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High-fat diet-induced metabolic changes are not restricted to the onset of cardiovascular diseases, but also include effects on brain functions related to learning and memory. This study aimed to evaluate mitochondrial markers and function, as well as cognitive function, in a rat model of metabolic dysfunction. Eight-week-old male Wistar rats were subjected to either a control diet or a two-hit protocol combining a high fat diet (HFD) with the nitric oxide synthase inhibitor L-NAME in the drinking water. HFD plus L-NAME induced obesity, hypertension, and increased serum cholesterol. These rats exhibited bioenergetic dysfunction in the hippocampus, characterized by decreased oxygen (O 2 ) consumption related to ATP production, with no changes in H 2 O 2 production. Furthermore, OPA1 protein expression was upregulated in the hippocampus of HFD + L-NAME rats, with no alterations in other morphology-related proteins. Consistently, HFD + L-NAME rats showed disruption of performance in the Morris Water Maze Reference Memory test. The neocortex did not exhibit either bioenergetic changes or alterations in H 2 O 2 production. Calcium uptake rate and retention capacity in the neocortex of HFD + L-NAME rats were not altered. Our results indicate that hippocampal mitochondrial bioenergetic function is disturbed in rats exposed to a HFD plus L-NAME, thus disrupting spatial learning, whereas neocortical function remains unaffected., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow-derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80 + ) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor - L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner., (© 2023. The Author(s).)

Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic ( Lepr db / Lepr db , referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

Cancer continues to be a leading cause of death worldwide, making the development of new treatment methods crucial in the fight against it. With cancer incidence rates increasing worldwide, ongoing research must focus on identifying new and effective ways to prevent and treat the disease. The combination of herbal extracts with chemotherapeutic agents has gained much interest as a novel strategy to combat cancer. Rumex obtusifolius L. is a wild plant known for its medicinal properties and is widely distributed worldwide. Our preclinical evaluations suggested that R. obtusifolius seed extracts possessed cancer-inhibiting properties and we also evaluated the beneficial effects of the arginase inhibitor NG-hydroxy-nor-L-arginine and nitric oxide inhibitor NG-nitro-L-arginine methyl ester in the treatment of breast cancer. The current study aimed to combine these observations and evaluate the antioxidant and antitumor properties of R. obtusifolius extracts alone and in combination with the arginase and nitric oxide synthase inhibitors. Metabolic characterization of the plant extract using a liquid chromatography/high-resolution mass spectrometry advanced system revealed the presence of 240 phenolic compounds many of which possess anticancer properties, according to the literature. In vitro studies revealed a significant cytotoxic effect of the R. obtusifolius extracts on the human colon (HT29) and breast cancer (MCF-7) cell lines. Thus, a new treatment approach of combining R. obtusifolius bioactive phytochemicals with the arginase and nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester and/or NG-hydroxy-nor-L-arginine, respectively, was proposed and could potentially be an effective way to treat breast cancer. Indeed, these combinations showed immunostimulatory, antiproliferative, antioxidant, anti-inflammatory, and antiangiogenic properties in a rat breast cancer model., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Hypertensive disorders complicating pregnancy (HDCP) are one of the most serious medical disorders during pregnancy., Objectives: To investigate the effects of hydrogen on the mitogen-activated protein kinase (MAPK) signaling pathway in preeclampsia (PE)., Material and Methods: The N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced PE model with Sprague Dawley (SD) rats was employed. An inhibitor of MAPK signaling pathways (SB203580) was used as a p38 MAPK inhibitor. The SD rats were randomized into 5 groups: non-pregnant (NP); normal pregnancy (P); pregnancy + L-NAME (L); pregnancy + L-NAME + hydrogen-rich saline (LH); and pregnancy + L-NAME + hydrogen-rich saline + SB203580 (LHS). The pregnancies were terminated on day 22 of gestation, and the placentas and kidneys were microscopically inspected. Tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and malondialdehyde (MDA) levels were assessed. The mean systolic blood pressure (SBP) and level of proteinuria were recorded. The p38 MAPK mRNA expression and p-p38 MAPK protein levels were measured using real-time polymerase chain reaction (RT-PCR) and western blot, respectively., Results: It was found that hydrogen-rich saline (LH group) decreased placental MDA, proteinuria, TNF-α, and IL-1β levels in the placental tissues compared with the L group (all p < 0.05). Additionally, hydrogen-rich saline (LH group) treatment significantly decreased the p38 MAPK mRNA expression and p-p38 MAPK protein levels compared with the L group (p < 0.05). The p38 MAPK inhibitor SB203580 (LHS group) further decreased the p38 MAPK mRNA expression and p-p38 MAPK protein levels compared with the LH group (p < 0.05)., Conclusions: Hydrogen can decrease the reactive oxygen species (ROS) content and inhibit the MAPK pathway. The protective effect of hydrogen may be associated with the inhibition of the p38 MAPK signaling pathway.

Focal segmental glomerulosclerosis (FSGS) is a major cause of end-stage renal disease and remains without specific treatment. To identify new events during FSGS progression, we used an experimental model of FSGS associated with nephroangiosclerosis in rats injected with L-NAME (N ω -nitro-L-arginine methyl ester). After transcriptomic analysis we focused our study on the role of Isthmin-1 (ISM1, an anti-angiogenic protein involved in endothelial cell apoptosis. We studied the renal expression of ISM1 in L-NAME rats and other models of proteinuria, particularly at the glomerular level. In the L-NAME model, withdrawal of the stimulus partially restored basal ISM1 levels, along with an improvement in renal function. In other four animal models of proteinuria, ISM1 was overexpressed and localized in podocytes while the renal function was degraded. Together these facts suggest that the glomerular expression of ISM1 correlates directly with the progression-recovery of the disease. Further in vitro experiments demonstrated that ISM1 co-localized with its receptors GRP78 and integrin αvβ5 on podocytes. Treatment of human podocytes with low doses of recombinant ISM1 decreased cell viability and induced caspase activation. Stronger ISM1 stimuli in podocytes dropped mitochondrial membrane potential and induced nuclear translocation of apoptosis-inducing factor (AIF). Our results suggest that ISM1 participates in the progression of glomerular diseases and promotes podocyte apoptosis in two different complementary ways: one caspase-dependent and one caspase-independent associated with mitochondrial destabilization.

Using dehydroabietic acid as the lead compound for structural modification, 25 dehydroabietic acid derivatives were synthesized. Among them, compound D1 not only showed the strongest relaxation effect on the aortic vascular ring in vitro (Emax = 99.5 ± 2.1%, EC 50  = 3.03 ± 0.96 µM), but also significantly reduced systolic and diastolic blood pressure in rats at a dose of 2.0 mg/kg in vivo. Next, the vascular protective effect of the best active D1 and its molecular mechanism were further investigated by HUVECs. The results showed that D1 induced endothelium-dependent diastole in the rat thoracic aorta in a concentration-dependent manner. Endothelium removal or aortic ring pretreatment with N G -nitro-l-arginine methylester (l-NAME), 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), and tetraethylammonium (TEA) significantly inhibited D1-induced relaxation. In addition, wortmannin, KT5823, triciribine, diltiazem, BaCl 2 , 4-aminopyridine, indomethacin, propranolol, and atropine attenuated D1-induced vasorelaxation. D1 increased the phosphorylation of eNOS in HUVECs Furthermore, D1 attenuated the expression of TNF-α-induced cell adhesion molecules such as ICAM-1 and VCAM-1. However, this effect was attenuated by the eNOS inhibitors l-NAME and asymmetric dimethylarginine (ADMA). The findings suggest that D1-induced vasorelaxation through the PI3K/Akt/eNOS/NO/cGMP/PKG pathway by activating the K Ca , K ir and K V channels or muscarinic and β-adrenergic receptors, and inhibiting the l-type Ca 2+ channels, which is closely related to the hypotensive action of the agent. Furthermore, D1 exhibits an inhibitory effect on vascular inflammation, which is associated with the observed vascular protective effects., 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 Inc. All rights reserved.)

Previous work has shown that dietary L-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400 µM) or 400 µM Arg + 0.5 mM N G -nitro-L-arginine methyl ester (L-NAME; an NOS inhibitor) for 48 h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P < 0.05) the oxidation of glucose and oleic acid to CO 2 in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400 µM also stimulated (P < 0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P < 0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P < 0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P > 0.05) on these variables in C2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

As a multifunctional phytohormone, melatonin (Mel) plays pivotal roles in plant responses to multiple stresses. However, its mechanism of action remains elusive. In the present study, we evaluated the role of NO and Ca 2+ signaling in Mel enhanced cold tolerance in winter turnip rape. The results showed that the NO content and concentration of intracellular free Ca 2+ ([Ca 2+ ] cyt ) increased by 35.42% and 30.87%, respectively, in the leaves of rape seedlings exposed to cold stress. Compared with those of the seedlings in cold stress alone, the NO content and concentration of [Ca 2+ ] cyt in rape seedlings pretreated with Mel increased further. In addition, the Mel-mediated improvement of cold tolerance was inhibited by L-NAME (a NO synthase inhibitor), tungstate (a nitrate reductase inhibitor), LaCl 3 (a Ca 2+ channel blocker), and EGTA (a Ca 2+ chelator), and this finding was mainly reflected in the increase in ROS content and the decrease in osmoregulatory capacity, photosynthetic efficiency and antioxidant enzyme activities, and expression levels of antioxidant enzyme genes. These findings suggest that NO and Ca 2+ are necessary for Mel to improve cold tolerance and function synergistically downstream of Mel. Notably, the co-treatment of Mel with L-NAME, tungstate, LaCl 3 , or EGTA also inhibited the Mel-induced expression of MAPK3/6 under cold stress. In conclusion, NO and Ca 2+ are involved in the enhancement of cold tolerance induced by Mel through activating the MAPK cascades in rape seedlings, and a crosstalk may exist between NO and Ca 2+ signaling., 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 Masson SAS. All rights reserved.)

Arterial hypertension (AH) leads to oxidative and inflammatory imbalance that contribute to fibrosis development in many target organs. Here, we aimed to highlight the harmful effects of severe AH in the cornea. Our experimental model was established by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) to C57BL/6 mice, which were monitored weekly for arterial blood pressure and intraocular pressure (IOP). Morphological studies of ocular tissues were accompanied by analyses of reactive oxygen species generation, and localization/expression of NAPDH oxidase isoforms (NOX1, NOX2, NOX4) and inflammatory biomarkers (PPARα, PPARγ, IL-1β, IL-6, IL-10, TNF-α, and COX-2). Masson's trichrome and Sirius Red staining were used to explore the fibrotic status of the cornea. The expression of collagen isoforms (COL1α1, COL1α2, COL3α1, COL4α1, COL4α2) and relevant metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were also quantified to evaluate the participation of collagen metabolism in AH-related corneal damage. Hypertensive animals showed an increase in IOP values, and a thinner cornea compared with normotensive controls. Moreover, AH increased NADPH oxidase activity and reactive oxygen species generation in the cornea, which was accompanied by transcriptional upregulation of NOX isoforms and inflammatory biomarkers, while reducing PPAR expression. L-NAME-treated animals also developed corneal fibrosis with overexpression of collagen isoforms and reduction of factors responsible for collagen degradation. This is the first study reporting structural changes in the cornea and elevated IOP in L-NAME-treated mice. Overexpression of the NADPH oxidase system and collagen deposition might play a substantial role in the pathogenic mechanisms contributing to ocular disturbances in a context of severe hypertension., (© 2022. The Author(s).)

Dibutyl phthalate (DBP) is an endocrine disruptor that has been widely used in various products of human use. DBP exposure has been associated with reproductive and cardiovascular diseases and metabolic disorders. Although dysfunction of the vascular endothelium is responsible for many cardiovascular and metabolic diseases, little is known about the effects of DBP on human endothelium. In this study, we investigated the effect of three concentrations of DBP (10 -6 , 10 -5 , and 10 -4  M) on angiogenesis in human endothelial cell (EC) line EA.hy926 after acute exposure. Tube formation assay was used to investigate in vitro angiogenesis, whereas qRT-PCR was employed to measure mRNA expression. The effect of DBP on extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt), and endothelial nitric oxide (NO) synthase (eNOS) activation was examined using Western blotting, whereas the Griess method was used to assess NO production. Results show that the 24-h-long exposure to 10 -4  M DBP increased endothelial tube formation, which was prevented by addition of U0126 (ERK1/2 inhibitor), wortmannin (PI3K-Akt inhibitor), and l-NAME (NOS inhibitor). Short exposure to 10 -4  M DBP (from 15 to 120 min) phosphorylated ERK1/2, Akt, and eNOS in different time points and increased NO production after 24 and 48 h of exposure. Application of nuclear estrogen receptor (ER) and G protein-coupled ER (GPER) inhibitors ICI 182,780 and G-15, respectively, abolished the DBP-mediated ERK1/2, Akt, and eNOS phosphorylation and increase in NO production. In this study, we report for the first time that DBP exerts a pro-angiogenic effect on human vascular ECs and describe the molecular mechanism involving ER- and GPER-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways., 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 B.V. All rights reserved.)

Background: The main cause of death among patients with malignant hypertension is a kidney failure. The promising field in essential and malignant hypertension therapy could be centered on the amelioration of oxidative stress using antioxidant molecules like resveratrol. Resveratrol is a potent antioxidative agent naturally occurred in many plants that possess health-promoting properties., Methods: In the present study, we investigated the therapeutic potential of resveratrol, a polyphenol with anti-oxidative activity, in N G -L-Arginine Methyl Ester (L-NAME) treated spontaneously hypertensive rats (SHR) - malignantly hypertensive rats (MHR)., Results: Resveratrol significantly improves oxidative damages by modulation of antioxidant enzymes and suppression of prooxidant factors in the kidney tissue of MHR. Enhanced antioxidant defense in the kidney improves renal function and ameliorates the morphological changes in this target organ. Besides, protective properties of resveratrol are followed by the restoration of the nitrogen oxide (NO) pathway. 4) Conclusion: Antioxidant therapy with resveratrol could represent promising therapeutical approach in hypertension, especially malignant, against kidney damage., Competing Interests: Conflict of interest statement None., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

This research aimed to explore the effects of the nitric oxide synthase (NOS) inhibitor (L-NAME) on mitochondria apoptosis in postmortem Gannan yak (Bos grunniens) longissimus dorsi (LD) muscle and to explore its effect on meat quality further. The Gannan yak meat samples were treated with the control group (0.9% NaCl) and L-NAME (20, 60, and 100 mM) for 24 h and then stored for 0, 1, 3, 5, and 7 days at 4°C. NOS activity and NO content were investigated, and the parameters of mitochondrial apoptosis of the postmortem Gannan yak meat were determined. Meanwhile, the meat quality such as the centrifugation loss, meat color, and myofibril fragmentation index (MFI) was evaluated. The results indicated that after treatment with L-NAME, NOS activity and NO content decreased, causing mitochondrial membrane damage, Bax protein, and Cyt-c levels increased, and resulted in increased activities of caspase-9 and -3, promoting the occurrence of mitochondrial apoptosis. Furthermore, it increased the tenderness and water retention of Gannan yak meat. The results indicated that NOS inhibitor played a regulatory role in postmortem Gannan yak meat quality by regulating mitochondria apoptosis during postmortem aging. PRACTICAL APPLICATIONS: The meat's tenderness is often considered the most important factor affecting consumers' willingness to repurchase. The relationship of caspases and MFI suggested that L-NAME played a regulatory role in postmortem Gannan yak meat quality by regulating mitochondria apoptosis during postmortem aging. This study provides valuable information for the development of the Gannan yak economy in Tibetan areas., (© 2022 Wiley Periodicals LLC.)

Background: Chronic inflammation and oxidative stress play important role in development of hypertension. Recently, we have reported novel fluorophenyl benzimidazole (FPD) for vasorelaxation and antihypertensive activity in SHRs. The present study envisaged the anti-inflammatory, anti-oxidant and cardio-protective properties of FPD in L-NAME model of hypertension with special emphasis on reversal of vascular remodeling, gene expression and restoration of hemodynamic., Methods: Antihypertensive activity of FPD was evaluated in L-NAME treated Wistar rats, and the parameters studied were anti-inflammatory activity, histomorphological changes, gene expression profile and anti-oxidant properties., Results: FPD at 50 and 100 mg kg -1 once daily for 15 days significantly reduced SBP, DBP and MAP in L-NAME treated rats and the values were well comparable to vehicle control group. Further, FPD treatment showed a significant increase in hepatic GSH content, SOD, catalase activity, decreased MDA level and restoration of pro and anti-inflammatory cytokine levels. The mRNA expression profile of genes associated with regulation of vascular tone, remodeling and inflammation showed a significant level of alteration by chronic L-NAME treatment and was dose-dependently restored upon treatment with FPD. Further, FPD treatment restored serum lipid profile, CK, CK-MB and LDH level and also reversed the histomorphological changes like intimal wall thickening, hyperplasia of cardiomyocytes and ventricular wall thickening., Conclusions: Taken together, FPD produced potent antihypertensive activity in L-NAME model through vasorelaxation, anti-oxidative and anti-inflammatory properties leading to restoration of serum lipid profile, cardiac biomarker, expression profile of target genes and reversal of histomorphological changes., (Copyright © 2022. Published by Elsevier B.V.)

Preeclampsia affects ∼2-8% of pregnancies worldwide. It is associated with increased long-term maternal cardiovascular disease risk. This study assesses the effect of the vasoconstrictor N(ω)-nitro-L-arginine methyl ester (L-NAME) in modelling preeclampsia in mice, and its long-term effects on maternal cardiovascular health. In this study, we found that L-NAME administration mimicked key characteristics of preeclampsia, including elevated blood pressure, impaired fetal and placental growth, and increased circulating endothelin-1 (vasoconstrictor), soluble fms-like tyrosine kinase-1 (anti-angiogenic factor), and C-reactive protein (inflammatory marker). Post-delivery, mice that received L-NAME in pregnancy recovered, with no discernible changes in measured cardiovascular indices at 1-, 2-, and 4-wk post-delivery, compared with matched controls. At 10-wk post-delivery, arteries collected from the L-NAME mice constricted significantly more to phenylephrine than controls. In addition, these mice had increased kidney Mmp9:Timp1 and heart Tnf mRNA expression, indicating increased inflammation. These findings suggest that though administration of L-NAME in mice certainly models key characteristics of preeclampsia during pregnancy, it does not appear to model the adverse increase in cardiovascular disease risk seen in individuals after preeclampsia., (© 2022 de Alwis et al.)

Background: Non-alcoholic steatohepatitis (NASH) is a progressive subtype of non-alcoholic fatty liver disease (NAFLD) that is closely related to cardiovascular disease (CVD). Nitric oxide (NO) plays a critical role in the control of various biological processes. Dysfunction of the NO signaling pathway is associated with various diseases such as atherosclerosis, vascular inflammatory disease, and diabetes. Recently, it has been reported that NO is related to lipid and cholesterol metabolism. Chronic NO synthase (NOS) inhibition accelerates NAFLD by increasing hepatic lipid deposition. However, the detailed relationship between NO and abnormal lipid and cholesterol metabolism in NAFLD/NASH has not been completely explained. We aimed to determine the effects of NOS inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), a NOS inhibitor, on NASH and CVD via lipid and cholesterol metabolism., Methods: Stroke-prone spontaneously hypertensive rats were fed a high-fat and high-cholesterol diet for 8 weeks and administered L-NAME for the last 2 weeks. Following blood and tissue sampling, biochemical analysis, histopathological staining, quantitative RT-PCR analysis, and western blotting were performed., Results: L-NAME markedly increased hepatic triglyceride (TG) and cholesterol levels by promoting TG synthesis and cholesterol absorption from the diet. L-NAME increased the mRNA levels of inflammatory markers and fibrotic areas in the liver. Cholesterol secretion from the liver was promoted in rats administered L-NAME, which increased serum cholesterol. L-NAME significantly increased the level of oxidative stress marker and lipid deposition in the arteries., Conclusions: NOS inhibition simultaneously aggravates NASH and atherosclerosis via hepatic lipid and cholesterol metabolism., (© 2022. The Author(s) under exclusive licence to Maj Institute of Pharmacology Polish Academy of Sciences.)

Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5-7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5-7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4-5), increased necrosis score (P < 0.05, n = 4-5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4-5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Introduction: Preeclampsia (PE) is associated with abnormal placental vascular structure. However, the volume density of fetoplacental vessels in PE remains unclear. Additionally, manually annotated CT angiography, which is widely used to analyze placental vessels, has issues regarding inaccuracy. Thus, computer-aided CT angiography for analyzing the volume density of fetoplacental vessels would facilitate the understanding of PE pathogenesis., Methods: We performed computer-aided CT angiography to compare differences in placentas among 17 women with PE and 34 normotensive women. The contrast ratio in CT angiography was significantly enhanced using a three-dimensional (3-D) Hessian matrix algorithm. The PE-like mouse model was established by administration of 125 mg/kg/day NG-nitro-l-arginine methyl ester (l-NAME) for 10 days. The presence of endothelial marker CD31 was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The expression of angiogenic factors (PlGF, VEGFA, and sFlt1) in placentas was detected using qRT-PCR and western blotting., Results: The volume density in fetoplacental vessels and CD31 expression were significantly reduced in women with PE and l-NAME-induced mice. Additionally, the downregulation of angiogenic factors (PlGF/VEGFA) and upregulation of an anti-angiogenic factor (sFlt1) were determined in a mouse model., Discussion: Contrast-enhanced CT angiography with the aid of a 3-D Hessian matrix algorithm was performed. PE significantly affects the formation of vascular vessels, resulting in a lower volume density of fetoplacental vessels in humans and mice. This may be explained by the abnormal release of angiogenic factors during PE., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Nitric oxide (NO) levels in brain nuclei, such as the hippocampus and brainstem, are involved in morphine analgesia, but the relationship between the dorsal hippocampus (dH) and the dorsolateral periaqueductal gray matter (dlPAG) needs to be clarified, which is our goal., Methods: Wistar rats were simultaneously equipped with a stereotaxic device with unilateral guide cannula at dH and dlPAG. After recovery, they were divided into control and experimental groups. Formalin (50 μl of 2.5%) was inoculated into the left hind paw of the rat. Morphine (6 mg/kg) was administered intraperitoneally (i.p.) 10 min before formalin injection. L-Arginine (0.25, 0.5, 1 and 2 μg/rat), and L-NAME (0.25, 0.5, 1 and 2 μg/rat), unrelatedly or with respect in the order of injection were used in the nuclei before morphine injection (i.p.). Activation of the neuronal NO synthase (nNOS) in the brains of all animals was measured using NADPH-diaphorase, a selective biochemical marker of nNOS., Results: Morphine reduced inflammatory pain in the early and late stages of the rat formalin test. The morphine response was attenuated before injection of single L-arginine but not L-NAME in the two target areas. However, the acute phase result was stopped due to L-NAME pretreatment. When L-NAME was injected into dlPAG before injecting L-arginine at dH, the morphine response did not decrease at all, indicating a modulatory role of NO in dlPAG, which was confirmed by NADPH-d staining., Conclusions: High levels of NO in dlPAG may regulate the pain process in downward synaptic interactions., Significance: Nitric oxide is involved in the dH and dlPAG in morphine-induced analgesia in the rat formalin test. Morphine has analgesic effects in both phases of the rat formalin test. The morphine response is reduced in two stages by injection of the NO precursor L-arginine but not the nNOS inhibitor L-NAME in the dH and dlPAG. By injecting L-NAME before L-arginine in both nuclei, the morphine-induced response returns in the early stages. Due to the initial injection of L-NAME into dlPAG and the subsequent injection of L-arginine at dH, morphine analgesia is not reduced at all, indicating NO modulation in the pain pathway from dH to dlPAG., (© 2022 European Pain Federation - EFIC®.)

Praeruptorin A (PA) is a natural coumarin compound from the roots of Radix Peucedani and is commonly used in the treatment of certain respiratory diseases and hypertension. Although previous studies identified relaxant effects of PA on tracheal and arterial preparations, little is known about its vasodilative effects and underlying mechanisms. Here, an organ bath system and tension recording methods were used to prepare and analyze isolated rat thoracic aorta artery rings. Aorta artery rings were pre-contracted with phenylephrine and then incubated with PA, and the possible mechanism of relaxation was investigated by adding inhibitors of nitric oxide synthase (NG-nitro-L-arginine methyl ester, L-NAME), endothelial nitric oxide synthase (L-NG-nitroarginine, L-NNA), cyclooxygenase (indomethacin), guanylyl cyclase (1 H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one, ODQ), and KCa channels (tetraethylammonium, TEA). Our study showed that PA-induced vasodilation was blocked by L-NAME, L-NNA, and ODQ, while CaCl 2 -induced vasoconstriction was countered by PA. Thus, PA may exert a vasodilatory effect by influencing the amounts of endothelium-derived relaxing factors through endothelial-dependent NO-cGMP and prostacyclin pathways (such as NO and prostacyclin 2). In the rat thoracic aorta, PA reduces vasoconstriction by inhibiting Ca 2+ inflow.

We previously reported that cytoprotective Heme oxygenase-1, HO-1 (HMOX1) gene-modified human placenta-derived mesenchymal stem cell (HO-1-PMSC) improved placental vascularization in vitro. In the current study, we explored the protective benefit of HO-1-PMSC transplantation in a preeclampsia (PE)-like rat model. A model of PE was successfully constructed by intraperitoneal injection of N-nitro-L-arginine methyl ester (L-NAME). Blood pressure and urinary protein levels were measured. Doppler ultrasound was examined to understand uteroplacental perfusion. ELISA was used to examine the serum levels of VEGF, PlGF, sFlt-1, and sEng. The placentas and fetuses were weighed to verify the improvement in pregnancy outcome. Immunohistochemical and H&E staining was used to detect microvessel density (MVD) in placental tissues and kidney pathology, respectively. The distribution of GFP-labeled PMSC in the placenta were observed under fluorescence microscopy. Blood pressure and proteinuria were reduced and kidney damage was improved. PE rat models treated with PMSC and HO-1-PMSC exhibited an increase in the quality of fetuses and placentas, MVD, VEGF, and PlGF expression, but substantially decreased expression of sFlt-1 and sEng. Doppler ultrasound showed that the placental perfusion was improved. Green fluorescent tracing experiments verified that the cells were successfully transplanted into the placenta and distributed in the blood vessels, indicating that the cells might participate in the process of angiogenesis. These results indicate that therapy with HO-1-PMSC could improve placental vascular dysplasia, increase placental perfusion, control PE symptoms, and promote pregnancy outcome by regulating the balance of angiogenic and antiangiogenic factors or directly participating in the repair of placental vessels in a PE-like rat model.

Background: Although hypothyroidism during pregnancy may develop grave outcomes for both mothers and offspring, management of which is still a challenge due to the insufficient understanding of this disease. The close correlation between hypothyroidism and preeclampsia is well documented, suggesting that preeclampsia is a potential risk factor for the development of maternal hypothyroidism. However, the exact role of preeclampsia in gestational hypothyroidism is still obscure., Objective: In this study, we explored the possible mechanisms of the effect of preeclampsia on thyroid function of maternal rats., Methods: Thirty pregnant rats were randomly divided into normal pregnancy control (NOP), preeclampsia (PE), and preeclampsia supplemented with amlodipine besylate (PEAml). NG-Nitro-L-arginine-methyl ester was used to induce preeclamptic symptoms. On gestational day 21, rats were sacrificed, and then, the ultrastructure of the thyroid gland, type 1 iodothyronine deiodinase (Dio1) expression, and serum-free thyroxine (FT 4 ), free triiodothyronine (FT 3 ), and thyroid stimulation hormones (TSH) were assessed., Results: Compared to NOP rats, results of PE rats showed that thyroid follicular cells' ultrastructure was damaged; both hepatic Dio1 mRNA and protein levels were decreased. Interestingly, these changes were ameliorated in PEAml rats. Additionally, FT 4 , FT 3 , and TSH levels have no significant differences among groups., Conclusion: These findings indicated that preeclampsia could disrupt synthesis, secretion, and metabolism function of thyroid hormones by damaging thyroid follicular cells and interfering Dio1 expression., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Yunlu Liu et al.)

Anti-oxidant and anti-inflammatory properties of caffeic acid (CA) have been reported recently. In this study, the therapeutic effects of CA on ethanol-induced ulcer and the roles of nitric oxide and cholinergic pathways in these effects were investigated. Ulcer was induced by ethanol via oral gavage. Ulcer induced rats were treated with either vehicle (ulcer group) or CA (100, 250 or 500 mg/kg, per oral gavage). Macroscopic evaluation showed that 250 mg/kg CA was the effective dose. To elucidate the action mechanism of CA, 10 mg/kg l-NAME or 1 mg/kg atropine sulfate was administered to 250 mg/kg CA treated groups. All rats were decapitated 1 h after ulcer induction and gastric samples were scored macroscopically and microscopically, and analyzed for myeloperoxidase (MPO), malondialdehyde (MDA), and glutathione (GSH) levels. ANOVA test was used for statistical analyses. Macroscopic and microscopic damage scores, MDA levels and MPO activity were increased while GSH levels were decreased in ulcer group. Treatment with 250 mg/kg and 500 mg/kg CA reduced macroscopic and microscopic damage scores, decreased MPO activity and MDA levels, and preserved the depleted glutathione significantly. l-NAME administration before CA treatment elevated MDA levels, MPO activity and depleted glutathione. However, atropine sulfate had no effect on biochemical parameters. We conclude that CA ameliorates ethanol-induced gastric mucosal damage, and NO pathway contributes to this effect. On the other hand, there is a lack of evidence for the contribution of the muscarinic cholinergic system., (Copyright © 2020 Elsevier B.V. All rights reserved.)

New Findings: What is the central question of this study? What mechanisms account for the hypotension observed during chronic elevations in circulating 5-hydroxytryptamine in rats? What is the main finding and its importance? Chronic 5-hydroxytryptamine-induced hypotension requires continued activation of the 5-HT 7 receptor subtype but does not require NO, an outcome that resolves previous conflicting results. Therapeutic interruption of the hypotensive actions of 5-HT under pathophysiological conditions can only be achieved through blockade of the 5-HT 7 receptor., Abstract: Low dose infusion of 5-hydroxytryptamine (5-HT) to rats causes both an acute and a chronic fall in arterial blood pressure. The 5-HT 7 receptor subtype plays a critical part in the observed hypotension. Acute (minutes to hours) 5-HT infusion shows no depressor role for nitric oxide (NO), but 5-HT depressor responses under chronic conditions suggest that NO production may be critical. We test the hypothesis that NO contributes to the chronic, but not the acute, depressor response to 5-HT. We compared the role of NO and 5-HT 7 receptors in 5-HT-induced hypotension under acute and chronic conditions in the same animal. Mean arterial pressure and heart rate were measured by radiotelemetry in conscious rats during 5 days of saline or 5-HT (25 μg kg -1  min -1 ; osmotic pump) infusion and for 2 days after infusion was stopped. To quantify the contributions of NO and the 5-HT 7 receptor to 5-HT-induced hypotension, the nitric oxide synthase (NOS) inhibitor l-NAME or the selective 5-HT 7 receptor antagonist SB-267790 were given at 1, 3 and 5 days of chronic infusion, and 1 day after 5-HT infusion pumps were removed. N ω -Nitro-l-arginine methyl ester (l-NAME) caused a pressor response of the same magnitude in the absence or presence of 5-HT infusion. Conversely, SB-269970 did not affect mean arterial pressure in the absence of 5-HT infusion and reversed the 5-HT-induced depressor response at each time point. Our findings demonstrate that acute and chronic 5-HT-induced hypotension does not require NOS activation but does require continued activation of the 5-HT 7 receptor., (© 2020 The Authors. Experimental Physiology © 2020 The Physiological Society.)

We characterized modes of action of NO-donor S-nitrosoglutathione (GSNO) and NO-synthase inhibitor l-NAME derived from dicrotic (DiN) and anacrotic (AnN) notches of rat arterial pulse waveform (APW) in the condition of increased/decreased NO bioavailability. The cross-relationship patterns of DiN and AnN with 34 hemodynamic parameters (HPs) induced by GSNO and l-NAME are presented. After GSNO bolus administration, approximate non-hysteresis relationships were observed in the difference between DiN-AnN (mmHg) blood pressure (BP) and other 19 HPs, suggesting that these HPs, i.e., their signaling pathways, responding to NO concentration, are directly connected. Hysteresis relationships were observed between DiN-AnN (mmHg) and other 14 HPs, suggesting that signaling pathways of these HPs are indirectly connected. The hysteresis relationships were only observed between the time interval DiN-AnN (ms) and other 34 HPs, indicating no direct connection of signaling pathways. The cross-relationship patterns of DiN-AnN (mmHg), but not DiN-AnN (ms), induced by l-NAME were in accordance to the increased NO bioavailability induced by GSNO. In conclusion, we found the non-hysteresis/hysteresis cross-relationship "patterns" of DiN-AnN intervals to other HPs in the presence of GSNO that revealed their direct or indirect signaling pathways connections. This may contribute to our understanding of biological effects of natural substances that modulate NO production and/or NO signaling pathways.

Prostaglandin E2 and its cognate EP1-4 receptors play important roles in blood pressure (BP) regulation. Herein, we show that endothelial cell-specific (EC-specific) EP4 gene-knockout mice (EC-EP4-/-) exhibited elevated, while EC-specific EP4-overexpression mice (EC-hEP4OE) displayed reduced, BP levels compared with the control mice under both basal and high-salt diet-fed conditions. The altered BP was completely abolished by treatment with l-NG-nitro-l-arginine methyl ester (l-NAME), a competitive inhibitor of endothelial nitric oxide synthase (eNOS). The mesenteric arteries of the EC-EP4-/- mice showed increased vasoconstrictive response to angiotensin II and reduced vasorelaxant response to acetylcholine, both of which were eliminated by l-NAME. Furthermore, EP4 activation significantly reduced BP levels in hypertensive rats. Mechanistically, EP4 deletion markedly decreased NO contents in blood vessels via reducing eNOS phosphorylation at Ser1177. EP4 enhanced NO production mainly through the AMPK pathway in cultured ECs. Collectively, our findings demonstrate that endothelial EP4 is essential for BP homeostasis.

Aff ;ective disorders, including anxiety and mood disorders, are a constellation of psychiatric diseases that aff ;ect over 10 % of the world's population. It has been proposed that drugs that change nicotinic acetylcholine receptor (nAChR) activity can affect mood- and anxiety-related behaviors. Also, neuronal nitric oxide synthase (nNOS) is closely associated with the pathophysiology of these disorders. To limit the potential adverse effects of alteration in cholinergic and nitric oxide (NO) systems, we investigated the combined efficacy of subthreshold doses of nAChR antagonist mecamylamine and NO ligands (L-arginine as agonist and l-NAME as an antagonist) on depression- and anxiety-related behaviors in male NMRI mice. Depression-related behaviors using the forced swim test (FST) and anxiety-like activity using the hole-board test were assessed. In our results, mecamylamine (3 mg/kg) showed antidepressant-like properties, and it also tended to have anxiolytic-like effects, though not significant. Concomitant treatment of subthreshold doses of mecamylamine (1 mg/kg) and l-arginine (25 mg/kg), l-NAME (1 mg/kg), or l-arginine/L-NAME were antidepressive. In contrast, l-arginine/L-NAME alone or in associated with mecamylamine showed anxiogenic-like efficacy. Isobolographic analysis exhibited an additive antidepressant effect of the combined subthreshold doses of mecamylamine and l-arginine, and a synergistic antidepressant effect of the combined subthreshold doses of mecamylamine and l-NAME. It should be noted that mecamylamine (3 mg/kg) elicited hypolocomotion. Our results suggest that mecamylamine produces a better antidepressant efficacy in combination with l-NAME than with l-arginine., (Copyright © 2020. Published by Elsevier B.V.)

Lupeol (1) was isolated from hexane branch extract of Maytenus salicifolia and the Lupeol stearate (2), Lupeol palmitate (3), Lupeol myristate (4), Lupeol laurate (5) and Lupeol caprylate (6) were obtained reacting 1 with an adequate carboxylic acid. Swiss mice were treated with vehicle, carbenoxolone or Lupeol esters before administration of ethanol/HCl or indomethacin. Additionally, the involvement of nitric oxide (NO), sulfhydryl compounds (NP-SH), α-2 adrenergic receptors (α2-AR) and prostaglandins (PGE) in antiulcer effects was investigated using appropriate inhibitors or antagonist. Oxidative and inflammatory parameters were measured after euthanasia and anti-secretory effects was evaluated in pylorus-ligated rats. Ethanol/HCl ulcerated the gastric mucosa by 64.45 ± 6.58 mm 2 , which the oral treatment with 1, 4 and 6 (10 mg/kg), and 3 and 5 (30 mg/kg) reduced the lesion area. Interestingly, 2 reduced the gastric ulcer by oral route in a potent and dose-dependent manner (ED 50  = 0.40 mg/kg), which was accompanied by the increase in reduced glutathione levels and by the reduction of lipids peroxidation and myeloperoxidase and superoxide dismutase activities. Moreover, 2 (0.1 mg/kg) also prevented the ulcerogenesis by intraperitoneal route. The participation of NO, NP-SH, α2-AR and PGE in 2-mediated gastroprotection was confirmed. In indomethacin-induced ulcer, 2 (1 mg/kg, p.o) also reduced the ulcer area and increased the PGE 2 levels. However, 2 did not alter the gastric acid secretion. Therefore, these findings indicate that the obtention of 2 potentiated the antiulcer activity of 1 and that this compound can elicit gastroprotective action due a diversified mode of action., 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 © 2020 Elsevier B.V. All rights reserved.)

New Findings: What is the central question of this study? What are the effects of exercise on Ang II-induced vasoconstriction in aortas of normotensive rats and how do these effects occur in two-kidney-one-clip hypertensive animals? What is the main finding and its importance? In two-kidney rats, exercise training improves the Ang II-induced vasoconstriction by endothelium-derived NO released through AT 2 R activation. This effect of exercise training on the Ang II-induced vasoconstriction is blunted in two-kidney-one-clip hypertensive animals, possibly as a consequence of oxidative stress., Abstract: This study investigated the effects of both acute exercise and training on the Ang II-induced vasoconstriction in aorta of normotensive (two-kidney; 2K) and two-kidney-one-clip (2K1C) hypertensive rats, focusing on endothelial mechanisms related to nitric oxide (NO) and prostanoids. Aorta rings of 2K and 2K1C male Wistar rats, sedentary and trained, killed at rest and after acute exercise, were challenged with Ang II in either the absence or the presence of PD 123,319, a selective angiotensin receptor subtype 2 (AT 2 R) antagonist; N ω -nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase; indomethacin, a non-selective inhibitor of cyclooxygenase; or Tiron, an analogue of superoxide dismutase. Aortas of sedentary and trained animals studied at rest were also submitted to histomorphometric analysis. Exercise training reduced the Ang II-induced vasoconstriction in aorta of 2K but not of 2K1C animals. This reduction of Ang II response in aortas of 2K animals was not found after endothelial removal or treatment with PD 123,319 or l-NAME. These results suggest that exercise training improves the modulation of Ang II-induced vasoconstriction in aorta of 2K animals, by endothelium-derived NO released due to the activation of AT 2 R. No exercise-induced change of Ang II response occurred in 2K1C animals, except in the presence of Tiron, which was evidence for reduction of such responses only in resting trained 2K1C animals. In 2K1C animals, NO modulation of Ang II-induced vasoconstriction might be suppressed by local oxidative stress. Moreover, exercise training slightly reduced the media layer thickness in the aortas of the 2K1C, but not 2K animals, which may indicate cardiovascular protection of these animals., (© 2020 The Authors. Experimental Physiology © 2020 The Physiological Society.)

Polyphenols consumption has been associated with a lower risk of cardiovascular diseases (CVDs) notably through nitric oxide (NO)- and estrogen receptor α (ERα)-dependent pathways. Among polyphenolic compounds, chalcones have been suggested to prevent endothelial dysfunction and hypertension. However, the involvement of both the NO and the ERα pathways for the beneficial vascular effects of chalcones has never been demonstrated. In this study, we aimed to identify chalcones with high vasorelaxation potential and to characterize the signaling pathways in relation to ERα signaling and NO involvement. The evaluation of vasorelaxation potential was performed by myography on wild-type (WT) and ERα knock-out (ERα-KO) mice aorta in the presence or in absence of the eNOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). Among the set of chalcones that were synthesized, four ( 3 , 8 , 13 and 15 ) exhibited a strong vasorelaxant effect (more than 80% vasorelaxation) while five compounds ( 6, 10, 11, 16, 17 ) have shown a 60% relief of the pre-contraction and four compounds ( 12 , 14 , 18 , 20 ) led to a lower vasorelaxation. We were able to demonstrate that the vasorelaxant effect of two highly active chalcones was either ERα-dependent and NO-independent or ERα-independent and NO-dependent. Thus some structure-activity relationships (SAR) were discussed for an optimized vasorelaxant effect.

Carbamazepine has been shown to exert analgesic effects in clinical and experimental pain situation. This study was conducted to evaluate its potential peripheral antinociceptive effects and the possible involvement of L-arginine/NO/cGMP/K ATP channel pathway and PPARγ receptors in an animal model of pain. The antinociceptive effect induced by intraplantar administration of carbamazepine (100-1 000 μg/paw) was assessed using the formalin test in rats. To evaluate the involvement of L-arginine/NO/cGMP/K ATP channel pathway in the antinociceptive action of carbamazepine, rats were pre-treated intraplantarlly with L-arginine (a nitric oxide precursor, 100 and 200 μg/paw), L-NAME (NOS inhibitor, 50 and 100 μg/paw), methylene blue (guanylyl cyclase inhibitor, 100 and 200 μg/paw), glibenclamide (K ATP channel blocker, 100 and 200 μg/paw), and diazoxide (400 μg/paw). Moreover, to investigate the possible involvement of PPARγ receptors, pioglitazone (10 μg/paw; a PPARγ agonist) alone or in combination with GW9662 (3 μg/paw; a PPARγ antagonist) were pre-treated with carbamazepine. The local ipsilateral, but not contralateral, administration of carbamazepine into the hind paw produced dose-related analgesia during both early and late phases of formalin test. Moreover, pre-treatment with L-NAME, methylene blue, and glibenclamide dose-dependently prevented carbamazepine (300 μg/paw)-induced antinociception in both phases of the test. In addition, administration of L-arginine and diazoxide before the sub-effective dose of carbamazepine (100 μg/paw) produced an antinociceptive effect. Also, antinociception induced by carbamazepine plus pioglitazone (10 μg/paw) was blocked by GW-9662 in both phases of the test. In conclusion, carbamazepine induced a peripheral antinociceptive effect through PPARγ receptors and L-arginine/NO/cGMP/K ATP channel pathway, with potential for a new topical analgesic drug., Competing Interests: The authors declare that there is no conflict of interest associated with this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

Pre-eclampsia (PE) is closely associated with perinatal morbidity and mortality and we want to investigate tetramethylpyrazine (TMP)'s effects on PE. Pregnant Sprague-Dawley rats were randomly divided into five groups: normal pregnant (PC), PE, PE+TMP 20 mg/kg, PE+TMP 40 mg/kg, and PE+TMP 60 mg/kg group. The PE rat model was established via L-NAME treatment. Systolic blood pressures (SBP) and urinary protein concentration were detected via the tail-cuff method and CBB kit, respectively. mRNA levels of key genes were analyzed via quantitative PCR and protein levels of key genes were measured by ELISA or western blot. TMP decreased SBP and urinary protein concentration of PE rats. TMP inhibited L-NAME-induced decrease in pups alive ratio, pups weight, and the ratio of pups/placenta weight and reversed L-NAME induced changes in placental histology, whereas it had little effect on placental weight. Urinary nephrin and podocin expressions were enhanced and serum placental growth factor level was decreased in PE rats, whereas TMP inhibited the above phenomena. TMP suppressed L-NAME-induced sFlt-1 upregulation in serums and kidneys of PE rats, whereas it downregulated IL-6 and MCP-1 expression in PE rats' serums, placentas and kidneys. TMP also suppressed the increase in placental sFlt-1 and vascular endothelial growth factor level caused by L-NAME. In addition, TMP inhibited CHOP and GRP78 expressions and decreased the ratio of p-elF2α/elF2α in PE rats. TMP attenuated the consequences of NO inhibition in pregnant rats., (© 2019 Wiley Periodicals, Inc.)

Background: Abnormalities of the L-arginine-nitric oxide pathway induce hypertension. 5-Lipoxygenase (5-LO) is the key enzyme involved in synthesis of leukotrienes (LTs). However, whether nitricoxide synthase dysfunction induces hypertensive vascular remodeling by regulating 5-LO activity and its downstream inflammatory metabolites remains unknown., Methods and Results: Six-week L-NAME treatment significantly induced hypertension and vascular remodeling in both wild-type (WT) and 5-LO-knockout (5-LO-KO) mice, and blood pressure in caudal and carotid arteries was lower in 5-LO-KO than WT mice with L-NAME exposure. On histology, L-NAME induced less media thickness, media-to-lumen ratio, and collagen deposition and fewer Ki-67-positive vascular smooth muscle cells (VSMCs) but more elastin expression in thoracic and mesenteric aortas of 5-LO-KO than L-NAME-treated WT mice. L-NAME significantly increased LT content, including LTB4 and cysteinyl LT (CysLTs), in plasma and neutrophil culture supernatants from WT mice. On immunohistochemistry, L-NAME promoted the colocalization of 5-LO and 5-LO-activating protein on the nuclear envelope of cultured neutrophils, which was accompanied by elevated LT content in culture supernatants. In addition, LTs significantly promoted BrdU incorporation, migration and phenotypic modulation in VSMCs., Conclusion: L-NAME may activate the 5-LO/LT pathway in immune cells, such as neutrophils, and promote the products of 5-LO metabolites, including LTB4 and CysLTs, which aggravate vascular remodeling in hypertension. 5-LO deficiency may protect against hypertension and vascular remodeling by reducing levels of 5-LO downstream inflammatory metabolites., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: We aimed to analyze whether a diet supplemented with a standard dose of copper (Cu) in the form of nanoparticles, as an alternative to carbonate, exerts beneficial effects within the vasculature and improves the blood antioxidant status., Methods: Male Wistar rats were fed for 8 weeks with a diet supplemented with Cu (6.5 mg Cu/kg in the diet) either as nanoparticles (40 nm diameter) or carbonate - the control group. Moreover, a negative control was not supplemented with Cu. At 12 weeks of age, blood samples, internal organs and thoracic aorta were taken for further analysis. Blood antioxidant mechanism was measured together with Cu and Zn., Results: Diet with Cu as nanoparticles resulted in an elevated catalase activity and ferric reducing ability of plasma, however decreased Cu (plasma), and ceruloplasmin (Cp) compared to carbonate. The participation of vasoconstrictor prostanoid was increased, as indomethacin did not modify the acetylcholine (ACh)-induced response. Arteries from Cu nanoparticle and carbonate rats exhibited a reduced maximal contraction to potassium chloride and an increased response to noradrenaline. The endothelium-dependent vasodilation to ACh was enhanced while exogenous NO donor, sodium nitroprusside, did not modify the vascular response. Down-regulation of BKCa channels influenced hyperpolarizing mechanism. The superoxide dismutase and HDL-cholesterol were decreased opposite to an increased lipid hydroperoxides, malondialdehyde, Cu (plasma and liver) and Cp., Conclusion: Despite the increased antioxidant capacity in blood of Cu nanoparticle fed rats, vasoconstrictor prostanoids and NO are involved in vascular regulation., (Copyright © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Introduction: Hepatic ischemic reperfusion injury occurs in multiple clinical settings. Novel potential protective agents are still needed to attenuate this injury. Apelin preconditioning protects against ischemic reperfusion injury in different organs. However, the protective mechanism of apelin on hepatic ischemic reperfusion injury is not yet clear., Aim: Evaluate the effect of apelin-13 preconditioning on hepatic ischemic reperfusion injury and clarify possible interactions between apelinergic, renin-angiotensin systems and endothelial nitric oxide synthase., Methods: In total, 60 rats were assigned to four groups: control sham-operated, ischemic reperfusion, apelin-treated ischemic reperfusion and apelin + N-nitro-L-arginine methyl ester-treated ischemic reperfusion. Apelin 2 µg/kg/day and N-nitro-L-arginine methyl ester 10 mg/kg/day were injected intraperitoneally daily for 3 days and 2 weeks respectively before hepatic ischemic reperfusion. Serum aminotransferase, aspartate aminotransferase, hepatic malondialdehyde, apelin, gene expression of caspase-3, endothelial nitric oxide synthase and angiotensin type 1 receptor and liver histopathology were compared between groups., Results: Apelin significantly reduced serum aminotransferase, aspartate aminotransferase, hepatic malondialdehyde, caspase-3 and angiotensin type 1 receptor expression, whereas hepatic apelin and endothelial nitric oxide synthase expression were significantly increased with improved hepatic histopathology. N-nitro-L-arginine methyl ester co-administration partially reversed this hepatoprotective effect., Conclusion: Apelin-13 reduced hepatic ischemic reperfusion injury. This protection could be related to the suppression of hepatic angiotensin type 1 receptor expression and elevation of hepatic apelin level and endothelial nitric oxide synthase expression, which counteracts the pathologic effects of Ang II/angiotensin type 1 receptor. An interaction exists between apelinergic, renin-angiotensin systems and endothelial nitric oxide synthase in hepatic ischemic reperfusion pathophysiology.

Myocardial infarction (MI) remains the leading cause of death worldwide. We aimed to investigate the effect of NO deficiency on selective biochemical parameters within discreet myocardial zones after experimentally induced MI. To induce MI, the left descending coronary artery was ligated in two groups of 16-week-old WKY rats. In one group, NO production was inhibited by L-NAME (20 mg/kg/day) administration four weeks prior to ligation. Sham operations were performed on both groups as a control. Seven days after MI, we evaluated levels of nitric oxide synthase (NOS) activity, eNOS, iNOS, NFҡB/p65 and Nrf2 in ischemic, injured and non-ischemic zones of the heart. Levels of circulating TNF-α and IL-6 were evaluated in the plasma. MI led to increased NOS activity in all investigated zones of myocardium as well as circulating levels of TNF-α and IL-6. L-NAME treatment decreased NOS activity in the heart of sham operated animals. eNOS expression was increased in the injured zone and this could be a compensatory mechanism that improves the perfusion of the myocardium and cardiac dysfunction. Conversely, iNOS expression increased in the infarcted zone and may contribute to the inflammatory process and irreversible necrotic changes.

Previous studies have investigated the cardiovascular activity of Gynura procumbens Merr. single-solvent extracts. The objective of this study was to evaluate the in vitro vasorelaxant properties and the underlying pharmacological mechanisms of serial extracts and fractions of Gynura procumbens (GP). The leaves of GP were serially extracted with petroleum ether, chloroform, methanol and water using the maceration method. Suspended aortic ring preparations were pre-contracted with phenylephrine (PE 1 µM), followed by cumulative addition of GP extracts (0.25-3 mg/mL). The petroleum ether extract (GPPE) was the most potent among the four extracts. Pre-incubation of endothelium-intact aorta with atropine (1 µM), indomethacin (10 µM), methylene blue (10 µM), propranolol (1 µM) and potassium channel blockers such as TEA (1 µM), glibenclamide (10 µM), 4-aminopyridine (1 µM) and barium chloride (10 mM) had no effect on GPPE-induced vasorelaxation. The vasorelaxant effect of GPPE was partly diminished by pretreatment of aortic rings preparations with L-NAME (10 µM) and even more so in endothelium-denuded aortic rings, indicating a minimal involvement of endothelium-dependent pathway in GPPE-induced vasorelaxation. The calcium-induced vasocontractions were antagonized significantly and concentration-dependently by GPPE in calcium free and high potassium medium. These results illustrate that Ca 2+ antagonizing actions of GPPE in rat isolated aorta are comparable to that of verapamil and may be mainly responsible for its vasodilation effect. The antioxidant activity of GPPE supports its vasorelaxant effect by attenuating the production of deleterious free radicals and reactive oxygen species in the vasculature.

GYY4137 is a novel hydrogen sulfide (H 2 S) releasing molecule with vasodilator activity. The objectives of this study were to investigate: (1) the pharmacological effect of GYY4137 on the reactivity of the corpus cavernosum (CC) from normal and diabetic rats; (2) the contribution of ATP-sensitive potassium (K-ATP) channels and nitric oxide (NO) pathway; (3) the reactivity to vasoactive agonists following ex vivo incubation of the diabetic rat CC with GYY4137. Longitudinal strips of CC from control and diabetic male Sprague-Dawley (SD) rats (n = 5-6 animals per group) were suspended in organ-baths. Responses to GYY4137, carbachol, or phenylephrine (PE) were determined by measurement of changes in isometric tension. The effects of acute incubation of the CC strips with L-NAME (NO synthase inhibitor) or glibenclamide (K-ATP channel inhibitor) on the relaxant responses to GYY4137 were examined. The effect of ex vivo incubation with GYY4137 (10 -5  M) on the responses of CC to carbachol or PE was evaluated. We found that GYY4137 provoked relaxation in the CC strips, which was significantly reduced in the presence of L-NAME or glibenclamide. Ex vivo incubation of diabetic CC with GYY4137 resulted in a significant improvement in the vascular responses to the added agonists. We conclude that GYY4137 is a relaxant agonist in SD rats CC, and the response is mediated, at least in part, by NO and K-ATP channels. Brief incubation of diabetic CC with GYY4137 markedly improved the impaired vascular reactivity, thus raising the question whether chronic in vivo treatment of diabetic animals with GYY4137 would have any protective effect, which is worth further investigation.

The cardioprotective and inotropic effects of standardized active natural substance based on high-molecular-weight compounds of humic origin were studied on the model of global ischemia (40 min) and reperfusion of isolated perfused rat heart. Preventive administration of the test substance (0.1 mg/ml) before ischemia/reperfusion modeling reduced reperfusion contracture and necrotic death of cardiomyocytes and promoted recovery of myocardial contractility. Blockade of NO synthase with L-NAME (100 μM) abolished the above effects of the test substance. It was hypothesized that NO synthase plays an important role in the development of the cardioprotective and inotropic effects of the test natural substance.

Dizocilpine is a highly selective and potent non-competitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. It is well known that dizocilpine has different neuroprotective effects in animal models of pain, epilepsy and oedema during trauma. The search for alternative antiinflammatory drugs is ongoing. We investigated the anti-oedematous effects of dizocilpine and the probable mechanism of action in a rat model that mimics local and persistent inflammation without tissue injury or damage. Male Wistar rats were injected with 100 μL of 0.5% carrageenan to the plantar surface of the hind paw. Anti-oedematous activity was assessed in the carrageenan-induced paw inflammatory oedema test with a plethysmometer. To assess possible mechanisms of dizocilpine action, we examined the effects of the selective inhibitor of neuronal [N-ω-propyl-L-arginine hydrochloride (L-NPA)] and inducible [S-methylisothiourea (SMT)] nitric oxide synthase (NOS). Dizocilpine after systemic (0.0005, 0.005 and 0.02 mg/kg, subcutaneous (s.c.)), but not after local peripheral administration, reduced the paw inflammatory oedema. The effect is not dose dependent, and the highest decrease by about 47% at the time of maximally developed oedema was achieved with 0.005 mg/kg. Intraperitoneally (i.p.) administered L-NPA (0.5, 1 and 2 mg/kg) or SMT (0.005, 0.01 and 0.015 mg/kg) before dizocilpine abolished or reduced the anti-oedematous effect of dizocilpine by about 70-85%. An acute single dose of dizocilpine administered before inducing oedema systemically reduced the development of inflammatory oedema. The mechanism of the anti-oedematous effect includes, at least partially, an increase in nitric oxide (NO) production.

: Background: Central nervous system function has been emerging as an approach to understand hypertension-mediated memory dysfunction, and chronic exercise is able to modulate the purinergic system., Method: Herein, we investigated the effect of chronic swimming training on the purinergic system in cortex and hippocampus of L-NAME-induced hypertensive rats. Male Wistar rats were divided into four groups: Control, Exercise, L-NAME and Exercise L-NAME. Inhibitory avoidance test was used to assess memory status. NTPDase, CD73 and adenosine deaminase activities and expression, and P2 receptors expression were analyzed. Data were analyzed using two-way ANOVA and Kruskal-Wallis tests, considering P less than 0.05., Results: Physical exercise reduced the blood pressure and prevented memory impairment induced by L-NAME model of hypertension. L-NAME treatment promoted an increase in NTPDase1, NTPDase3 and CD73 expression and activity in the cortex. A2A expression is increased in hippocampus and cortex in the hypertension group and exercise prevented this overexpression., Conclusion: These changes suggest that hypertension increases adenosine generation, which acts through A2A receptors, and exercise prevents these effects. These data may indicate a possible mechanism by which exercise may prevent memory impairment induced by L-NAME.

Aim: This study aims to investigate the effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Ex) on placental tissue and angiogenesis in rats with preeclampsia (PE)., Method: The expression of MSC surface markers were identified by flow cytometry. Alizarin red staining and oil red O staining were used to examine osteogenic and adipogenic differentiation of hucMSCs. Western blotting was used to determine expressions of CD63 and CD81 in hucMSC-Ex. PE rat models were established using endothelial nitric oxide synthase, eNOS)N G -Nitro-l-arginine Methyl Ester, which were then treated with exosome (Exo) of low dosage (L-Exo), Exo of medium dosage (M-Exo) and Exo of high dosage (H-Exo). The blood pressure at the 15d, 17d and 19d of pregnancy and 24-h urinary protein were measured. TUNEL staining and immunohistochemistry were applied to detect the cell apoptosis and micro-vascular density (MVD) in placental tissues, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to examine serum levels of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase receptor-1 (sFlt1)., Results: In vitro cultured hucMSCs showed expression of MSC surface markers (CD29, CD90 and CD105), and no expression of CD34 and CD45. Besides, the isolated exosomes expressed the exosome markers (CD63 and CD81). In response to the treatment of L-Exo, M-Exo and H-Exo, the blood pressure of PE rat models on the 17 d and the 19 d as well as the 24-h urinary protein were substantially decreased. Moreover, at the 21 d, PE rat models treated with L-Exo, M-Exo and H-Exo exhibited an increase in the number and quality of fetuses, placenta quality, MVD and VEGF expression, but substantial decreased cell apoptosis and expression of sFlt1. The influence of Exos was exerted in a dosage dependent manner., Conclusion: hucMSC-Ex, in a dose-dependent manner, can improve the morphology of placental tissue in rats with PE, by inhibiting cell apoptosis and promoting angiogenesis in placental tissue., (Copyright © 2018. Published by Elsevier Masson SAS.)

New Findings: What is the central question of this study? Gastric slow waves originating from the interstitial cells of Cajal-smooth muscle syncytium are usually studied in culture or in tissue segments, but nobody has described recordings of slow waves from awake, freely moving mice. Can radiotelemetry be used to record slow waves, and do they respond predictably to drug treatment? What is the main finding and its importance? Radiotelemetry can be used to record slow waves from awake, freely moving mice, permitting an examination of drug actions in vivo, which is crucial to drug discovery projects for characterizing the effects of drugs and metabolites on gastrointestinal function., Abstract: The mouse is the most commonly used species in preclinical research, and isolated tissues are used to study slow waves from the interstitial cells of Cajal-smooth muscle syncytium of the gastrointestinal tract. The aim of this study was to establish a radiotelemetric technique in awake mice to record gastric myoelectric activity from the antrum to gain insight into the effects of endogenous modulatory systems on slow waves. Under general anaesthesia, two biopotential wires from a telemetry transmitter were sutured into the antrum of male ICR (imprinting control region) mice. The animals were allowed 1 week to recover from surgery before the i.p. administration of drugs to stimulate or inhibit slow waves. The basal dominant frequency of slow waves was 6.96 ± 0.43 c.p.m., and the percentages of power in the bradygastric, normogastric and tachygastric ranges were 6.89 ± 0.98, 37.32 ± 1.72 and 34.38 ± 0.77%, respectively (n = 74). Nicotine at 1 mg kg -1 increased normogastric power, but at 3 mg kg -1 it increased bradygastric power (P < 0.05). Metoclopramide at 10 mg kg -1 increased normogastric power; sodium nitroprusside at 10 mg kg -1 had latent effects on tachygastric power (P < 0.05); and l-NAME at 10 mg kg -1 had no effect (P > 0.05). Nicotine and bethanechol also caused varying degrees of hypothermia (>1°C reductions; P < 0.05). In conclusion, radiotelemetry can be used to record slow waves from awake, freely moving mice. In light of our findings, we recommend that studies assessing slow waves should also assess body temperature simultaneously., (© 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.)

Exposure of barley roots to mM concentrations of L-NAME for 30 min caused a considerable root growth inhibition in a dose-dependent manner. The inhibition of root growth was higher in seedlings co-treated with Cd and L-NAME, compared with roots treated with Cd alone, despite the fact that L-NAME markedly reduced the uptake of Cd by roots. Treatment of roots with L-NAME evoked a decrease in NO level in both control and Cd-treated root tips only after a relatively long lag period, which overlaps with an increase in superoxide and H 2 O 2 levels and peroxynitrite generation. L-NAME-induced root growth inhibition is alleviated not only by the application of the NO donor SNP but also by the ROS and peroxynitrite scavengers. Our results indicate that L-NAME, a NOS inhibitor in the animal kingdom, indeed evokes NO depletion also in the plant tissues; however, it does not occur due to the action of L-NAME as an inhibitor of NOS or NOS-like activity, but as a consequence of L-NAME-induced enhanced superoxide generation, leading to increased peroxynitrite level in the root tips due to the reaction between superoxide and NO., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Purpose: Nicorandil has dual properties and acts as a nitric oxide donor and an ATP-sensitive potassium (K ATP ) channel opener. Considering its pharmacological profile, nicorandil might exert protective effects on the brain as well as on the heart. The purpose of this study was to directly evaluate the effect of nicorandil on cerebral blood flow (CBF) in mice using a transcranial Doppler method., Methods: Under general anesthesia, the nicorandil groups received a single-bolus intraperitoneal injection of the respective doses of nicorandil (1, 5, or 10 mg/kg), while the control group received vehicle only. CBF was measured using a transcranial Doppler flowmeter. N G -nitro-L-arginine methyl ester and glibenclamide were used to elucidate the underlying mechanisms., Results: A single-bolus injection of 1 mg/kg of nicorandil increased the CBF (11.6 ± 3.6 vs. 0.5 ± 0.7%, p < 0.001) without affecting the heart rate and blood pressure. On the contrary, 5 and 10 mg/kg of nicorandil significantly decreased the cerebral blood flow by decreasing the mean blood pressure below the cerebral autoregulation range. The positive effect of 1 mg/kg of nicorandil on the cerebral blood flow was inhibited by co-administration of either N G -nitro-L-arginine methyl ester or glibenclamide., Conclusions: A clinical dose of nicorandil increases CBF without affecting systemic hemodynamics. The positive effect of nicorandil on CBF is most likely caused via both the nitric oxide pathway and K ATP channel opening.

Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S -nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions., Competing Interests: The authors declare no conflict of interest.

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection and allow recognition of an original pathophysiologic mechanism underlying gastrointestinal diseases as well as identification of novel therapeutic targets.

After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids, and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Separate treatments with an insect cytokine, 2 biogenic amines, and an eicosanoid lead to a single result, hemocyte spreading, understood in terms of intracellular cross-talk among these signaling systems. This study focuses on the cross-talk between NO and eicosanoid signaling in our model insect, Spodoptera exigua. Bacterial injection increased NO concentrations in the larval hemocytes and fat body, and RNA interference (RNAi) of the S. exigua NO synthase (NOS) gene suppressed NO concentrations. RNAi treatment also led to a significant reduction in hemocyte nodulation following bacterial injection. Similar RNAi treatments led to significantly reduced PLA2 activities in the hemocytes and fat body compared to control larvae. Injection of L-NAME also prevented the induction of PLA2 activity following bacterial challenge. An injected NO donor, S-nitroso-N-acetyl-DL-penicillamine, increased PLA2 activity in a dose-dependent manner. However, eicosanoids did not influence NO concentrations in immune-challenged larvae. We infer that NO and eicosanoid signaling operate via cross-talk mechanisms in which the elevated NO concentrations activate PLA2 and eicosanoid biosynthesis, which finally mediates various immune responses., (© 2017 S. Karger AG, Basel.)

S-palmitoylation is an important post-translational modification that affects the translocation and the activity of target proteins in a variety of cell types including cardiomyocytes. Since endothelial nitric oxide synthase (eNOS) is known to be palmitoylated and the activity of eNOS is essential in fatty acid-dependent β-oxidation in muscle, we aimed to test whether palmitoylation of eNOS is involved in palmitic acid (PA) regulation of left ventricular (LV) myocyte contraction from healthy (sham) and hypertensive (HTN) rats. Our results showed that PA, a predominant metabolic substrate for cardiac β-oxidation, significantly increased contraction and oxygen consumption rate (OCR) in LV myocytes from sham. Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) or eNOS gene deletion prevented PA regulation of the myocyte contraction or OCR, indicating the pivotal role of eNOS in mediating the effects of PA in cardiac myocytes. PA increased the palmitoylation of eNOS in LV myocytes and depalmitoylation with 2-bromopalmitate (2BP; 100 μM) abolished the increment. Furthermore, although PA did not increase eNOS-Ser 1177 , 2BP reduced eNOS-Ser 1177 with and without PA. Intriguingly, PA-induced increases in contraction and OCR were unaffected by 2BP treatment. In HTN, PA did not affect eNOS palmitoylation, eNOS-Ser 1177 , or myocyte contraction. However, 2BP diminished eNOS palmitoylation and eNOS-Ser 1177 in the presence and absence of PA but did not change myocyte contraction. Collectively, our results confirm eNOS palmitoylation in LV myocytes from sham and HTN rats and its upregulation by PA in sham. However, such post-transcriptional modification plays negligible role in PA regulation of myocyte contraction and mitochondrial activity in sham and HTN.