP-6 (30 µM) or collagen (30 µg/ml) all produced robust aggregation responses in WP and PRP respectively which was largely unaffected by the addition of 10nM DEA-NONOate and 4nM PGI₂ or 3 µM PAM. However, the combination of all three (NO, PGI₂ and PAM) resulted in almost complete inhibition of platelet aggregation. Isobolographic analysis of the data showed that the interaction between DEA/NONOate, PGI₂ and PAM in WP was strongly synergistic (isoboles curved away from the predicted linear line for an additive relationship). <h3>Conclusions</h3> These data confirm that activation of platelet P2Y₁₂ receptors by secreted ADP limits the anti-platelet effects of both NO and PGI_2, suggesting that P2Y₁₂ activation may be an important mechanism for haemostasis. In addition, we have demonstrated that the interaction between P2Y₁₂ receptors and vascular mediators is strongly synergistic. Potentiation of the effects of endogenous NO and PGI₂ may represent an important mechanism for how P2Y₁₂ inhibitors produce anti-thrombotic protection.

Endogenous nitric oxide induces activation of apoptosis signal-regulating kinase 1 via S-nitrosylation in rat hippocampus during cerebral ischemia–reperfusion

Apoptosis signal-regulating kinase 1 (ASK1) is a general mediator of cell death in response to a variety of stimuli, including reactive oxygen species, tumor necrosis factor α, lipopolysaccharide, endoplasmic reticulum stress, calcium influx and ischemia. Here we reported ASK1 was activated by nitric oxide (NO) through S-nitrosylation during cerebral ischemia–reperfusion. The reagents that abrogate neuronal nitric oxide synthase (nNOS) activity such as nNOS inhibitor 7NI and N-methyl-d-aspartate receptor antagonist MK801 prevented ASK1 activation via decreasing ASK1 S-nitrosylation. In HEK293 cells, over-expressed ASK1 could be S-nitrosylated by both exogenous and endogenous NO and Cys869 was identified as the site of ASK1 S-nitrosylation. S-nitrosylation increased the level of ASK1 phosphorylation at Thr845, which represents ASK1 activation. Our results further confirmed that S-nitrosylation led to the increment of ASK1 dimerization. S-nitrosylation of ASK1 also activated the downstream JNK signaling and JNK-mediated nucleic pathway. The exogenous NO (SNP and GSNO) reversed the effect of endogenous NO by suppressing S-nitrosylation of ASK1 and exerted neuroprotection during ischemia–reperfusion. These results suggest that inhibiting ASK1 S-nitrosylation may be a novel approach for stroke therapy.

11 Effect of magnetic-photo stimulating the system of nitric oxide in arterial hypertension

Materials and Methods: The main goal of this study was to investigate the effects of endogenous nitric oxide on mitochondrial respiration and to study the effects of blue (442 nm) and green (532 nm) low-power radiation on mitochondrial respiration in experimental endotoxic shock. We used a model of experimental endotoxic shock produced by intraperitoneal injection of lipopolysaccharide B. Results: Low-power laser radiation has an appreciable effect on the rate of mitochondrial respiration in states of 3 and 4, both in normal and in endotoxic shock. The result of blue laser radiation (442 nm) was an increase of mitochondrial respiratory rate V3 in the control group by 12% at dose of 6 J/cm2. Increasing the irradiation dose lead to gradual decrease in rate V3. In the case of green laser (532 nm), statistically significant deviations from control in the rate V3 is not revealed. The greatest increase in respiratory rate in state of V3 (about 30%) was observed in LPS-treated animals at dose of 6 J/cm2 (442 nm). Oxygen consumption rate in the state of 4 irradiation at dose of 10 J/cm2 increased compared to the control all sources of radiation. In an experimental endotoxic shock rate of oxygen consumption by mitochondria V4 also increased (approximately 40%) for all sources, but this effect was more pronounced than in the control group. To evaluate the mitochondria membrane potential in experimental endotoxic shock a JC-1 fluorescent probe was applied. It was found that statistically significant difference (approx. 10%) exists in the mitochondria membrane potential of in LPS-treated and untreated rats. This phenomenon could be due to the membrane damage in endotoxic shock development. Conclusion: Study of the effect of laser radiation on mitochondrial respiration showed that in experiments using blue laser (442 nm), an increase rate V3 and V4, which can be attributed to the destruction of nitrosyl complexes of the respiratory chain. This tendency was more pronounced in an experimental endotoxic shock.

Glioma Stem Cell Proliferation and Tumor Growth Are Promoted by Nitric Oxide Synthase-2

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Rapid Upregulation of Cytoprotective Nitric Oxide in Breast Tumor Cells Subjected to a Photodynamic Therapy‐like Oxidative Challenge

Many tumor cells produce nitric oxide (NO) as an antiapoptotic/progrowth molecule which also promotes antiogenesis and tumor expansion. This study was designed to examine possible antagonistic effects of endogenous NO on tumor eradication by photodynamic therapy (PDT). Using COH-BR1 breast cancer cells sensitized in mitochondria with 5-aminolevulinic acid (ALA)-generated protoporphyrin IX as a model for ALA-based PDT, we found that caspase-9 activation and apoptotic death following irradiation were strongly enhanced by 1400W, an inhibitor of inducible nitric oxide synthase (iNOS). RT-PCR and Western analyses revealed a substantial upregulation of both iNOS mRNA and protein, beginning ca 4 h after irradiation and persisting for at least 20 h. Accompanying this was a strong 1400W-inhibitable increase in intracellular NO, as detected with the NO probe, DAF-2-DA. Short hairpin RNA-based iNOS knockdown in COH-BR1 cells dramatically reduced NO production under photostress while enhancing caspase-9 activation and apoptosis. These findings suggest that cytoprotective iNOS/NO induction in PDT-treated tumor cells could reduce treatment efficacy, and point to pharmacologic intervention with iNOS inhibitors for counteracting this.