S-nitroso-N-acetyl-DL-penicillamine Research Articles

Propionate exerts neuroprotective and neuroregenerative effects in the peripheral nervous system

In inflammatory neuropathies, oxidative stress results in neuronal and Schwann cell (SC) death promoting early neurodegeneration and clinical disability. Treatment with the short-chain fatty acid propionate showed a significant immunoregulatory and neuroprotective effect in multiple sclerosis patients. Similar effects have been described for patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Therefore, Schwann cell's survival and dorsal root ganglia (DRG) outgrowth were evaluated invitro after propionate treatment and application of H2O2 or S-nitroso-N-acetyl-D-L-penicillamine (SNAP) to evaluate neuroprotection. In addition, DRG resistance was evaluated by the application of oxidative stress by SNAP exvivo after invivo propionate treatment. Propionate treatment secondary to SNAP application on DRG served as a neuroregeneration model. Histone acetylation as well as expression of the free fatty acid receptor (FFAR) 2 and 3, histone deacetylases, neuroregeneration markers, and antioxidative mediators were investigated. β-hydroxybutyrate was used as a second FFAR3 ligand, and pertussis toxin was used as an FFAR3 antagonist. FFAR3, but not FFAR2, expression was evident on SC and DRG. Propionate-mediated activation of FFAR3 and histone 3 hyperacetylation resulted in increased catalase expression and increased resistance to oxidative stress. In addition, propionate treatment resulted in enhanced neuroregeneration with concomitant growth-associated protein 43 expression. We were able to demonstrate an antioxidative and neuroregenerative effect of propionate on SC and DRG mediated by FFAR3-induced histone acetylases expression. Our results describe a pathway to achieve neuroprotection/neuroregeneration relevant for patients with immune-mediated neuropathies.

Impact of Exogenous Nitric Oxide Treatment on Vascularization of a Subcutaneous Device for Cell Transplantation

Subcutaneous polymer scaffolds have shown potential for creating an optimal transplantation site in cellular replacement therapy, e.g., when transplanting insulin-producing cells to cure type 1 diabetes. Imperative for these scaffolds is a high degree of vascularization to guarantee long-term functional cellular survival. In this study, the effect of the nitric oxide (NO) donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) on the vascularization degree of a subcutaneous poly(d,l-lactide-co-ε-caprolactone) (PDLLCL) scaffold was investigated. To this end, scaffolds were implanted under the skin of C57BL/6 mice. Each mouse received a control scaffold and a scaffold containing SNAP. At day 7, 14, and 28, the oxygen percentage within the scaffolds was measured and at day 28, the vascularization degree was determined with lectin infusion and gene expression analysis. We measured lower oxygen percentages within the scaffolds containing the NO-donor up to day 14 compared to the control scaffolds, but no differences were found at day 28. Although blood vessels in the scaffolds were well perfused, no differences between the groups were found in the lectin staining and gene expression of vascular markers, such as CD31, CD105, and VEGFa. To conclude, in this biomaterial setting, addition of a NO-donor did not improve the vascularization degree of the subcutaneous scaffold.

Cu(II)-functionalized silk fibroin films for the catalytic generation of nitric oxide.

In situ release of nitric oxide (NO) has been suggested to be a potential functionalization strategy for blood-contacting implants. In this study, the NO generation capability catalyzed by the copper ion-incorporated silk fibroin (SF) films in the presence of S-nitroso-N-acetyl-dl-penicillamine (SNAP) is demonstrated. Cu(II) is effectively bound to the surface of the SF film based on metal-protein coordination. The x-ray photoelectron spectroscopy results indicate that copper ions may exist on the surface of the SF film in the form of Cu(II)/Cu(I) coexistence. The degradation behavior showed that the bound copper ions on the surface of the SF films can maintain a slow release in phosphate-buffered saline (PBS) or collagenase IA solution for 7 days. There was no significant difference in the release of copper ions between PBS degradation and enzyme degradation. The loading of copper ions significantly improved the release of NO from SNAP through catalysis. Based on the biological effects of copper ions and the ability to catalyze the release of NO from S-nitrosothiols, copper ion loading provides an option for the construction of bioactive SF biomaterials.

−808 nm-activated Ca2+ doped up-conversion nanoparticles that release no inducing liver cancer cell (HepG2) apoptosis

A near-infrared (NIR) light-triggered release method for nitric oxide (NO) was developed utilizing core/shell NaYF4: Tm/Yb/Ca@NaGdF4: Nd/Yb up-conversion nanoparticles (UCNPs) bearing a mesoporous silica (mSiO2) shell loaded with the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP). To avoid overheating in biological samples, Nd3+ was chosen as a sensitizer, Yb3+ ions as the bridging sensitizer, and Tm3+ ions as UV-emissive activator while co-doping with Ca2+ was done to enhance the luminescence of the activator Tm3+. NO release from SNAP was triggered by an NIR-UV up-conversion process, initiated by 808 nm light absorbed by the Nd3+ ions. NO release was confirmed by the Griess method. Under 808 nm irradiation, the viability of the liver cancer cell line HepG2 significantly decreased with increasing UCNPs@mSiO2-SNAP concentration. For a UCNPs@mSiO2-SNAP concentration of 200 μg ml−1, the cell survival probability was 47%. These results demonstrate that UCNPs@mSiO2-SNAP can induce the release of apoptosis-inducing NO by NIR irradiation.

Endoplasmic Reticulum Stress Promotes iNOS/NO and Influences Inflammation in the Development of Doxorubicin-Induced Cardiomyopathy.

Doxorubicin (Dox) is known to cause heart failure in some cancer patients. Despite extensive studies over the past half century, the subcellular basis of Dox-induced cardiomyopathy (DIC) is still elusive. Earlier, we suggested that Dox causes a delayed activation of unfolded protein response (UPR) which may promote mitochondrial Bax activity leading to cardiomyocyte death. As a follow up, using NO donor, S-Nitroso-N-acetyl-d,l-penicillamine (SNAP), and/or NOS inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), we now show that endoplasmic reticulum (ER) stress promotes inflammation through iNOS/NO-induced TLR2 activation. In vivo Dox treatment increased mitochondrial iNOS to promote ER stress as there was an increase in Bip (Grp78) response, proapoptotic CHOP (DDIT3) and ER-mediated Caspase 12 activation. Increased iNOS activity is associated with an increase in TLR2 and TNF-α receptor associated factor 2 (TRAF2). These two together with NF-κB p105/50 expression and a synergistic support through ER stress, promote inflammatory response in the myocardium leading to cell death and ultimately fostering DIC conditions. In the presence of NOS inhibitor, such detrimental effects of Dox were inhibited, suggesting iNOS/NO as key mediators of Dox-induced inflammatory as well as apoptotic responses.

Repurposing Riociguat to Target a Novel Paracrine Nitric Oxide-TRPC6 Pathway to Prevent Podocyte Injury.

Increased expression and activity of the Ca2+ channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca2+ influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.

Role of nitric oxide in regulating epidermal permeability barrier function.

Nitric oxide (NO), a free radical molecule synthesized by nitric oxide synthases (NOS), regulates multiple cellular functions in a variety of cell types. These NOS, including endothelial NOS (eNOS), inducible NOS (iNOS) and neural NOS (nNOS), are expressed in keratinocytes. Expression levels of both iNOS and nNOS decrease with ageing, and insufficient NO has been linked to the development of a number of disorders such as diabetes and hypertension, and to the severity of atherosclerosis. Conversely, excessive NO levels can induce cellular oxidative stress, but physiological levels of NO are required to maintain the normal functioning of cells, including keratinocytes. NO also regulates cutaneous functions, including epidermal permeability barrier homeostasis and wound healing, through its stimulation of keratinocyte proliferation, differentiation and lipid metabolism. Topical applications of a diverse group of agents which generate nitric oxide (called NO donors) such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP) can delay permeability barrier recovery in barrier-disrupted skin, but iNOS is still required for epidermal permeability barrier homeostasis. This review summarizes the regulatory role that NO plays in epidermal permeability barrier functions and the underlying mechanisms involved.

Expression of soybean plant hemoglobin gene family under abiotic stresses

Many abiotic stresses induce the generation of nitric oxide (NO) in plant tissues, where it functions as a signal molecule in stress responses. Plants modulate NO by oxidizing it to NO3 - with plant hemoglobin (GLB), because excess NO is toxic to cells. At least eight genes encoding GLB have been identified in soybean, in three clades: GLB1, GLB2, and GLB3. However, it is still unclear which GLB genes are responsible for NO regulation under abiotic stress in soybean. We exposed soybean roots to flooding, salt, and two NO donors-sodium pentacyanonitrosylferrate (III) dihydrate (SNP) and S-nitroso-N-acetyl-d,l-penicillamine (SNAP)-and analyzed expression of GLB genes. GmGLB1, one of two GLB1 genes of soybean, significantly responded to both SNP and SNAP, and its induction was almost completely repressed by a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. GmGLB1 responded to flooding but not to salt, suggesting that it is responsible for NO regulation under NO-inducing abiotic stresses such as flooding. GmGLB3, one of two GLB3 genes of soybean, did not respond to NO donors at all but did respond to flooding, at a lower level than GmGLB1. These results suggest that flooding induces not only NO but also unknown factor(s) that induce GmGLB3 gene in soybean.

Enhanced Hemocompatibility and In Vivo Analytical Accuracy of Intravascular Potentiometric Carbon Dioxide Sensors via Nitric Oxide Release.

In this letter, the innate ability of nitric oxide (NO) to inhibit platelet activation/adhesion/thrombus formation is employed to improve the hemocompatibility and in vivo accuracy of an intravascular (IV) potentiometric PCO2 (partial pressure of carbon dioxide) sensor. The catheter-type sensor is fabricated by impregnating a segment of dual lumen silicone tubing with a proton ionophore, plasticizer, and lipophilic cation-exchanger. Subsequent filling of bicarbonate and strong buffer solutions and placement of Ag/AgCl reference electrode wires within each lumen, respectively, enables measurement of the membrane potential difference across the inner wall of the tube, with this potential changing as a function of the logarithm of sample PCO2. The dual lumen device is further encapsulated within a S-nitroso-N-acetyl-DL-penicillamine (SNAP)-doped silicone tube that releases physiological levels of NO. The NO releasing sensor exhibits near-Nernstian sensitivity toward PCO2 (slope = 59.31 ± 0.78 mV/decade) and low drift rates (<2 mV/24 h after initial equilibration). In vivo evaluation of the NO releasing sensors, performed in the arteries and veins of anesthetized pigs for 20 h, shows enhanced accuracy (vs non-NO releasing sensors) when benchmarked to measurements of discrete blood samples made with a commercial blood gas analyzer. The accurate, continuous monitoring of blood PCO2 levels achieved with this new IV NO releasing PCO2 sensor configuration could help better manage hospitalized patients in critical care units.

Contributions of Nitric Oxide to AHR-Ligand-Mediated Keratinocyte Differentiation.

Activation of the aryl hydrocarbon receptor (AHR) in normal human epidermal keratinocytes (NHEKs) accelerates keratinocyte terminal differentiation through metabolic reprogramming and reactive oxygen species (ROS) production. Of the three NOS isoforms, NOS3 is significantly increased at both the RNA and protein levels by exposure to the very potent and selective ligand of the AHR, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition of NOS with the chemical N-nitro-l-arginine methyl ester (l-NAME) reversed TCDD-induced cornified envelope formation, an endpoint of terminal differentiation, as well as the expression of filaggrin (FLG), a marker of differentiation. Conversely, exposure to the NO-donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), increased the number of cornified envelopes above control levels and augmented the levels of cornified envelopes formed in response to TCDD treatment and increased the expression of FLG. This indicates that nitric oxide signaling can increase keratinocyte differentiation and that it is involved in the AHR-mediated acceleration of differentiation. As the nitrosylation of cysteines is a mechanism by which NO affects the structure and functions of proteins, the S-nitrosylation biotin switch technique was used to measure protein S-nitrosylation. Activation of the AHR increased the S-nitrosylation of two detected proteins of about 72 and 20 kD in size. These results provide new insights into the role of NO and protein nitrosylation in the process of epithelial cell differentiation, suggesting a role of NOS in metabolic reprogramming and the regulation of epithelial cell fate.

AB0068 NOVEL CHONDROPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF THE SELECTIVE HUMAN MELANOCORTIN MC3 RECEPTOR AGONIST PG-990 ON SNAP ACTIVATED CHONDROCYTES

Background:Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4].Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear.Objectives:This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes.Methods:The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M ofn=4 samples repeated in triplicate. #p≤0.05vscontrol or *p≤0.05vsstimulus.Results:Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (#p≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (*p≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (*p≤0.05). GAG content was significantly reduced by SNAP by 29% (#p≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (*p≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (*p≤0.05).Conclusion:The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line.

Reactive Nitrogen Species Releasing Hydrogel for Enhanced Wound Healing.

Poor proliferation and migration of fibroblast, keratinocyte and endothelial cells delays the wound healing in diabetic patients and results into chronicity of wounds. Slow or decreased formation of blood vessels is another issue that increases the chronicity of non-healing wounds. These chronic wounds turn into an ulcer that may lead to limb amputation. Recently, nitric oxide (NO) has emerged as a potential agent for accelerating cell migration and proliferation to enhance wound healing. It increases the expression of necessary angiogenic growth factors which stimulates the proliferation and migration of major cell types involved in wound repair. Here we report the synthesis of chitosan (CS), polyvinyl alcohol (PVA) and a NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) to enhance the wound healing activities in chronic wounds. A three-fold increase in the proliferation of 3T3 cells was observed with NO-releasing CS-PVA hydrogels. In vitro cell migration assay demonstrated a four-fold faster migration of cells to the scratched area compared to the control group. The results depict that the use of CS-PVA hydrogel impregnated with the NO donor (SNAP) can be a promising material for promoting cell migration and subsequent accelerated healing of the chronic wounds in burns and diabetic patients.

Nitric oxide releasing chitosan-poly (vinyl alcohol) hydrogel promotes angiogenesis in chick embryo model

The lack of angiogenic activity is one of the serious complications of chronic wounds associated with delayed wound closure, chronic ulceration, and subsequent limb amputation. Multiple lines of evidence suggest that nitric oxide (NO) produced endogenously by nitric oxide synthase pathway plays a significant role in angiogenic activity and accelerates wounds closure. In this work, chitosan (CS), polyvinyl alcohol (PVA) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) hydrogel was fabricated to accelerate angiogenesis and promote healing in chronic wounds due to better wound closure potential of CS-PVA hydrogel and angiogenic properties of SNAP. The developed CS-PVA hydrogels loaded with SNAP produced a continuous and sustained supply of NO. 3T3 and HaCaT cells showed a significant increase in cell proliferation with 5‰ SNAP loaded CS-PVA hydrogel compared to the control group. Wound scratch assay resulted in four-fold faster recovery of the scratched wound area and an enhanced degree of angiogenic activity was observed in the chick embryo model with the SNAP incorporated CS-PVA hydrogel compared to the control group. The results depict that the use of CS-PVA hydrogel impregnated with SNAP could be a promising material for promoting angiogenesis followed by accelerated healing of the chronic wounds in burns and diabetic patients.

Nitric oxide accelerates germination via the regulation of respiration in chickpea.

Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.

A Novel Nitric Oxide Donor, S-Nitroso-NPivaloyl-D-Penicillamine, Activates a Non-Neuronal Cardiac Cholinergic System to Synthesize Acetylcholine and Augments Cardiac Function.

In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS. To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO₂ and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system. Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group. SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.

Maternal titanium dioxide nanomaterial inhalation exposure compromises placental hemodynamics

The fetal consequences of gestational engineered nanomaterial (ENM) exposure are unclear. The placenta is a barrier protecting the fetus and allowing transfer of substances from the maternal circulation. The purpose of this study was to determine the effects of maternal pulmonary titanium dioxide nanoparticle (nano-TiO2) exposure on the placenta and umbilical vascular reactivity. We hypothesized that pulmonary nano-TiO2 inhalation exposure increases placental vascular resistance and impairs umbilical vascular responsiveness. Pregnant Sprague-Dawley rats were exposed via whole-body inhalation to nano-TiO2 with an aerodynamic diameter of 188 ± 0.36 nm. On gestational day (GD) 11, rats began inhalation exposures (6 h/exposure). Daily lung deposition was 87.5 ± 2.7 μg. Animals were exposed for 6 days for a cumulative lung burden of 525 ± 16 μg. On GD 20, placentas, umbilical artery and vein were isolated, cannulated, and treated with acetylcholine (ACh), angiotensin II (ANGII), S-nitroso-N-acetyl-DL-penicillamine (SNAP), or calcium-free superfusate (Ca2+-free). Mean outflow pressure was measured in placental units. ACh increased outflow pressure to 53 ± 5 mmHg in sham-controls but only to 35 ± 4 mmHg in exposed subjects. ANGII decreased outflow pressure in placentas from exposed animals (17 ± 7 mmHg) compared to sham-controls (31 ± 6 mmHg). Ca2+-free superfusate yielded maximal outflow pressures in sham-control (63 ± 5 mmHg) and exposed (30 ± 10 mmHg) rats. Umbilical artery endothelium-dependent dilation was decreased in nano-TiO2 exposed fetuses (30 ± 9%) compared to sham-controls (58 ± 6%), but ANGII sensitivity was increased (−79 ± 20% vs −36 ± 10%). These results indicate that maternal gestational pulmonary nano-TiO2 exposure increases placental vascular resistance and impairs umbilical vascular reactivity.

Chronic Ethanol Consumption Impairs the Tactile-Evoked Long-Term Depression at Cerebellar Molecular Layer Interneuron-Purkinje Cell Synapses in vivo in Mice

The cerebellum is sensitive to ethanol (EtOH) consumption. Chronic EtOH consumption impairs motor learning by modulating the cerebellar circuitry synaptic transmission and long-term plasticity. Under in vitro conditions, acute EtOH inhibits both parallel fiber (PF) and climbing fiber (CF) long-term depression (LTD). However, thus far it has not been investigated how chronic EtOH consumption affects sensory stimulation-evoked LTD at the molecular layer interneurons (MLIs) to the Purkinje cell (PC) synapses (MLI-PC LTD) in the cerebellar cortex of living animals. In this study, we investigated the effect of chronic EtOH consumption on facial stimulation-evoked MLI-PC LTD, using an electrophysiological technique as well as pharmacological methods, in urethane-anesthetized mice. Our results showed that facial stimulation induced MLI–PC LTD in the control mice, but it could not be induced in mice with chronic EtOH consumption (0.8 g/kg; 28 days). Blocking the cannabinoid type 1 (CB1) receptor activity with AM-251, prevented MLI-PC LTD in the control mice, but revealed a nitric oxide (NO)-dependent long-term potentiation (LTP) of MLI–PC synaptic transmission (MLI-PC LTP) in the EtOH consumption mice. Notably, with the application of a NO donor, S-nitroso-N-Acetyl-D, L-penicillamine (SNAP) alone prevented the induction of MLI–PC LTD, but a mixture of SNAP and AM-251 revealed an MLI-PC LTP in control mice. In contrast, inhibiting NO synthase (NOS) revealed the facial stimulation-induced MLI-PC LTD in EtOH consumption mice. These results indicate that long-term EtOH consumption can impair the sensory stimulation-induced MLI–PC LTD via the activation of a NO signaling pathway in the cerebellar cortex in vivo in mice. Our results suggest that the chronic EtOH exposure causes a deficit in the cerebellar motor learning function and may be involved in the impaired MLI–PC GABAergic synaptic plasticity.

Stably Transformed Lotus japonicus Plants Overexpressing Phytoglobin LjGlb1-1 Show Decreased Nitric Oxide Levels in Roots and Nodules as Well as Delayed Nodule Senescence.

The class 1 phytoglobin, LjGlb1-1, is expressed in various tissues of the model legume Lotus japonicus, where it may play multiple functions by interacting with nitric oxide (NO). One of such functions is the onset of a proper symbiosis with Mesorhizobium loti resulting in the formation of actively N2-fixing nodules. Stable overexpression lines (Ox1 and Ox2) of LjGlb1-1 were generated and phenotyped. Both Ox lines showed reduced NO levels in roots and enhanced nitrogenase activity in mature and senescent nodules relative to the wild-type (WT). Physiological and cytological observations indicated that overexpression of LjGlb1-1 delayed nodule senescence. The application to WT nodules of the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP) or the phytohormones abscisic acid (ABA) and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) repressed nitrogenase activity, induced the expression of three senescence-associated genes and caused cytological changes evidencing nodule senescence. These effects were almost completely reverted by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Our results reveal that overexpression of LjGlb1-1 improves the activity of mature nodules and delays nodule senescence in the L.japonicus-M.loti symbiosis. These beneficial effects are probably mediated by the participation of LjGlb1-1 in controlling the concentration of NO that may be produced downstream in the phytohormone signaling pathway in nodules.

Individual variations in platelet reactivity towards ADP, epinephrine, collagen and nitric oxide, and the association to arterial function in young, healthy adults

IntroductionPlatelet aggregation and secretion can be induced by a large number of endogenous activators, such as collagen, adenosine diphosphate (ADP) and epinephrine. Conversely, the blood vessel endothelium constitutively release platelet inhibitors including nitric oxide (NO) and prostacyclin. NO and prostacyclin are also well-known vasodilators and contribute to alterations in local blood flow and systemic blood pressure. Materials and methodsIn this study we investigated individual variations in platelet reactivity and arterial functions including blood pressure and flow-mediated vasodilation (FMD) in 43 young, healthy individuals participating in the Lifestyle, Biomarkers and Atherosclerosis (LBA) study. Platelet aggregation and dense granule secretion were measured simultaneously by light transmission and luminescence. FMD was measured with ultrasound. ResultsThe platelet function assay showed inter-individual differences in platelet reactivity. Specifically, a sub-group of individuals had platelets with an increased response to low concentrations of ADP and epinephrine, but not collagen. When the NO-donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) was combined with high doses of these platelet activators, the results indicated for sub-groups of NO-sensitive and NO-insensitive platelets. The individuals with NO-sensitive platelets in response to SNAP in combination with collagen had a higher capacity of FMD of the arteria brachialis. ConclusionsPlatelet reactivity towards ADP, epinephrine and NO differs between young, healthy individuals. Some individuals have a more effective response towards NO, both in the aspect of platelet inhibition ex vivo, as well as vasodilation in vivo.

Influence of NO and [Ca2+]o on [Ca2+]i homeostasis in rat ventricular cardiomyocytes

In this study, we explored the effect of NO-induced changes in [Ca2+]i homeostasis in rat ventricular cardiomyocytes through variation in extracellular Ca2+ and application of SNAP (S-nitroso-N-acetyl-d,l-penicillamine) as an NO-donor. The Fura-2 signal dynamics and phalloidin intensity profile of z-disks in rat cardiomyocytes served as endpoints to describe the mechanisms involved in the control of the intracellular Ca2+ levels, depending on the kinetics of distribution of the SNAP-produced NO. The results demonstrated that SNAP caused small phalloidin intensity profile changes between the z-lines in the presence of [Ca2+]o. However, in the absence of [Ca2+]o, SNAP in a concentration of 300 μmol/L induced a significant decrease in the distance between z-lines. This SNAP-induced decrease was reflected as a decrease in the phalloidin fluorescence intensity in the middle of the sarcomere, due to the preferential imaging of greater fluorescence in the bulk of the thin filaments. Based on the Ca2+ fluorescence intensity profile, we could suggest that intracellular Ca2+ is mainly affected by the mechanisms of Ca2+ outflow, rather than the mechanisms of Ca2+ inflow in the presence of NO. Actually, through variation in the electrochemical gradient of Ca2+, we induced mechanisms of faster/slower cytosolic Ca2+ emptying. The obtained data showed that the delay in the cytosolic Ca2+ emptying in the presence of [Ca2+]o is due to the increased electrochemical gradient of Ca2+.