Prospects for the Personalized Multimodal Therapy Approach to Pain Management via Action on NO and NOS

Chronic pain syndromes are an important medical problem generated by various molecular, genetic, and pathophysiologic mechanisms. Back pain, neuropathic pain, and posttraumatic pain are the most important pathological processes associated with chronic pain in adults. Standard approaches to the treatment of them do not solve the problem of pain chronicity. This is the reason for the search for new personalized strategies for the prevention and treatment of chronic pain. The nitric oxide (NO) system can play one of the key roles in the development of peripheral pain and its chronicity. The purpose of the study is to review publications devoted to changes in the NO system in patients with peripheral chronical pain syndromes. We have carried out a search for the articles published in e-Library, PubMed, Oxford Press, Clinical Case, Springer, Elsevier, and Google Scholar databases. The search was carried out using keywords and their combinations. The role of NO and NO synthases (NOS) isoforms in peripheral pain development and chronicity was demonstrated primarily from animal models to humans. The most studied is the neuronal NOS (nNOS). The role of inducible NOS (iNOS) and endothelial NOS (eNOS) is still under investigation. Associative genetic studies have shown that single nucleotide variants (SNVs) of NOS1, NOS2, and NOS3 genes encoding nNOS, iNOS, and eNOS may be associated with acute and chronic peripheral pain. Prospects for the use of NOS inhibitors to modulate the effect of drugs used to treat peripheral pain syndrome are discussed. Associative genetic studies of SNVs NOS1, NOS2, and NOS3 genes are important for understanding genetic predictors of peripheral pain chronicity and development of new personalized pharmacotherapy strategies.     

An Overview of NO Signaling Pathways in Aging

Nitric Oxide (NO) is a potent signaling molecule involved in the regulation of various cellular mechanisms and pathways under normal and pathological conditions. NO production, its effects, and its efficacy, are extremely sensitive to aging-related changes in the cells. Herein, we review the mechanisms of NO signaling in the cardiovascular system, central nervous system (CNS), reproduction system, as well as its effects on skin, kidneys, thyroid, muscles, and on the immune system during aging. The aging-related decline in NO levels and bioavailability is also discussed in this review. The decreased NO production by endothelial nitric oxide synthase (eNOS) was revealed in the aged cardiovascular system. In the CNS, the decline of the neuronal (n)NOS production of NO was related to the impairment of memory, sleep, and cognition. NO played an important role in the aging of oocytes and aged-induced erectile dysfunction. Aging downregulated NO signaling pathways in endothelial cells resulting in skin, kidney, thyroid, and muscle disorders. Putative therapeutic agents (natural/synthetic) affecting NO signaling mechanisms in the aging process are discussed in the present study. In summary, all of the studies reviewed demonstrate that NO plays a crucial role in the cellular aging processes.     

Design and Synthesis of New Acridone-Based Nitric Oxide Fluorescent Probe

Nitric oxide (NO) is an important signaling molecule involved in a wide range of physiological and pathological processes. Fluorescent imaging is a useful tool for monitoring NO concentration, which could be essential in various biological and biochemical studies. Here, we report the design of a novel small-molecule fluorescent probe based on 9(10H)acridone moiety for nitric oxide sensing. 7,8-Diamino-4-carboxy-10-methyl-9(10H)acridone reacts with NO in aqueous media in the presence of O₂, yielding a corresponding triazole derivative with fivefold increased fluorescence intensity. The probe was shown to be capable of nitric oxide sensing in living Jurkat cells.     

Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses

Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.     

Recent Developments in Nitric Oxide Donors and Delivery for Antimicrobial and Anti-Biofilm Applications

The use of nitric oxide (NO) is emerging as a promising, novel approach for the treatment of antibiotic resistant bacteria and biofilm infections. Depending on the concentration, NO can induce biofilm dispersal, increase bacteria susceptibility to antibiotic treatment, and induce cell damage or cell death via the formation of reactive oxygen or reactive nitrogen species. The use of NO is, however, limited by its reactivity, which can affect NO delivery to its target site and result in off-target effects. To overcome these issues, and enable spatial or temporal control over NO release, various strategies for the design of NO-releasing materials, including the incorporation of photo-activable, charge-switchable, or bacteria-targeting groups, have been developed. Other strategies have focused on increased NO storage and delivery by encapsulation or conjugation of NO donors within a single polymeric framework. This review compiles recent developments in NO drugs and NO-releasing materials designed for applications in antimicrobial or anti-biofilm treatment and discusses limitations and variability in biological responses in response to the use of NO for bacterial eradiation.     

Effects of Siqi Decoction on Concentration of Nitric Oxide and Activity of Nitric Oxide Synthase in Blood Serum of Rats with Myocardial Ischemia

To study the effects of Siqi decoction on rats with Myocardial Ischemia, the model of Myocardium Ischemia was made for the Wistar rats cured with posterior pituitary injection through vein in tail. Siqi decoction, Diaoxinxuekang（DK） and Fufangdanshenpian（FD）, the latter two drugs of which were effective TCM drugs of anti-Myocardial Ischemia at present, were administrated to the rats with Myocardium Ischemia for 5 days to compare the effect of anti-Myocardium Ischemia as reference drugs by measuring the changes of NO concentration and activity of NOS in the rat blood serum with Myocardial Ischemia. There was a remarkable increase in the NO concentration and activity of NOS in serum in Siqi decoction groups compared with those in control group（p〈0.05）. The results of the prevention group in experiment of Siqi decoction are better than those of the cure group. Siqi decoction was really fit for Myocardium Ischemia via increasing NO concentration by stimulating the activity of NOS in serum. The effect of Siqi decoction against Myocardium Ischemia in preventive group is better than the curative that of Siqi decoction in the curative group.     

两种含锌化合物对慢性染铅大鼠海马NADPH-d阳性神经元影响的比较

为探讨硫酸锌(ZnSO4)和牛磺酸锌(TZC)拮抗铅对学习记忆的损害的不同，采用NADPH-黄递酶(NADPH-d)组化，观察了饮用含0.2g／L醋酸铅(PbAc)饮水和含不同剂量(5．0，15．0g／kg)ZnSO4、(5．9、17．7g／kg)TZC饲料喂养的大鼠海马一氧化氮合酶(NOS)活性的变化。结果表明，各补锌组均不同程度地缓解铅致NOS活力减低，铅 低TZC组、铅 高ZnSO4组和铅 低ZnSO4组大鼠海马神经元的NOS活性明显高于染铅组，其中铅 低TZC组的保护作用最显著，而铅 高TZC组的NOS活性明显低于对照组，而接近染铅组水平。结论是锌对抗铅的毒性作用与锌的化学结合形式、锌的剂量密切相关。     

Reductive decomposition of a diazonium intermediate by dithiothreitol affects the determination of NOS turnover rates

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure-function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed.     

Anti-Inflammatory Activity of Bryophytes Extracts in LPS-Stimulated RAW264.7 Murine Macrophages

Bryophytes produce rare and bioactive compounds with a broad range of therapeutic potential, and many species are reported in ethnomedicinal uses. However, only a few studies have investigated their potential as natural anti-inflammatory drug candidate compounds. The present study investigates the anti-inflammatory effects of thirty-two species of bryophytes, including mosses and liverworts, on Raw 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) or recombinant human peroxiredoxin (hPrx1). The 70% ethanol extracts of bryophytes were screened for their potential to reduce the production of nitric oxide (NO), an important pro-inflammatory mediator. Among the analyzed extracts, two moss species significantly inhibited LPS-induced NO production without cytotoxic effects. The bioactive extracts of Dicranum majus and Thuidium delicatulum inhibited NO production in a concentration-dependent manner with IC₅₀ values of 1.04 and 1.54 µg/mL, respectively. The crude 70% ethanol and ethyl acetate extracts were then partitioned with different solvents in increasing order of polarity (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions were screened for their inhibitory effects on NO production stimulated with LPS at 1 ng/mL or 10 ng/mL. The NO production levels were significantly affected by the fractions of decreasing polarity such as n-hexane and diethyl ether ones. Therefore, the potential of these extracts to inhibit the LPS-induced NO pathway suggests their effective properties in attenuating inflammation and could represent a perspective for the development of innovative therapeutic agents.     

The Nitric Oxide (NO) Donor Sodium Nitroprusside (SNP) and Its Potential for the Schizophrenia Therapy: Lights and Shadows

Schizophrenia is a severe psychiatric disorder affecting up to 1% of the worldwide population. Available therapy presents different limits comprising lack of efficiency in attenuating negative symptoms and cognitive deficits, typical features of schizophrenia and severe side effects. There is pressing requirement, therefore, to develop novel neuroleptics with higher efficacy and safety. Nitric oxide (NO), an intra- and inter-cellular messenger in the brain, appears to be implicated in the pathogenesis of schizophrenia. In particular, underproduction of this gaseous molecule is associated to this mental disease. The latter suggests that increment of nitrergic activity might be of utility for the medication of schizophrenia. Based on the above, molecules able to enhance NO production, as are NO donors, might represent a class of compounds candidates. Sodium nitroprusside (SNP) is a NO donor and is proposed as a promising novel compound for the treatment of schizophrenia. In the present review, we intended to critically assess advances in research of SNP for the therapy of schizophrenia and discuss its potential superiority over currently used neuroleptics.     

Nanomolar Nitric Oxide Concentrations in Living Cells Measured by Means of Fluorescence Correlation Spectroscopy

Measurement of the nitric oxide (NO) concentration in living cells in the physiological nanomolar range is crucial in understanding NO biochemical functions, as well as in characterizing the efficiency and kinetics of NO delivery by NO-releasing drugs. Here, we show that fluorescence correlation spectroscopy (FCS) is perfectly suited for these purposes, due to its sensitivity, selectivity, and spatial resolution. Using the fluorescent indicators, diaminofluoresceins (DAFs), and FCS, we measured the NO concentrations in NO-producing living human primary endothelial cells, as well as NO delivery kinetics, by an external NO donor to the immortal human epithelial living cells. Due to the high spatial resolution of FCS, the NO concentration in different parts of the cells were also measured. The detection of nitric oxide by means of diaminofluoresceins is much more efficient and faster in living cells than in PBS solutions, even though the conversion to the fluorescent form is a multi-step reaction.     

Calix[4]arene‐Based Bis(Nitric Oxide) Complexes: Synthesis,Physical Properties,and Structural Characterization

Calix[4]arene‐based molecules hold great promise as candidate sensors and storage materials for nitric oxide (NO), owing to their unprecedented binding affinity for NO. However, the structure of calix[4]arene is complicated by the availability of four possible conformers: 1,3‐alternate, 1,2‐alternate, cone, and partial cone (paco). Whilst complexes of NO with several of these conformers have previously been established, the 1,2‐alternate conformer complex, that is, [1,2‐alter ? NO]⁺, has not been previously reported. Herein, we determine the crystal structure of the NO complex with the 1,2‐alternate conformer for the first time. In addition, we have also found that the 1,2‐alternate and 1,3‐alternate conformers can combine with two NO molecules to form stable bis(nitric oxide) complexes. These new complexes, which exhibit remarkable binding capacity for the construction of NO‐storage molecules, were characterized by using X‐ray crystallography and NMR, IR, and UV/Vis spectroscopy. These findings will extend our understanding of the interactions between nitric oxide and cofacially and non‐cofacially arrayed aromatic rings, and we expect them to aid in the design and development of new supramolecular sensors and storage materials for NO with high capacity and efficacy.     

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.     

Electrochemical Sensors for Nitric Oxide Detection in Biological Applications

Nitric oxide (NO) plays an important role in physiological processes and it has been confirmed some human diseases are related to its biological function. Electrochemical sensors provide an efficient way to explore the NO function in biological processes. This review details different kinds of electrochemical sensors used for NO concentration detection between 2008 and 2013 together with their application in biological samples. Four commonly used electrodes and different assisted analysis membranes used for contributions towards the development of the novel sensors were summarized. Electrochemical sensors employed to measure NO concentration in a single cell, cell culture, tissue homogenate, organ, in vivo, human being, as well as in plant were also detailed. The trends of developing novel NO sensors were outlooked in the last part.     

Photolytic Measurement of Tissue S-Nitrosothiols in Rats and Humans In Vivo

S-nitrosothiols are labile thiol-NO adducts formed in vivo primarily by metalloproteins such as NO synthase, ceruloplasmin, and hemoglobin. Abnormal S-nitrosothiol synthesis and catabolism contribute to many diseases, ranging from asthma to septic shock. Current methods for quantifying S-nitrosothiols in vivo are suboptimal. Samples need to be removed from the body for analysis, and the S-nitrosothiols can be broken down during ex vivo processing. Here, we have developed a noninvasive device to measure mammalian tissue S-nitrosothiols in situ non-invasively using ultraviolet (UV) light, which causes NO release in proportion to the S-nitrosothiol concentration. We validated the assay in vitro; then, we applied it to measure S-nitrosothiols in vivo in rats and in humans. The method was sensitive to 0.5 µM, specific (did not detect other nitrogen oxides), and was reproducible in rats and in humans. This noninvasive approach to S-nitrosothiol measurements may be applicable for use in human diseases.     

巯基乙酸自组装膜DNA电化学传感器对转基因NOS的定量检测

以转基因植物中常用的根癌农杆菌终止子(NOS)为检测对象, 将巯基乙酸自组装于金电极表面形成巯基乙酸自组装单分子膜, 再利用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)的活化作用将NOS探针ssDNA序列固定于金电极表面形成NOS电化学生物传感器, 以亚甲基蓝(MB)为杂交指示剂, 对NOS靶基因相关序列进行了定量检测.     

Vasodilator Effect of Ethanolic Extract of Mulberry Leaves (Morus alba L.) in Rat and Rabbit

The effects of ethanolic extracts of Mulberry Leaves (Morus alba L.) on nitric oxide (NO) serum level in rat and diameter of blood vessels in rabbit ear were investigated. NO level of ethanolic extract of mulberry leaves at dose of 100 mg/kg bw rats gave significant difference compared to negative control (P<0,05) at minute 30 after extract administration (4,51 ± 4,31 μM). Ethanolic extract of mulberry leaves at dose of 200 mg/kg bw had no significant difference in NO serum level. However, ethanolic extract of mulberry leaves at dose of 400 mg/kg bw rats increased NO serum level significantly compared to negative control (P<0,05) at minute 0, 10, 30, 60, and 90 after extract administration. The maximum serum level of NO of 400 mg/kg bw mulberry extract was 4,62 ± 3,05 μM, and it is the highest among other groups. Therefore this dose was choosen for vasodilatation assay in rabbit. Observation on blood vessels diameter in rabbit ears showed that ethanolic extract of mulberry leaves were able to dilatate the big vessels and small vessels of rabbit ears significantly compared to negative control (P<0,05) at minute 60 after extract administration. In conclusion, ethanolic extract of mulberry leaves at dose of 400 mg/kg bw rats or 202.67 mg/kg bw rabbits has a vasodilator effect, probably due to an increase of NO serum level.     

Nitric Oxide Disproportionation Mediated by a Rh Complex with an Asymmetric Pincer Ligand: Spectroscopic Detection of a Dinitrosyl Intermediate

The disproportionation of nitric oxide was studied by FTIR with two different Rh(I)PCN complexes as mediators (PCN 1-((diethylamino)methyl)-3-((di-tertbutylphosphino)methyl)-benzene). As had already been reported for a PCP analogue, reaction yielded a Rh(PCN)(NO)(NO₂) complex along with gaseous N₂O in both cases. However, when [Rh(PCN)(NO)]⋅ was used as a reactant, FTIR monitoring allowed for the detection of signals of a reaction intermediate, coherent with the expected but seldom reported dinitrosyl species Rh(PCN)(NO)(NO). DFT studies on this species revealed that pincer hemilability of the amino arm is involved in its stabilization, therefore accounting for the differences observed in reactivity between PCN and PCP.     

The iromycins, a new family of pyridone metabolites from Streptomyces sp. I. Structure, NOS inhibitory activity, and biosynthesis

The iromycins A-D are members of a new family of rare alpha-pyridone metabolites. The isolation and structure elucidation of these microbial secondary metabolites from Streptomyces sp. Dra 17 revealed a N-heterocyclic core structure with two unusual side chains. Iromycins act as inhibitors of nitric oxide synthases (NOS), a protein family, which produces the crucial second messenger nitric oxide (NO). Importantly, these compounds inhibit selectively endothelial NOS rather than neuronal NOS and thus set prospects for both medical therapy and basic research. Feeding experiments with 13C- and 15N -labeled precursors indicated an uncommon type of polyketide biosynthesis and clearly ruled out an isoprenoid origin. A detoxification pathway of a particular secondary metabolite in the host strain is a rare observation and here we demonstrate it with the iromycin family.     

Increased arginase II activity contributes to endothelial dysfunction through endothelial nitric oxide synthase uncoupling in aged mice

The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (ArgII) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting ArgII with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against ArgII. These results might be associated with increased L-arginine bioavailability. Collectively, these results suggest that ArgII may be a valuable target in age-dependent vascular diseases.