scholarly journals Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

2020 ◽  
Vol 21 (6) ◽  
pp. 2093
Author(s):  
Sara Miguel-Jiménez ◽  
Melissa Carvajal-Serna ◽  
Silvia Calvo ◽  
Adriana Casao ◽  
José Álvaro Cebrián-Pérez ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Acrosome Reaction ◽  
No Production ◽  
Sperm Capacitation ◽  
Synthase Regulation ◽  
Oxide Synthase ◽  
Nos Isoforms ◽  
Ram Sperm

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

scholarly journals Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Hiroshi Ishida ◽  
Radharaman Ray ◽  
Jack Amnuaysirikul ◽  
Keiko Ishida ◽  
Prabhati Ray
Keyword(s):  
Nitric Oxide ◽  
Wound Healing ◽  
Nitric Oxide Synthase ◽  
Gene Transfer ◽  
Sulfur Mustard ◽  
No Production ◽  
Skin Injury ◽  
Inos Gene ◽  
Oxide Synthase

Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury.

Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

2002 ◽  
Vol 282 (4) ◽  
pp. C762-C767 ◽  
Author(s):  
Tatsuya Takizawa ◽  
Hiroshi Yoshikawa ◽  
Miho Yamada ◽  
Hidetoshi Morita
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Pcr Analysis ◽  
No Production ◽  
Epr Spectrum ◽  
Pregnant Rats ◽  
Paramagnetic Resonance ◽  
Triplet Signal ◽  
Oxide Synthase ◽  
Nos Isoforms

Nitric oxide (NO) production in the rat placenta was monitored and quantified by electron paramagnetic resonance (EPR) spectroscopy with hemoglobin and an Fe- N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trapping reagents. Expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative RT-PCR analysis. The EPR spectrum of the placenta with hemoglobin trapping showed a three-line hyperfine structure ( g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOS inhibitor l-NAME was administered to pregnant rats. Therefore, the specific signal was definitely identified as being derived from endogenous NO spin-trapped by hemoglobin, and the EPR spectrum showed that the NO adduct existed as a pentacoordinate α-NO heme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal ( g = 2.038) derived from an NO-Fe-DTCS complex. The height of the triplet signal did not vary significantly with gestational stage during the last few days of gestation. At the gestational stages examined, the level of NOS II mRNA expression was significantly higher than that of NOS III mRNA. NOS II expression in term ( day 21.5) placenta was significantly increased compared with that in preterm ( day 19.5) placenta ( P < 0.01, n = 4 or 5). These results suggest that NOS II is the predominant producer of NO in the placenta and that NOS II-generated NO plays significant roles in the maintenance of placental functions immediately before birth.

scholarly journals Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport

2005 ◽  
Vol 386 (3) ◽  
pp. 567-574 ◽  
Author(s):  
Chunying LI ◽  
Wei HUANG ◽  
M. Brennan HARRIS ◽  
Jonathan M. GOOLSBY ◽  
Richard C. VENEMA
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Adaptor Protein ◽  
No Production ◽  
Arginine Transport ◽  
No Release ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

eNOS (endothelial nitric oxide synthase) catalyses the conversion of L-arginine into L-citrulline and NO. Evidence has been presented previously that eNOS is associated with the CAT (cationic amino acid transporter)-1 arginine transporter in endothelial caveolae, and it has been proposed that eNOS–CAT-1 association facilitates the delivery of extracellular L-arginine to eNOS. Definitive proof of a protein–protein interaction between eNOS and CAT-1 is lacking, however, and it is also unknown whether the two proteins interact directly or via an adaptor protein. In the present study, we raised a polyclonal antibody against CAT-1, and show using reciprocal co-immunoprecipitation protocols that eNOS and CAT-1 do indeed form a complex in BAECs (bovine aortic endothelial cells). In vitro binding assays with GST (glutathione S-transferase)–CAT-1 fusion proteins and eNOS show that the two proteins interact directly and that no single CAT-1 intracellular domain is sufficient to mediate the interaction. Overexpression of CAT-1 in BAECs by adenoviral-mediated gene transfer results in significant increases in both L-arginine uptake and NO production by the cells. However, whereas increased L-arginine transport is reversed completely by the CAT-1 inhibitor, L-lysine, increased NO release is unaltered, suggesting that NO production in this in vitro model is independent of CAT-1-mediated transport. Furthermore, eNOS enzymic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased phosphorylation of eNOS at Ser-1179 and Ser-635, and decreased association of eNOS with caveolin-1. Taken together, these data suggest that direct interaction of eNOS with CAT-1 enhances NO release by a mechanism not involving arginine transport.

scholarly journals Nitric oxide in Tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2 expression.

1996 ◽  
Vol 184 (2) ◽  
pp. 557-567 ◽  
Author(s):  
N M Anstey ◽  
J B Weinberg ◽  
M Y Hassanali ◽  
E D Mwaikambo ◽  
D Manyenga ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Cerebral Malaria ◽  
Disease Severity ◽  
Asymptomatic Infection ◽  
No Production ◽  
Subclinical Infection ◽  
Oxide Synthase

Nitric oxide (NO)-related activity has been shown to be protective against Plasmodium falciparum in vitro. It has been hypothesized, however, that excess NO production contributes to the pathogenesis of cerebral malaria. The purpose of this study was to compare markers of NO production [urinary and plasma nitrate + nitrite (NOx)], leukocyte-inducible nitric oxide synthase type 2 (NOS2), and plasma TNF-alpha and IL-10 levels with disease severity in 191 Tanzanian children with and without malaria. Urine NOx excretion and plasma NOx levels (corrected for renal impairment) were inversely related to disease severity, with levels highest in subclinical infection and lowest in fatal cerebral malaria. Results could not be explained by differences in dietary nitrate ingestion among the groups. Plasma levels of IL-10, a cytokine known to suppress NO synthesis, increased with disease severity. Leukocyte NOS2 antigen was detectable in all control children tested and in all those with subclinical infection, but was undetectable in all but one subject with cerebral malaria. This suppression of NO synthesis in cerebral malaria may contribute to pathogenesis. In contrast, high fasting NOx levels and leukocyte NOS2 in healthy controls and asymptomatic infection suggest that increased NO synthesis might protect against clinical disease. NO appears to have a protective rather than pathological role in African children with malaria.

Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia

2001 ◽  
Vol 280 (5) ◽  
pp. C1242-C1254 ◽  
Author(s):  
Ragnar Henningsson ◽  
Per Alm ◽  
Ingmar Lundquist
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Heme Oxygenase ◽  
Insulin Response ◽  
Glucagon Secretion ◽  
No Production ◽  
Compensatory Mechanisms ◽  
Oxide Synthase

We investigated, by a combined in vivo and in vitro approach, the temporal changes of islet nitric oxide synthase (NOS)-derived nitric oxide (NO) and heme oxygenase (HO)-derived carbon monoxide (CO) production in relation to insulin and glucagon secretion during acute endotoxemia induced by lipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP and cGMP content after in vitro incubation, the insulin response to glucose in vivo and in vitro, and the insulin and glucagon responses to the adenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) in islet tissue. Immunocytochemistry revealed a marked expression of iNOS in many β-cells, but only in single α-cells after LPS. Moreover, biochemical analysis showed a time dependent and markedly increased production of NO and CO in these islets. Addition of a NOS inhibitor to such islets evoked a marked potentiation of glucose-stimulated insulin release. Finally, after incubation in vitro, a marked suppression of NO production by both exogenous CO and glucagon was observed in control islets. This effect occurred independently of a concomitant inhibition of guanylyl cyclase. We suggest that the impairing effect of increased production of islet NO on insulin secretion during acute endotoxemia is antagonized by increased activities of the islet cAMP and HO-CO systems, constituting important compensatory mechanisms against the noxious and diabetogenic actions of NO in endocrine pancreas.

Nitric oxide synthase regulation during embryonic implantation

1997 ◽  
Vol 9 (5) ◽  
pp. 557 ◽  
Author(s):  
V. Novaro ◽  
E. González ◽  
A. Jawerbaum ◽  
V. Rettori ◽  
G. Canteros ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Receptor Antagonist ◽  
Early Pregnancy ◽  
Embryo Implantation ◽  
Competitive Inhibitor ◽  
No Production ◽  
Physiological Relevance ◽  
Synthase Regulation ◽  
Oxide Synthase

It has previously been demonstrated that uterine nitric oxide synthase (NOS) activity increases before embryonic implantation in rats. The aim of the present work was to investigate the regulation and the physiological relevance of the nitric oxide (NO) system in ovoimplantation. The increase in NOS activity in early pregnancy was found to be independent of the presence of embryos in the uterus. Whereas the Ca2+-dependent isoform of NOS increased gradually in the preimplantation days, the Ca2+-independent isoform increased just at the beginning of implantation (Day 5, 1800 hours); then the activity of both isoforms declined. Oestradiol, whose concentration peaks before implantation, might be regulating NOS activity in the uterus, since treatment of rats with tamoxifen, a receptor antagonist, reduces the activity of both isoforms to preimplantation levels. Intraluminal injections of L-NAME (0·5 mg kg-1), a competitive inhibitor of NOS, reduced by 50% the number of implanted embryos; this suggests that the NO system plays a role during implantation. The data suggest that oestradiol might be a modulator of NOS activity during nidation and that NO production is necessary to achieve a successful embryo implantation.

Modulation of Neuronal Nitric Oxide Synthase (NOS1) Sensitized NOS1 -/- Mice to Total Body Irradiaton.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4597-4597
Author(s):  
Brandon Stone ◽  
Malolan Rajagopalan ◽  
Jean-Claude Rwigema ◽  
Umar Salimi, Medical ◽  
Tracy Dixon ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Bone Marrow ◽  
Nitric Oxide Synthase ◽  
Stromal Cells ◽  
Total Body Irradiation ◽  
No Production ◽  
Significant Difference ◽  
Total Body ◽  
Oxide Synthase

Abstract Abstract 4597 Ionizing irradiation results in increased superoxide and nitric oxide (NO) production. These products combine to form peroxynitrite which, through reactions with DNA, lipids, proteins, and cell membranes, initiates apoptotic cell death. Removal of nitric oxide synthase should reduce NO production and limit peroxynitrite formation thereby limiting irradiation induced apoptosis. To test this hypothesis, bone marrow stromal cell lines were derived from long term bone marrow cultures established from NOS1 -/-, NOS2 -/-, NOS3 -/-, or control background strain C57BL/6NHsd mice. Irradiation survival curves using doses ranging from 0 to 8 Gy, and scoring 7 day colonies of greater than 50 cells revealed that NOS1 -/- bone marrow stromal cells were radioresistant compared to all others showing an increased shoulder with a Ñ = 20.8 ± 5.6 compared to 8.3 ± 2.4 for C57BL/6NHsd mice (p = 0.0356). There were no significant differences between the NOS 2 -/- and NOS3 -/- stromal cells and the C57BL/6NHsd stromal cells. To determine whether inhibition of nitric oxide synthase in vivo protected mice from irradiation, groups of C57BL/6NHsd mice had Alzet osmotic pumps containing either the NOS inhibitor, L-NAME, or control phosphate buffered saline (PBS) placed subcutaneously. Three days later, at a time when NO production had been inhibited, mice were irradiated to the LD 50/30 dose of 9.5 Gy total body irradiation, and followed for expected development of the hematopoietic syndrome. Mice with pumps delivering L-NAME showed significantly increased survival (p = 0.0011), compared to control PBS pump containing mice (50% survival at 14 days compared to 8 days). Since L-NAME inhibits all NOS isoforms, NOS1 -/- cell lines were radioresistant in vitro, and NOS1 -/- mice demonstrate in vivo bladder radioresistance (Kanai, Epperly, Pearce et al, American Journal of Physiology-Heart & Circulatory Physiology, 286:H13-H21, 2004) we sought to establish that NOS1-/- mice would be relatively radioresistant to total body irradiation. Groups of female NOS1 -/-, NOS2 -/-, NOS3 -/-, and C57BL/6NHsd mice were total body irradiated to 9.5 Gy. Unexpectedly, NOS1 -/- mice were significantly more radiosensitive (50% survival at 9 days) (p = 0.0006) compared to other groups (50% survival at 25 days for C57BL/6NHsd, 19 days for NOS2 -/-, and greater than 30 days for NOS3 -/-). To determine the mechanism of rapid death in irradiated NOS1-/- mice, peripheral blood was analyzed before and 6 days after 9.5 Gy irradiation. Freshly removed bone marrow CFU-GEMM was tested for clonagenic radiation survival in vitro. There was no significant difference in radiation response of hematocrit, white cell or platelet counts, or marow CFU-GEMM between strains. While NOS1 -/- mice show reduced density of enteric neurons and associated developmental non-morbid pyloric stenosis and gastoesophageal dilation, there was no significant detectable difference in post irradiation histopathology of the esophagus, duodenum, jejunum, ileum, cecum, or colon. There was also no difference in numbers or density of intestinal crypt cells. Intestinal transit studies in irradiated mice demonstrated no significant difference in transit times. Therefore, NOS1 -/- mice display a novel total body irradiation sensitivity that is independent of hematopoietic or gastrointestinal syndromes. Supported by NIAID/NIH grant U19AI068021 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene transfer of recombinant endothelial nitric oxide synthase to liver in vivo and in vitro

2000 ◽  
Vol 279 (5) ◽  
pp. G1023-G1030 ◽  
Author(s):  
Vijay Shah ◽  
Alex F. Chen ◽  
Sheng Cao ◽  
Helen Hendrickson ◽  
Deb Weiler ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Enos Gene ◽  
No Production ◽  
Vasomotor Function ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding β-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, β-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the α1-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.

scholarly journals Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

2018 ◽  
Vol 60 (No. 8) ◽  
pp. 359-366
Author(s):  
J. Li ◽  
B. Shi ◽  
S. Yan ◽  
L. Jin ◽  
Y. Guo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Weaned Piglets ◽  
Inos Activity ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 &micro;g chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P &lt; 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P &lt; 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P&nbsp;&lt; 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P &lt; 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

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