Nitric Oxide Signaling Pathway Research Articles

Cell autonomous functions of CD47 in regulating cellular plasticity and metabolic plasticity.

CD47 is a ubiquitously expressed cell surface receptor, which is widely known for preventing macrophage-mediated phagocytosis by interacting with signal regulatory protein α (SIRPα) on the surface of macrophages. In addition to its role in phagocytosis, emerging studies have reported numerous noncanonical functions of CD47 that include regulation of various cellular processes such as proliferation, migration, apoptosis, differentiation, stress responses, and metabolism. Despite lacking an extensive cytoplasmic signaling domain, CD47 binds to several cytoplasmic proteins, particularly upon engaging with its secreted matricellular ligand, thrombospondin 1. Indeed, the regulatory functions of CD47 are greatly influenced by its interacting partners. These interactions are often cell- and context-specific, adding a further level of complexity. This review addresses the downstream cell-intrinsic signaling pathways regulated by CD47 in various cell types and environments. Some of the key pathways modulated by this receptor include the PI3K/AKT, MAPK/ERK, and nitric oxide signaling pathways, as well as those implicated in glucose, lipid, and mitochondrial metabolism. These pathways play vital roles in maintaining tissue homeostasis, highlighting the importance of understanding the phagocytosis-independent functions of CD47. Given that CD47 expression is dysregulated in a variety of cancers, improving our understanding of the cell-intrinsic signals regulated by this molecule will help advance the development of CD47-targeted therapies.

Involvement of ObRb receptor, nitric oxide, and BKCa channel signaling pathways in leptin-induced relaxation of pregnant mouse uterus

The purpose of this study was to determine the receptor subtype and the underlying mechanisms involved in the relaxant effect to leptin in mid- and late-pregnant mouse uterus. We determined the relative mRNA expression of receptor subtypes, eNOS, and BKCa channel by quantitative PCR and also the overall receptor expression by immunohistochemistry. Isometric tension studies were conducted to evaluate the effects of leptin and to delineate its mechanisms. A selective siRNA for the ObRb receptor was used to determine the participation of the receptor subtype in biochemical and molecular effects of leptin.The relaxant response to leptin was greater in mid-pregnancy compared to late pregnancy and was mediated by the activation of BKCa channels by eNOS-derived nitric oxide in an ObRb receptor-dependent manner. In comparison to mid-pregnancy, expression of short forms (mainly ObRa receptor) of the receptor was significantly increased in late pregnancy, whereas ObRb receptor expression was similar in both phases.The results of the study suggest that ObRb receptor mediates leptin-induced increase in eNOS expression and NO synthesis. Leptin-induced eNOS expression and activation cause cGMP-independent stimulation of BKCa channels causing uterine relaxation. Increased short forms of the receptors and reduced BKCa channels exert a negative effect on uterine relaxation in late pregnancy. Leptin may have a physiological role in maintaining uterine quiescence in mid-pregnancy and its reduced relaxant response in late gestation may facilitate labor. Further, ObRb receptor agonists may be useful in the management of preterm labor.

Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance.

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.

FABP4 as potential indicator for GDM-induced fetal endothelial dysfunction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG project no. 288034826—International Research Training Group (IRTG) 2251 Background Gestational diabetes mellitus (GDM) is linked to an elevated risk of developing cardiovascular diseases (CVD) both for the mother and the baby in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. FABP4 is known to promote an inflammatory response and vascular permeability, induce oxidative stress in endothelial cells (ECs), and impair the nitric oxide signaling pathway, thereby promoting endothelial dysfunction. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. However, the role that FABP4 plays in the fetal endothelial function remains poorly understood. Purpose Thus, our aim is to understand the role of FABP4 in fetal endothelial dysfunction and its regulation to mitigate the impact of GDM on the vasculature. Methods Fetoplacental vessels and primary human umbilical vein endothelial cells (HUVECs) were isolated from placentas and umbilical cords obtained from both normoglycemic (NG) and GDM pregnancies. We also obtained the clinical data from the mothers and collected the cord blood. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression, while gene expression analysis in the vessels was conducted using RT-qPCR. Additionally, NG HUVECs were pooled and cultured under high laminar flow conditions (30 dyn/cm²) or subjected to various treatments, including insulin (5nM), glucose (25mM), simvastatin (10nm), or VEGF-A stimulation. Subsequent RT-qPCRs were performed to analyze the gene expression of several genes of interest. Results The bulk RNA sequencing data analysis revealed FABP4 as an overexpressed gene in GDM HUVECs compared to NG. Furthermore, the analysis of data through Gene Set Enrichment Analysis provided a list of different gene sets that are enriched in GDM samples. Among these gene sets are pathways or processes implicated in endothelial dysfunction, such as angiogenesis, PI3K/AKT/mTOR signaling, reactive oxygen species pathway, or inflammatory response. In vitro experiments provided evidence for an elevated FABP4 mRNA expression after VEGF treatment of HUVECs. Conversely, both simvastatin treatment and exposure to laminar flow reduced FABP4 mRNA expression and increased eNOS expression, suggesting a potential inhibitory effect of nitric oxide on FABP4. Conclusion We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. Shear stress and statins might protect the endothelium against the negative impact of FABP4.

Anti-pruritic effect of L-carnitine against chloroquine-induced pruritus mediated via nitric oxide pathway

BackgroundPruritus, or itching, is a distressing symptom associated with various dermatological and systemic diseases. L-carnitine (βeta hydroxy-γ-tri methyl amino-butyric acid), is a naturally occurring substance, it controls numerous physiological processes. The present research aims to identify L-carnitine for its anti-pruritic effect via nitric oxide-dependent mechanism.MethodsChloroquine-induced pruritus serves as an experimental model to investigate possible therapeutic interventions. In this study, we evaluated the efficacy of L-carnitine in combating oxidative stress, nitric oxide, and inflammatory cytokines in a chloroquine-induced pruritus model.ResultsL-carnitine treatment significantly reduced scratching behavior compared to the disease group (***P < 0.001 vs. chloroquine group), indicating its antipruritic potential. The markers of oxidative stress, GST, GSH, Catalase, and LPO were dysregulated in the disease model, but administration of L-carnitine restored GST, GSH, and Catalase levels and decreased LPO levels (***P < 0.001 vs. chloroquine group), thereby alleviating oxidative stress. L-carnitine also reduced nitric oxide synthase (NOS) activity, suggesting that it modulates nitric oxide signaling pathways involved in pruritus. In addition, L-carnitine lowered levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), inflammatory marker nuclear factor kappa B (p-NFκB) and also reduces an inflammatory enzyme, cyclooxygenase-2 (COX-2), determined by ELISA (Enzyme-Linked Immunosorbent Assay) (***P < 0.001 vs. chloroquine group). It downregulates nNOS mRNA expression confirmed by real-time polymerase chain reaction (RT-PCR).ConclusionThese findings highlight the therapeutic effects of L-carnitine in alleviating chloroquine-induced pruritus.

RBC aggregation, deformation and adhesion to endothelium: Role of nitric oxide derived from L-Arginine and sodium nitroprusside

Red blood cells (RBCs) are the most abundant human blood cells. RBC aggregation and deformation strongly determine blood viscosity which impacts hemorheology and microcirculation. In turn, RBC properties depend on different endogenous and exogenous factors. One such factor is nitric oxide (NO), which is mainly produced by endothelial cells (EC) from L-arginine amino acid in the circulatory system. Since the mechanisms of the RBC-endothelium interplay are not clear up to date and considering its possible clinical importance, the aims of this study are to investigate in vitro: (1) The effect of L-arginine induced NO on RBC aggregation and adhesion to endothelium; (2) the NO effect on RBC aggregation and deformation induced by L-arginine and sodium nitroprusside without the presence of endothelium in the samples. The RBC aggregation and adhesion to a monolayer of EC were studied using optical tweezers (OT). The RBC deformability and aggregation without endothelium in the samples were studied using the flow chamber method and Myrenne aggregometer. We confirmed that NO increases deformability and decreases aggregation of RBCs. We showed that the soluble guanylate cyclase pathway appears to be the only NO signaling pathway involved. In the samples with the endothelium, the “bell-shaped” dependence of RBC aggregation force on L-arginine concentration was observed, which improves our knowledge about the process of NO production by endothelium. Additionally, data related to L-arginine accumulation by endothelium were obtained: Necessity of the presence of extracellular L-arginine stated by other authors was put under question. In our study, NO decreased the RBC-endothelium adhesion, however, the tendency appeared to be weak and was not confirmed in another set of experiments. To our knowledge, this is the first attempt to measure the forces of RBC adhesion to endothelium monolayer with OT.

Abstract 4454: Manipulation of the amino acid metabolism promotes therapeutic efficacy through targeting lysosome in glioblastoma

Abstract Nutrients are converted by the body to smaller molecules, which are utilized for both anabolic and catabolic reactions. Cooperative regulation of these metabolic processes is critical for maintaining tissue homeostasis. Based on these findings, our group has been focusing on how the regulation of nutrient-driven metabolism controls the malignancy of cancer cells. Metabolic regulation has been considered crucial for cellular adaptation to environmental changes and promoting survival and proliferation. Recent studies showed the successful applications of targeting specific nutrients for cancer therapy. In this study, we developed a new lysosome-targeting therapeutic strategy, centered around the manipulation of the amino acid metabolism for GBM. Using patient-derived GBM cells, we found that lysosomal proteolytic activity is a unique metabolic biomarker representing malignant phenotypes of GBM and that it is closely correlated with the efficacy of current conventional therapy for GBM patients. Furthermore, the MiT/TFE family members, TFEB and TFE3, which are the master regulators of lysosomal biogenesis, controlled malignant progression and therapeutic efficacy in vitro and vivo. Combinatory analyses with clinical patient samples and xenograft models demonstrated that the MiT/TFE family plays a crucial role as a regulator of malignant properties in GBM. Analysis of The Cancer Genome Atlas (TCGA) glioma dataset, which includes data from patients with Grade 2, 3, and 4 (GBM) gliomas, showed that higher expression of the MiT/TFE family was significantly associated with shorter survival. Interestingly, we discovered that an essential amino acid controls lysosomal biogenesis and membrane integrity by regulation of nitric oxide signaling pathway. Finally, we screened the potential anti-cancer drugs, including temozolomide, exhibiting the synergy effect in amino acid-restriction conditions by conducting a drug screening using a clinically available anti-cancer drug library. Our study demonstrated that lysosomal function, supported by amino acid metabolism, plays a critical role in the regulation of GBM malignant status and proposed precision nutrition as a promising therapeutic approach. Citation Format: Yongwei Jing, Masahiko Kobayashi, Atsushi Hirao. Manipulation of the amino acid metabolism promotes therapeutic efficacy through targeting lysosome in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4454.

Sodium nitroprusside enhances stepping test performance and increases medium spiny neurons responsiveness to cortical inputs in a rat model of Levodopa-induced dyskinesias.

Levodopa (L-DOPA) is the classical gold standard treatment for Parkinson's disease. However, its chronic administration can lead to the development of L-DOPA-induced dyskinesias (LIDs). Dysregulation of the nitric oxide-cyclic guanosine monophosphate pathway in striatal networks has been linked to deficits in corticostriatal transmission in LIDs. This study investigated the effects of the nitric oxide (NO) donor sodium nitroprusside (SNP) on behavioural and electrophysiological outcomes in sham-operated and 6-hydroxydopamine-lesioned rats chronically treated with vehicle or L-DOPA, respectively. In sham-operated animals, systemic administration of SNP increased the spike probability of putative striatal medium spiny neurons (MSNs) in response to electrical stimulation of the primary motor cortex. In 6-hydroxydopamine-lesioned animals, SNP improved the stepping test performance without exacerbating abnormal involuntary movements. Additionally, SNP significantly increased the responsiveness of putative striatal MSNs in the dyskinetic striatum. These findings highlight the critical role of the NO signalling pathway in facilitating the responsiveness of striatal MSNs in both the intact and dyskinetic striata. The study suggests that SNP has the potential to enhance L-DOPA's effects in the stepping test without exacerbating abnormal involuntary movements, thereby offering new possibilities for optimizing Parkinson's disease therapy. In conclusion, this study highlights the involvement of the NO signalling pathway in the pathophysiology of LIDs.

A Comprehensive Review of Inhaled Nitric Oxide Therapy: Current Trends, Challenges, and Future Directions.

This comprehensive review explores the multifaceted landscape of inhaled nitric oxide (iNO) therapy, tracing its historical evolution, mechanisms of action, clinical applications, challenges, and future directions. The nitric oxide signaling pathway, characterized by vasodilatory effects and anti-inflammatory properties, forms the foundation of iNO's therapeutic efficacy. Clinical applications are found in neonatal respiratory distress syndrome, pulmonary hypertension, and acute respiratory distress syndrome, showcasing its versatility. However, challenges, including cost considerations, technical intricacies, safety concerns, and resistance, highlight the nuanced landscape surrounding iNO therapy. Implications for clinical practice underscore the need for a tailored and evidence-based approach, considering individual patient characteristics and indications. Recommendations for future research emphasize ongoing exploration, novel indications, and the development of targeted therapies. In conclusion, this review positions iNO as a dynamic and adaptable intervention, poised to reshape therapeutic strategies and enhance patient outcomes in critical care.

Impact of intravascular hemolysis on functional and molecular alterations in the urinary bladder: implications for an overactive bladder in sickle cell disease.

Patients with sickle cell disease (SCD) display an overactive bladder (OAB). Intravascular hemolysis in SCD is associated with various severe SCD complications. However, no experimental studies have evaluated the effect of intravascular hemolysis on bladder function. This study aimed to assess the effects of intravascular hemolysis on the micturition process and the contractile mechanisms of the detrusor smooth muscle (DSM) in a mouse model with phenylhydrazine (PHZ)-induced hemolysis; furthermore, it aimed to investigate the role of intravascular hemolysis in the dysfunction of nitric oxide (NO) signaling and in increasing oxidative stress in the bladder. Mice underwent a void spot assay, and DSM contractions were evaluated in organ baths. The PHZ group exhibited increased urinary frequency and increased void volumes. DSM contractile responses to carbachol, KCl, α-β-methylene-ATP, and EFS were increased in the PHZ group. Protein expression of phosphorylated endothelial NO synthase (eNOS) (Ser-1177), phosphorylated neuronal NO synthase (nNOS) (Ser-1417), and phosphorylated vasodilator-stimulated phosphoprotein (VASP) (Ser-239) decreased in the bladder of the PHZ group. Protein expression of oxidative stress markers, NOX-2, 3-NT, and 4-HNE, increased in the bladder of the PHZ group. Our study shows that intravascular hemolysis promotes voiding dysfunction correlated with alterations in the NO signaling pathway in the bladder, as evidenced by reduced levels of p-eNOS (Ser-1177), nNOS (Ser-1417), and p-VASP (Ser-239). The study also showed that intravascular hemolysis increases oxidative stress in the bladder. Our study indicates that intravascular hemolysis promotes an OAB phenotype similar to those observed in patients and mice with SCD.

Homocysteine impairs the anticontractile/vasorelaxing activity of perivascular adipose tissue surrounding human internal mammary artery.

Perivascular adipose tissue (PVAT) surrounding human internal mammary artery (IMA) possesses anticontractile property. Its function under pathological conditions is barely studied. We previously reported that homocysteine impairs the vasodilator function of IMA through endothelium and smooth muscle-dependent mechanisms. This study investigated the effect of homocysteine on the function of PVAT and the associated mechanisms. Residual IMA tissues were collected from patients undergoing coronary artery bypass grafting. Vasoreactivity was studied using myograph. Adiponectin was measured by ELISA. Expressions of adiponectin receptors (AdipoRs), eNOS and p-eNOS were determined by RT-qPCR and Western blot. Exposure to homocysteine augmented the contractile responses of PVAT-intact IMA to U46619 and potassium chloride, regardless with or without endothelium. Such augmentation was also observed in skeletonized IMA with transferred, homocysteine-exposed PVAT. Homocysteine attenuated the relaxant response of PVAT-intact while endothelium-denuded vessels to acetylcholine. Homocysteine lowered adiponectin content in the PVAT, downregulated the expression of AdipoR1 and AdipoR2 as well as eNOS and p-eNOS in skeletonized IMA. The relaxant response of skeletonized IMA to AdipoR agonist AdipoRon was blunted by homocysteine or eNOS inhibitor, and homocysteine significantly attenuated the inhibitory effect of eNOS inhibitor on AdipoRon-induced relaxation. Homocysteine impairs the anticontractile/vasorelaxing activity of PVAT surrounding the IMA through inhibiting adiponectin/AdipoR/eNOS/nitric oxide signalling pathway.

Nitric oxide signaling pathways in the normal and pathological bladder: Do they provide new pharmacological pathways?-ICI-RS 2023.

The nitric oxide (NO•)/soluble guanylate cyclase/cyclic-GMP (cGMP) signaling pathway is ubiquitous and regulates several functions in physiological systems as diverse as the vascular, nervous, and renal systems. However, its roles in determining normal and abnormal lower urinary tract functions are unclear. The aim was to identify potential therapeutic targets associated with this pathway to manage lower urinary tract functional disorders. This review summarizes a workshop held under the auspices of ICI-RS with a view to address these questions. Four areas were addressed: NO• signaling to regulate neurotransmitter release to detrusor smooth muscle; its potential dual roles in alleviating and exacerbating inflammatory pathways; its ability to act as an antifibrotic mediator; and the control by nitrergic nerves of lower urinary tract vascular dynamics and the contractile performance of muscular regions of the bladder wall. Central to much of the discussion was the role of the NO• receptor, soluble guanylate cyclase (sGC) in regulating the generation of the enzyme product, the second messenger cGMP. The redox state of sGC is crucial in determining its enzymic activity and the role of a class of novel agents, sGC activators, to optimize activity and to potentially alleviate the consequences of lower urinary tract disorders was highlighted. In addition, the consequences of a functional relationship between nitrergic and sympathetic nerves to regulate vascular dynamics was discussed. Several potential NO•-dependent drug targets in the lower urinary tract were identified that provide the basis for future research and translation to clinical trials.

Brassinosteroid enhances salt tolerance via S-nitrosoglutathione reductase and nitric oxide signaling pathway in mangrove Kandelia obovata.

Brassinosteroid (BR) has been shown to modulate plant tolerance to various stresses. S-nitrosoglutathione reductase (GSNOR) is involved in the plant response to environment stress by fine-turning the level of nitric oxide (NO). However, whether GSNOR is involved in BR-regulated Na+ /K+ homeostasis to improve the salt tolerance in halophyte is unknown. Here, we firstly reported that high salinity increases the expression of BR-biosynthesis genes and the endogenous levels of BR in mangrove Kandelia obovata. Then, salt-induced BR triggers the activities and gene expressions of GSNOR and antioxidant enzymes, thereafter decrease the levels of malondialdehyde, hydrogen peroxide. Subsequently, BR-mediated GSNOR negatively regulates NO contributions to the reduction of reactive oxygen species generation and induction of the gene expression related to Na+ and K+ transport, leading to the decrease of Na+ /K+ ratio in the roots of K. obovata. Finally, the applications of exogenous BR, NO scavenger, BR biosynthetic inhibitor and GSNOR inhibitor further confirm the function of BR. Taken together, our result provides insight into the mechanism of BR in the response of mangrove K. obovata to high salinity via GSNOR and NO signaling pathway by reducing oxidative damage and modulating Na+ /K+ homeostasis.

Suppressive effect of nitric oxide synthase (NOS) inhibitor L-NMMA acetate on choroidal fibrosis in experimental myopic guinea pigs through the nitric oxide signaling pathway

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.

Biphasic inflammatory response induced by intra-plantar injection of L-cysteine: Role of CBS-derived H2S and S1P/NO signaling

This study investigates the inflammatory response to intra-plantar injection of L-cysteine in a murine model. L-cysteine induces a two-phase response: an early phase lasting 6 h and a late phase peaking at 24 h and declining by 192 h. The early phase shows increased neutrophil accumulation at 2 h up to 24 h, followed by a reduction at 48 h. On the other hand, the late phase exhibits increased macrophage infiltration peaking at 96 h. Inhibition of cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway, significantly reduces L-cysteine-induced edema, suggesting its dependence on CBS-derived hydrogen sulfide (H2S). Sequential formation of sphingosine-1-phosphate (S1P) preceding nitric oxide (NO) generation suggests the involvement of a CBS/S1P/NO axis in the inflammatory response. Inhibition of de novo sphingolipid biosynthesis, S1P1 receptor, and endothelial NO synthase (eNOS) attenuates L-cysteine-induced paw edema. These findings indicate a critical role of the CBS/H2S/S1P/NO signaling pathway in the development and maintenance of L-cysteine-induced inflammation. The co-presence of H2S and NO is necessary for inducing and sustaining the inflammatory response, as NaHS or L-arginine alone do not replicate the marked and prolonged inflammatory effect observed with L-cysteine. This study enhances our understanding of the complex molecular mechanisms of the interplay between NO and H2S pathways in inflammation and identifies potential therapeutic targets for inflammatory disorders.

Identification of a New Pyrrolyl Pyridoindole Alkaloid, Melpyrrole, and Flazin from Honey and Their Cough-Suppressing Effect in Guinea Pigs.

The cough-suppressing effect of honey was demonstrated for the first time using a guinea pig model whereby cough was induced by citric acid and capsaicin, and a new pyrrolyl pyridoindole, 1-(5-(hydroxymethyl)-1H-pyrrol-2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1), named melpyrrole, and flazin (2) were identified as the active principle components. The structures of 1 and 2 were estimated using a combination approach of an activity-guided survey and LC-MS/MS multivariate analysis and were finally established by total synthesis of 1 and comparison with an authentic standard for 2. Both compounds showed antitussive activity comparable to that of dextromethorphan in guinea pigs. Their antitussive effects were unaffected by an opioid antagonist and reversed by a nitric oxide (NO) synthase inhibitor, indicating that these natural products do not act directly on opiate receptors but through the NO signaling pathway.

Neuroprotective Effect of NO-Delivery Dinitrosyl Iron Complexes (DNICs) on Amyloid Pathology in the Alzheimer's Disease Cell Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment, memory loss, and behavioral deficits. β-amyloid1-42 (Aβ1-42) aggregation is a significant cause of the pathogenesis in AD. Despite the numerous types of research, the current treatment efficacy remains insufficient. Hence, a novel therapeutic strategy is required. Nitric oxide (NO) is a multifunctional gaseous molecule. NO displays a neuroprotective role in the central nervous system by inhibiting the Aβ aggregation and rescuing memory and learning deficit through the NO signaling pathway. Targeting the NO pathway might be a therapeutic option; however, NO has a limited half-life under the biological system. To address this issue, a biomimetic dinitrosyl iron complex [(NO)2Fe(μ-SCH2CH2COOH)2Fe(NO)2] (DNIC-COOH) that could stably deliver NO was explored in the current study. To determine whether DNIC-COOH exerts anti-AD efficacy, DNIC-COOH was added to neuron-like cells and primary cortical neurons along with Aβ1-42. This study found that DNIC-COOH protected neuronal cells from Aβ-induced cytotoxicity, potentiated neuronal functions, and facilitated Aβ1-42 degradation through the NO-sGC-cGMP-AKT-GSK3β-CREB/MMP-9 pathway.

Protection by Means of Perinatal Oral Sodium Thiosulfate Administration against Offspring Hypertension in a Rat Model of Maternal Chronic Kidney Disease.

Hydrogen sulfide (H2S) and related reactive sulfur species are implicated in chronic kidney disease (CKD) and hypertension. Offspring born to CKD-afflicted mothers could develop hypertension coinciding with disrupted H2S and nitric oxide (NO) signaling pathways as well as gut microbiota. Thiosulfate, a precursor of H2S and an antioxidant, has shown anti-hypertensive effects. This study aimed to investigate the protective effects of sodium thiosulfate (STS) in a rat model of maternal CKD-induced hypertension. Before mating, CKD was induced through feeding 0.5% adenine chow for 3 weeks. Mother rats were given a vehicle or STS at a dosage of 2 g/kg/day in drinking water throughout gestation and lactation. Perinatal STS treatment protected 12-week-old offspring from maternal CKD-primed hypertension. The beneficial effects of STS could partially be explained by the enhancement of both H2S and NO signaling pathways and alterations in gut microbiota. Not only increasing beneficial microbes but maternal STS treatment also mediates several hypertension-associated intestinal bacteria. In conclusion, perinatal treatment with STS improves maternal CKD-primed offspring hypertension, suggesting that early-life RSS-targeting interventions have potential preventive and therapeutic benefits, awaiting future translational research.

BIO18: Hemoglobin and Heme Scavenger Proteins Reduce Free Hemoglobin During Venovenous Extracorporeal Circulation and Preserves Microvascular Function.

Extracorporeal circulation procedures often utilize a blood pump to drive flow through an external circuit, which can cause hemolysis due to high shear conditions and foreign surface contact. The purpose of this study was to determine whether priming fluids containing proteins to sequester hemoglobin (Hb) and heme could be used in venovenous extracorporeal circulation (VV-ECC) to reduce their toxicity. A day prior to the experiment, male Golden Syrian hamsters were instrumented with a dorsal window chamber to monitor the microcirculation. The day of the experiment, both jugular veins were then cannulated under isoflurane anesthesia. Baseline (BL) microvascular oxygenation was measured using a hyperspectral camera. The animals were then heparinized (2.5 IU/g) and connected to the VV-ECC circuit, consisting of a peristaltic pump and bubble trap. The ECC circuit was primed with either 5% human serum albumin (HSA), ApoHb-Hp (62.01 mg/mL), or a protein solution containing hemopexin, transferrin, and Hp (protein cocktail, 84.50 mg/mL). Haptoglobin (Hp), hemopexin, and transferrin are native proteins that bind to Hb, heme, and iron respectively. Apohemoglobin (apoHb) is a synthetic scavenger produced by removing heme from Hb. ApoHb binds to haptoglobin (Hp) forming the apoHb-Hp complex that retains its ability to bind heme and scavenge free Hb. The experiment lasted a total of 2 hrs (Table 1), in which first the animals were hemodiluted, and the flow rate through the circuit was ramped up to 50% of the animal’s cardiac output. This max flow rate was then maintained for 35 minutes before being ramped down to 0.2 mLPM. The circuit was maintained at this flow rate for 15 minutes, allowing the animal and the circuit to equilibrate before the circuit flow was stopped. While mean arterial pressure (MAP, Figure 1a) showed differences at baseline (BL) between HSA and both other groups, there was no significance observed after hemodilution and the ramp up phase of the experiment. This difference in MAP between HSA and the two other solutions returned at all timepoints after the ramp up phase. Hb oxygen saturation (sO2, Figure 2) within the microvasculature showed a drop for both arterioles and venules in both the protein cocktail and ApoHb-Hp groups at the full VV-ECC timepoint. The plasma Hb (Figure 3) for the protein cocktail and ApoHb-Hp groups were significantly lower than the HSA control from the start of VV-ECC to the equilibration stage when compared to the HSA control. These results confirm that the ApoHb and protein cocktail solutions have the capability to bind and sequester free Hb, which has the potential to reduce the extravasation of Hb from the circulatory compartment into tissues, preserves the vascular nitric oxide (NO) signaling pathway, and reduce Hb and heme toxicity. Future work is needed to determine impact on organs, especially the kidneys, and to quantify the binding efficiency of these proteins to free hemoglobin.Figure 1. Hemodynamic parameters measured via the carotid artery: a) Mean arterial pressure (MAP) and b) heart rate (HR). bl = baseline, dil = hemodilution, up = ramp up, ecc = VV-ECC at max flow rate (50% of cardiac output), down = ramp down, equ = equilibration, off = off pump. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups. N = 6 for all groups.Figure 2. Changes in microvascular hemoglobin oxygen saturation. The top row shows arterioles, while the bottom row shows venules. Arterioles and venules were analyzed for 4 hamsters per group. bl = baseline, dil = hemodilution, up = ramp up, ecc = VV-ECC at max flow rate (50% of cardiac output), down = ramp down, equ = equilibration, off = off pump. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 between groups. † P<0.05 compared to baseline (BL).Figure 3. a) Hematocrit (Hct) and b) plasma hemoglobin (pHb). 0 = baseline (BL), 25 = hemodilution, 45 = VV-ECC (start), 75 = VV-ECC (end), 105 = equilibrate, 120 = off pump. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 between groups. † P<0.05 compared to BL. Table 1. - Experimental timeline highlight the blood flow through the circuit. Baseline Hemodilution Ramp Up VV-ECC Ramp Down Equilibrate Off Pump Duration 15 min 15 min 15 min 35 min 15 min 15 min 10 min Blood Flow 0 mLPM 0.2 mLPM 0.2 mLPM → 50% CO 50% CO 50% CO → 0.2 mLPM 0.2 mLPM 0 mLPM Hyperspectral images were taken every 5 minutes. mLPM = milliliters per minute, CO = cardiac output, VV-ECC = full venovenous extracorporeal circulation.

Acute and Sub-chronic Anticonvulsant Effects of Edaravone on Seizure Induced by Pentylenetetrazole or Electroshock in Mice, Nitric Oxide Involvement.

Edaravone is an anti-stroke medication that may have nitric oxide (NO) modulating properties. This study evaluated the role of NO in the acute and sub-chronic anticonvulsant effects of edaravone in murine models of seizures induced by intraperitoneal (IP) or intravenous (IV) injections of pentylenetetrazole (PTZ) or electroshock (maximal electroshock seizure [MES]). 132 male albino mice were randomly divided into 22 groups (n=6) and given IP injections of vehicle or edaravone either acutely or for eight days (sub-chronically). The seizure was induced by electroshock or PTZ (IP or IV). The following edaravone doses were used: 7.5, 10, 12.5 (acute); 5, 7.5, 10 (sub-chronic) in IP PTZ model; 5, 7.5, 10 in IV PTZ model; and 5, 10 mg/Kg in the MES. To evaluate NO involvement, 216 mice were randomly divided into 36 groups (n=6) and pretreated with vehicle, edaravone, a non-specific nitric oxide synthase (NOS) inhibitor: N(ω)-nitro-L-arginine methyl ester (L-NAME) (5 mg/Kg), a specific nNOS inhibitor: 7-nitroindazole (7-NI) (60 mg/Kg), or a combination of edaravone plus L-NAME or 7-NI, either acutely or for eight days before seizure induction. Doses of edaravone were as follows: in IP PTZ model: 12.5 (acute) and 10 (sub-chronic); in IV PTZ model: 10; and in the MES: 5 mg/Kg. Data were analyzed using the one-way analysis of variance (ANOVA) followed by Tukey's test (SPSS 18). P≤0.05 was considered statistically significant. In the IP PTZ model, edaravone increased time latencies to seizures (P<0.001), prevented tonic seizures, and death. Edaravone increased the seizure threshold (P<0.001) in the IV PTZ model and shortened the duration of tonic hind-limb extension (THE) in the MES model (P<0.001). In comparison to mice treated with edaravone alone, adding L-NAME or 7-NI reduced seizure time latencies (P<0.001), reduced seizure threshold (P<0.001), and increased THE duration (P<0.001). Edaravone (acute or sub-chronic) could prevent seizures by modulating NO signaling pathways.