Nitric Oxide Release In Response Research Articles

Intracellular infection-responsive release of NO and peptides for synergistic bacterial eradication.

Bacteria have the ability to invade and survive in host cells to form intracellular bacteria (ICBs), and challenges remain in the intracellular delivery of sufficient antibiotics to remove ICBs. Herein, antimicrobial peptide of epsilon-poly-l-lysine (ePL) and nitric oxide (NO) donors are integrated into nanoparticles (NPs) for ICB treatment without using any antibiotics. ePL was grafted with dodecyl alcohol through ethyl dichlorophosphate to prepare ePL-C12, followed by conjugation of nitrate-functionalized NO donors to obtain ePL-C12NO. PNO/C NPs were prepared from mixtures of ePL-C12NO and ePL-C12 and the optimal ePL-C12NO ratio was 7% in terms of bactericidal effect and macrophage toxicity. Once being engulfed by bacteria-infected macrophages (BIMs), NPs are disintegrated when encountering with ICB-secreted phosphatase, and the NP degradation accelerates intracellular NO release in response to the elevated glutathione levels in BIMs. The selective and abrupt release of NO and ePL with different antimicrobial mechanisms exhibits synergistic eradication of ICBs and no apparent toxicity to macrophages. ICB-infected mice show persistent weight loss and 100% of mortality rate after treatment with ePL-C12 NPs for 7days, while PNO/C treatment causes entire survival of infected mice and full recovery of body weights to normal values. ICB-infected mice are also accompanied with apparent hepatomegaly and splenomegaly, which are only eliminated by PNO/C treatment without associated any pathological abnormality. PNO/C treatment reduces bacterial burdens in livers (2.45 log), spleens (2.16 log) and kidneys (3.46 log) and restores hepatic and renal function to normal levels. Thus, this study provides a feasible strategy to selectively release NO and cationic peptides in response to intracellular infection-derived signals, achieving synergistic eradication of ICBs and function restoration of the main tissues.

Role of Endothelial STAT3 in Cerebrovascular Function and Protection from Ischemic Brain Injury.

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood–brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke.

Nitric oxide production by glomerular podocytes

Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration. However, whether changes in NO occur in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. We showed here that podocytes produce NO, and further hypothesized that hypertensive animals would exhibit reduced NO production in these cells in response to various paracrine factors, which might contribute to the damage of glomeruli filtration barrier and development of proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed a low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks) diets and loaded podocytes with either a combination of NO and Ca2+ fluorophores (DAF-FM and Fura Red, respectively) or DAF-FM alone. Changes in fluorescence were observed with confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and hydrogen peroxide (H2O2). Application of Ang II resulted in activation of both NO and intracellular calcium ([Ca2+]i) transients. In contrast, ATP promoted [Ca2+]i transients, but did not have any effects on NO production. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production: the response to Ang II or H2O2 in podocytes of glomeruli isolated from SS rats fed a HS diet was significantly reduced compared to rats fed a LS diet. Therefore, glomerular podocytes from hypertensive rats showed a diminished NO release in response to Ang II or oxidative stress, suggesting that podocytic NO signaling is dysfunctional in this condition and likely contributes to the development of kidney injury.

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μM, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μM RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.

Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function.

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.

Omega-3 fatty acids supplementation improves endothelial function and maximal oxygen uptake in endurance-trained athletes

The study aimed to evaluate the effects of a 3-week n-3 polyunsaturated fatty acids (n-3 PUFA) supplementation on serum nitric oxide (NO), asymmetric dimethyloarginine (ADMA), ultrasound indices of endothelial function and maximal oxygen uptake () of elite cyclists. The effects of dietary supplementation (n-3 PUFA at a dose of 1.3 g twice daily for 3 weeks) and placebo administration on flow-mediated dilatation (FMD), pulse wave velocity, serum markers (NO, ADMA), lipid profile, and were analysed in 13 cyclists both before and after dietary protocols. Significant differences between pre- and post-intervention baseline NO levels were observed after n-3 PUFA dietary protocol (13.9 ± 4.2 vs. 23.5 ± 3.6 µmol·l−1; P < 0.001). Higher post-intervention baseline NO level was observed after n-3 PUFA diet compared with placebo (23.5 ± 3.6 vs. 15.3 ± 3.0 µmol·l −1; P < 0.01, respectively). The n-3 PUFA increased baseline NO concentration (ΔNO) by 6.7 ± 3.8 µmol·l−1 and placebo by 1.6 ± 4.4 µmol·l−1. The positive correlation was observed between baseline post-intervention NO concentration and maximal oxygen uptake (r = 0.72; P < 0.01) and also between ΔNO and (r = 0.54; P < 0.05) in response to omega-3 fatty acids supplementation. There was an association between a 5.25% higher FMD (P < 0.05) and higher (P < 0.001) after n-3 PUFA diet compared with lower values of placebo (r = 0.68; P < 0.05). These findings suggest that an increase in NO release in response to n-3 PUFA supplementation may play a central role in cardiovascular adaptive mechanisms and enhanced exercise performance in cyclists.

Abstract 15: Novel Functions of Small GTPase Rap1 in Regulating Endothelial Homeostasis: Control of Nitric Oxide Release, Vascular Function and Blood Pressure

Endothelial dysfunction, resulting from decreased nitric oxide (NO) bioavailability is a pathology linked to endothelial vasomotor dysfunction and hypertension, inflammation and atherosclerosis, perturbed endothelial barrier and progression of diabetes. In blood vessels, NO is produced by the endothelial NO synthase (eNOS), the activity of which is regulated by Ca2+/calmodulin, binding of regulatory cofactors, and posttranslational modifications, including phosphorylation events on Ser1177, which stimulate NO production. Rap1 is a ubiquitously expressed small GTPase implicated in promoting vascular barrier. We have shown that endothelial cell (EC)-specific Rap1 deletion leads to defective angiogenesis in vivo due to faulty VEGFR2 activation and signaling. Importantly, EC-specific Rap1 knockout mice developed hypertension and pathological left ventricular hypertrophy. The objective of the study was to determine the role of small G protein Rap1 in regulating endothelial NO production and endothelial-dependent vasorelaxation in vivo and ex vivo. Using ex vivo myography and tamoxifen-inducible, endothelial-specific Rap1-knockout mice (Cadh5-CreERT2+/0;Rap1f/f), we demonstrate that Rap1 deficiency completely abrogates NO-dependent vasodilation and attenuates NO production. Mechanistically, we show that Rap1 is rapidly activated in response to receptor agonists that activate eNOS via Ca2+/calmodulin- dependent pathway and in response to shear flow, which modules eNOS activity by its phosphorylation. Rap1 deletion in human ECs, in vitro, leads to deficient NO release in response to both these stimuli, and interferes with PI3K/Akt pathway and eNOS Ser1177 phosphorylation. Further, we demonstrate Rap1 is required for transducing signals from the endothelial mechanosensing complex comprising PECAM-1, VE-cadherin and VEGFR2 in response to shear flow, leading to ligand-independent VEGFR2 activation and signaling to stimulate NO production. We conclude that Rap1 in endothelium is critically required for endothelial homeostasis and NO production, thereby affecting vascular tone and regulation of blood pressure. Furthermore, this study establishes Rap1 as a novel regulator of mechanotransduction in response to shear flow.

Potential of Tissue Engineered Blood Vessel as Model to Study Effect of Flow and Wall Thickness on Cellular Communication

In physiology, blood vessel function is maintained mainly through nitric oxide (NO)-mediated cross-talk between endothelial cells (ECs) and smooth muscle cells (SMCs), which is compromised in pathology. Lack of an appropriate in vitro model hampers the study of vascular disease progression mechanisms. This study attempted to use tissue engineered blood vessel (TEBV) as a model system to understand the effect of wall thickness and shear stress on EC to SMC communication. Differentiated ECs and SMCs obtained by in vitro culture of sheep peripheral blood mononuclear cells (PBMNCs) were seeded on biodegradable €-polycaprolactone (PCL) conduits of different wall thicknesses and exposed to shear stress in a two-channel bioreactor to construct functional TEBV. Phenotypes of ECs and SMCs were studied in terms of nitric oxide synthase (eNOS) and basic calponin expressions respectively, using real time polymerase chain reaction. Endothelial to SMC cross-talk under the influence of wall thickness and shear stress was interrelated to NO and cyclic GMP (cGMP) production. Shear stress accelerates, but wall thickness has no influence on endothelial NO production. Increased release of NO in response to shear stress resulted in augmented cGMP production, but only when the wall thickness was lower. Both wall thickness and shear stress affect cGMP production and smooth muscle contractile phenotype. From this study, it is also suggested that TEBV may be a suitable model to study various risk factors on vessel integrity. Keywords: Blood vessel wall thickness, cGMP synthesis in SMC, endothelial nitric oxide synthetase, functional tissue engineering, nitric oxide synthesis, shear stress, tissue engineered blood vessel.

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm2 shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.

Quantitative nitric oxide release to study the effects on dorsal root ganglion cells

Nitric oxide (NO) has shown to play important and diverse biological roles. NO is suspected to be beneficial at low levels for a variety of cells, including retinal epithelial cells, lymphatic epithelial cells, and dorsal root ganglion cells, but can be detrimental at high levels or at certain periods of time in their growth. Currently, the beneficial or harmful levels and durations of NO have not been determined. We have developed an NO-generating polydimethylsiloxane (PDMS) that contains S-nitroso-N-acetyl- d -penicillamine (SNAP) covalently linked to its backbone which is capable of stable and dynamic control of NO release in response to light. A solid core waveguide device has also been fabricated to propagate controllable levels of light along its surfaces. By coating this surface with the photosensitive SNAP-PDMS and growing cells on an additional polymer layer, a device has been developed that allows for specified variation in NO release and duration the cultured cells experience, without exposing the cells directly to light. The waveguide system allows precise levels of NO to be generated that will allow the determination of beneficial and damaging levels of NO the cells experience. In these experiments dorsal root ganglion (DRG) cells were cultured with different levels of NO in order to determine cellular response. Future work includes varying the level and duration of NO release the cells are exposed to. This can lead to a better understanding of the effects of NO during spinal cord injury so that nerve growth and regeneration can become a possibility.

Oxymatrine attenuates bleomycin-induced pulmonary fibrosis in mice via the inhibition of inducible nitric oxide synthase expression and the TGF-β/Smad signaling pathway

Oxymatrine (OM) is an alkaloid extracted from the Chinese herb Sophora flavescens Ait. with a variety of pharmacological activities. The aim of this study was to investigate the preventive effects of OM on bleomycin (BLM)-induced pulmonary fibrosis (PF) and to further explore the underlying mechanisms. C57BL/6 mice were randomly assigned to five groups: the saline sham group; the BLM group, in which mice were endotracheally instilled with BLM (3.0mg/kg); and the BLM plus OM groups, in which OM was given to mice daily (10, 20 or 40mg/kg) one day after BLM instillation for 21days. The bronchoalveolar lavage fluid (BALF) and lung tissues were collected at 15 and 22days post BLM administration, respectively. Lung tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. Levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and nitric oxide (NO) in mouse BALF were measured, as well as myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in lung homogenates. The inducible nitric oxide synthase (iNOS) expression in the lung tissues was determined by immunohistochemical staining, quantitative real-time PCR and western blot analysis. Moreover, the expression of transforming growth factor (TGF)-β1, Smad2, Smad3, p-Smad2 and p-Smad3 were also detected. We found that OM improved BLM-induced lung pathological changes, inhibited MPO activity and reduced MDA levels in a dose-dependent manner. OM also dose-dependently inhibited the release of TNF-α and IL-6, and decreased the expression of iNOS in lung tissues and thus prevented NO release in response to BLM challenge. In addition, OM decreased the expression of TGF-β1, p-Smad2 and p-Smad3, which are all important members of the TGF-β/Smad signaling pathway. Our study provides evidence that OM significantly ameliorated BLM-induced PF in mice via the inhibition of iNOS expression and the TGF-β/Smad pathway.

Autologous transplantation of adipose-derived mesenchymal stem cells attenuates cerebral ischemia and reperfusion injury through suppressing apoptosis and inducible nitric oxide synthase

The purpose of this study was to investigate whether autologous transplantation of adipose-derived mesenchymal stem cells (ADMSCs) has a neuroprotective effect against cerebral ischemia/reperfusion (I/R) injury in rats and to explore the possible underlying mechanisms. Adult male Sprague-Dawley rats were randomly assigned into the sham, I/R injury model (I/R), and model plus autologous transplantation of ADMSCs (ADMSC) groups. Cerebral I/R injury was induced by 2 h middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. Rats in the I/R and ADMSC groups were intravenously injected with culture medium and ADMSCs (2.0x10(6)), respectively, at the onset of reperfusion and 12 h after reperfusion. Cerebral infarct volume was detected by triphenyltetrazolium chloride (TTC) staining. The histopathological changes and neuronal apoptosis in the ischemic penumbra were evaluated with H&E staining and the TUNEL assay, respectively. The nitric oxide (NO) content, caspase-3 activity and the Bax/Bcl-2 protein ratio were also measured. Moreover, the inducible nitric oxide synthase (iNOS) expression in the ischemic regions of rats was determined by immunohistochemical staining, quantitative real-time RT-PCR and western blot analysis. We found that autologous transplantation of ADMSCs significantly reduced the cerebral infarct volume, improved the I/R injury-induced brain damages and inhibited the neuronal apoptosis. ADMSC implantation also decreased caspase-3 activity and the Bax/Bcl-2 protein ratio, and markedly downregulated the expression of iNOS and thus prevented NO release in response to cerebral I/R injury. Taken together, our results demonstrated that autologous transplantation of ADMSCs can protect the brain against cerebral I/R injury via the inhibition of neuronal apoptosis and iNOS expression.

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm 2 shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell–matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.

EDF-1 contributes to the regulation of nitric oxide release in VEGF-treated human endothelial cells

Vascular endothelial growth factor (VEGF) induces nitric oxide (NO) release by triggering multiple intracellular signals, among others the calcium/calmodulin pathway and the activation of Akt, events which induce endothelial NO synthase (eNOS) activity. Because Endothelial Differentiation-related Factor (EDF)-1 is a calmodulin binding protein and plays a role in modulating endothelial functions, we evaluated whether EDF-1 is implicated in the regulation of eNOS activity in VEGF-treated human endothelial cells. While VEGF does not modulate the total amounts of EDF-1, it promotes the dissociation of calmodulin from EDF-1 which correlates with the increase of calmodulin bound to eNOS and the induction of NO release. To better characterize the contribution of EDF-1 to the regulation of VEGF-induced NO release, we stably silenced EDF-1 in endothelial cells. We here show that endothelial cells silencing EDF-1 produce more NO than controls and do not increase NO release in response to VEGF. The insensitivity to VEGF results from the incapability of cells silencing EDF-1 to phosphorylate eNOS Ser1177, even though Akt is activated. Interestingly, okadaic acid, a pharmacologic inhibitor of the serine/threonine phosphatase PP2A, which preferentially dephosphorylates eNOS Ser1177, restores NO release and eNOS Ser1177 phosphorylation in cells silencing EDF-1. Our results suggest EDF-1 as a novel contributor to the complex regulation of eNOS activity in human endothelial cells.

Effect of the 894G&gt;T polymorphism of the endothelial nitric oxide synthase on vascular reactivity following maximal dynamic exercise

Considering that the role of nitric oxide as a vasodilator is increased after an acute bout of exercise and that the 894G>T polymorphism of the endothelial nitric oxide synthase seems to reduce the nitric oxide release in response to shear stress, the present study investigated the 894G>T polymorphism in relation to vascular reactivity following maximal dynamic exercise. We studied 110 healthy volunteers (wild-type group 45.5% and polymorphic group 54.5%). The protocol included vascular reactivity assessment at baseline and during reactive hyperemia, before, 10, 60 and 120 min after a maximal cardiopulmonary exercise test. Genomic DNA was extracted from blood samples to determine the 894G>T polymorphism. There were no differences between the wild-type and polymorphic groups concerning anthropometric, metabolic and hemodynamic characteristics. Blood flow, before maximal exercise, was similar between the wild-type and the polymorphic groups. The polymorphic group presented lower vascular reactivity regardless of time (P = 0.019 for group main effect), and posthoc analysis revealed that polymorphic patients had lower values than wild-type only at the 120 min measurement (P = 0.002). Concerning within-group analysis, vascular reactivity increased at 10 min after exercise (P = 0.029) returning to baseline at 120 min (P = 0.005) in the polymorphic group. Patients with the 894G>T polymorphism had lower vascular reactivity after a single bout of exercise.

Levosimendan induces NO production through p38 MAPK, ERK and Akt in porcine coronary endothelial cells: role for mitochondrial KATPchannel

Levosimendan acts as a vasodilator through the opening of ATP-sensitive K(+) channels (K(ATP)) channels. Moreover, the coronary vasodilatation caused by levosimendan in anaesthetized pigs has recently been found to be abolished by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester, indicating that nitric oxide (NO) has a role in the vascular effects of levosimendan. However, the intracellular pathway leading to NO production caused by levosimendan has not yet been investigated. Thus, the purpose of the present study was to examine the effects of levosimendan on NO production and to evaluate the intracellular signalling pathway involved. In porcine coronary endothelial cells (CEC), the release of NO in response to levosimendan was examined in the presence and absence of N(omega)-nitro-L-arginine methyl ester, an adenylyl cyclase inhibitor, K(ATP) channel agonists and antagonists, and inhibitors of intracellular protein kinases. In addition, the role of Akt, ERK, p38 and eNOS was investigated through Western blot analysis. Levosimendan caused a concentration-dependent and K(+)-related increase of NO production. This effect was amplified by the mitochondrial K(ATP) channel agonist, but not by the selective plasma membrane K(ATP) channel agonist. The response of CEC to levosimendan was prevented by the K(ATP) channel blockers, the adenylyl cyclase inhibitor and the Akt, ERK, p38 inhibitors. Western blot analysis showed that phosphorylation of the above kinases lead to eNOS activation. In CEC levosimendan induced eNOS-dependent NO production through Akt, ERK and p38. This intracellular pathway is associated with the opening of mitochondrial K(ATP) channels and involves cAMP.

Endothelial Nitric Oxide Synthase in Human Intestine Resected for Necrotizing Enterocolitis

To determine the expression and function of endothelial nitric oxide synthase (eNOS) in submucosal arterioles harvested from human intestine resected for necrotizing enterocolitis (NEC) or congenital bowel disease. eNOS expression was determined by using immunohistochemistry. The arteriolar diameter was measured in vitro at pressures of 10 to 40 mm Hg and also in response to the eNOS agonist acetylcholine (ACh), the exogenous nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine, and the smooth muscle relaxant papaverine. Arteriolar release of NO in response to ACh was determined with a Sievers NOAnalyzer. Hemodynamics were also determined at flow rates of 50 and 100 microL/min. eNOS was present in microvessels from both groups, but NEC arterioles failed to demonstrate physiological evidence of eNOS function: they constricted in response to pressure, failed to dilate or generate NO in response to ACh, and failed to dilate in response to flow. However, they dilated in response to exogenous NO and papaverine, indicating functional vascular smooth muscle and vasodilator reserve. eNOS-derived NO, a vasodilator in the newborn intestine, did not contribute to vasoregulation in arterioles harvested from intestine resected for NEC. These vessels were constricted; lack of eNOS-derived NO may contribute to this vasoconstriction.

Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb.

Endothelin-1 (ET-1) acutely inhibits NaCl reabsorption by the thick ascending limb (THAL) by activating the ET(B) receptor, stimulating endothelial nitric oxide synthase (eNOS), and releasing nitric oxide (NO). In nonrenal tissue, chronic exposure to ET-1 stimulates eNOS expression via the ET(B) receptor and activation of phosphatidylinositol 3-kinase (PI3K). We hypothesized that ET-1 increases eNOS expression in the THAL by binding to ET(B) receptors and stimulating PI3K. In primary cultures of medullary THALs treated for 24 h, eNOS expression increased by 36 +/- 18% with 0.01 nM ET-1, 123 +/- 30% with 0.1 nM (P < 0.05; n = 5), and 71 +/- 30% with 1 nM, whereas 10 nM had no effect. BQ-788, a selective ET(B) receptor antagonist, completely blocked stimulation of eNOS expression caused by 0.1 nM ET-1 (12 +/- 25 vs. 120 +/- 40% for ET-1 alone; P < 0.05; n = 5). BQ-123, a selective ET(A) receptor antagonist, did not affect the increase in eNOS caused by 0.1 nM ET-1. Sarafotoxin c (S6c; 0.1 microM), a selective ET(B) receptor agonist, increased eNOS expression by 77 +/- 30% (P < 0.05; n = 6). Wortmannin (0.01 microM), a PI3K inhibitor, completely blocked the stimulatory effect of 0.1 microM S6c (77 +/- 30 vs. -28 +/- 9%; P < 0.05; n = 6). To test whether the increase in eNOS expression heightens activity, we measured NO release in response to simultaneous treatment with l-arginine, ionomycin, and clonidine using a NO-sensitive electrode. NO release by control cells was 337 +/- 61 and 690 +/- 126 pA in ET-1-treated cells (P < 0.05; n = 5). Taken together, these data suggest that ET-1 stimulates THAL eNOS, activating ET(B) receptors and PI3K and thereby increasing NO production.

Pulse of nitric oxide release in response to activation of N-methyl-D-aspartate receptors in the rat striatum: rapid desensitization, inhibition by receptor antagonists, and potentiation by glycine.

Increased activity of glutamate N-methyl-d-aspartate (NMDA) receptors is the dominant mechanism by which nitric oxide (NO.) is generated. By using a selective direct-current amperometry method, we characterized real time NO* release in vivo in response to chemical stimulation of NMDA receptors in the rat striatum. The application of NMDA caused the appearance of a sharp and transient oxidation signal. Concentration-response studies (10-500 microM) indicated an EC(50) of 48 microM. The NMDA-induced amperometric signal was suppressed by focal application of the nitric-oxide synthase inhibitor L-nitro-arginine methyl ester (L-NAME, 100 microM) or D-(-)-2-amino-5-phosphonopentanoic acid (AP-5, 100 microM) or by systemic injection of dizocilpine (1 mg/kg i.p.), drugs that, when given alone, had no effect on baseline oxidation current. Repeated injections of NMDA at short intervals (approximately 80 s) resulted in a progressive reduction of the amperometric signal with a decay half-life of 1.36 min. Sixty min after the last NMDA application the amperometric response was restored to initial levels. Finally, the coapplication of glycine (50 or 100 microM), which, when given alone had no effect, potentiated the NMDA-induced response. Thus, NMDA receptor-mediated activation of striatal NO* system shuts off quickly and undergoes rapid desensitization, suggesting a feedback inhibition of NMDA receptor function. To the extent of NO* release can represent a correlate of NMDA receptor activity, its amperometric detection could be useful to assess in vivo the state of excitatory transmission under physiological, pharmacological, or pathological conditions.

Reduced perivascular PO2 increases nitric oxide release from endothelial cells.

Many studies have suggested that endothelial cells can act as "oxygen sensors" to large reductions in oxygen availability by increasing nitric oxide (NO) production. This study determined whether small reductions in oxygen availability enhanced NO production from in vivo intestinal arterioles, venules, and parenchymal cells. In vivo measurements of perivascular NO concentration ([NO]) were made with NO-sensitive microelectrodes during normoxic and reduced oxygen availability. During normoxia, intestinal first-order arteriolar [NO] was 397 +/- 26 nM (n = 5), paired venular [NO] was 298 +/- 34 nM (n = 5), and parenchymal cell [NO] was 138 +/- 36 nM (n = 3). During reduced oxygen availability, arteriolar and venular [NO] significantly increased to 695 +/- 79 nM (n = 5) and 534 +/- 66 nM (n = 5), respectively, whereas parenchymal [NO] remained unchanged at 144 +/- 34 nM (n = 4). During reduced oxygenation, arteriolar and venular diameters increased by 15 +/- 3% and 14 +/- 5%, respectively: NG-nitro-L-arginine methyl ester strongly suppressed the dilation to lower periarteriolar Po2. Micropipette injection of a CO2 embolus into arterioles significantly attenuated arteriolar dilation and suppressed NO release in response to reduced oxygen availability. These results indicated that in rat intestine, reduced oxygen availability increased both arteriolar and venular NO and that the main site of NO release under these conditions was from endothelial cells.