Epithelial Nitric Oxide Synthase Research Articles

An ACE-inhibitory peptide derived from maize germ antagonizes the Angiotensin II-induced dysfunction of HUVECs via the PI3K/Akt/eNOS signaling pathway

Our previous work identified an ACE inhibitory peptide (ACEIP) with a sequence of LPGP from maize germ protein. In this study, by employing Ang II-induced Human Umbilical Vein Endothelial Cells as a cell model, we aimed to explore the anti-vascular endothelial dysfunction (VED) effects of LPGP and its mechanisms of action. Our results elucidated that LPGP rescued the Ang II-induced cell injury by exerting cytoprotective effects, reducing apoptosis, relieving oxidative stress, and balancing the secretion of vasodilatory and vasoconstrictive factors. Moreover, we found that LPGP stimulated the expression of phosphorylated serine/threonine kinase B (p-Akt) and epithelial Nitric Oxide Synthase (p-eNOS). Finally, we proved that LPGP resists Ang II-induced injury through the PI3K/Akt/eNOS pathway. To our knowledge, we linked the VED repairing effects of a food-borne ACEIP to the PI3K/Akt/eNOS pathway for the first time, which opened a new avenue to verify and understand the potential anti-hypertension effects of food-derived ACEIPs.

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

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.

N-Carbamylglutamate and l-arginine supplementation improve hepatic antioxidant status in intrauterine growth-retarded suckling lambs.

The influence of dietary supplementation of l-arginine (Arg) or N-carbamylglutamate (NCG) on the hepatic antioxidant status in intrauterine-growth-retarded (IUGR) suckling lambs remains unclear. The current work aimed to investigate the regulatory mechanisms whereby dietary Arg or NCG alter hepatic antioxidant status in suckling lambs suffering from IUGR. Forty-eight newborn Hu lambs of normal birth weight (CON) and IUGR were allocated randomly into four groups of 12 animals each: CON (4.25 ± 0.14 kg), IUGR (3.01 ± 0.12 kg), IUGR + 1% Arg (2.99 ± 0.13 kg), or IUGR + 0.1% NCG (3.03 ± 0.11 kg). All lambs were raised for a period of 21 days from 7 to 28 days after birth. Compared with the IUGR suckling animals, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and reduced glutathione (GSH) content were greater (P < 0.05), and protein carbonyl and malondialdehyde (MDA) levels were reduced (P < 0.05) in the livers of both IUGR + 1% Arg and 0.1% NCG suckling animals. Relative to IUGR suckling lambs, supplementing with Arg or NCG markedly reduced (P < 0.05) reactive oxygen species (ROS) levels, apoptosis, and necrosis in liver. Relative to IUGR suckling lambs, protein and mRNA expression of GSH-Px1, SOD2, catalase (CAT), heme oxygenase-1 (HO-1), inducible nitric oxide (NO) synthase (iNOS), and epithelial NO synthase (eNOS) increased in IUGR animals receiving Arg or NCG (P < 0.05). Both Arg and NCG can protect neonates from IUGR-induced hepatic oxidative damage through promoting the expression of antioxidative enzymes (including SOD, CAT, and GSH-Px), phase II metabolizing enzymes, and activation of the NO pathway.

Promotion of angiogenesis by M13 phage and RGD peptide in vitro and in vivo

One of the most important goals of regenerative medicines is to generate alternative tissues with a developed vascular network. Endothelial cells are the most important cell type required in angiogenesis process, contributing to the blood vessels formation. The stimulation of endothelial cells to initiate angiogenesis requires appropriate extrinsic signals. The aim of this study was to evaluate the effects of M13 phage along with RGD peptide motif on in vitro and in vivo vascularization. The obtained results demonstrated the increased cellular proliferation, HUVECs migration, cells altered morphology, and cells attachment to M13 phage-RGD coated surface. In addition, the expression of Vascular Endothelial Growth Factor A (VEGF-A), VEGF Receptors 2 and 3, Matrix Metalloproteinase 9 (MMP9), and epithelial nitric oxide synthase (eNOS) transcripts were significantly upregulated due to the HUVECs culturing on M13 phage-RGD coated surface. Furthermore, VEGF protein secretion, nitric oxide, and reactive oxygen species (ROS) production were significantly increased in cells cultured on M13 phage-RGD coated surface.

Relaxation Effects of Eucomiae Cortex in Isolated Rabbit Corpus Cavernosum Smooth Muscle

This study was aimed to investigate the relaxation effects of Eucomiae Cortex (EC) extract in isolated rabbit corpus cavernosum smooth muscle and its mechanism. To evaluate the relaxation of EC extract in rabbit corpus cavernosum, EC extract was treated in corporal strips which were precontracted with phenylephrine(PE). To study its mechanism, Nω-nitro-L-arginine (L-NNA) was pretreated after infuse of EC extract and compared with non-treated. In calcium chloride (CaSUP2+/SUP) -free krebs solution, EC extract and CaSUP2+/SUP 1 mM were infused by turns after CaSUP2+/SUP 1 mM was treated into corporal strips contracted by PE. Cell ability, nitric oxide (NO) and epithelial nitric oxide synthase (eNOS) on human umbilical vein endothelial cell (HUVEC) were measured by MTT assay, Griess reagent system and histochemical, immunohistochemical methods. EC extract showed a significant relaxation effects on the corporal strips, this effects were inhibited by pretreatment of L-NNA. EC extract inhibited the increase of contraction by CaSUP2+/SUP influx in CaSUP2+/SUP-free krebs solution, and eNOS positive reaction in corpus cavernosum, NO production in HUVEC increased by treatment of EC extract. These result suggest that the relaxation effects of EC extract in isolated corpus cavernosum smooth muscle are involved in increase of eNOS and NO production, blocking of extracellular CaSUP2+/SUP influx.

An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability.

Background. Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. Methods. This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. Results. Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. Conclusions. These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome.

The value of FeNO measurement in childhood asthma: uncertainties and perspectives

Asthma is considered an heterogeneous disease, requiring multiple biomarkers for diagnosis and management. Fractional exhaled nitric oxide in exhaled breath (FeNO) was the first useful non-invasive marker of airway inflammation in asthma and still is the most widely used. The non-invasive nature and the relatively easy use of FeNO technique make it an interesting tool to monitor airway inflammation and rationalize corticosteroid therapy in asthmatic patients, together with the traditional clinical tools (history, physical examination and lung function tests), even if some controversies have been published regarding the use of FeNO to support the management of asthma in children. The problem of multiple confounding factors and overlap between healthy and asthmatic populations preclude the routine application of FeNO reference values in clinical practice and suggest that it would be better to consider an individual “best”, taking into account the context in which the measurement is obtained and the clinical history of the patient. Besides, there is still disagreement about the role of FeNO as a marker of asthma control, due to the complexity of balance among the different items involved in its determination and the lack of homogeneity in the population groups studied in the few studies conducted so far. Heterogeneity of problematic severe asthma greatly limits utility of FeNO alone as a biomarker of inflammation to optimize the disease management on an individual basis. None of the studies conducted so far demonstrated that the use of FeNO was better than current asthma guidelines in controlling asthma exacerbations. In summary, there is a large variation in FeNO levels between individuals, which may reflect the natural heterogeneity in baseline epithelial nitric oxide synthase activity and/or the contribution of other noneosinophilic factors to epithelial nitric oxide synthase activity. FeNO is a promising biomarker, but at present some limits are highlighted. We would recommend that further research can be carried out by organizing studies aimed to obtain reliable reference values of FeNO and in order to better interpret FeNO measurements in clinical settings, taking also into account the influence of genetic and environmental factors.

Sıçanlarda Pentilentetrazol ile Uyarılan Kindling Gelişimi Üzerine Atorvastatinin Antiepileptogenik Etkisinin Değerlendirilmesi; İndüklenebilir Nitrik Oksit ve Metalloproteinaz 2'nin Sorumluluğu

Objective: Recently, studies indicated that metalloproteinase and inducible nitric oxide synthase (iNOS), a key participant in neuronal death and neuroinflammation, are upregulated in experimental epilepsy models. Statins are inhibitors of HMG-CoA reductase that have been have been show to have anti-inflammatory and neuroprotective effects. Atorvastatin (AT) was shown to possess antiepileptic effects, but the mechanism is not clear. Nitric oxide (NO) and matrixmetalloproteinases (MMP) may be responsible for this effect. The purpose of this study was to assess the effect of AT against development of kindling-induced changes of endothelial nitric oxide synthase (NOS), inducible nitric oxide synthase (iNOS) and MMP2 and MMP9 expression in pentylenetetrazole (PTZ)-induced kindling. Material and Methods: In male Wistar rats, kindling (epileptogenesis model) was induced by repeated administration of a sub-convulsive dose (40 mg/kg, intraperitoneal) of PTZ to give rise to kindling. AT was given (5 mg/kg, intraperitoneal) every day and before 30 min from PTZ. After each PTZ injection, the convulsive behavior was observed for 60 minutes. NOS and MMP activities in hippocampus were studied using immunohistochemistry. Results: PTZ kindling induced the expression of iNOS and MMP2 and did not affect MMP9, but decreased eNOS expression. AT pretreatment suppressed formation of kindling, increased epithelial nitric oxide synthase, decreased iNOS and MMP2 immune reaction. Conclusion: iNOS and MMP2 may be regarded as proconvulsant substances while MMP9 has no effect in development of PTZ kindling. Antiepileptogenic effect of AT may result from a reduction in the activity of MMP2, iNOS with its anti-inflammatory effect. These findings can shed light on future studies, which aim to investigate therapeutic interventions for preventing epileptogenesis.

The value of FeNO measurement in childhood asthma: uncertainties and perspectives.

Asthma is considered an heterogeneous disease, requiring multiple biomarkers for diagnosis and management. Fractional exhaled nitric oxide in exhaled breath (FeNO) was the first useful non-invasive marker of airway inflammation in asthma and still is the most widely used. The non-invasive nature and the relatively easy use of FeNO technique make it an interesting tool to monitor airway inflammation and rationalize corticosteroid therapy in asthmatic patients, together with the traditional clinical tools (history, physical examination and lung function tests), even if some controversies have been published regarding the use of FeNO to support the management of asthma in children. The problem of multiple confounding factors and overlap between healthy and asthmatic populations preclude the routine application of FeNO reference values in clinical practice and suggest that it would be better to consider an individual “best”, taking into account the context in which the measurement is obtained and the clinical history of the patient. Besides, there is still disagreement about the role of FeNO as a marker of asthma control, due to the complexity of balance among the different items involved in its determination and the lack of homogeneity in the population groups studied in the few studies conducted so far. Heterogeneity of problematic severe asthma greatly limits utility of FeNO alone as a biomarker of inflammation to optimize the disease management on an individual basis. None of the studies conducted so far demonstrated that the use of FeNO was better than current asthma guidelines in controlling asthma exacerbations. In summary, there is a large variation in FeNO levels between individuals, which may reflect the natural heterogeneity in baseline epithelial nitric oxide synthase activity and/or the contribution of other noneosinophilic factors to epithelial nitric oxide synthase activity. FeNO is a promising biomarker, but at present some limits are highlighted. We would recommend that further research can be carried out by organizing studies aimed to obtain reliable reference values of FeNO and in order to better interpret FeNO measurements in clinical settings, taking also into account the influence of genetic and environmental factors.

Effects of acute treatment with dexamethasone on hemodynamic and histopathological changes in rats

We assessed the time-dependent effects of intraperitoneal (i.p.) and intravenous (i.v.) application of dexamethasone (Dexa) on the mean arterial blood pressure (MAP), heart rate (HR) and total blood volume (TBV). We evaluated also the relation between the effects and immunoreactivities of transforming growth factor-beta (TGF-β), epithelial nitric oxide synthase (eNOS), interleukin-1 beta (IL1-β) and vascular endothelial growth factor (VEGF) in rat brain, lung and kidney tissues. Rats were anesthetized and while still breathing spontaneously, a tracheotomy and femoral vein and artery catheterizations were performed. To determine TBV using the hemodilution method, 2 ml albumin-electrolyte solutions were applied by i.v. injection. Group 1 (control group) received a 1 ml bolus injection of physiologic saline, Group 2 received 15 mg/kg and Group 3 received 75 mg/kg Dexa i.p. The hematocrit was measured at 10, 20, 60 and 120 min. For each animal, the values of MAP, HR and TBV were measured within 2 h. For immunohistochemical evaluation, anti-TGF-β, anti-eNOS, anti-IL1-β and anti-VEGF primary antibodies were tested using the avidin-biotin-peroxidase method. TBV was decreased in Group 1 and the increase in MAP was statistically significant. HR values increased slightly. None of the values changed significantly in Group 2. Although TBV was unchanged in Group 3, the decrease in MAP was statistically significant. HR values increased, but the increase was not statistically significant. Mild IL1-β immunoreactivity and moderate TGF-β, eNOS and VEGF immunoreactivities were observed in the brain, lung and kidney samples in Group 1. Increased eNOS immunoreactivity in the kidney samples were observed in Group 2. eNOS immunoreactivity was as strong in the brain and the kidney samples in Group 3. Decreased VEGF immunoreactivity was observed in the lung and kidney tissues in Group 3. Significantly decreased TGF-β immunoreactivity was observed in all tissue samples in Group 3. The decreased MAP values in Group 3 differed from those in Groups 1 and 2. Despite increased eNOS immunoreactivity, especially in brain and kidney, the decrease in VEGF immunoreactivity in Group 3, especially lung and kidney, were consistent with a drop in blood pressure.

Angiotensin AT1 receptor-associated protein Arap1 in the kidney vasculature is suppressed by angiotensin II

Arap1 is a protein that interacts with angiotensin II type 1 (AT(1)) receptors and facilitates increased AT(1) receptor surface expression in vitro. In the present study, we assessed the tissue localization and regulation of Arap1 in vivo. Arap1 was found in various mouse organs, with the highest expression in the heart, kidney, aorta, and adrenal gland. Renal Arap1 protein was restricted to the vasculature and to glomerular mesangial cells and was absent from tubular epithelia. A similar localization was found in human kidneys. To test the hypothesis that angiotensin II may control renal Arap1 expression, mice were subjected to various conditions to alter the activity of the renin-angiotensin system. A high-salt diet (4% NaCl, 7 days) upregulated Arap1 expression in mice by 47% compared with controls (0.6% NaCl, P = 0.03). Renal artery stenosis (7 days) or water restriction (48 h) suppressed Arap1 levels compared with controls (-64 and -62% in the clipped and contralateral kidney, respectively; and -50% after water restriction, P < 0.01). Angiotensin II infusion (2 μg·kg(-1)·min(-1), 7 days) reduced Arap1 mRNA levels compared with vehicle by 29% (P < 0.01), whereas AT(1) antagonism (losartan, 30 mg·kg(-1)·day(-1), 7 days) enhanced Arap1 mRNA expression by 52% (P < 0.01); changes in mRNA were paralleled by Arap1 protein abundance. Experiments with hydralazine and epithelial nitric oxide synthase-/- mice further suggested that Arap1 expression changed in parallel with angiotensin II, rather than with blood pressure per se. Similar to in vivo, Arap1 mRNA and protein were suppressed by angiotensin II in a time- and dose-dependent manner in cultured mesangial cells. In summary, Arap1 is highly expressed in the renal vasculature, and its expression is suppressed by angiotensin II. Thus Arap1 may serve as a local modulator of vascular AT(1) receptor function in vivo.

Resting Tension Affects eNOS Activity in a Calcium-Dependent Way in Airways

The alteration of resting tension (RT) from 0.5 g to 2.5 g increased significantly airway smooth muscle contractions induced by acetylcholine (ACh) in rabbit trachea. The decrease in extracellular calcium concentration [Ca2+]o from 2 mM to 0.2 mM reduced ACh-induced contractions only at 2.5 g RT with no effect at 0.5 g RT. The nonselective inhibitor of nitric oxide synthase (NOS), NG-nitro-L-arginine methyl ester (L-NAME) increased ACh-induced contractions at 2.5 g RT. The inhibitor of inducible NOS, S-methylsothiourea or neuronal NOS, 7-nitroindazole had no effect. At 2.5 g RT, the reduction of [Ca2+]o from 2 mM to 0.2 mM abolished the effect of L-NAME on ACh-induced contractions. The NO precursor L-arginine or the tyrosine kinase inhibitors erbstatin A and genistein had no effect on ACh-induced contractions obtained at 2.5 g RT. Our results suggest that in airways, RT affects ACh-induced contractions by modulating the activity of epithelial NOS in a calcium-dependent, tyrosine-phosphorylation-independent way.

Distribution of oxidation enzyme eNOS and myeloperoxidase in primary open angle glaucoma

Genetic factors and the influence of superoxide are known to play roles in the etiology of glaucoma. We evaluated the association between primary open angle glaucoma (POAG) and two polymorphisms in the epithelial nitric oxide synthase (eNOS) gene, and one polymorphism in the myeloperoxidase (MPO) gene. We enrolled 66 patients with POAG and 100 healthy volunteers in this study. The polymorphisms in the eNOS and the polymorphism MPO -463 G-to-A in the MPO gene were resolved by polymorphism polymerase chain reaction (PCR). There were no significant differences in the distribution of the eNOS intron -4 (P=0.481), eNOS promotor -786 (P=0.555), and MPO -463 (P=0.292) gene polymorphisms between the POAG patients and the volunteers (P-values=0.481, 0.555, and 0.292, respectively). None of the alleles from either gene differed between the groups (P-values=0.483, 0.554, and 0.183, respectively). Superoxide is closely related to glaucoma, and eNOS and MPO are two important enzymes in the free radical pathway. However, polymorphisms of the eNOS intron-4, eNOS promotor -786, and MPO -463 gene polymorphisms did not reveal significant differences between POAG patients and controls in our study. The use of these agents and other superoxide-related genes for clinical applications requires further investigation.

Functional characterization of HUVEC-CS: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production.

While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell. We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel. HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production. Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA2, and COX-1 and -2. Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI2 production is minimal. Receptors were detected for angiotensin II (AII), bradykinin, ATP and growth factors. ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ([Ca2+]i). Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin. AII, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in [Ca2+]i in a subset of the cells. ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min. L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation. In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC. These cells will prove a valuable tool for future studies in both basic and therapeutic sciences.

Nitric oxide synthase expression in AT2 receptor–deficient mice after DOCA-salt

Angiotensin II type 2 receptor-deficient mice (AT(2)-/y) provide an opportunity to study the relationship between the angiotensin II type 1 receptor (AT(1)) and nitric oxide synthase (NOS) isoforms without concomitant AT(2) receptor-related effects. To test this relationship, the expression of renal NOS isoforms (neural, inducible, and endothelial) in AT(2)-/y and AT(2)+/y mice was examined. The mice were challenged with deoxycorticosterone acetate (DOCA)-salt to stimulate NO generation. Gene expression analyses by real-time polymerase chain reaction (PCR) (TaqMan) were performed in kidneys to characterize neuronal nitric oxide synthase (nNOS), epithelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and the AT(1) receptor. Pressure-natriuresis experiments were done to determine the physiologic background. AT(2)-/y mice showed nNOS and iNOS up-regulation. DOCA-salt increased iNOS expression more in AT(2)-/y mice than in AT(2)+/y mice. Immunohistochemistry localized the iNOS expression with DOCA-salt mainly in the glomeruli. eNOS was not different between the groups, and was not affected by DOCA-salt. DOCA-salt increased mean arterial pressure more in AT(2)-/y mice than in AT(2)+/y mice. Concomitantly, the pressure-natriuresis relationship was shifted to the right in AT(2)-/y and AT(2)+/y mice after DOCA-salt. DOCA-salt decreased renal blood flow (RBF) and glomerular filtration rate (GFR) in both groups. iNOS blockade did not lower blood pressure. We conclude that AT(2) receptor deletion and concomitant up-regulation of the AT(1) receptor is associated with up-regulation of nNOS and iNOS. Under DOCA-salt, renal iNOS expression was further increased. Because iNOS inhibition did not change blood pressure, iNOS may not be involved in the hemodynamics, but may contribute to organ damage.

The Protective Role of Epithelium-derived Nitric Oxide in Isolated Bovine Trachea

Airway epithelial cells from bovine airways can release relaxant factors such as nitric oxide (NO) and prostaglandin E2 and the removal of airway epithelium results in an increased responsiveness of smooth muscle to spasmogen stimuli. In this study, we assessed whether or not epithelial NO modulates the contractile response of bovine trachea in vitro.Cumulative concentration–response curves to acetylcholine (ACh), histamine (Hist) and 5-hydroxytryptamine (5-HT) were obtained in both intact and epithelium denuded tracheal strips in the presence of indomethacin (10μM).In intact, but not in epithelium denuded strips, preincubation with the NO synthase inhibitor L-NG-Nitro-arginine methyl ester (L-NAME), but not with D-NAME, shifted to the left the concentration–response curve to ACh (pD2 values in the absence and in the presence of L-NAME were 3.47±0.1 and 4.60±0.1, respectively; P<0.05) and to Hist (pD2 in the absence and in the presence of L-NAME: 3.89±0.1 and 4.54±0.1, respectively; P<0.05). This effect was reversed by L-arginine (1mM), but not by D-arginine. The contractile response to 5-HT was not affected by L-NAME in either intact or epithelium denuded strips.These data suggest that NO is an epithelial relaxant factor modulating airway cholinergic and histaminergic contraction of bovine trachea and that the activation of the epithelial NO synthase is a mediator-specific process.

Role of airway nitric oxide on the regulation of pulmonary circulation by carbon dioxide.

The effects of hypercapnia (CO(2)) confined to either the alveolar space or the intravascular perfusate on exhaled nitric oxide (NO), perfusate NO metabolites (NOx), and pulmonary arterial pressure (Ppa) were examined during normoxia and progressive 20-min hypoxia in isolated blood- and buffer-perfused rabbit lungs. In blood-perfused lungs, when alveolar CO(2) concentration was increased from 0 to 12%, exhaled NO decreased, whereas Ppa increased. Increments of intravascular CO(2) levels increased Ppa without changes in exhaled NO. In buffer-perfused lungs, alveolar CO(2) increased Ppa with reductions in both exhaled NO from 93.8 to 61.7 (SE) nl/min (P < 0.01) and perfusate NOx from 4.8 to 1.8 nmol/min (P < 0.01). In contrast, intravascular CO(2) did not affect either exhaled NO or Ppa despite a tendency for perfusate NOx to decline. Progressive hypoxia elevated Ppa by 28% from baseline with a reduction in exhaled NO during normocapnia. Alveolar hypercapnia enhanced hypoxic Ppa response up to 50% with a further decline in exhaled NO. Hypercapnia did not alter the apparent K(m) for O(2), whereas it significantly decreased the V(max) from 66.7 to 55.6 nl/min. These results suggest that alveolar CO(2) inhibits epithelial NO synthase activity noncompetitively and that the suppressed NO production by hypercapnia augments hypoxic pulmonary vasoconstriction, resulting in improved ventilation-perfusion matching.

Regulation of NO-producing function of the lungs with salmeterol.

The state of epithelial nitric oxide synthase, bronchomotor reaction, and concentration of NO metabolites (NO(2)(-) and NO(3)(-)) in bronchoalveolar lavage fluid were studied in healthy rats and rats with experimental bronchial asthma induced by inhalations of long-acting beta(2)-agonist salmeterol. The effects of salmeterol on NO-producing function of the lung in healthy animals and in animals with bronchial asthma were studied.

Anticonvulsion Effect of Acupuncture Might Be Related to the Decrease of Neuronal and Inducible Nitric Oxide Synthases

To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of theses levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric Oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (P<0.01) and inducible nitric oxide synthase (iNOS) mRNA significantly emerged during epilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (P<0.01) and iNOS (P<0.01) correspondingly but had no effect on the amount of eNOS mRNA. The data suggest that penicillin-induced epilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

A non-nucleotide–bridged DNA decoy inhibits renal epithelial nitric oxide synthase expression

A non-nucleotide-bridged DNA decoy inhibits renal epithelial nitric oxide synthase expression. The expression of inducible nitric oxide synthase (iNOS) is subject to strict tissue-specific transcriptional control. In mouse renal epithelium, an interferon-gamma (IFN-gamma)-induced signaling protein, IFN-gamma regulatory factor 1 (IRF-1), appears to mediate the induction of iNOS expression by cytokines and bacterial lipopolysaccharide (LPS). We used a novel technique, namely, blockade of cytosolic IRF-1 activity with a triethyleneglycol-bridged decoy DNA oligonucleotide (ODN) containing the IRF-1 consensus binding sequences present in the iNOS promoter to inhibit iNOS gene expression. Cultured mouse renal epithelial cells were treated with a combination of LPS (1 microg/mL) and IFN (100 U/mL) in the absence or presence of IRF-1 decoy ODN followed by determinations of NO production and iNOS protein and mRNA expression. Treatment with IRF-1 decoy ODN resulted in concentration-dependent inhibition of NO production and a marked reduction in iNOS protein and mRNA levels. A scrambled ODN failed to affect LPS/IFN-stimulated NO production or iNOS protein and mRNA levels. Transcriptional assays showed that the IRF-1 decoy ODN inhibited transcriptional activity of an iNOS promoter-CAT gene construct. Decoy ODN-based techniques effectively inhibit iNOS expression in renal epithelium and represent a potentially useful approach for selective blockade of this enzyme in pathologic conditions associated with excessive NO production.