Inhibitor L-NAME Research Articles

Role of AMPK in Adiponectin‐Mediated Restoration of Nitric Oxide‐Dependent Flow Induced Dilation in the Human Microvasculature

Flow‐induced dilation (FID), the ability of an arteriole to dilate in response to increased flow, is primarily mediated by endothelial‐derived nitric oxide (NO) in healthy individuals, however is replaced by hydrogen peroxide (H2O2) in patients with coronary artery disease (CAD). Our laboratory has shown that adiponectin restores NO‐mediated FID in microvessels from patients with CAD. Adiponectin activates multiple signaling pathways that promote NO formation, including activation of neutral ceramidase to hydrolyze ceramide, a sphingolipid that in elevated amounts promotes FID mediated by H2O2. Our prior work has shown that both NO and H2O2 contribute as mediators of FID in diseased vessels treated with adiponectin and ceranib‐1, an inhibitor of neutral ceramidase. As adiponectin is also capable of activating AMP kinase (AMPK) to increase cellular NO levels, we tested the hypothesis that AMPK activation is required to restore NO‐mediated FID in diseased arterioles. Human microvessels (80–200μm) were dissected from adipose tissue collected from patients diagnosed with CAD. Following cannulation and equilibration, vessels were constricted with endothelin‐1. Using videomicroscopy, vessel diameters were measured in response to graded increases in intraluminal flow. To investigate whether activation of AMPK is a key mechanistic step in adiponectin‐induced restoration of NO‐mediated signaling, vessels from patients diagnosed with CAD were first treated with the AMPK inhibitor Compound C (1μM, 4hrs) followed by adiponectin treatment (2μg/mL, 16–20hrs). Dilation to flow was maintained during exposure to the nitric oxide synthase (NOS) inhibitor L‐NAME (100μM) (69.8% max dilation±2.7, n=2) as well as in the presence of the PEG‐Catalase (500U) (64.5%±21.2, n=4) compared to compound C and adiponectin alone (61.0%±9.4, n=4). To determine whether ceramide hydrolysis via neutral ceramidase contributes to the maintained dilation despite NOS inhibition, FID was also assessed in diseased arterioles treated with both compound C and ceranib‐1 prior to treatment with adiponectin. Both NO and H2O2 contributed to dilation under both AMPK and neutral ceramidase inhibition as dilation was observed in the presence of L‐NAME (33.6%±15.9, n=3) or PEG‐Catalase (51.7%±23.9, n=3). However, FID was decreased during exposure to both L‐NAME and PEG‐Catalase (11.6%±12.4, n=2). These preliminary data suggest that adiponectin promotes NO formation through multiple pathways and contributes to the redundancy of endothelial‐derived mediators in order to maintain FID.Support or Funding InformationThis research was supported by National Institute of Health (NHLBI) K08HL141562 (J.K.F.) and National Institute of Health 5R38HL143561‐02 (M.E.W.).

Is Blue Light the Next Little Blue Pill?: Effects of Light‐Stimulation on Erectile Dynamics

INTRODUCTIONMany major risk factors for erectile dysfunction (ED) are associated with depressed nitric oxide (NO) availability. Phosphodiesterase type 5 inhibitors are the current first line therapy for ED and are ineffective in 30–50% of patients, largely due to impaired NO production. New therapeutic approaches for ED are needed under such conditions. The phenomenon of light‐stimulated vasodilation has recently been described in aortic tissue, which is enhanced by G‐protein coupled receptor kinase 2 (GRK2) inhibition and particular for blue light. The objective of this project was to determine if this GRK2 sensitive photorelaxation response is active in erectile tissue and the contribution of the NO signaling pathway to the response.METHODSYoung male mice (n = 4–8 / experiment) were sacrificed and erectile tissue was harvested from the shaft of the penis. Ex vivo constriction and relaxation responses were assessed in a muscle strip myograph. The GRK2‐sensitive photorelaxation response was assessed ± 1 μM GRK2 inhibitor using nine different wavelengths (365–700 nm) of light at constant intensity (100 Lux), and then at nine different light intensities (1–1000 Lux) at 455 nm. The contribution of the NO signaling pathway to the GRK2‐sensitive response was tested with stimulations of 20 and 100 Lux at 455 nm with 1 μM GRK2 inhibitor ± the NO synthase inhibitor L‐NAME, the soluble guanylate cyclase (sGC) inhibitor ODQ, the protein kinase G (PKG) inhibitor Rp‐8‐Br‐cGMPS, or the NO scavenger cPTIO, as well as in tissue from mice with a deletion of endothelial + neuronal NO synthase. Differences in responses were determined by two‐way repeated measures ANOVA followed by Sidak’s post hoc test where appropriate.RESULTSMaximal relaxation responses at 455 nm increased with increasing light intensity in the absence (2–10%) and presence (57–103%) of the GRK2 inhibitor, demonstrating a stark increase in photorelaxation under GRK2 inhibition. However, this response waned more rapidly at intensities > 100 Lux. Significant GRK2‐sensitive responses were observed at 365, 395, 430, and 455 nm at 100 Lux intensity, with no significant responses observed in the 490–700 nm range. Both L‐NAME and deletion of endothelial/neuronal NO synthase increased the magnitude of relaxation at all time‐points at 20 Lux, and prolonged maximal relaxation at 100 Lux. CPTIO prolonged the relaxation response at both intensities. ODQ drastically blunted the photorelaxation response at all time points and intensities. Rp‐8‐Br‐cGMPS had no effect on photorelaxation.CONCLUSIONSGRK2‐sensitive photorelaxation was determined to be optimal in the 430–455 nm (violet‐blue) range at ~100 Lux. Depletion of NO by scavenging, enzyme inhibition or genetic deletion enhanced the response, which is sGC‐dependent but PKG‐independent. These findings have implications for treatment of ED due to low NO availability if the GRK2‐sensitive photorelaxation pathway can be harnessed therapeutically.

Initiation of the Gut Microbiome Targeted Compound 3,3‐Dimethyl‐1‐butanol at Mid‐life Prevents Age‐related Vascular Dysfunction

Aging induces vascular dysfunction (arterial stiffening and endothelial dysfunction) mediated by superoxide‐driven oxidative stress and reduced nitric oxide (NO) bioavailability, which increases risk of cardiovascular (CV) diseases. A potential intervention target is the gut microbiome‐derived metabolite trimethylamine N‐oxide (TMAO), which increases in circulation with advancing age, is associated with higher CV risk, and can be inhibited by the food‐derived compound 3,3‐dimethyl‐1‐butanol (DMB).PurposeTo determine if DMB initiated at mid‐life (i.e., prior to the onset of dysfunction) prevents the development of age‐related vascular dysfunction.MethodsMiddle‐aged (18 mo) male C57BL/6 mice received drinking water with (n=24) or without (control: n=27) 1% DMB until sacrifice at 21, 24, or 27 mo of age (n=7–13/age/treatment). Arterial stiffness was assessed serially in vivo by aortic pulse wave velocity (aPWV). Endothelial function (ex vivo carotid artery endothelium‐dependent dilation [EDD] to acetylcholine), plasma TMAO (LC‐MS) and aortic superoxide (electron paramagnetic resonance spectroscopy) were assessed post‐sacrifice. To determine the prevention of age‐related vascular dysfunction with DMB, functional measures were made in a reference group of young (5 mo) male C57BL/6 mice (YC, n=12).ResultsPlasma TMAO increased with age vs YC (3.1±0.5 μM), but tended to be lower in DMB (5.6±0.5 μM) vs control (6.9±0.8 μM) mice at 24 and 27 mo (p=0.15). In control mice, aPWV was similar to YC (356±14 cm/s) at 18/21 mo (359±12/368±11 cm/s, both p>0.54), but increased with age (24 mo: 386±12 cm/s, p=0.06 vs 18 mo; 27 mo: 432±10 cm/s, p<0.001 vs 18 mo); this increase was attenuated by ~50% in DMB mice (aPWV at 24 mo: 359±9 cm/s; 27 mo: 388±9 cm/s, both p=0.01 vs control). Age‐related declines in peak EDD were apparent in control mice at 24 mo (79±3 vs YC 93±2%, p<0.001) and 27 mo (76±3%, p<0.0001 vs YC) and were fully prevented by DMB (24 mo: 91±3%; 27 mo: 93±1%; both p<0.05 vs control). The effects of DMB at 24 and 27 mo (data combined across these ages) were mediated by reduced aortic superoxide production (control 4.7±0.5 vs DMB 3.1±0.4 AU, p=0.01), which reduced tonic superoxide‐associated suppression of EDD (normalization of EDD with superoxide dismutase mimetic TEMPOL) and restored NO‐mediated dilation (the difference in peak EDD in the absence vs presence of the NO synthase inhibitor L‐NAME: control 45±6 vs DMB 69±4%, p<0.01). There were no differences in smooth muscle sensitivity to NO (peak dilation to sodium nitroprusside; 96±1 vs 98±1%, p=0.12), indicating improvements in EDD were endothelium‐specific.ConclusionSupplementation with DMB from mid‐life through old age in mice prevented the development of arterial stiffening and endothelial dysfunction. DMB may be a promising therapeutic strategy for preventing age‐related vascular dysfunction and thereby reducing CV risk with aging in humans.Support or Funding InformationR01 HL1348870‐02S1, R21 AG060884, F32 HL140875

Reactive oxygen species and nitric oxide imbalances lead to in vivo and in vitro arrhythmogenic phenotype in acute phase of experimental Chagas disease.

Chagas Disease (CD) is one of the leading causes of heart failure and sudden death in Latin America. Treatments with antioxidants have provided promising alternatives to ameliorate CD. However, the specific roles of major reactive oxygen species (ROS) sources, including NADPH-oxidase 2 (NOX2), mitochondrial-derived ROS and nitric oxide (NO) in the progression or resolution of CD are yet to be elucidated. We used C57BL/6 (WT) and a gp91PHOX knockout mice (PHOX-/-), lacking functional NOX2, to investigate the effects of ablation of NOX2-derived ROS production on the outcome of acute chagasic cardiomyopathy. Infected PHOX-/- cardiomyocytes displayed an overall pro-arrhythmic phenotype, notably with higher arrhythmia incidence on ECG that was followed by higher number of early afterdepolarizations (EAD) and 2.5-fold increase in action potential (AP) duration alternans, compared to AP from infected WT mice. Furthermore, infected PHOX-/- cardiomyocytes display increased diastolic [Ca2+], aberrant Ca2+ transient and reduced Ca2+ transient amplitude. Cardiomyocyte contraction is reduced in infected WT and PHOX-/- mice, to a similar extent. Nevertheless, only infected PHOX-/- isolated cardiomyocytes displayed significant increase in non-triggered extra contractions (appearing in ~75% of cells). Electro-mechanical remodeling of infected PHOX-/—cardiomyocytes is associated with increase in NO and mitochondria-derived ROS production. Notably, EADs, AP duration alternans and in vivo arrhythmias were reverted by pre-incubation with nitric oxide synthase inhibitor L-NAME. Overall our data show for the first time that lack of NOX2-derived ROS promoted a pro-arrhythmic phenotype in the heart, in which the crosstalk between ROS and NO could play an important role in regulating cardiomyocyte electro-mechanical function during acute CD. Future studies designed to evaluate the potential role of NOX2-derived ROS in the chronic phase of CD could open new and more specific therapeutic strategies to treat CD and prevent deaths due to heart complications.

Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment

BackgroundBacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30–50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure. Objectiveto evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor l-NAME. ResultsIn human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9+ tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production.Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8+ and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8+-infiltrating cells and induced a systemic increase in CD8+ and a reduction in Treg. BCG combined with l-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8+-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8+ and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected. ConclusionThe present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.

Resveratrol and its dimers ε-viniferin and δ-viniferin in red wine protect vascular endothelial cells by a similar mechanism with different potency and efficacy.

Red wine compounds have been reported to reduce the rate of atherosclerosis by inducing nitric oxide (NO) production and antioxidant enzyme expression in vascular endothelial cells (VECs). The present study compared the effects of the three red wine compounds resveratrol and its dimers, ε-viniferin and δ-viniferin, on VECs function for the first time. Both 5 μM ε-viniferin and δ-viniferin, but not 5 μM resveratrol, significantly stimulated wound repair of VECs. Increased levels of wound repair induced by 10 and 20 μM ε-viniferin were significantly higher than those stimulated by 10 and 20 μM resveratrol, respectively. These stimulatory effects of the three compounds were suppressed by the NO synthase inhibitor L-NAME. When VECs were exposed to each compound, endothelial NO synthase was activated and the expression of sirtuin 1 (SIRT1) and HO-1 was induced. Addition of the SIRT1 and HO-1 inhibitors EX527 and ZnPPiX, respectively, suppressed wound repair stimulated by the three compounds, demonstrating that SIRT1 and HO-1 are involved in these wound repair processes. Furthermore, each compound induced the suppression of H2 O2 -dependent reduction of cell viability as well as the expression of the antioxidant enzyme catalase. These data suggest that not only resveratrol, but also its dimers, ε-viniferin and δ-viniferin, may be effective in preventing atherosclerosis by a similar molecular mechanism with different potency and efficacy.

Skeletal restoration by phosphodiesterase 5 inhibitors in osteopenic mice: Evidence of osteoanabolic and osteoangiogenic effects of the drugs.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides and thereby regulate diverse cellular functions. The reports on the skeletal effects of PDE inhibitors are conflicting. Here, we screened 17 clinically used non-xanthine PDE inhibitors (selective and non-selective) using mouse calvarial osteoblasts (MCO) where the readout was osteoblast differentiation. From this screen, we identified sildenafil and vardenafil (both PDE5 inhibitors) having the least osteogenic EC50. Both drugs significantly increased vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expressions in MCO and the nitric oxide synthase inhibitor L-NAME completely blocked VEGF expression induced by these drugs. Sunitinib, a tyrosine receptor kinase inhibitor that also blocks VEGFR2 blocked sildenafil-/vardenafil-induced osteoblast differentiation. At half of their human equivalent doses, i.e. 6.0mg/kg sildenafil and 2.5mg/kg vardenafil, the maximum bone marrow level of sildenafil was 32% and vardenafil was 21% of their blood levels. At these doses, both drugs enhanced bone regeneration at the femur osteotomy site and completely restored bone mass, microarchitecture, and strength in OVX mice. Furthermore, both drugs increased surface referent bone formation and serum bone formation marker (P1NP) without affecting the resorption marker (CTX-1). Both drugs increased the expression of VEGF and VEGFR2 in bones and osteoblasts and increased skeletal vascularity. Sunitinib completely blocked the bone restorative and vascular effects of sildenafil and vardenafil in OVX mice. Taken together, our study suggested that sildenafil and vardenafil at half of their adult human doses completely reversed osteopenia in OVX mice by an osteogenic mechanism that was associated with enhanced skeletal vascularity.

Interactive role of exogenous 24 Epibrassinolide and endogenous NO in Brassica juncea L. under salinity stress: Evidence for NR-dependent NO biosynthesis

The present study unravels origin of nitric oxide (NO) and the interaction between 24-Epibrassinolide (EBL) and nitrate reductase (NR) for NO production in Indian mustard (Brassica juncea L.) under salinity stress. Two independent experiments were performed to check whether (i) Nitrate reductase or Nitric oxide synthase takes part in the biosynthesis of endogenous NO and (ii) EBL has any regulatory effect on NR-dependent NO biosynthesis in the alleviation of salinity stress. Results revealed that NR-inhibitor tungstate significantly (P ≤ 0.05) decreased the NR activity and endogenous NO content, while NOS inhibitor l-NAME did not influence NO biosynthesis and plant growth. Under salinity stress, inhibition in NR activity decreased the activities of antioxidant enzymes, increased H2O2, MDA, protein carbonyl content and caused DNA damage, implying that antioxidant defense might be related to NO signal. EBL supplementation enhanced the NR activity but did not influence NOS activity, suggesting that NR was involved in endogenous NO production. EBL supplementation alleviated the inhibitory effects of salinity stress and improved the plant growth by enhancing nutrients, photosynthetic pigments, compatible osmolytes, and performance of AsA-GSH cycle. It also decreased the superoxide ion accumulation, leaf epidermal damages, cell death, DNA damage, and ABA content. Comet assay revealed significant (P ≤ 0.05) enhancement in tail length and olive tail moment, while flow cytometry did not showed any significant (P ≤ 0.05) changes in genome size and ploidy level under salinity stress. Moreover, EBL supplementation increased the G6PDH activity and S-nitrosothiol content which further boosted the antioxidant responses under salinity stress. Taken together, these results suggested that NO production in mustard occurred in NR-dependent manner and EBL in association with endogenous NO activates the antioxidant system to counter salinity stress.

Nitric Oxide Synthase Is Involved in Follicular Development via the PI3K/AKT/FoxO3a Pathway in Neonatal and Immature Rats.

It is assumed that nitric oxide synthase and nitric oxide are involved in the regulation of female reproduction. This study aimed to assess the roles of nitric oxide synthase (NOS) in follicular development. The endothelial NOS (eNOS) inhibitor L-NAME, inducible NOS (iNOS) inhibitor S-Methylisothiourea (SMT) and NOS substrate L-arginine (L-Arg) were used in the NOS inhibition models in vivo. Neonatal female rats were treated with phosphate buffer saline (PBS, control), L-NAME (L-NG-Nitroarginine Methyl Ester, 40 mg/kg), SMT (S-Methylisothiourea, 10 mg/kg), L-NAME + SMT, or L-Arg (L-arginine, 50 mg/kg) via subcutaneous (SC) injection on a daily basis for 19 consecutive days, with the samples being collected on specific postnatal days (PD5, PD10, and PD19). The results indicated that the number of antral follicles, the activity of total-NOS, iNOS, neuronal NOS (nNOS), and eNOS, and the content of NO in the ovary were significantly (p < 0.05) increased in the L-Arg group at PD19, while those in L + S group were significantly (p < 0.05) decreased. Meanwhile, the ovarian expression in the L-Arg group in terms of p-AKT, p-FoxO3a, and LC3-II on PD19 were significantly (p < 0.05) upregulated, while the expressions of PTEN and cleaved Caspase-3 were (p < 0.05) downregulated as a result of NOS/NO generation, respectively. Therefore, the results suggest that NOS is possibly involved in the maturation of follicular development to puberty via the PI3K/AKT/FoxO3a pathway, through follicular autophagia and apoptosis mechanisms.

Head-to-head comparison of inorganic nitrate and metformin in a mouse model of cardiometabolic disease

Background/PurposeUnhealthy dietary habits contribute to the increasing incidence of metabolic syndrome and type 2 diabetes (T2D), which is accompanied by oxidative stress, compromised nitric oxide (NO) bioavailability and increased cardiovascular risk. Apart from lifestyle changes, biguanides such as metformin are the first-line pharmacological treatment for T2D. Favourable cardiometabolic effects have been demonstrated following dietary nitrate supplementation to boost the nitrate-nitrite-NO pathway. Here we aim to compare the therapeutic value of inorganic nitrate and metformin alone and their combination in a model of cardiometabolic disease. Experimental approachMice were fed control or high fat diet (HFD) for 7 weeks in combination with the NO synthase (NOS) inhibitor l-NAME to induce metabolic syndrome. Simultaneously, the mice were treated with vehicle, inorganic nitrate, metformin or a combination of nitrate and metformin in (drinking water). Cardiometabolic functions were assessed in vivo and tissues were collected/processed for analyses. Key resultsHFD + L-NAME was associated with cardiometabolic dysfunction, compared with controls, as evident from elevated blood pressure, endothelial dysfunction, impaired insulin sensitivity and compromised glucose clearance as well as liver steatosis. Both nitrate and metformin improved insulin/glucose homeostasis, whereas only nitrate had favourable effects on cardiovascular function and steatosis. Mechanistically, metformin and nitrate improved AMPK signalling, whereas only nitrate attenuated oxidative stress. Combination of nitrate and metformin reduced HbA1c and trended to further increase AMPK activation. Conclusion/ImplicationsNitrate and metformin had equipotent metabolic effects, while nitrate was superior regarding protection against cardiovascular dysfunction and liver steatosis. If reproduced in future clinical trials, these findings may have implications for novel nutrition-based strategies against metabolic syndrome, T2D and associated complications.

Poor glycemic control impairs the cardioprotective effects of red blood cells on myocardial ischemia/reperfusion injury

Red blood cells (RBCs) play an important role in the cardiac ischemia/reperfusion (I/R) injury. Cardiovascular risk factors impair the RBC function in an endothelial nitric oxide synthase (eNOS) dependent manner. However, it is unclear whether the protective role of RBCs can be rescued by modifying cardiovascular risk factors or by pharmacologic intervention. RBCs obtained from elderly patients with or without diabetes as well as from young volunteers were treated with vehicle, eNOS inhibitor l-NAME and/or arginase inhibitor nor-NOHA before loading to the coronary system of isolated murine hearts in a Langendorff system before 40 min of global ischemia. RBCs from young and healthy volunteers as well as from aged persons and elderly diabetes patients with satisfying blood glucose control improved left ventricular function upon 60 min of reperfusion with Krebs-Henseleit buffer and reduced the infarct size compared to buffer treated controls. This cardioprotective effect was abolished in RBCs from aged diabetes patients with poor blood glucose control. Treatment of RBCs from elderly diabetes patients with nor-NOHA partly rescued the cardioprotective function. Thus, effective glucose control in aged diabetes patients rescues RBC-dependent cardioprotection in an ex-vivo model of myocardial I/R injury.

Stenosis coexists with compromised \u03b11-adrenergic contractions in the ascending aorta of a mouse model of Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a rare disorder caused by a heterozygous deletion of 26–28 contiguous genes that affects the brain and cardiovascular system. Here, we investigated whether WBS affects aortic structure and function in the complete deletion (CD) mouse model harbouring the most common deletion found in WBS patients. Thoracic aortas from 3–4 months-old male CD mice and wild-type littermates were mounted in wire myographs or were processed for histomorphometrical analysis. Nitric oxide synthase (NOS) isoforms and oxidative stress levels were assessed. Ascending aortas from young adult CD mice showed moderate (50%) luminal stenosis, whereas endothelial function and oxidative stress were comparable to wild-type. CD mice showed greater contractions to KCl. However, α1-adrenergic contractions to phenylephrine, but not with a thromboxane analogue, were compromised. Decreased phenylephrine responses were not affected by selective inducible NOS blockade with 1400 W, but were prevented by the non-selective NOS inhibitor L-NAME and the selective neuronal NOS inhibitor SMTC. Consistently, CD mice showed increased neuronal NOS expression in aortas. Overall, aortic stenosis in CD mice coexists with excessive nNOS-derived NO signaling that compromises ascending aorta α1-adrenergic contractions. We suggest that increased neuronal NOS signaling may act as a physiological ‘brake’ against the detrimental effects of stenosis.

Pretreatment with cilnidipine attenuates hypoxia/reoxygenation injury in HL-1 cardiomyocytes through enhanced NO production and action potential shortening.

Myocardial ischemia/reperfusion injury worsens in the absence of nitric oxide synthase (NOS). Cilnidipine, a Ca2+ channel blocker, has been reported to activate endothelial NOS (eNOS) and increases nitric oxide (NO) in vascular endothelial cells. We examined whether pretreatment with cilnidipine could attenuate cardiac cell deaths including apoptosis caused by hypoxia/reoxygenation (H/R) injury. HL-1 mouse atrial myocytes as well as H9c2 rat ventricular cells were exposed to H/R, and cell viability was evaluated by an autoanalyzer and flow cytometry; eNOS expression, NO production, and electrophysiological properties were also evaluated by western blotting, colorimetry, and patch clamping, respectively, in the absence and presence of cilnidipine. Cilnidipine enhanced phosphorylation of eNOS and NO production in a concentration-dependent manner, which was abolished by siRNAs against eNOS or an Hsp90 inhibitor, geldanamycin. Pretreatment with cilnidipine attenuated cell deaths including apoptosis during H/R; this effect was reproduced by an NO donor and a xanthine oxidase inhibitor. The NOS inhibitor L-NAME abolished the protective action of cilnidipine. Pretreatment with cilnidipine also attenuated H9c2 cell death during H/R. Additional cilnidipine treatment during H/R did not significantly enhance its protective action. There was no significant difference in the protective effect of cilnidipine under normal and high Ca2+ conditions. Action potential duration (APD) of HL-1 cells was shortened by cilnidipine, with this shortening augmented after H/R. L-NAME attenuated the APD shortening caused by cilnidipine. These findings indicate that cilnidipine enhances NO production, shortens APD in part by L-type Ca2+ channel block, and thereby prevents HL-1 cell deaths during H/R.

The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71-257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase-dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase-positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

Role of Nitric Oxide in the Antipruritic Effect of WIN 55,212-2, a Cannabinoid Agonist.

Introduction:For centuries, cannabinoids are known to be effective in pain relief. Itch is an unpleasant sensation that provokes a desire to scratch. Since itch and pain are two sensations sharing a lot in common, we aimed to investigate whether the cannabinoid agonist WIN 55,212-2 reduces serotonin-induced scratching behavior and also observe whether modulation of Nitric Oxide (NO) production mediates the antipruritic effect of WIN 55,212-2.Methods:Scratching behavior is induced by intradermal injection of serotonin (50 μg/50 μL/mouse) to BALB/c mice. The cannabinoid agonist WIN 55,212-2 (1, 3, 10 mg/kg, IP) was given 30 min before serotonin injection. To observe the effect of NO modulation on the antipruritic effect of cannabinoids, the endothelial nitric oxide synthase (NOS) inhibitor L-NAME (3 mg/kg, IP), the neuronal NOS inhibitor 7-nitroindazole (3 mg/kg, IP), and the NO precursor L-arginine (100 mg/kg, IP) were administered together with WIN 55,212-2.Results:WIN 55,212-2 reduced serotonin-induced scratches at higher doses (3, 10 mg/ kg; P<0.0001). The endothelial NOS inhibitor L-NAME, the neuronal NOS inhibitor 7-nitroindazole, and the nitric oxide precursor L-arginine did not influence the antipruritic action of WIN 55,212-2. When NO modulators were used alone, only the neuronal NOS inhibitor 7-nitroindazole attenuated serotonin-induced scratches (P<0.0001).Conclusion:Our findings indicate that exogenous cannabinoids may attenuate serotonininduced scratches and NO does not mediate the antipruritic effect of WIN 55,212-2. On the other hand, neuronal NOS inhibition may play a role in the production of serotonin-induced scratches.

Effects of NO/cGMP inhibitors in a rat model of anaphylactoid shock

Anaphylactic shock can be defined as an acute syndrome, and it is the most severe clinical manifestation of allergic diseases. Anaphylactoid reactions are similar to anaphylactic events but differ in the pathophysiological mechanism. Nitric oxide (NO) inhibitors during anaphylaxis suggest that NO might decrease the signs and symptoms of anaphylaxis but exacerbate associated vasodilation. Therefore, blocking the effects of NO on vascular smooth muscle by inhibiting the guanylate cyclase (GC) would be a reasonable strategy. This study aimed to investigate the effects of NO/cGMP pathway inhibitors methylene blue (MB), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and indigo carmine (IC) in shock induced by compound 48/80 (C48/80) in rats. The effect was assessed by invasive blood pressure measurement. Shock was initiated by C48/80 intravenous bolus injection 5 min before (prophylactic) or after (treatment) the administration of the inhibitors MB (3 mg/kg), L-NAME (1 mg/kg), and IC (3 mg/kg). Of the groups that received drugs as prophylaxis for shock, only the IC group did not present the final systolic blood pressure (SBP) better than the C48/80 group. Regarding shock treatment with the drugs tested, all groups had the final SBP similar to the C48/80group. Altogether, our results suggested that inhibition of GC and NO synthase in NO production pathway was not sufficient to revert hypotension or significantly improve survival.

The basal release of endothelium-derived catecholamines regulates the contractions of Chelonoidis carbonaria aorta caused by electrical-field stimulation.

ABSTRACTThe contractions of Chelonoidis carbonaria aortic rings induced by electrical field stimulation (EFS) are not inhibited by blockade of the voltage-gated sodium channels by tetrodotoxin but almost abolished by the α1/α2-adrenoceptor antagonist phentolamine. The objective of this study was to identify the mediator(s) responsible for the EFS-induced contractions of Chelonoidis carbonaria aortic rings. Each ring was suspended between two wire hooks and mounted in isolated 10 ml organ chambers filled with oxygenated and heated Krebs-Henseleit's solution. Dopamine, noradrenaline and adrenaline concentrations were analysed by liquid chromatography coupled to tandem mass spectrometry. The contractions caused by dopamine and EFS were done in absence and presence of the nitric oxide (NO) synthesis inhibitor L-NAME, the NO-sensitive guanylyl cyclase inhibitor ODQ, the D1-like receptor antagonist SCH-23390, the D2-like receptor antagonists risperidone, quetiapine, haloperidol, and the tyrosine hydroxylase inhibitors salsolinol and 3-iodo-L-tyrosine. Basal concentrations of dopamine, noradrenaline and adrenaline were detected in Krebs-Henseleit solution containing the aortic rings. The catecholamine concentrations were significantly reduced in endothelium-denuded aortic rings. L-NAME and ODQ significantly potentiated the dopamine-induced contractions. The D2-like receptor antagonists inhibited the EFS-induced contractions of the aortic rings treated with L-NAME, whereas SCH 23390 had no effect. Similar results were observed in the contractions induced by dopamine in L-NAME treated aortic rings. These results indicate that catecholamines released by endothelium regulate the EFS-induced contractions. This may constitute a suitable mechanism by which reptilia modulate specific organ blood flow distribution.This paper has an associated First Person interview with the first author of the article.

The study of the endothelial protective properties of the L-norvaline combination with mexidol in the simulation of L-NAME-induced NO deficiency

Introduction: The endothelium is considered as a target for the prevention and treatment of pathological processes leading to cardiovascular diseases.&#x0D; Materials and methods: Endothelial dysfunction was simulated in male rats using an e-NOS inhibitor L-NAME (25 mg/kg/day intraperitoneally, 7 days). This pathology was then corrected by administering mexidol at a dose of 60 mg/kg and L-norvaline at a dose of 10 mg/kg, as well as the combination of both at the same doses, intragastrically, through an atraumatic probe, once a day.&#x0D; Results and discussion: A combination of L-norvaline at a dose of 10 mg/kg and mexidol at a dose of 60 mg/kg against the background of L-NAME-induced endothelial dysfunction has a pronounced endothelial protective effect. This is reflected in the prevalence of endothelial-dependent vascular relaxation, a significant decrease in the coefficient of endothelial dysfunction, as well as the prevention of a decline in the concentrations of stable nitric oxide metabolites and of a decrease in the level of the inflammatory changes marker in the vascular wall. The combination of L-norvaline and mexidol, with different pathogenetic action mechanisms, has a pronounced endothelial protective effect because of their antioxidant properties (mexidol) and donation of nitric oxide (L-arginine).&#x0D; Conclusion: The investigated drugs showed pronounced endothelial protective activity and can be recommended for further pre-clinical studies.

Fibrauretine reduces ischemia/reperfusion injury via RISK/eNOS activation.

Current studies have shown that fibrauretine can be used in the treatment of cardiovascular diseases; however, the protective mechanism of fibrauretine in cardiovascular diseases is unclear. The aim of this study was to investigate the effect and mechanism of fibrauretine in acute myocardial ischemia-reperfusion injury. We investigated the effects of glucocorticoid receptor/oestrogen receptor (GR/ER)-mediated Akt phosphorylation, extracellular regulated protein kinase (ERK1/2) activation and nitric oxide (NO) on the treatment of acute myocardial ischemia-reperfusion injury by fibrauretine. Myocardial ischemia-reperfusion (I/R) injury models were established in rats and gene-knockout mice, and the infarct size was measured. We detected the expression and phosphorylation of phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), glucocorticoid receptor, oestrogen receptor, lactate dehydrogenase (LDH), creatine phosphokinase (CK-MB), stress-activated protein kinase (JNK), P38 protein kinase (P38 MAPK) and nitric oxide synthase (NOS) with or without the inhibitors to investigate the protective effect of fibrauretine on the heart. The results showed that fibrauretine can significantly reduce the myocardial infarction area in myocardial I/R injury, inhibit the activities of LDH and CK-MB in the serum, and increase the content of NO. However, the effects of fibrauretine on the reduction of the myocardial infarction area were eliminated by the PI3K inhibitor LY294002, Akt inhibitor IV, GR inhibitor RU468, ER inhibitor tamoxifen, eNOS inhibitor L-NAME and ERK1/2 inhibitor U0126. Moreover, in the case of WT mice and gene-knockout eNOS and iNOS mice, fibrauretine was able to significantly reduce the myocardial infarction area in iNOS-/- and wild type mice. However, there was no significant protective effect of fibrauretine in eNOS-/- mice. It is suggested that eNOS plays an important role in the protective effect of fibrauretine on the heart. Therefore, the results of this study show that the protective effect of fibrauretine on myocardial I/R injury is closely associated with eNOS expression, GR/ER-induced Akt phosphorylation and ERK1/2 activation.

Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G

Microglia population is primarily determined by a finely-regulated proliferation process during early development of the central nervous system (CNS). Nitric oxide (NO) is known to inhibit proliferation in numerous cell types. However, how NO signaling regulates microglia proliferation remains elusive. Using wildtype (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice, this study investigated the role and underlying mechanisms of iNOS/NO signaling in microglia proliferation. Here we reported that iNOS-/- mice displayed significantly more BrdU-labeled proliferating microglia in the cortex than that in WT mice at postnatal day 10. Compared to microglia isolated from WT mouse cortex, significantly more iNOS-/- microglia displayed the specific cell-cycle markers Ki67 and phospho-histone H3 (pH3) in their nuclei. In addition, treating WT microglia with the NOS inhibitor LNAME drastically increased the percentage of cells expressing Ki67 and pH3, whereas treating iNOS-/- microglia with NOC18, a slow-release NO-donor, significantly decreased the percentage of microglia expressing the two cell-cycle markers. Moreover, inhibition of protein kinase-G (PKG) in WT microglia increased the proportion of microglia expressing Ki67 and pH3, whereas activation of PKG signaling using 8Br-cGMP in iNOS-/- microglia significantly decreased the fraction of microglia displaying Ki67 and pH3. Interestingly, in the presence of a PKG inhibitor, NOC18 increased the quantity of iNOS-/- microglia expressing Ki67 and pH3. Together, these results indicate that basal activity of iNOS/NO signaling impedes microglial cell-cycle progression and attenuates proliferation through activation of the cGMP-PKG pathway. However, NO increases microglia cell-cycle progression in the absence of cGMP-PKG signaling.