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Nitric oxide (NO) has been defined as the "miracle molecule" due to its essential pleiotropic role in living systems. Besides its implications in physiologic functions, it is also involved in the development of several disease states, and understanding this ambivalence is crucial for medicinal chemists to develop therapeutic strategies that regulate NO production without compromising its beneficial functions in cell physiology. Although nitric oxide synthase (NOS), i.e., the enzyme deputed to the NO biosynthesis, is a well-recognized druggable target to regulate NO bioavailability, some issues have emerged during the past decades, limiting the progress of NOS modulators in clinical trials. In the present review, we discuss the most promising advancements in the research of small molecules that are able to regulate NOS activity with improved pharmacodynamic and pharmacokinetic profiles, providing an updated framework of this research field that could be useful for the design and development of new NOS modulators.

The transcriptional regulator nuclear factor-κB (NF-κB) is a mediator of endothelial dysfunction. Inhibiting NF-κB with salsalate is used to investigate inflammatory mechanisms contributing to accelerated cardiovascular disease risk. However, in the absence of disease, inhibition of NF-κB can impact redox mechanisms, resulting in paradoxically decreased endothelial function. This study aimed to measure microvascular endothelial function during inhibition of the transcriptional regulator NF-κB in reproductive-aged healthy women. In a randomized, single-blind, crossover, placebo-controlled design, nine healthy women were randomly assigned oral salsalate (1,500 mg, twice daily) or placebo treatments for 5 days. Subjects underwent graded perfusion with the endothelium-dependent agonist acetylcholine (ACh, 10 -10 to 10 -1 M, 33°C) alone and in combination with 15 mM N -nitro-l-arginine methyl ester [l-NAME; nonselective nitric oxide (NO) synthase inhibitor] through intradermal microdialysis. Laser-Doppler flux was measured over each microdialysis site, and cutaneous vascular conductance (CVC) was calculated as flux divided by mean arterial pressure and normalized to site-specific maximum (CVC G -nitro-l-arginine methyl ester [l-NAME; nonselective nitric oxide (NO) synthase inhibitor] through intradermal microdialysis. Laser-Doppler flux was measured over each microdialysis site, and cutaneous vascular conductance (CVC) was calculated as flux divided by mean arterial pressure and normalized to site-specific maximum (CVC %max ; 28 mM sodium nitroprusside + 43°C). The l-NAME sensitive component was calculated as the difference between the areas under the dose-response curves. During the placebo and salsalate treatments, the l-NAME sites were reduced compared with the control sites (both P < 0.0001). Across treatments, there was a significant difference between the control and l-NAME sites, where both sites shifted upward following salsalate treatment (both P < 0.0001), whereas the l-NAME-sensitive component was not different ( P The transcription factor nuclear factor-κB (NF-κB) regulates multiple aspects of innate and adaptive immunity by encoding for genes that participate in inflammation and impact endothelial function following NF-κB inhibition with salsalate treatment. Our results show that cutaneous microvascular function is increased through non-nitric oxide (NO)-dependent mechanisms following salsalate treatment in reproductive-aged healthy women.NEW & NOTEWORTHY The transcription factor nuclear factor-κB (NF-κB) regulates multiple aspects of innate and adaptive immunity by encoding for genes that participate in inflammation and impact endothelial function following NF-κB inhibition with salsalate treatment. Our results show that cutaneous microvascular function is increased through non-nitric oxide (NO)-dependent mechanisms following salsalate treatment in reproductive-aged healthy women.

Soluble epoxide hydrolase (sEH) is an enzyme targeted for the treatment of inflammation and cardiovascular diseases. Activated inflammatory cells produce nitric oxide (NO), which induces oxidative stress and exacerbates inflammation. We identify an inhibitor able to suppress sEH and thus NO production. Five flavonoids 1 - 5 isolated from Inula britannica flowers were evaluated for their abilities to inhibit sEH with IC 50 values of 12.1 ± 0.1 to 62.8 ± 1.8 µM and for their effects on enzyme kinetics. A simulation study using computational chemistry was conducted as well. Furthermore, five inhibitors ( 1 - 5 ) were confirmed to suppress NO levels at 10 µM. The results showed that flavonoids 1 - 5 exhibited inhibitory activity in all tests, with compound 3 exhibiting the most significant efficacy. Thus, in the development of anti-inflammatory inhibitors, compound 3 is a promising natural candidate.

Close to 19% of the world population suffers from anxiety. Current medications for this chronic mental disorder have improved treatment over the last half century or more, but the newer anxiolytics have proved disappointing, and enormous challenges remain. Nitric oxide (NO), an intra- and inter-cellular messenger in the brain, is involved in the pathogenesis of anxiety. In particular, excessive NO production might contribute to its pathology. This implies that it might be useful to reduce nitrergic activity; therefore, molecules aiming to downregulate NO production such as NO synthase inhibitors (NOSIs) might be candidates. Here, it was intended to critically review advances in research on these emerging molecules for the treatment of anxiety disorders. Current assessment indicates that, although NOSIs are implicated in anxiety, their potential anti-anxiety action remains to be established.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a protein kinase that inactivates eEF2, a protein that mediates a peptidyl-tRNA translocation during an elongation step of protein synthesis. We have previously shown that eEF2K was involved in pathogenesis of essential and pulmonary hypertension. A484954 (7-amino-1-cyclopropyl-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d] pyrimidine-6-carboxamide), a selective eEF2K inhibitor, is a membrane permeable small molecule. We have previously shown that A484954 lowered blood pressure and induced diuretic effects in spontaneously hypertensive rats (SHR) due to an increase in renal blood flow. Here we aimed to reveal mechanisms underlying the diuretic effects of A484954 in SHR. A484954-induced diuresis and increase in urinary Na + excretion were inhibited by N (G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor. A484954 increased mRNA expression of angiotensin type 2 receptor (AT2R) and nuclear factor-erythroid 2-related factor 2 (Nrf2). In summary, we for the first time revealed that A484954 induces diuresis in SHR at least partly via the activation of NO/Nrf2/AT2R pathway.

Ischemic stroke (IS) is a life-threatening disease worldwide. Nitric oxide (NO) derived from l-arginine catalyzed by NO synthase (NOS) is closely associated with IS. Three isomers of NOS (nNOS, eNOS and iNOS) produce different concentrations of NO, resulting in quite unlike effects during IS. Of them, n/iNOSs generate high levels of NO, detrimental to brain by causing nerve cell apoptosis and/or necrosis, whereas eNOS releases small amounts of NO, beneficial to the brain via increasing cerebral blood flow and improving nerve function. As a result, a large variety of NO regulators (NO donors or n/iNOS inhibitors) have been developed for fighting IS. Regrettably, up to now, no review systematically introduces the progresses in this area. This article first outlines dynamic variation rule of NOS/NO in IS, subsequently highlights advances in NO regulators against IS, and finally presents perspectives based on concentration-, site- and timing-effects of NO production to promote this field forward., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Myopia is one of the most prevalent eye diseases that seriously threaten the eyesight of children and adolescents worldwide. However, the pathogenesis is still unclear, and effective drugs are still scarce. In the present study, the guinea pigs were randomly divided into a normal control (NC) group, a lens-induced myopia (LIM) group, a NOS inhibitor (L-NMMA) injection group, and a NOS inhibitor solvent phosphate-buffered saline (PBS) group and the animals received relevant treatments. After 2- and 4-week different treatments, we noted that the refraction and choroidal thickness in the LIM group decreased compared with the NC group, whereas the ocular axial length increased significantly, and the choroid showed a fibrotic trend. The expression of NOS1, NOS3, TGF-β1, COLI, and α-SMA at gene and protein levels was increased significantly in the choroid (all P < 0.05). After intravitreal injection of NOS inhibitor L-NMMA, we found that compared with the LIM group, the refraction and the choroidal thickness significantly increased, whereas the axial length reduced significantly, accompanied by an increase of choroidal thickness and an improvement of choroidal fibrosis. The expression levels of choroidal NOS1, NOS3, TGF-β, COLI, and α-SMA were significantly reduced (all P < 0.05). In conclusion, the trend of choroidal fibrosis in LIM guinea pigs is positively correlated with the increase in axial length. The NOS inhibitor L-NMMA can alleviate the process of choroidal fibrosis in myopic guinea pigs by inhibiting NO signaling pathway., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We examined the effects of nobiletin, a polymethoxyflavonoid, on the retinal microvascular diameter to determine if they depend on the endothelium and/or smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded diametric responses to nobiletin. The retinal arterioles dilated in a nobiletin concentration-dependent (100 pM-10 μM) manner and decreased by 50% after endothelial removal. The nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME), reduced nobiletin-induced vasodilation comparable to denudation. Blockade of soluble guanylyl cyclase by 1H-[1,2,4] oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) produced a similar inhibitory effect as that by L-NAME. Nobiletin-induced vasodilation was also inhibited by the nonselective potassium channel inhibitor, tetraethylammonium (TEA), and the voltage-gated K (Kv) inhibitor, 4-aminopyridine. Co-administration of L-NAME and TEA almost eliminated nobiletin-induced vasodilation. Nobiletin elicits both endothelium-dependent and -independent dilation of retinal arterioles mediated by NO release and Kv channel activation, respectively., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

While numerous studies have suggested the involvement of cerebrovascular dysfunction in the pathobiology of blast-induced traumatic brain injury (bTBI), its exact mechanisms and how they affect the outcome of bTBI are not fully understood. Our previous study showed the occurrence of cortical spreading depolarization (CSD) and subsequent long-lasting oligemia/hypoxemia in the rat brain exposed to a laser-induced shock wave (LISW). We hypothesized that this hemodynamic abnormality is associated with shock wave-induced generation of nitric oxide (NO). In this study, to verify this hypothesis, we used an NO-sensitive fluorescence probe, diaminofluorescein-2 diacetate (DAF-2 DA), for real-time in vivo imaging of male Sprague-Dawley rats' brain exposed to a mild-impulse LISW. We observed the most intense fluorescence, indicative of NO production, along the pial arteriolar walls during the period of 10-30 min post-exposure, parallel with CSD occurrence. This post-exposure period also coincided with the early phase of hemodynamic abnormalities. While the changes in arteriolar wall fluorescence measured in rats receiving pharmacological NO synthase inhibition by nitro-L-arginine methyl ester (L-NAME) 24 h before exposure showed a temporal profile similar to that of changes observed in LISW-exposed rats with CSD, their intensity level was considerably lower; this suggests partial involvement of NOS in shock wave-induced NO production. To the best of our knowledge, this is the first real-time in vivo imaging of NO in rat brain, confirming the involvement of NO in shock-wave-induced hemodynamic impairments. Finally, we have outlined the limitations of this study and our future research directions., (© 2023 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

The review summarises the data of the last 50 years on the effectiveness of the amino acid L-arginine in therapeutic practice in conditions accompanied by different-origin hypoxia. The aim of this review was to analyse the literature and our research data on the role of nitric oxide in the modulation of individual physiological reactivity to hypoxia. The review considers the possibility of eliminating methodological conflicts in the case of L-arginine, which can be solved by taking into account individual physiological reactivity (or the hypoxia resistance factor). Considerable attention is paid to genetic and epigenetic mechanisms of adaptation to hypoxia and conditions of adaptation in different models. The article presents data on the clinical effectiveness of L-arginine in cardiovascular system diseases (hypertension, atherosclerosis, coronary heart disease, etc.) and stress disorders associated with these diseases. The review presents a generalised analysis of techniques, data on L-arginine use by athletes, and the ambiguous role of NO in the physiology and pathology of hypoxic states shown via nitric oxide synthesis. Data on the protective effects of adaptation in the formation of individual high reactivity in sportsmen are demonstrated. The review demonstrates a favourable effect of supplementation with L-arginine and its application depending on mitochondrial oxidative phosphorylation processes and biochemical indices in groups of individuals with low and high capacity of adaptation to hypoxia. In individuals with high initial anti-hypoxic reserves, these favourable effects are achieved by the blockade of NO-dependent biosynthesis pathways. Therefore, the methodological tasks of physiological experiments and the therapeutic consequences of treatment should include a component depending on the basic level of physiological reactivity.

Young non-Hispanic Black adults have reduced microvascular endothelial function compared with non-Hispanic White counterparts, but the mechanisms are not fully elucidated. The purpose of this study was to investigate the effect of endothelin-1 A receptor (ET A R) and superoxide on cutaneous microvascular function in young non-Hispanic Black ( n = 10) and White ( n = 10) adults. Participants were instrumented with four intradermal microdialysis fibers: 1 ) lactated Ringer's (control), 2 ) 500 nM BQ-123 (ET A R antagonist), 3 ) 10 μM tempol (superoxide dismutase mimetic), and 4 ) BQ-123 + tempol. Skin blood flow was assessed via laser-Doppler flowmetry (LDF), and each site underwent rapid local heating from 33°C to 39°C. At the plateau of local heating, 20 mM l-NAME [nitric oxide (NO) synthase inhibitor] was infused to quantify NO-dependent vasodilation. Data are means ± standard deviation. NO-dependent vasodilation was decreased in non-Hispanic Black compared with non-Hispanic White young adults ( P < 0.01). NO-dependent vasodilation was increased at BQ-123 sites (73 ± 10% NO) and at BQ-123 + tempol sites (71 ± 10%NO) in non-Hispanic Black young adults compared with control (53 ± 13%NO, P = 0.01). Tempol alone had no effect on NO-dependent vasodilation in non-Hispanic Black young adults (63 ± 14%NO, P = 0.18). NO-dependent vasodilation at BQ-123 sites was not statistically different between non-Hispanic Black and White (80 ± 7%NO) young adults ( P = 0.15). ET A R contributes to reduced NO-dependent vasodilation in non-Hispanic Black young adults independent of superoxide, suggesting a greater effect on NO synthesis rather than NO scavenging via superoxide. NEW & NOTEWORTHY Endothelin-1 A receptors (ET A Rs) have been shown to reduce endothelial function independently and through increased production of superoxide. We show that independent ET A R inhibition increases microvascular endothelial function in non-Hispanic Black young adults. However, administration of a superoxide dismutase mimetic alone and in combination with ET A R inhibition had no effect on microvascular endothelial function suggesting that, in the cutaneous microvasculature, the negative effects of ET A R in non-Hispanic Black young adults are independent of superoxide production.

Nitric oxide (NO) participates in processes such as endothelium-dependent vasodilation and neurotransmission/neuromodulation. The role of NO in epilepsy is controversial, attributing it to anticonvulsant but also proconvulsant properties. Clarification of this dual effect of NO might lead to the development of new antiepileptic drugs. Previous results in our laboratory indicated that this contradictory role of NO in seizures could depend on the nitric oxide synthase (NOS) isoform involved, which could play opposite roles in epileptogenesis, one of them being proconvulsant but the other anticonvulsant. The effect of convulsant drugs on neuronal NO (nNO) and endothelial NO (eNO) levels was investigated. Considering the distribution of neuronal and endothelial NOS in neurons and astrocytes, resp., primary cultures of neurons and astrocytes were used as a study model. The effects of convulsant drugs pentylenetetrazole, thiosemicarbazide, 4-aminopyridine and bicuculline on NO levels were studied, using a spectrophotometric method. Their effects on NO levels in neurons and astrocytes depend on the concentration and time of treatment. These convulsant drugs caused an increase in nNO, but a decrease in eNO was proportional to the duration of treatment in both cases. Apparently, nNO possesses convulsant properties mediated by its effect on the glutamatergic and GABAergic systems, probably through GABA A receptors. Anticonvulsant properties of eNO may be the consequence of its effect on endothelial vasodilation and its capability to induce angiogenesis. Described effects last as seizures do. Considering the limitations of these kinds of studies and the unexplored influence of inducible NO, further investigations are required., (© 2023 Lourdes A. Vega Rasgado et al., published by Sciendo.)

Background: Nitric oxide is a free radical bioregulator controlling homeostasis, vasodilation, and inhibition of platelet aggregation, significantly implicated in the nervous and immune system functionality. In vivo it is produced by nitric oxide synthases (NOSs)., Objective: Overproduction of nitric oxide is linked to several inflammatory, immunological, and neurodegenerative diseases and for that, various compounds have been synthesized as inhibitors of NOSs. In this review, the QSAR analyses were summarized in a variety of compounds as potent inhibitors of NOSs, and the models derived through 1D, 2D and 3D QSAR analyses., Conclusion: Ten groups of various NOS inhibitors and 17 1D, 2D, and 3D QSAR models and analyses were presented and discussed. A lack of hydrophobic terms was noticed in most of the cases. Chemical substituents were selected considering the increase either of the hydrophilicity and/or of hydrophobicity, bulkiness supported steric interactions, and point to potent inhibitors. CMR (Calculated Molar Refractivity) a steric variable, with a negative sign, underlines the critical effects participating on (in) an active site on the enzymes. Indicator variables imply the influence of specific structural moieties. Electronic parameters were found to be significant., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The dorsomedial periaqueductal gray (dmPAG) is a mesencephalic area and has numerous functions including cardiovascular regulation. Because nitric oxide (NO) is present in the dmPAG, here we investigate, the probable cardiovascular effect of NO in the dmPAG., Methods: Five groups (n = 6 for each group) were used as follows: (1) control; (2) L-NAME (N G -nitro-L-arginine methyl ester, a NO synthase inhibitor, 90 nmol); (3) L-arginine (L-Arg, a precursor for NO, 60 nmol); (4) Sodium nitroprusside (SNP, a NO donor, 27 nmol); and (5) L-Arg + L-NAME. The cardiovascular parameters were recorded by a Power Lab device after cannulation of the femoral artery. Drugs were injected using a stereotaxic instrument. The changes (∆) in systolic blood pressure (SBP), mean arterial pressure (MAP), and heart rate (HR) were calculated at different times and compared to the control group., Results: Microinjection of L-NAME significantly increased ∆SBP, ∆MAP, and ∆HR more than saline (from p < 0.05 to p < 0.001). L-Arg only significantly increased ∆HR (p < 0.05). In the L-Arg + L-NAME group, the above parameters also significantly increased (from p < 0.01 to p < 0.05) but not as significantly as with L-NAME alone. Microinjection of SNP significantly decreased ∆SBP and ∆MAP more than in the control and L-NAME groups (from p < 0.01 to p < 0.001), but ∆HR did not change significantly., Conclusion: The results indicated that NO in dmPAG has an inhibitory effect on cardiovascular responses in anesthetized rats., (© 2022 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Neuronal nitric oxide synthase (nNOS) is an enzyme constitutively expressed in the mammalian brain and skeletal muscles. The excessive activation of nNOS in the neurons results in oxidative and nitrosative stress associated with neuronal loss in various neurological disorders. Several nNOS inhibitors have been reported to limit the excessive activation of nNOS. In the present work, we have designed and carried the synthesis of benzo[d]thiazol-2-yl-methyl-4-(substituted)-piperazine-1-carbothioamide as novel neuronal nitric oxide inhibitors (5-28, twenty-four compounds). Stably transfected HEK 293 cells expressing NOS isoforms treated with the compounds (5-28) showed that the eight compounds exhibited > 95% cell survival in the MTT assay. nNOS inhibition assay of the eight compounds illustrated that the compound 18 was most selective for nNOS (nNOS=66.73 ± 1.51; eNOS=28.70 ± 1.39; iNOS =13.26 ± 1.01) in HEK 293 cells expressing NOS isoforms. 6-OHDA-induced unilaterally lesioned rats treated with the compound 18 showed the improvement in motor and non-motor functions. Furthermore, the compound 18 showed the increased levels of dopamine and decreased levels of glutamate and nitrite ions in the isolated rat brain. In the docking analysis, the compound 18 showed the significant binding affinity with the nNOS binding site (the ∆G value = - 9.0 kcal/mol). Overall results demonstrated that the N-(benzo[d]thiazol-2-ylmethyl)-4-(4-nitrophenyl) piperazine-1-carbothioamide (the compound 18) possessed significant nNOS inhibiting activity and neuroprotecting potential in 6-OHDA-induced unilaterally lesioned rat model of PD and more work will be required to establish the role of the compound 18 in the therapy of PD and other neurodegenerative disorders., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Pratibha Mehta Luthra reports financial support was provided by Department of Science and Technology. Dr Pratibha Mehta Luthra reports financial support was provided by Council of Scientific & Industrial Research. Dr Pratibha Mehta Luthra reports financial support was provided by University of Delhi., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Myogenic and flow-induced reactivity contribute to cerebral autoregulation, with potentially divergent roles for smaller versus larger arteries. The present study tested the hypotheses that compared with first-order (1A) branches of the middle cerebral artery, second- and third-order branches (2A and 3A, respectively) exhibit greater myogenic reactivity but reduced flow-induced constriction. Furthermore, nitric oxide synthase (NOS) inhibition may amplify myogenic reactivity and abolish instances of flow-induced dilation. Isolated porcine cerebral arteries mounted in a pressure myograph were exposed to incremental increases in intraluminal pressure (40-120 mmHg; n = 41) or flow (1-1,170 µL/min; n = 31). Intraluminal flows were adjusted to achieve 5, 10, 20, and 40 dyn/cm 2 of wall shear stress at 60 mmHg. Myogenic tone was greater in 3A versus 1A arteries ( P < 0.05). There was an inverse relationship between myogenic reactivity and passive arterial diameter ( P < 0.01). NOS inhibition increased basal tone to a lesser extent in 3A versus 1A arteries ( P < 0.01) but did not influence myogenic reactivity ( P = 0.49). Increasing flow decreased luminal diameter ( P ≤ 0.01), with increased vasoconstriction at 10-40 dyn/cm 2 of shear stress ( P < 0.01). However, relative responses were similar between 1A, 2A, and 3A arteries ( P = 0.40) with and without NOS inhibition conditions ( P ≥ 0.29). Whereas NOS inhibition increases basal myogenic tone, and myogenic reactivity was less in smaller versus larger arteries (range = ∼100-550 µM), neither NOS inhibition nor luminal diameter influences flow-induced constriction in porcine cerebral arteries. NEW & NOTEWORTHY This study demonstrated size-dependent heterogeneity in myogenic reactivity in porcine cerebral arteries. Smaller branches of the middle cerebral artery exhibited increased myogenic reactivity, but attenuated NOS-dependent increases in myogenic tone compared with larger branches. Flow-dependent regulation does not exhibit the same variation; diameter-independent flow-induced vasoconstrictions occur across all branch orders and are not affected by NOS inhibition. Conceptually, flow-induced vasoconstriction contributes to cerebral autoregulation, particularly in larger arteries with low myogenic tone.

Nitric oxide (NO) is a signalling molecule that controls a multitude of regulatory functions including neurotransmission, vascular tone, immune response, and angiogenesis. Regulating NO concentrations in cells using small molecules is an active area of research in the treatment of several pathologies such as cardiovascular disease, cancer, and inflammatory conditions. Small molecule-inhibition of critical NO regulatory enzymes, NO synthase (NOS), arginase, and dimethylarginine dimethyaminohydrolase-1 (DDAH1), has shown therapeutic benefits as well as limitations and is a focus of current research.In recent years, DDAH1 has been explored as a potential target to indirectly regulate NO in diseases characterized by excessive NO production. This review discusses the biological and pathophysiological role of the NO pathway, the existing inhibitors of NOS, arginase and DDAH1, and the conventional and structure-guided structure-activity relationship studies involved in their discovery. The key structural elements of amino acid-derived inhibitors responsible for selective inhibition of each enzyme, and the chemical features responsible for dual enzyme inhibition are also discussed. Finally, a synthetic scheme for developing both selective and dual inhibitors using common starting materials is provided, offering unique insights in the quest for the rational design of novel NO pathway inhibitors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood-brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (K i  = 46 nM) and human nNOS (K i  = 48 nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (P e  = 17.3 × 10 -6 cm s -1 ) through a parallel artificial membrane permeability assay, a model for blood-brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood-brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure-activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The "astrocyte-to-neuron lactate shuttle" (ANLS) mechanism is part of the central inhibitory pathway to modulate sodium intake. An interaction between the GABAergic neurons and nitric oxide (NO) in the subfornical organ (SFO) in salt-appetite inhibition has been suggested. In addition, NO is a key molecule involved in astrocytic energy metabolism and lactate production. In the present study, we hypothesized there is an interaction between astrocytic lactate and central NO to negatively modulate water and sodium intake through the ANLS mechanism. The results showed that central Nω-nitro-L-arginine methyl ester (L-NAME, NO-synthase inhibition) induced an increase in water and sodium intake. These responses were attenuated by previous central microinjection of fluorocitrate (FCt, a reversible glial inhibitor). Interestingly, L-NAME-induced water and sodium intake were also decreased by previous microinjection of lactate but did not change after inhibition of the ANLS mechanism by α-cyano 4-hydroxycinnamic acid (α-CHCA), an inhibitor of the MCT lactate transporter. Our results suggest a central interaction between NO, glial cells, and lactate to modulate water and sodium intake., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H 2 O 2 around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H 2 O 2 -alone treatment kills catalase-deficient (katEG) mutants similar to H 2 O 2 +NO treatment. However, the Fenton reaction, in addition to H 2 O 2 , requires Fe(II), which H 2 O 2 excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H 2 O 2 is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H 2 O 2 -alone and H 2 O 2 +NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H 2 O 2 -alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H 2 O 2 and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitric oxide (NO)-dependent pathways may play a significant role in the decline of synaptic and cognitive functions in Alzheimer's disease (AD). However, whether NO in the hippocampal dentate gyrus (DG) is involved in the spatial learning and memory impairments of AD by affecting the glutamate (Glu) response during these processes is not well-understood. Here, we prepared an AD rat model by long-term i.p. of D-galactose into ovariectomized rats, and then the effects of L-NMMA (a NO synthase inhibitor) on Glu concentration and amplitude of field excitatory postsynaptic potential (fEPSP) were measured in the DG region during the Morris water maze (MWM) test in freely-moving rats. During the MWM test, compared with the sham group, the escape latency was increased in the place navigation trial, and the percentage of time spent in target quadrant and the number of platform crossings were decreased in the spatial probe trial, in addition, the increase of fEPSP amplitude in the DG was significantly attenuated in AD group rats. L-NMMA significantly attenuated the spatial learning and memory impairment in AD rats, and reversed the inhibitory effect of AD on increase of fEPSP amplitude in the DG during the MWM test. In sham group rats, the Glu level in the DG increased significantly during the MWM test, and this response was markedly enhanced in AD rats. Furthermore, the response of Glu in the DG during spatial learning was recovered by microinjection of L-NMMA into the DG. Our results suggest that NO in the DG impairs spatial learning and memory and related synaptic plasticity in AD rats, by disturbing the Glu response during spatial learning., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The Frank-Starling response of the heart is known to be mediated by nitric oxide (NO) signaling, which is regulated by reduced glutathione (GSH) and hydrogen sulfide (H 2 S). We hypothesized that stimulation of endogenous H 2 S or GSH synthesis would improve the Frank-Starling response. Wistar male rats were injected with propargylglycine (PAG; 11.3 mg/kg, 40 min, n  = 12), an inhibitor of H 2 S-producing enzyme (cystationine-γ-lyase), and l-cysteine (121 mg/kg, 30 min, n  = 20), a precursor of H 2 S and GSH. Pretreatment with PAG or l-cysteine separately slightly improved the pressure-volume (P-V) dependence of the isolated rat heart, but the combination of PAG and l-cysteine ( n  = 12) improved heart contractile activity. H 2 S content, Ca 2+ -dependent NOS activity (cNOS) activity, nitrate reductase activity, and nitrite content increased by 2, 3.83, 2.5, and 1.3 times in cardiac mitochondria, and GSH and oxidized glutathione (GSSG) levels increased by 2.24 and 1.86 times in the heart homogenates of the PAG + l-cysteine group compared with the control (all P  < 0.05). Inhibition of glutathione with DL-buthionine-sulfoximine (BSO; 22.2 mg/kg, 40 min, n  = 6) drastically decreased Frank-Starling response of the heart and prevented PAG + l-cysteine-induced increase of GSH and GSSG levels (BSO + PAG + l-cysteine, n  = 9). Inhibition of NOS, N-nitro-l-arginine-methylester hydrochloride (l-NAME; 40 min, 27 mg/kg) abolished positive inotropy induced by PAG+l-cysteine pretreatment (l-NAME + PAG + l-cysteine, n  = 7). Thus, PAG + l-cysteine administration improves the Frank-Starling response by upregulating mitochondrial H 2 S, glutathione, and NO synthesis, which may be a promising approach in the treatment of myocardial dysfunction.

Dry cough has been reported in patients receiving statin therapy. However, the underlying mechanism or other possible alterations in the airways induced by statins remain unknown. Thus, the aim of this study was to evaluate whether simvastatin promotes alterations in airways, such as bronchoconstriction and plasma extravasation, as well as the mechanism involved in these events. Using methods to detect alterations in airway resistance and plasma extravasation, we demonstrated that simvastatin [20 mg/kg, intravenous (i.v.)] caused plasma extravasation in the trachea (79.8 + 14.8 μg/g/tissue) and bronchi (73.3 + 8.8 μg/g/tissue) of rats, compared to the vehicle (34.2 + 3.6 μg/g/tissue and 29.3 + 5.3 μg/g/tissue, respectively). N G -nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, intraperitoneal), a nitric oxide (NO) synthase inhibitor, Icatibant [HOE 140, 10 nmol/50 μl, intratracheal (i.t.)], a bradykinin B 2 antagonist, and capsazepine (100 nmol/50 μl, i.t.), a TRPV1 antagonist, attenuated simvastatin-induced plasma extravasation. Simvastatin (5, 10 and 20 mg/kg) did not cause bronchoconstriction per se, but exacerbated the bronchoconstrictive response to bradykinin (30 nmol/kg, i.v.), a B 2 agonist (0.7 + 0.1 ml/H2O), or capsaicin (30 nmol/kg, i.v.), a TRPV1 agonist (0.8 + 0.1 ml/H2O), compared to the vehicle (0.1 + 0.04 ml/H2O and 0.04 + 0.01 ml/H2O, respectively). The bronchoconstriction elicited by bradykinin (100 nmol/kg, i.v.) in simvastatin non-treated rats was inhibited by L-NAME. The exacerbation of bronchoconstriction induced by bradykinin or capsaicin in simvastatin-treated rats was inhibited by L-NAME, HOE 140 or capsazepine. These results suggest that treatment with simvastatin promotes the release of bradykinin, which, via B 2 receptors, releases NO that can then activate the TRPV1 to promote plasma extravasation and bronchoconstriction., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with several human malignancies. KSHV lytic replication promotes the spread of infection and progression of KSHV-associated malignancies; however, the mechanism regulating KSHV lytic replication remains unclear. In this study, we investigated the role of nitric oxide (NO) in KSHV lytic replication. In the TREx BCBL1-RTA KSHV lytic replication cell system, induction of KSHV lytic replication increased intracellular and extracellular NO. Chemical inhibition of NO production resulted in a lower level of KSHV lytic replication as shown by a reduced level of infectious virions, and decreased levels of viral lytic transcripts and proteins. In a second KSHV lytic replication system of iSLK-RGB-BAC16 cells, we confirmed that KSHV lytic replication increased NO production. Chemical inhibition of NO production resulted in reduced numbers of cells expressing enhanced green fluorescent protein and blue fluorescent protein, two reporters that closely track the expression of KSHV early and late genes, respectively. Consistent with these results, inhibition of NO production resulted in reduced levels of infectious virions, and viral lytic transcripts and proteins. Importantly, exogenous addition of a NO donor was sufficient to enhance the full KSHV lytic replication program. These results demonstrate that NO is required for efficient KSHV lytic replication, and NO plays a crucial role in the KSHV life cycle and KSHV-induced malignancies., (© 2021 Wiley Periodicals LLC.)

We previously reported that inhibition of nitric oxide (NO) production promotes rat reconnected facial nerve regeneration. However, the underlying mechanism is obscure. Microtubule assembly is known to be essential to axon regeneration; nevertheless, tubulins and microtubule-associated proteins (MAPs) have been demonstrated as targets for NO and peroxynitrite. Thus, we hypothesized that NO and/or peroxynitrite may affect facial nerve regeneration via influencing on microtubule assembly. First, tubulins and tau (a MAP) were extracted from facial nerves of normal rats, treated with NO donor or peroxynitrite, and processed for microtubule assembly assay. We found that peroxynitrite, DEA NONOate, and Angeli's salt reduced the tubulin polymerization activity to a greater extent than GSNO, SIN-1, and SNAP. Additionally, SIN-1, peroxynitrite, and Angeli's salt impaired the ability of tau to promote microtubule assembly. Next, nitrosative stress biomarkers 3-nitrotyrosine (3-NT) and S-nitrosylated cysteine (SNO-Cys) were immunolabeled in facial nerves. Both biomarkers were highly upregulated in proximal and distal stumps of reconnected facial nerves at 3 days and 1 week after neurorrhaphy. Notably, the expression of 3-NT was greatly reduced at 2 weeks, whereas that of SNO-Cys was maintained. Conversely, inhibition of NO production with L-NAME prevented the upregulation of SNO-Cys. Further, we used tubulins and tau extracted from facial nerves of sham-operated, nerve suture + vehicle treatment, and nerve suture + L-NAME treatment rats to perform microtubule assembly assay. We found that L-NAME treatment enhanced polymerization activity of tubulins and ability of tau to promote microtubule assembly. It is noteworthy that α-tubulin plays a more important role than β-tubulin since the activity of microtubule assembly using α-tubulin extracted from L-NAME-treated rats was greatly elevated, whereas that using β-tubulin extracted from L-NAME-treated rats was not. Overall, our findings support that inhibition of NO production reduces nitrosative stress, and may thus facilitate microtubule assembly and facial nerve regeneration., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The protein deacetylase sirtuin 1 (SIRT1) and adenosine monophosphate-dependent protein kinase (AMPK) play important roles in the development of insulin resistance. In glomerular podocytes, crosstalk between these two enzymes may be altered under hyperglycemic conditions. SIRT1 protein levels and activity and AMPK phosphorylation decrease under hyperglycemic conditions, with concomitant inhibition of the effect of insulin on glucose uptake into these cells. Nitric oxide (NO)-dependent regulatory signaling pathways have been shown to be downregulated under diabetic conditions. The present study examined the involvement of the NO synthase (NOS)/NO pathway in the regulation of SIRT1-AMPK signaling and glucose uptake in podocytes. We examined the effects of NOS/NO pathway alterations on SIRT1/AMPK signaling and glucose uptake using pharmacological tools and a small-interfering transfection approach. We also examined the ability of the NOS/NO pathway to protect podocytes against high glucose-induced alterations of SIRT1/AMPK signaling and insulin-dependent glucose uptake. Inhibition of the NOS/NO pathway reduced SIRT1 protein levels and activity, leading to a decrease in AMPK phosphorylation and blockade of the effect of insulin on glucose uptake. Treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prevented high glucose-induced decreases in SIRT1 and AMPK activity and increased GLUT4 protein expression, thereby improving glucose uptake in podocytes. These findings suggest that inhibition of the NOS/NO pathway may result in alterations of the effects of insulin on glucose uptake in podocytes. In turn, the enhancement of NOS/NO pathway activity may prevent these deleterious effects of high glucose concentrations, thus bidirectionally stimulating the SIRT1-AMPK reciprocal activation loop., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aerobic training (AT) promotes several health benefits that may attenuate the progression of obesity associated diabetes. Since AT is an important nitric oxide (NO - ) inducer mediating kidney-healthy phenotype, the present study is aimed at investigating the effects of AT on metabolic parameters, morphological, redox balance, inflammatory profile, and vasoactive peptides in the kidney of obese-diabetic Zucker rats receiving L-NAME (N(omega)-nitro-L-arginine methyl ester). Forty male Zucker rats (6 wk old) were assigned into four groups ( n = 10, each): sedentary lean rats (CTL-Lean), sedentary obese rats (CTL-Obese), AT trained obese rats without blocking nitric oxide synthase (NOS) (Obese+AT), and obese-trained with NOS block (Obese+AT+L-NAME). AT groups ran 60 min in the maximal lactate steady state (MLSS), five days/wk/8 wk. Obese+AT rats improved glycemic homeostasis, SBP, aerobic capacity, renal mitochondria integrity, redox balance, inflammatory profile (e.g., TNF- α , CRP, IL-10, IL-4, and IL-17a), and molecules related to renal NO - metabolism (klotho/FGF23 axis, vasoactive peptides, renal histology, and reduced proteinuria). However, none of these positive outcomes were observed in CTL-Obese and Obese+AT+L-NAME ( p < 0.0001) groups. Although Obese+AT+L-NAME lowered BP (compared with CTL-Obese; p < 0.0001), renal damage was observed after AT intervention. Furthermore, AT training under conditions of low NO - concentration increased signaling pathways associated with ACE-2/ANG1-7/MASr. We conclude that AT represents an important nonpharmacological intervention to improve kidney function in obese Zucker rats. However, these renal and metabolic benefits promoted by AT are dependent on NO - bioavailability and its underlying regulatory mechanisms., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Rodrigo Vanerson Passos Neves et al.)

Narirutin is one of the most common flavanones found in citrus fruits. The vascular effects of its analogues naringenin and naringin have been reported but its effects on the cardiovascular system are largely unknown. In this study, relaxation effect of narirutin and its mechanisms of action were investigated by measuring isometric tension in rat mesenteric arteries. Patch-clamping was also used to study the effect of narirutin on potassium channels in vascular smooth muscle cells. Moreover, its effects on phosphorylation of endothelial nitric oxide synthase, cAMP level and phosphodiesterase activity in rat mesenteric arteries were studied by Western blot and biochemical assays. The results showed that pre-incubation of rat mesenteric arteries with narirutin had no influence on acetylcholine-induced endothelial-dependent relaxation. However, narirutin caused a direct concentration-dependent relaxation in rat mesenteric arteries. This relaxation effect was comparable to that of narirutin's structural analogue naringenin. Narirutin-induced relaxation was reduced by the removal of endothelium, N G -nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor), and 4-aminopyridine (a voltage-gated potassium channel blocker). In addition, narirutin increased the phosphorylation of endothelial nitric oxide synthase and increased the voltage-dependent potassium current in mesenteric arterial smooth muscle cells. These effects were abolished by protein kinase A inhibitor. Furthermore, narirutin could increase cAMP level and inhibit phosphodiesterase activity in rat mesenteric arteries. In conclusion, narirutin has vasorelaxing effect and the mechanism involves the inhibition of phosphodiesterase, which increases intracellular cAMP, thereby stimulating the endothelial nitric oxide synthase and activating the voltage-gated potassium channels in vascular smooth muscle cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, the role of nitric oxide (NO) in the pathogenesis of hydatidosis and the interaction with effects of anthelmintic drugs, albendazole and praziquantel, were examined in larval infection caused by protoscolices obtained from hydatid cysts of sheep liver in Albino Balb/c mice. Animals were divided into ten groups including controls and infected groups. Larval infection was established with intraperitoneal injection of protoscolices. Eight months after infection with protoscolices, the infected animals were divided into 6 groups. The infected animals were given a selective inhibitor of inducible nitric oxide synthase (iNOS) L-N 6 -(1-Iminoethyl) lysine-hydrochloride (L-NIL), NO donor sodium nitroprusside (SNP), albendazole and praziquantel as anthelmintic drugs for 7 days. In addition, control groups were composed of intact group, control, anthelmintic drugs + L-NIL, and anthelmintic drugs + SNP. The liver and blood samples were taken for cytological, histological, immunohistochemical and biochemical analyses 7 days after treatments at the end of experiment. The animals injected with protoscolices showed histopathological changes including inflammation areas, infiltration and accumulation of leukocytes, dilation of sinusoids, and damage in endothelial cells and hepatocytes at light microscopy. Electron microscopy were revealed severe damage in sinusoidal endothelial cells, leukocytes especially eosinophils in sinusoid lumens and disorganization in endoplasmic reticulum and nuclear membrane. Endothelial nitric oxide synthase (eNOS) and iNOS reactions were increased in the tissue. Anthelmintic drugs decreased inflammation areas and damages; however, it did not change NOS reactions in the animals given protoscolices. L-NIL and SNP diminished both iNOS and eNOS reactions. Unlike the group administered the inhibitor, SNP treated group exhibited less inflammation areas. Combination of these substances and drugs resulted in decreased inflammation areas. eNOS and iNOS reactions decreased in the drugs and SNP administered group, while only iNOS reaction was decreased in L-NIL given infection group. In addition, the infected groups which received SNP displayed expanded sinusoids and hepatocytes with vacuoles, intriguingly. While levels of serum nitrite/nitrate elevated only in the infection group given drugs and SNP, it decreased in the L-NIL administered group. Tissue level of malondialdehyde increased in infection groups with drugs and SNP. In conclusion, the results indicated that NO plays an important role in the pathogenesis of hydatidosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The heme enzyme myeloperoxidase (MPO) is a key mediator of endothelial dysfunction and a therapeutic target in cardiovascular disease. During inflammation, MPO released by circulating leukocytes is internalized by endothelial cells and transcytosed into the subendothelial extracellular matrix of diseased vessels. At this site, MPO mediates endothelial dysfunction by catalytically consuming nitric oxide (NO) and producing reactive oxidants, hypochlorous acid (HOCl) and the nitrogen dioxide radical ( • NO 2 ). Accordingly, there is interest in developing MPO inhibitors that effectively target endothelial-localized MPO. Here we studied a series of piperidine nitroxides conjugated to polyamine moieties as novel endothelial-targeted MPO inhibitors. Electron paramagnetic resonance analysis of cell lysates showed that polyamine conjugated nitroxides were efficiently internalized into endothelial cells in a heparan sulfate dependent manner. Nitroxides effectively inhibited the consumption of MPO's substrate hydrogen peroxide (H 2 O 2 ) and formation of HOCl catalyzed by endothelial-localized MPO, with their efficacy dependent on both nitroxide and conjugated-polyamine structure. Nitroxides also differentially inhibited protein nitration catalyzed by both purified and endothelial-localized MPO, which was dependent on • NO 2 scavenging rather than MPO inhibition. Finally, nitroxides uniformly inhibited the catalytic consumption of NO by MPO in human plasma. These studies show for the first time that nitroxides effectively inhibit local oxidative reactions catalyzed by endothelial-localized MPO. Novel polyamine-conjugated nitroxides, ethylenediamine-TEMPO and putrescine-TEMPO, emerged as efficacious nitroxides uniquely exhibiting high endothelial cell uptake and efficient inhibition of MPO-catalyzed HOCl production, protein nitration, and NO oxidation. Polyamine-conjugated nitroxides represent a versatile class of antioxidant drugs capable of targeting endothelial-localized MPO during vascular inflammation.

Nine previously undescribed butyrolactone and sesquiterpene derivatives, named cyclopentanone A (1), subamolides F and G (2 and 3), secosubamolide F (4), rupestonic acids J - L (5-7), linderaguaianols A and B (8 and 9), together with six known ones 10-15 were isolated from the roots of Lindera glauca. Their structures, including their absolute configurations were elucidated by extensive spectroscopic analysis, quantum chemical calculations, and Mo 2 (AcO) 4 -induced circular dichroism. Compound 1 that possessed a unique five-membered cyclopentane skeleton with a side chain was rarely found from natural sources. The biogenetic pathway for 1-4 was postulated. Secosubamolide F (4) inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 cells with IC 50 value of 1.73 ± 0.18 μM and also significantly suppressed the production of iNOS. The binding interactions between 4 and iNOS were investigated using docking analyses., (Copyright © 2021. Published by Elsevier Inc.)

Whether hypoxic acclimation influences nitric oxide (NO)-mediated control of fish cardiac function is not known. Thus, we measured the function/performance of myocardial strips from normoxic- and hypoxic-acclimated (40% air saturation; ∼8 kPa O 2 ) trout at several frequencies (20-80 contractions·min -1 ) and two muscle strain amplitudes (8% and 14%) when exposed to increasing concentrations of the NO donor sodium nitroprusside (SNP) (10 -9 to 10 -4 M). Further, we examined the influence of 1 ) nitric oxide synthase (NOS) produced NO [by blocking NOS with 10 -4 M N G -monomethyl-l-arginine (l-NMMA)] and 2 ) soluble guanylyl cyclase mediated, NOS-independent, NO effects (i.e., after blockade with 10 -4 M ODQ), on myocardial contractility. Hypoxic acclimation increased twitch duration by 8%-10% and decreased mass-specific net power by ∼35%. However, hypoxic acclimation only had minor impacts on the effects of SNP and the two blockers on myocardial function. The most surprising finding of the current study was the degree to which contraction frequency and strain amplitude influenced NO-mediated effects on myocardial power. For example, at 8% strain, 10 -4 SNP resulted in a decrease in net power of ∼30% at 20 min -1 but an increase of ∼20% at 80 min -1 , and this effect was magnified at 14% strain. This research suggests that hypoxic acclimation has only minor effects on NO-mediated myocardial contractility in salmonids, is the first to report the high frequency- and strain-dependent nature of NO effects on myocardial contractility in fishes, and supports previous work showing that NO effects on the heart (myocardium) are finely tuned spatiotemporally.

Astrocyte activation is characterized by hypertrophy with increased glial fibrillary acidic protein (GFAP), whose expression may involve pro-inflammatory cytokines. In this study, the effects of pro-inflammatory IL-6 and TNF-α and anti-inflammatory cytokines IL-4 and IL-10 on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling, intracellular calcium concentration ([Ca 2+ ] i ) and GFAP expression were investigated. In human glioblastoma astrocytoma U-373 MG cells, IL-6 and TNF-α, but not IL-4 or IL-10, increased iNOS, cGMP, [Ca 2+ ] i and GFAP expression. The inhibitors of iNOS (1400 W), soluble guanylyl cyclase (ODQ) and IP3 receptors (ryanodine and 2-APB) reversed the increase in cGMP or [Ca 2+ ] i , respectively, and prevented GFAP expression. In rat striatal slices, IL-6 and TNF-α, at variance with IL-4 and IL-10, promoted a concentration-dependent increase in Ca 2+ efflux, an effect prevented by 1400 W, ODQ and RY/2APB. These data were confirmed by in vivo studies, where IL-6, TNF-α or the NO donor DETA/NO injected in the striatum of anaesthetised rats increased cGMP levels and increased GFAP expression. The present findings point to NO/cGMP-dependent calcium signalling as part of the mechanism mediating IL-6- and TNF-α-induced GFAP expression. As this process plays a fundamental role in driving neurotoxicity, targeting NO/cGMP-dependent calcium signalling may constitute a new approach for therapeutic interventions in neurological disorders.

Ultrasound-mediated microbubble cavitation improves perfusion in chronic limb and myocardial ischemia. The purpose of this study was to determine the effects of ultrasound-mediated microbubble cavitation in acute limb ischemia and investigate the mechanism of action. The animal with acute hindlimb ischemia was established using male Sprague-Dawley rats. The rats were randomly divided into three groups: intermittent high-mechanical-index ultrasound pulses combined with microbubbles (ultrasound [US] + MB group), US alone (US group) and MB alone (MB group). Both hindlimbs were treated for 10 min. Contrast ultrasound perfusion imaging of both hindlimbs was performed immediately and 5, 10, 15, 20 and 25 min after treatment. The role of the nitric oxide (NO) pathway in increasing blood flow in acutely ischemic tissue was evaluated by inhibiting endothelial nitric oxide synthase (eNOS) with N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME). In the US + MB group, microvascular blood volume and microvascular blood flow of the ischemic hindlimb were significantly increased after treatment (both p values <0.05), while the microvascular flux rate (β) increased, but not significantly (p > 0.05). The increases were observed immediately after treatment, and had dissipated by 25 min. Changes in the US and MB groups were minimal. Inhibitory studies indicated cavitation increased phospho-eNOS concentration in ischemic hindlimb muscle tissue, and the increase was significantly inhibited by L-NAME (p < 0.05). Ultrasound-mediated microbubble cavitation transiently increases local perfusion in acutely ischemic tissue, mainly by improving microcirculatory perfusion. The eNOS/NO signaling pathway appears to be an important mediator of the effect., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Lipopolysaccharide (LPS) as the major component of the outer membrane of Gram-negative bacteria activates macrophages to produce a high level of pro-inflammatory cytokines which is considered as a cause of liver dysfunction. Overproduction of nitric oxide (NO) has been suggested to have a role in hepatic injury. The aim of the present study was to explore the protective effects of aminoguanidine (AG) as inducible nitric oxide synthase (iNOS) inhibitor against LPS -induced liver dysfunction in rat. The animals were divided into five groups: (1) control (2) LPS (3) LPS-AG50, (4) LPS-AG100 and (5) LPS-AG150. LPS (1 mg/kg) was injected for 5 weeks and AG (50, 100 and 150 mg/kg) was administered 30 min before LPS. Drugs were injected intraperitoneally. LPS induced liver dysfunction presented by increasing the serum level of alkaline phosphatase (ALK-P), alanine aminotransferase (ALT), aspartate aminotransferase (AST). Pretreatment with AG restored harmful effects of LPS on liver function. In addition, LPS resulted in hepatotoxicity, accompanied by enhancing the level of interleukin (IL)-6, malondialdehyde (MDA) and nitric oxide (NO) metabolites and decreasing the content of total thiol groups and superoxide dismutase (SOD) and catalase (CAT) activity. Injection of AG before LPS attenuated LPS-induced hepatotoxicity through decreasing the level of IL-6, MDA and NO metabolites and increasing total thiols and SOD and CAT activity. Considering the protective effect of AG which was seen in the present study, it seems that increased levels of NO due to activation of iNOS has a role in LPS-induced hepatic injury.

Eight new sesquiterpene derivatives (2, 4-6 and 10-13), along with five known analogues were isolated from the mangrove endophytic fungus Phomopsis sp. SYSU-QYP-23. Their structures of new compounds were established by spectroscopic methods, and the absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental ECD spectra. The absolute configuration of the side chain in 1 was first defined by modified Mosher's method. Compounds 1-7 showed potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharides (LPS) induced RAW 264.7 cells with IC 50 values ranging from 8.6 to 14.5 μM. The molecular docking results implied that the bioactive sesquiterpenes may directly bind with targeting residues in the active cavity of iNOS protein., (Published by Elsevier Inc.)

The present study aimed to evaluate the effects of dexamethasone on the redox status, parasitemia evolution, and survival rate of Plasmodium berghei-infected mice. Two-hundred and twenty-five mice were infected with Plasmodium berghei and subjected to stimulation or inhibition of NO synthesis. The stimulation of NO synthesis was performed through the administration of L-arginine, while its inhibition was made by the administration of dexamethasone. Inducible NO synthase (iNOS) inhibition by dexamethasone promoted an increase in the survival rate of P. berghei-infected mice, and the data suggested the participation of oxidative stress in the brain as a result of plasmodial infection, as well as the inhibition of brain NO synthesis, which promoted the survival rate of almost 90% of the animals until the 15th day of infection, with possible direct interference of ischemia and reperfusion syndrome, as seen by increased levels of uric acid. Inhibition of brain iNOS by dexamethasone caused a decrease in parasitemia and increased the survival rate of infected animals, suggesting that NO synthesis may stimulate a series of compensatory redox effects that, if overstimulated, may be responsible for the onset of severe forms of malaria.

Cardiovascular diseases are often characterized by dysfunctional endothelium. To compensate for the related lack of nitric oxide (NO), a class of soluble guanylate cyclase (sGC) stimulators and activators have been developed with the purpose of acting downstream of NO in the NO-sGC-cGMP cascade. These drugs have been discovered using photoaffinity labeling of sGC and high-throughput screening of a vast number of chemical compounds. Therefore, an understanding of the integrated physiological effects of these drugs in vivo is necessary on the path to clinical application. We have characterized the integrated hemodynamic impact of the sGC stimulator riociguat and the activator cinaciguat in different NO-states in healthy juvenile pigs (n = 30). We assessed the vascular effects in both systemic and pulmonary circulation, the contractile effects in the right and left ventricles, and the effects on diastolic cardiac functions. Nitric oxide-tone in these pigs were set by using the NO-blocker l-NAME and by infusion of nitroglycerine. The studies show a more pronounced vasodilatory effect in the systemic than pulmonary circulation for both drugs. Riociguat acts integrated with NO in an additive manner, while cinaciguat, in principle, completely blocks the endogenous NO effect on vascular control. Neither compound demonstrated pronounced cardiac effects but had unloading effect on both systolic and diastolic function. Thus, riociguat can potentially act in various disease states as a mean to increase NO-tone if systemic vasodilation can be balanced. Cinaciguat is a complicated drug to apply clinically due to its almost complete lack of integration in the NO-tone and balance.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role., (Copyright © 2020. Published by Elsevier Inc.)

A continuous state of oxidative stress during inflammation contributes to the development of 25% of human cancers. Epithelial and inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can damage DNA. ROS/RNS have biological implications in both chemoresistance and tumor recurrence. As several clinically employed anticancer drugs can generate ROS/RNS, we have addressed herein how inducible nitric oxide synthase and nitric oxide (iNOS/ • NO) affect the molecular pathways implicated in the tumor response to oxidative stress. To mimic the oxidative stress associated with chemotherapy, we used a photosensitizer (pheophorbide a) that can generate ROS/RNS in a controlled manner. We investigated how iNOS/ • NO modulates the tumor response to oxidative stress by involving the NF-κB and Nrf2 molecular pathways. We found that low levels of iNOS induce the development of a more aggressive tumor population, leading to survival, recurrence and resistance. By contrast, high levels of iNOS/ • NO sensitize tumor cells to oxidative treatment, causing cell growth arrest. Our analysis showed that NF-κB and Nrf2, which are activated in response to oxidative stress, communicate with each other through RKIP. For this critical role, RKIP could be an interesting target for anticancer drugs. Our study provides insight into the complex signaling response of cancer cells to oxidative treatments as well as new possibilities for the rational design of new therapeutic strategies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Nitric oxide (NO) is presumed to be a regulator of metamorphosis in many invertebrate species, and although NO pathways have been comparatively well-investigated in gastropods, annelids and crustaceans, there has been very limited research on the effects of NO on metamorphosis in bivalve shellfish., Results: In this paper, we investigate the effects of NO pathway inhibitors and NO donors on metamorphosis induction in larvae of the Pacific oyster, Crassostrea gigas. The nitric oxides synthase (NOS) inhibitors s-methylisothiourea hemisulfate salt (SMIS), aminoguanidine hemisulfate salt (AGH) and 7-nitroindazole (7-NI) induced metamorphosis at 75, 76 and 83% respectively, and operating in a concentration-dependent manner. Additional induction of up to 54% resulted from exposures to 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, with which NO interacts to catalyse the synthesis of cyclic guanosine monophosphate (cGMP). Conversely, high concentrations of the NO donor sodium nitroprusside dihydrate in combination with metamorphosis inducers epinephrine, MK-801 or SMIS, significantly decreased metamorphosis, although a potential harmful effect of excessive NO unrelated to metamorphosis pathway cannot be excluded. Expression of CgNOS also decreased in larvae after metamorphosis regardless of the inducers used, but intensified again post-metamorphosis in spat. Fluorescent detection of NO in competent larvae with DAF-FM diacetate and localisation of the oyster nitric oxide synthase CgNOS expression by in-situ hybridisation showed that NO occurs primarily in two key larval structures, the velum and foot. cGMP was also detected in the foot using immunofluorescent assays, and is potentially involved in the foot's smooth muscle relaxation., Conclusion: Together, these results suggest that the NO pathway acts as a negative regulator of metamorphosis in Pacific oyster larvae, and that NO reduction induces metamorphosis by inhibiting swimming or crawling behaviour, in conjunction with a cascade of additional neuroendocrine downstream responses.

Insular cortex is a brain structure involved in the modulation of autonomic activity and cardiovascular function. The nitric oxide/cyclic guanosine-3',5'-monophosphate pathway is a prominent signaling mechanism in the central nervous system, controlling behavioral and physiological responses. Nevertheless, despite evidence regarding the presence of nitric oxide-synthesizing neurons in the insular cortex, its role in the control of autonomic and cardiovascular function has never been reported. Thus, the present study aimed to investigate the involvement of nitric oxide/cyclic guanosine-3',5'-monophosphate pathway mediated by neuronal nitric oxide synthase (nNOS) activation within the insular cortex in the modulation of baroreflex responses in unanesthetized rats. For this, we evaluated the effect of bilateral microinjection of either the nitric oxide scavenger carboxy-PTIO, the selective neuronal nitric oxide synthase inhibitor N ω -Propyl-l-arginine or the soluble guanylate cyclase inhibitor ODQ into the insular cortex on the bradycardia evoked by blood pressure increases in response to intravenous infusion of phenylephrine, and the tachycardia caused by blood pressure decreases evoked by intravenous infusion of sodium nitroprusside. Bilateral microinjection of either NPLA or carboxy-PTIO into the insular cortex increased the reflex bradycardic response, whereas the reflex tachycardia was decreased by these treatments. Bilateral microinjection of the soluble guanylate cyclase inhibitor into the insular cortex did not affect any parameter of baroreflex function evaluated. Overall, our findings provide evidence that insular cortex nitrergic signaling, acting via neuronal nitric oxide synthase, plays a prominent role in control of baroreflex function. However, control of reflex responses seems to be independent of soluble guanylate cyclase activation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Key Points: Preclinical models have demonstrated that nitric oxide is a key component of neurovascular coupling; this has yet to be translated to humans. We conducted two separate protocols utilizing intravenous infusion of a nitric oxide synthase inhibitor and isovolumic haemodilution to assess the influence of nitric oxide on neurovascular coupling in humans. Isovolumic haemodilution did not alter neurovascular coupling. Intravenous infusion of a nitric oxide synthase inhibitor reduced the neurovascular coupling response by ∼30%, indicating that nitric oxide is integral to neurovascular coupling in humans., Abstract: Nitric oxide is a vital neurovascular signalling molecule in preclinical models, yet the mechanisms underlying neurovascular coupling (NVC) in humans have yet to be elucidated. To investigate the contribution of nitric oxide to NVC in humans, we utilized a visual stimulus paradigm to elicit an NVC response in the posterior cerebral circulation. Two distinct mechanistic interventions were conducted on young healthy males: (1) NVC was assessed during intravenous infusion of saline (placebo) and the non-selective competitive nitric oxide synthase inhibitor N G -monomethyl-l-arginine (l-NMMA, 5 mg kg -1 bolus & subsequent 50 μg kg -1 min -1 maintenance dose; n = 10). The order of infusion was randomized, counterbalanced and single blinded. A subset of participants in this study (n = 4) underwent a separate intervention with phenylephrine infusion to independently consider the influence of blood pressure changes on NVC (0.1-0.6 μg kg -1 min -1 constant infusion). (2) NVC was assessed prior to and following isovolumic haemodilution, whereby 20% of whole blood was removed and replaced with 5% human serum albumin to reduce haemoglobin concentration (n = 8). For both protocols, arterial and internal jugular venous blood samples were collected at rest and coupled with volumetric measures of cerebral blood flow (duplex ultrasound) to quantify resting cerebral metabolic parameters. l-NMMA elicited a 30% reduction in the peak (P = 0.01), but not average (P = 0.11), NVC response. Neither phenylephrine nor haemodilution influenced NVC. Nitric oxide signalling is integral to NVC in humans, providing a new direction for research into pharmacological treatment of humans with dementia., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Introduction: Cardiovascular diseases are coupled to decreased nitric oxide (NO) bioavailability, and there is a constant search for novel and better NO-donors. Here we synthesized and characterized the cardiovascular effects of the new organic nitrate 2-nitrate-1,3-dioctanoxypropan (NDOP)., Methods: A combination of in vitro and in vivo experiments was performed in C57BL/6 mice and Wistar rats. Thus, the ability of NDOP in donating NO in a cell-free system and in vascular smooth muscles cells (VSMC) and its ability to induce vasorelaxation in aortic rings from mice were evaluated. In addition, changes in blood pressure and heart rate to different doses of NDOP were evaluated in conscious rats. Finally, acute pre-clinical toxicity to oral administration of NDOP was assessed in mice., Results: In cell-free system, NDOP increased NO levels, which was dependent on xanthine oxidoreductase (XOR). NDOP also increased NO levels in VSMC, which was not influenced by endothelial NO synthase. Furthermore, incubation with the XOR inhibitor febuxostat blunted the vasorelaxation in aortic ring preparations. In conscious rats, NDOP elicited dose-dependent reduction in blood pressure accompanied with increased heart rate. In vessel preparations, NDOP (10 -8 -10 -3  mol/L) induced endothelium-independent vasorelaxation, which was inhibited by the NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and hydroxocobalamin or by inhibition of soluble guanylyl cyclase using H- [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one. To investigate if NDOP acts through potassium channels, selective blockers were used. Inhibition of BK Ca , K v or K ATP subtypes of potassium channels had no effect, but inhibition of inward-rectifier potassium channels (K IR ) significantly reduced NDOP-mediated vasorelaxation. Lastly, NDOP showed low toxicity (LD 50 ~5000 mg/kg)., Conclusion: Bioactivation of NDOP involves functional XOR, and this new organic nitrate elicits vasorelaxation via NO-cGMP-PKG signaling and activation of K IR channels. Future studies should further characterize the underlying mechanism and evaluate the therapeutic benefits of chronic NDOP treatment in relevant cardiovascular disease models., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The adenosine (Ado) system may participate in regulation of kidney function in diabetes mellitus (DM), therefore we explored its role and interrelation with NO in the control of renal circulation and excretion in normoglycemic (NG) and streptozotocin-diabetic (DM) rats. Effects of theophylline (Theo), a non-selective Ado receptor antagonist, were examined in anaesthetized NG or in streptozotocin induced diabetic (DM) rats, untreated or after blockade of NO synthesis with l-NAME. We measured arterial blood pressure (MABP), whole kidney blood flow and renal regional flows: cortical and outer- and inner-medullary (IMBF), determined as laser-Doppler fluxes. Renal excretion of water, total solutes and sodium and in situ renal tissue NO signal (selective electrodes) were also determined. Theo experiments disclosed minor baseline vasoconstrictor and vasodilator tone in the kidney of NG and DM rats, respectively. NO blockade increased baseline MABP and decreased renal haemodynamics, similarly in NG and DM rats, indicating comparable vasodilator influence of NO in the two groups. Unexpectedly, in all rats with intact NO synthesis, Ado receptor blockade increased kidney tissue NO. In NO-deficient NG and DM rats, Ado receptor blockade induced comparable renal vasodilatation, suggesting similar vasoconstrictor influence of the Ado system. However, DM rats showed an unexplained association of decreased MABP and IMBF and increased NO signal. Higher baseline renal excretion in DM rats indicated inhibition of renal tubular reabsorption due to the prevalence of natriuretic A2 over antinatriuretic A1 receptors. In conclusion, the experiments provided new insights in functional interrelation of adenosine and NO in normoglycaemia and streptozotocin-diabetes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glutaric acidaemia type I (GA-I) is a cerebral organic disorder characterized by the accumulation of glutaric acid (GA) and seizures. As seizures are precipitated in children with GA-I and the mechanisms underlying this disorder are not well established, we decided to investigate the role of nitric oxide (NO) in GA-induced convulsive behaviour in pup rats. Pup male Wistar rats (18-day-old) were anesthetized and placed in stereotaxic apparatus for cannula insertion into the striatum for injection of GA. The experiments were performed 3 days after surgery (pup rats 21-day-old). An inhibitor of NO synthesis (N-G-nitro-l-arginine methyl ester-L-NAME, 40 mg/kg) or saline (vehicle) was administered intraperitoneally 30 min before the intrastriatal injection of GA (1 µl, 1.3 µmol/striatum) or saline. Immediately after the intrastriatal injections, the latency and duration of seizures were recorded for 20 min. The administration of L-NAME significantly increased the latency to the first seizure episode and reduced the duration of seizures induced by GA in pup rats. The administration of the NO precursor l-arginine (L-ARG; 80 mg/kg) prevented the effects of L-NAME. Besides, GA significantly increased nitrate and nitrite (NOx) levels in the striatum of pup rats and the preadministration of L-NAME prevented this alteration. L-ARG blocked the reduction of striatal NOx provoked by L-NAME. These results are experimental evidence that NO plays a role in the seizures induced by GA in pup rats, being valuable in understanding the physiopathology of neurological signs observed in children with this organic acidaemia and to develop new therapeutic strategies., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Objectives: To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy., Methods: In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100-300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10 -6 M), and subsequently dilated in acetylcholine (ACh, 10 -6 M - 10 -4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10 -4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10 -6 M) followed by ACh (10 -5 M). The Ach-induced vasodilation and NO release were recorded simultaneously., Results: ACh (10 -6 M - 10 -4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10 -4 M) significantly attenuated the vasodilatation and the intracellular NO release., Conclusions: This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

Repeated column chromatography of Syringa dilatata flowers, a native shrub to Korea, led to the isolation of eight new oleoside-type secoiridoids, syringoleosides A-H ( 1 - 8 ), as well as five known secoiridoids ( 9 - 13 ). The new chemical structures were identified through spectroscopic data analysis, as well as the application of chemical methods. Compounds 1 , 2 , 6 , 7 , 11 , and 13 showed suppression effects on NO production in LPS-induced RAW 264.7 cells, with IC 50 values ranging from 32.5 ± 9.8 to 65.7 ± 11.0 μ M, and no visible toxicity. The content of the major secoiridoids in S. dilatata flowers, compounds 1 , 4 , 5 , 8 , 9 , 12 , and 13 , were determined through HPLC analysis.

Aim: Empathy is defined as the capability to comprehend and simulate the feelings of others. Though it has been considered as a human feature, recent studies have demonstrated empathy-like behaviors in other animals including rats. The objective of the current study was to evaluate the role of nitric oxide system in cognition and nociception changes following observation of cagemates in pain. Material and methods: Adult male Wistar rats were used ( n  = 8 for each group). One sibling received formalin injection into the hindpaw five times within a nine-day period and the other sibling observed the pain while being pretreated with saline, L-NAME or L-arginine (10 mg/kg, i.p.). Nociception, anxiety-like behavior and locomotion, balance, muscle strength, spatial and fear learning were evaluated. Results: Observing a family member (sibling) in pain increased anxiety-like behavior, led to a hyperalgesia in the observer and disruption of spatial memory. Nitric oxide system modulated these changes, so that in some paradigms the activation of NO and in some others inhibition of NO dampened the effect of observing pain in a cagemate on the evaluated features. Conclusions: Results in the current study demonstrated a modulating effect of NO on empathy induced changes in nociception, motor function and spatial memory. Further studies addressing the specific brain regions and other neurotransmitters involved are recommended.

Background Basal release of nitric oxide (NO) from the vascular endothelium regulates the tone of muscular arteries and resistance vasculature. Effects of NO on muscular arteries could be particularly important during exercise when shear stress may stimulate increased NO synthesis. Methods and Results We investigated acute effects of NO synthase inhibition on exercise hemodynamics using N G -monomethyl-l-arginine (l-NMMA), a nonselective NO synthase -inhibitor. Healthy volunteers (n=10, 5 female, 19-33 years) participated in a 2-phase randomized crossover study, receiving l-NMMA (6 mg/kg, iv over 5 minutes) or placebo before bicycle exercise (25-150 W for 12 minutes). Blood pressure, cardiac output (measured by dilution of soluble and inert tracers) and femoral artery diameter were measured before, during, and after exercise. At rest, l-NMMA reduced heart rate (by 16.2±4.3 bpm relative to placebo, P <0.01), increased peripheral vascular resistance (by 7.0±1.4 mmHg per L/min, P <0.001), mean arterial blood pressure (by 8.9±3.5 mmHg, P <0.05), and blunted an increase in femoral artery diameter that occurred immediately before exercise (change in diameter: 0.14±0.04 versus 0.32±0.06 mm after l-NMMA and placebo, P <0.01). During/after exercise l-NMMA had no significant effect on peripheral resistance, cardiac output, or on femoral artery diameter. Conclusions These results suggest that NO plays little role in modulating muscular artery function during exercise but that it may mediate changes in muscular artery tone immediately before exercise.