Intracellular Nitric Oxide Production Research Articles

Red blood cell eNOS: a major player in regulating cardiovascular health.

Red blood cells (RBCs) have traditionally been seen as simple carriers of gases and nutrients in the body. One important non-canonical function of RBCs in the cardiovascular system is the regulation of nitric oxide (NO) metabolism. It was shown that RBCs can scavenge NO, transport NO metabolites, and produce NO in hypoxic conditions, thereby inducing hypoxic vasodilation. RBCs also express an endothelial nitric oxide synthase (eNOS). However, its physiological significance has been controversial for many years. This review article provides a comprehensive overview of the experimental research on RBC eNOS signaling in vivo. These data show that RBC eNOS signaling modulates intracellular NO production and NO-heme levels, as well as extracellular paracrine NO metabolite signaling, which contributes to regulating peripheral vascular resistance, blood pressure, and cardioprotection. Additionally, this article explores the potential intracellular molecular mechanisms and the implications of RBC eNOS in cardiovascular health and disease.

Neuroprotective effects of hesperetin on H2O2-induced damage in neuroblastoma SH-SY5Y cells.

Oxidative stress is a fundamental neurodegenerative disease trigger that damages and decimates nerve cells. Neurodegenerative diseases are chronic central nervous system disorders that progress and result from neuronal degradation and loss. Recent studies have extensively focused on neurodegenerative disease treatment and prevention using dietary compounds. Heseperetin is an aglycone hesperidin form with various physiological activities, such as anti-inflammation, antioxidant, and antitumor. However, few studies have considered hesperetin's neuroprotective effects and mechanisms; thus, our study investigated this in hydrogen peroxide (H2O2)-treated SH-SY5Y cells. SH-SY5Y cells were treated with H2O2 (400 µM) in hesperetin absence or presence (10-40 µM) for 24 h. Three-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assays detected cell viability, and 4',6-diamidino-2-phenylindole staining allowed us to observe nuclear morphology changes such as chromatin condensation and apoptotic nuclei. Reactive oxygen species (ROS) detection assays measured intracellular ROS production; Griess reaction assays assessed nitric oxide (NO) production. Western blotting and quantitative polymerase chain reactions quantified corresponding mRNA and proteins. Subsequent experiments utilized various non-toxic hesperetin concentrations, establishing that hesperetin notably decreased intracellular ROS and NO production in H2O2-treated SH-SY5Y cells (P < 0.05). Furthermore, hesperetin inhibited H2O2-induced inflammation-related gene expression, including interluekin-6, tumor necrosis factor-α, and nuclear factor kappa B (NF-κB) p65 activation. In addition, hesperetin inhibited NF-κB translocation into H2O2-treated SH-SY5Y cell nuclei and suppressed mitogen-activated protein kinase protein expression, an essential apoptotic cell death regulator. Various apoptosis hallmarks, including shrinkage and nuclear condensation in H2O2-treated cells, were suppressed dose-dependently. Additionally, hesperetin treatment down-regulated Bax/Bcl-2 expression ratios and activated AMP-activated protein kinase-mammalian target of rapamycin autophagy pathways. These results substantiate that hesperetin activates autophagy and inhibits apoptosis and inflammation. Hesperetin is a potentially potent dietary agent that reduces neurodegenerative disease onset, progression, and prevention.

Palmitic acid promotes endothelial-to-mesenchymal transition via activation of the cytosolic DNA-sensing cGAS-STING pathway

Elevated levels of plasma free fatty acids (FFAs) lead to endothelial dysfunction, a process that is involved in the pathogenesis of atherosclerosis. Endothelial-to-mesenchymal transformation (EndMT) has been reported to accelerate endothelial dysfunction during the process of atherosclerosis. However, the underlying mechanisms of EndMT remain poorly understood. The present study aimed to investigate the role of the cytosolic DNA-sensing cyclic GMP-AMP synthase-stimulator interferon gene (cGAS-STING) pathway in palmitic acid (PA)-induced EndMT. Human aortic endothelial cells (HAECs) were exposed to different concentrations of PA, and subsequently its effects on EndMT and the cGAS-STING pathway were assessed. To investigate the role of cGAS-STING pathway on PA-induced EndMT, RNA interference was used to knockdown the expression of cGAS in HAECs prior to their exposure to PA. First, it was observed that PA reduced cell viability and intracellular nitric oxide production, and increased migratory capacity of the HAECs as well as the cellular oxidative stress response, leading to EndMT. Moreover, it was observed that the cGAS-STING pathway was activated in PA-exposed primary HAECs. Activating cGAS-STING pathway via mtDNA directing lead to EndMT in HAECs. Interestingly, cGAS knockdown by RNA interference attenuated PA-induced inflammation, oxidative stress and EndMT in HAECs. Taken together, the results of the present study suggested that the cytosolic DNA-sensing cGAS-STING pathway may have important roles in PA-induced EndMT in endothelial cells.

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells.

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitativereal-time polymerase chain reaction(qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.

Nicotinamide Attenuates UV-Induced Stress Damage in Human Primary Keratinocytes from Cancerization Fields

UVB radiation directly damages DNA, increases ROS and nitric oxide (NO) release, and promotes inflammation leading to genomic instability and cell death. Nicotinamide (NAM) is the precursor of NAM adenine dinucleotide, essential for cell energy production and DNA damage repair. NAM protects HaCat cells from UV-induced impairment; however, little is known about its effects on human primary keratinocytes and those isolated from field cancerization (i.e., field cancerization human primary keratinocytes [FC-HPKs]). We examined the role of NAM against UV-induced oxidative stress damages in FC-HPKs, isolated from precancerous lesions and skin cancers, and in normal human epidermal keratinocytes. Cells were treated for 18, 24, and 48 hours with NAM (5, 25, and 50 μM, respectively) before UVB irradiation. FC-HPK showed four-fold higher basal ROS levels than normal human epidermal keratinocytes; NAM downregulated ROS production only in irradiated FC-HPKs, which showed a greater sensibility to UV rays. UV exposure increased OGG1, inducible nitric oxide synthase, and IL-1β expression, an effect counteracted by NAM pretreatment. Intracellular nitric oxide production and DNA damages were inhibited by NAM exposure before irradiation. Collectively, our findings indicate that pretreatment with 25 μM NAM 24 hours before UVB irradiation effectively prevents oxidative stress formation, DNA damage, and inflammation in both normal human epidermal keratinocytes and FC-HPKs.

Large magnitude of force leads to NO-mediated cell shrinkage in single osteocytes implying an initial apoptotic response

Damage accumulation in the bone under continuous daily loading causes local mechanical overloading known to induce osteocyte apoptosis, which promotes bone resorption to repair bone damage. However, only a few studies have investigated the mechanism of apoptosis in mechanically overloaded osteocytes. As mechanically stimulated osteocytes produce nitric oxide (NO), which triggers apoptosis in various cell types, we aimed to elucidate the mechanism underlying apoptosis in mechanically overloaded osteocytes, focusing on intracellular NO. To investigate the effects of force magnitude on apoptosis and intracellular NO production, we isolated osteocytes from DMP1-EGFP mice and subjected them to quantitative local forces via fibronectin-coated micro beads targeting integrin on the cell surface using a magnetic tweezer. Cell shrinkage was microscopically examined, and intracellular NO production was visualized using DAR-4 M. Mechanical stimulation revealed relationships between force magnitude, apoptosis, and intracellular NO production. The application of a smaller force resulted in no significant cell shrinkage or intracellular NO production; however, a larger force caused a rapid increase in intracellular NO production followed by cell shrinkage. Besides, intracellular NOS (NO synthase) inhibition and NO donation revealed the pro-apoptotic roles of NO in osteocytes. L-NAME (NOS inhibitor)-treated cells displayed no significant shrinkage under a larger force, whereas SNP (NO donor)-treated cells showed cell shrinkage and Annexin V fluorescence, indicating apoptosis. Collectively, our study demonstrates that larger force leads to NO production-mediated osteocyte shrinkage, implying an initial apoptotic response and highlighting the importance of NO production in bone damage.

A Sulfated Polysaccharide from Saccharina japonica Suppresses LPS-Induced Inflammation Both in a Macrophage Cell Model via Blocking MAPK/NF-κB Signal Pathways In Vitro and a Zebrafish Model of Embryos and Larvae In Vivo.

Inflammation is a complicated host-protective response to stimuli and toxic conditions, and is considered as a double-edged sword. A sulfated Saccharina japonica polysaccharide (LJPS) with a sulfate content of 9.07% showed significant inhibitory effects against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells and zebrafish. Its chemical and structural properties were investigated via HPLC, GC, FTIR, and NMR spectroscopy. In vitro experiments demonstrated that LJPS significantly inhibited the generation of nitric oxide (NO) and prostaglandin E2 (PGE2) via the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and suppressed pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β production via the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways in LPS-induced RAW 264.7 cells. Moreover, LJPS showed strong protective effects against LPS-induced inflammatory responses in zebrafish, increasing the survival rate, reducing the heart rate and yolk sac edema size, and inhibiting cell death and the production of intracellular reactive oxygen species (ROS) and NO. Its convenience for large-scale production and significant anti-inflammatory activity indicated the potential application of LJPS in functional foods, cosmetics, and pharmaceutical industries.

β-hydroxybutyrate (β-HB) exerts anti-inflammatory and antioxidant effects in lipopolysaccharide (LPS)-stimulated macrophages in Liza haematocheila

Poly-β-hydroxybutyrate (PHB) can be hydrolyzed to β-hydroxybutyrate (β-HB) in the intestinal tract of animals, and dietary PHB supplementation could enhance the immunity and disease resistance of aquatic animals. Antioxidant system is responsive to PHB stimuli via MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. However, the precise immunopotentiation mechanism needs further study. In this study, macrophages from spleen in Liza haematocheila was used to study the effect of β-HB on cell viability and antioxidant function to illustrate the immunopotentiation mechanism of PHB. The results showed that β-HB (100 μg/mL) promoted the viability of macrophages and balanced the production of reactive oxygen species, but inhibited the excessive production of intracellular nitric oxide. In order to further explore the immunopotentiation mechanism of β-HB, LPS (100 μg/mL) was used to induce the inflammation and investigated the inhibitory effect of β-HB on inflammation. The results showed that LPS could induce inflammation successfully, and β-HB exerted anti-inflammatory and antioxidant effects in LPS-stimulated macrophages. Compared with LPS stimuli alone, the expression of anti-inflammatory genes NF-κBIA, MAP3K8 and TLR5 in β-HB pretreatment group was up-regulated, and the expression of pro-inflammatory genes TNFSF6, TNF-α, PI3K, NF-κB and TLR1 down-regulated. It suggested that β-HB inhibited the inflammatory response by up-regulation of anti-inflammatory genes such as NF-κBIA, thereby enhancing the immunity of the body.

Natural products with skin - Whitening effects

UV radiation is widely considered as a major cause of skin pigmentation. Upon exposure to UV radiation, the melanocytes increase the production of intracellular nitric oxide, which triggers signal transduction cascades to initiate melanogenesis by tyrosinase. UV radiation also influences melanogenesis through a paracrine regulation process involving the keratinocytes. Although a number of hypopigmenting products have been developed, those from natural sources are preferred and will predominate in the cosmetics market. More active compounds such as phenols, flavonoids, coumarins and other derivatives have been identified from natural sources. This article summarized natural skin whitening products involving tyrosinase blockers like phenols and polyphenols, and non-tyrosinase blockers like α-MSH, melanosome transferase and cytokine inhibitors. In addition, the skin-whitening effects and the mechanisms of these natural products were also reviewed. Most of the compounds achieve the hypopigmenting effect by inhibiting tyrosinase; however, some interesting compounds blocking the upstream regulation points of melanogenesis are extraordinarily promising for developing novel whitening agents.

Chemometrics-Assisted Identification of Anti-Inflammatory Compounds from the Green Alga Klebsormidium flaccidum var. zivo.

The green alga Klebsormidium flaccidum var. zivo is a rich source of proteins, polyphenols, and bioactive small-molecule compounds. An approach involving chromatographic fractionation, anti-inflammatory activity testing, ultrahigh performance liquid chromatography-mass spectrometry profiling, chemometric analysis, and subsequent MS-oriented isolation was employed to rapidly identify its small-molecule anti-inflammatory compounds including hydroxylated fatty acids, chlorophyll-derived pheophorbides, carotenoids, and glycoglycerolipids. Pheophorbide a, which decreased intracellular nitric oxide production by inhibiting inducible nitric oxide synthase, was the most potent compound identified with an IC50 value of 0.24 µM in lipopolysaccharides-induced macrophages. It also inhibited nuclear factor kappaB activation with an IC50 value of 32.1 µM in phorbol 12-myristate 13-acetate-induced chondrocytes. Compared to conventional bioassay-guided fractionation, this approach is more efficient for rapid identification of multiple chemical classes of bioactive compounds from a complex natural product mixture.

Local Delivery of Dual MicroRNAs in Trilayered Electrospun Grafts for Vascular Regeneration.

Globally growing problems related to cardiovascular diseases lead to a considerable need for synthetic vascular grafts. For small-caliber vascular prosthesis, it remains essential to fulfill rapid endothelialization, inhibit intimal hyperplasia, and prevent calcification for keeping patency. To modulate vascular regeneration, herein, we developed a bioactive trilayered tissue-engineered vascular graft encapsulating both microRNA-126 and microRNA-145 in the fibrous inner and middle layers, respectively. In vitro cell activities demonstrated that the trilayered electrospun membranes had significant biological advantages in enhanced growth and intracellular nitric oxide production of vascular endothelial cells, modulation of phenotypes of vascular smooth muscle cells (SMCs), and restraint of calcium deposition through fast-releasing microRNA-126 and slow-releasing microRNA-145. Histological and immunofluorescent analyses of in vivo implantation in a rat abdominal aorta interposition model suggested that the dual-microRNA-loading trilayered electrospun graft exerted a positive effect on accelerating endothelialization, improving contractile SMC regeneration, and promoting normal extracellular matrix formation. Meanwhile, the local bioactivity of microRNA-126 and microRNA-145 in the trilayered vascular graft could regulate inflammation and depress calcification possibly by facilitating transformation of macrophages into the anti-inflammatory M2 phenotype. These findings indicated that the trilayered electrospun graft by local delivery of dual microRNAs could be possibly used as a bioactive substitute for replacement of artificial small-caliber blood vessels.

Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte.

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host.OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated.METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry.FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines.MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Viability and intracellular nitric oxide generation in the umbilical cord blood CD34+CD133- and CD34+CD133+ cell populations exposed to local anaesthetics.

Local anesthetics (LAs) are capable of influencing cell viability in systemic immunity and may also modify metabolism of those present in umbilical cord blood (UCB) following obstetric neuraxial analgesia and anaesthesia. Data regarding UCB immature cells, important for the neonate and critical for putative UCB transplantations, are lacking. LAs are capable of stimulating intracellular nitric oxide (NO) in human neutrophils; no information is available concerning newly perpetuated cells and its potential association with viability. The study aimed at assessing the LAs influence on the cell viability and intracellular NO production by UCB CD34+CD133– and CD34+ CD133+ cell populations. Mononuclear cells separated from UCB samples (n = 19) were incubated with bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM), and ropivacaine (0.0007, 0.007, 1.4 mM) for 4 h. Flow cytometry was applied for the assessment of cell viability and intracellular NO generation in CD34+CD133– and CD34+CD133+ cell populations using annexinV/7-AAD and DAF-2DA stainings, respectively. CD34+CD133+ cells showed less pronounced late apoptosis and necrosis as compared to CD34+CD133-population. Intracellular NO generation was comparable between both cell populations studied. LAs neither influenced cell viability nor changed NO production in either population. LAs do not interfere with viability and intracellular NO generation in the UCB CD34+CD133– and CD34+CD133+ cell populations.

Measuring stimulation and inhibition of intracellular nitric oxide production in SIM-A9 microglia using microfluidic single-cell analysis.

Chronic neuroinflammation has long been considered to be a central factor in accelerating the progression of neurodegenerative diseases such as Alzheimer's diseases, Parkinson's disease and chronic traumatic encephalopathy. Under pathological conditions microglia produce inflammatory signaling molecules, such as nitric oxide (NO), that can damage DNA and proteins and ultimately induce neuronal apoptosis. One strategy for treating neurodegenerative diseases is to specifically target NO production through inhibition of inducible nitric oxide synthase (iNOS). However, accurately measuring changes in microglial NO production in response to potential therapeutics is challenging due to NO's short half-life and microglial heterogeneity. In this paper we report the application of a microfluidic device for the high-throughput measurement of intracellular NO in SIM-A9 microglial cells. NO production was measured in response to treatment with lipopolysaccharides (LPS) and interferon gamma (IFN-γ) with and without a potent iNOS inhibitor (1400 W dihydrochloride). Cells were labeled with a fluorogenic NO probe, 4-amino-5-methylamino-2',7'-difluorofluoescein diacetate (DAF-FM DA), and 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. Separation and quantitation of intracellular NO was achieved using microchip electrophoresis and laser induced fluorescence detection (LIF). Statistical analysis suggests that the populations fit a lognormal distribution and are better represented by their geometric mean values. Comparison of the geometric means indicated a 1.6-fold increase in NO production between untreated and stimulated cells and a decrease by a factor of approximately 0.5 comparing stimulated and inhibited cells. Additionally, we report experimental data demonstrating the improvement in the sensitivity of our integrated optical fiber-based detection system through the use of refractive index matching gel.

Role of Nox4 in Angiotensin II‐Mediated Changes in Volume Dynamics and Nitric Oxide Production in Podocytes

Nitric oxide (NO) is a powerful vasodilator that serves important functions in the kidney such as regulation of renal hemodynamics, pressure natriuresis, and diuresis. Significant reduction in NO production has been found to play a role in the pathogenesis of hypertension. Our recent studies revealed that Angiotensin II (Ang II), in addition to causing a release of calcium, stimulates NO production in the glomerular podocytes. This signaling pathway can be also triggered by extracellular application of hydrogen peroxide (H2O2) and is significantly reduced with the development of salt‐sensitive hypertension and kidney injury in Dahl salt‐sensitive (SS) rats (Palygin et al., Nitric Oxide, 2018). Recently, using a knock out of NADPH oxidase 4 (Nox4) in SS rats (SSNox4−/−), one of the primary source of renal reactive oxygen species (ROS) and specifically H2O2 we described the reduction in intracellular calcium activation in podocytes as well as H2O2 production in isolated glomeruli in response to Ang II (Ilatovskaya et al., J Am Soc Nephrol. 2018). Overall, SSNox4−/− rats displayed a protective phenotype with a significant reduction of albuminuria, glomeruli injury and reduction in the elevation of blood pressure under high salt challenge (Cowley et al., Hypertension, 2016). Here we used this model to characterize the effects of alterations in the balance in the relationship between ROS and NO production in podocytes and establish the relation between Nox4 and glomerular/podocyte volume dynamics. The in vivo concentration of H2O2, measured by biosensors amperometry as catalase sensitive current, was significantly reduced in both cortex and medullar parts of the SSNox4−/− rat kidney compared to wild type SS rats, indicating a direct association between Nox4 and the production of renal ROS. We found that acute application of Ang II promoted opposite changes in glomeruli and podocyte volumes, where glomeruli volume decreased, and podocyte volume increased approximately by 18% in SS rats. Overall, SSNox4−/− rats exhibited an increase in total NO production (by 16% to compare with SS), calculated using confocal microscopy as a total integral of NO fluorescent transient (DAF‐FM dye) in the population of podocytes within a freshly isolated glomerulus. In SSNox4−/− rats the elevation in NO together with declining in H2O2 levels directly correlated with an increase of podocyte volume in response to Ang II (13% vs. SS control). We anticipate a rapid podocyte volume change dependent on intracellular NO production and reflecting the ability of these cells to dynamically regulate the permeability of glomeruli filtration barrier. Thus far, we can conclude that in the absence of Nox4 the podocytes shift the ROS production in response to Ang II towards the increase of intracellular NO bioavailability and a decrease of renal H2O2 production. This mechanism may play a key role in the protection of glomeruli form oxidative stress and elevation of blood pressure during the development of salt‐sensitive hypertension.Support or Funding InformationNHLBI R35 HL135749; P01 HL116264; AHA 18PRE34030127; ADA 1‐15‐BS‐172; DK‐020595 Pilot &amp; Feasibility projectThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

167 Improvement in SHIM Scores with the iNOS Stimulator, Revactin®

Revactin®, a mixture of three components found in nature (ginger root, muira puama and Paullinia cupana) combined with the amino acid, L-citrulline, has been shown both in vivo and in vitro to act directly on the corporal smooth muscle cells (CSMC) to upregulate the production of the inducible nitric oxide synthase (iNOS) enzyme. This results in an increase in the intracellular production of nitric oxide (NO) and cGMP which has been shown in the rat to act in an anti-apoptotic and anti-oxidative manner and, when given long-term, leads to an improvement in the CSMC to collagen ratio and an improvement in erectile function . A recently reported short-term pilot study conducted in men primarily in their 7th decade of life demonstrated that the Revactin® was not only safe but surprisingly also elicited some early improvement in their erectile function. To examine the efficacy of Revactin® on early erectile function among middle-aged men with erectile dysfunction.

Genotoxic and anti-proliferative effects of aminoguanidine on gamma-irradiated MCF-7 breast cancer cells.

The intracellular production of nitric oxide is studied as a relevant phenomenon in exposure to ionizing radiation. There is evidence of local nitric oxide production in solid tumours. Its effects were observed on the relationship between their presence with tumor progression, linked to the emergence of potential genotoxic or cytotoxic damage, or loss of proliferative capacities of tumour cells. The study evaluated the effects of the administration of aminoguanidine, a selective inhibitor of an isoform of nitric oxide synthase on the frequency of genotoxic damage, loss of clonogenic potential, induction of cytotoxicity and nitrite production after exposure to ionizing radiation in radiotherapeutic doses. Human breast tumor (MCF7) cells were treated with aminoguanidine (1 or 2 mM) and irradiated by gamma radiation at doses between 0.5 and 8Gy. The study used a well stablished technique with some modifications for evaluation of genotoxic damage by frequency of micronuclei in binucleated cells. In cultures treated with 1 mM, we observed increased cytotoxicity and genotoxicity, and reduction of the clonogenic potential of the colonies. Alternatively, 2 mM aminoguanidine produced the opposite effect, apparently protecting cultures from the effects of exposures. The experiments suggested that the administration of aminoguanidine may reduce the in vitro radiossensitivity of tumors due to the increase of the frequency of genotoxic damage.

Modulation of Common Carotid Arterial Function by Exercise: A Hemodynamics Study

Common carotid arteries (CCAs) are the major arteries supplying blood to the brain, and the hemodynamic variables in which are closely associated with the cardiovascular diseases. Exercise can induce the hemodynamic responses in the CCAs, including variations in blood pressure, circumferential stretch, and wall shear stress (WSS). Mechanosensors in the endothelial cells (ECs) are able to sense and distinguish these variations as mechanical signal, and transmit them into the interior of cells to affect cellular morphology and gene expression. Notably, reasonable exercises improve arterial structure and function, while unreasonable exercises cause endothelial dysfunction. Therefore, studies on the modulation of common carotid arterial structure and function by exercises are quite necessary, and it’s significant to choose reasonable exercise modalities for improving arterial structure and function and preventing cardiovascular diseases. In this work, firstly, we studied the acute and chronic effects of different exercise modalities on the carotid arterial elasticity and hemodynamic variables. The results showed that the acute exercise caused the increases in arterial elastic modulus, blood pressure and the magnitude and frequency of WSS, and led to the decrease of arterial diameter; moreover, the changes in these hemodynamic variables exhibited an exercise-intensity-dependent manner. Additionally, the responses of intracellular nitric oxide (NO), reactive oxygen species (ROS) and the autophagy flux to WSS waveforms induced by different intensity exercise were also studied in a multi-component parallel-plate flow chamber system. The experimental results indicated that autophagy regulated intracellular NO and ROS production, and the magnitude and frequency of WSS induced by the moderate intensity exercise were more beneficial to improve arterial endothelial function than the high intensity exercise. Finally, the feasibility of quantitative regulation of the intracellular Ca2+ concentration in ECs by WSS was preliminarily investigated and confirmed in a microfluidic chip. In summary, our work indicated that it is feasible to choose reasonable exercise modalities to accurately modulate the hemodynamic variables, including blood pressure, blood flow and WSS in the CCAs, and then to improve the structure and function of the CCAs.

Intracellular NO-Generator Based on Enzyme Trigger for Localized Tumor-Cytoplasm Rapid Drug Release and Synergetic Cancer Therapy.

Nitric oxide (NO) is an important biological messenger implicated in tumor therapy. However, current NO release systems suffer from some disadvantages, such as hydrolysis during blood circulation, poor specificity, and robust irradiation for stimuli. Accordingly, we constructed an intracellular enzyme-triggered NO-generator to achieve tumor cytoplasm-specific disruption and localized rapid drug release. Diethylamine NONOate (DEA/NO) was used as a NO donor and conjugated with hyaluronic acid (HA) to form self-assembly micelle (HA-DNB-DEA/NO), and encapsulate chemotherapeutic agent (doxorubicin (DOX)) into its hydrophobic core (DOX@HA-DNB-DEA/NO). After HA receptor mediated internalization into tumor cells, HA shell would undergo digestion into small conjugated pieces by hyaluronidase. Meanwhile, DOX@HA-DNB-DEA/NO also responded to the intratumoral overexpressed glutathion and glutathione S-transferase π, leading to the intracellular NO production and controlled DOX rapid release. In vitro and in vivo results proved the enzyme-dependent and enhanced targeting delivery profile, and demonstrated that NO and DOX could colocate in specific tumor site, which provided a precondition for exerting their synergistic efficacy. Moreover, expression of p53 protein was upregulated in tumor tissue after treatment, indicating that NO induced cell apoptosis mediated by tumor suppressor gene p53. Overall, this intelligent drug loaded NO-generator might perform as an enhancer to realize better clinical outcomes.

Application of flow cytometry with a fluorescent dye to measurement of intracellular nitric oxide in plant cells

Main conclusionA simple and rapid method involving flow cytometry and NO-specific probe (DAF-FM DA) proved useful for detection and determination of intracellular NO production in Medicago truncatula suspension cells and leaves as well as in cells of Avena fatua, Amaranthus retroflexus embryos and leaves.The measurement of nitric oxide (NO) in plant material is important for examining the regulatory roles of endogenous NO in various physiological processes. The possibility of detecting and determining intracellular NO production by flow cytometry (FCM) with 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM DA), an NO-specific probe in Medicago truncatula cells in suspension and leaves as well as in cells of embryos and leaves of Avena fatua L. or Amaranthus retroflexus L. was explored. To detect and measure NO production by cell suspension or embryos and leaves, the recommended DAF-FM DA concentration is 5 or 10 µM, respectively, applied for 30 min. Exogenous NO increased the intensity of the fluorescent signal in embryos and leaves of both plants, while carboxy-PTIO (cPTIO), an NO scavenger, decreased it. Thus, these results demonstrate that NO can be detected and an increase and a decrease of its intracellular level can be estimated. Wounding was observed to increase the fluorescence signal, indicating an increase in the intracellular NO level. In addition, the levels of exogenous and endogenous ascorbic acid were demonstrated to have no effect on the NO-related fluorescence signal, indicating the signal’s specificity only in relation with NO. The applicability of the proposed method for detection and determination of NO was confirmed (1) by in situ NO imaging in cell suspensions and (2) by determining the NO concentration in embryos and leaves using the Griess reagent. In view of the data obtained, FCM is recommended as a rapid and simple method with which to detect and determine intracellular NO production in plant cells.