Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.     

Chelation of intracellular iron enhances endothelial barrier function: A role for vitamin C?

Ascorbic acid improves endothelial barrier function by decreasing the permeability of endothelial cells cultured on semi-porous membrane filters. This decrease was not due to enhanced collagen synthesis and was mimicked by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoic acid (EDHB). Since EDHB is known to chelate intracellular free iron, the effects of two membrane-permeant iron chelators were tested on endothelial permeability. Both 2,2′-dipyridyl and desferrioxamine decreased trans-endothelial permeability in a concentration-dependent manner. Increasing intracellular iron with a chelate of 8-hydroxyquinoline and ferric iron prevented effects of both EDHB and intracellular ascorbate. That EDHB and ascorbate did in fact chelate intracellular iron was supported by finding that they both decreased the cellular fluorescence quenching of the iron-sensitive dye Phen green SK. These results show that chelation of intracellular iron decreases endothelial barrier permeability and implicate this mechanism in the ability of EDHB and possibly intracellular ascorbate to tighten the endothelial barrier.     

Desflurane inhibits endothelium-dependent vasodilation more than sevoflurane with inhibition of endothelial nitric oxide synthase by different mechanisms

The effects of desflurane on endothelium-dependent vasodilation remain uncertain, whereas sevoflurane is known to inhibit it. Endothelium-dependent vasodilation is mainly mediated by endothelial nitric oxide synthase. The effects of desflurane on endothelium-dependent vasodilation were compared with those of sevoflurane, and inhibition mechanisms, including phosphorylation of endothelial nitric oxide synthase and the calcium pathway, were evaluated for the two anesthetics. We hypothesized that desflurane would inhibit endothelium-dependent vasodilation in a concentration-dependent manner more than sevoflurane, with inhibition of a calcium pathway.Isolated rat aortic rings were randomly assigned to treatment with desflurane or sevoflurane for measurements of the vasodilation ratio. To determine NO production with desflurane and sevoflurane, an in vitro assay was performed with cultured bovine aortic endothelial cells. These cells were also used for measurement of intracellular calcium or Western blotting.For endothelium-dependent vasodilation, the ratio of vasodilation was more significantly inhibited by 11.4% desflurane than by 4.8% sevoflurane. Inhibition did not between 5.7% desflurane and 2.4% sevoflurane. No inhibitory effect of desflurane or sevoflurane was observed in endothelium-denuded aorta. Desflurane inhibited nitric oxide production caused by stimulation of bradykinin significantly more than sevoflurane. Desflurane had a greater suppressive effect on the bradykinin-induced increase in intracellular calcium concentration than did sevoflurane. Sevoflurane, but not desflurane, inhibited phosphorylation of the serine 1177 residue by bradykinin stimulation.Desflurane inhibited endothelium-dependent vasodilation more than sevoflurane through inhibition of a calcium pathway. Sevoflurane inhibited endothelium-dependent vasodilation by inhibition of phosphorylation of the serine 1177 residue of endothelial nitric oxide synthase.     

Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway

Bradykinin (BK) acutely increases endothelial nitric oxide (NO) production by activating endothelial NO synthase (eNOS), and this increase is in part correlated with enhanced phosphorylation/dephosphorylation of eNOS by several protein kinases and phosphatases. However, the signaling mechanisms producing this increase are still controversial. In an attempt to delineate the acute effect of BK on endothelial NO production, confluent bovine aortic endothelial cells were incubated with BK, and NO production was measured by NO-specific chemiluminescence. Significant increase in NO levels was detected as early as 1 min after BK treatment, with concomitant increase in the phosphorylation of Ser(1179) (bovine sequence) site of eNOS (eNOS-Ser(1179)). This acute effect of BK on both increases was blocked only by treatment of protein kinase A inhibitor H-89, but not by the inhibitors of calmodulin-dependent kinase II and protein kinase B, suggesting that the rapid increase in NO production by BK is mediated by the PKA-dependent phosphorylation of eNOS-Ser(1179).     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.     

Activation by Nitric Oxide of Guanylate Cyclase in Endothelial Cells from Brain Capillaries

Endothelial cells (ECs) from brain microvessels respond to exogenous nitric oxide (NO) donor molecules (N-ethoxycarbonyl-3-morpholinosydnonimine and sodium nitroprusside) with large (greater than 15-fold) increases in cyclic GMP (cGMP) levels. Comparable actions of sodium nitroprusside were observed in vascular smooth muscle cells and in neuroblastoma cells. Coculturing brain capillary ECs in the presence of N1E-115 neuroblastoma cells increased their cGMP levels fourfold. A further increase was observed in the presence of 50 nM neurotensin, although brain capillary ECs lack receptor sites for neurotensin. The neuroblastoma cell-dependent formation of cGMP was suppressed by 0.1 mM L-NG-monomethylarginine, indicating that NO, produced by N1E-115 cells in response to neurotensin, activated guanylate cyclase in brain capillary ECs. Similarly, culturing brain capillary ECs in the presence of aortic ECs increased their cGMP content in a manner that was amplified by bradykinin and that was inhibited by L-NG-monomethylarginine. Bradykinin had no action in pure cultures of brain capillary ECs. It is concluded that brain capillary ECs express high levels of guanylate cyclase activity that could be activated by exogenous NO donor molecules and by NO produced by neuroblastoma cells and by aortic ECs in response to specific agonists. Brain capillary ECs are thus potential target cells for brain-derived NO.     

Nitric oxide signaling in invertebrates

Nitric oxide (NO) is an unconventional neurotransmitter and neuromodulator molecule that is increasingly found to have important signaling functions in animals from nematodes to mammals. NO signaling mechanisms in the past were identified largely through experiments on mammals, after the discovery of NO's vasodilatory functions. The use of gene knock out mice has been particularly important in revealing the functions of the several isoforms of nitric oxide synthase (NOS), the enzyme that produces NO. Recent studies have revealed rich diversity in NO signaling. In addition to the well-established pathway in which NO activates guanylyl cyclase and cGMP production, redox mechanisms involving protein nitrosylation are important contributors to modulation of neurotransmitter release and reception. NO signaling studies in invertebrates are now generating a wealth of comparative information. Invertebrate NOS isoforms have been identified in insects and molluscs, and the conserved and variable amino acid sequences evaluated. Calcium-calmodulin dependence and cofactor requirements are conserved. NADPH diaphorase studies show that NOS is found in echinoderms, coelenterates, nematodes, annelids, insects, crustaceans and molluscs. Accumulating evidence reveals that NO is used as an orthograde transmitter and cotransmitter, and as a modulator of conventional transmitter release. NO appears to be used in diverse animals for certain neuronal functions, such as chemosensory signalin, learning, and development, suggesting that these NO functions have been conserved during evolution. The discovery of NO's diverse and unconventional signaling functions has stimulated a plethora of enthusiastic investigations into its uses. We can anticipate the discovery of many more interesting and some surprising NO signaling functions.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO-mediated pathways. Fluorescently labeled mitochondria were studied using time-lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re-oxygenation or to diethyleneamine/nitric oxide adduct, DETA-NO (100-500 microm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 +/- 1.3% at 0 h and 7.4 +/- 1.7% at 1 h of re-oxygenation, versus 25.6 +/- 1.4% in controls (p < 0.05). Mean mitochondrial velocity (microm s(-1)) decreased from 0.374 +/- 0.01 in controls to 0.146 +/- 0.01 at 0 h and 0.177 +/- 0.02 at 1 h of re-oxygenation (p < 0.001). Exposure to DETA-NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG-nitro-L-arginine methyl ester (L-NAME) prevented the hypoxia-induced decrease in mitochondrial movement at 0 h (30.1 +/- 1.6%) and at 1 h (26.1 +/- 9%) of re-oxygenation. Exposure to either hypoxia/re-oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO-mediated pathway is at least partially involved.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness

Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.     

Nitric oxide-induced oxidant stress in endothelial cells: amelioration by ascorbic acid

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.     

Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats

We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. l-Nitroarginine methyl ester (l-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.     

神经型一氧化氮合酶的活性调控

一氧化氮是重要的信使分子,在生物体内参与众多生理及病理过程。生物体内存在着复杂的一氧化氮合酶活性调控机制以精确调控一氧化氮的生成。在神经系统中,一氧化氮主要由神经型一氧化氮合酶催化生成。神经型一氧化氮合酶的活性主要受到翻译后水平上钙离子和钙调蛋白的调控,其调控方式包括二聚化、多位点的磷酸化和去磷酸化,以及主要由PDZ结构域介导的蛋白质-蛋白质相互作用。一氧化氮本身对其合酶的活性具有负反馈调控作用。近年来的研究提示,细胞质膜上的脂筏微区在神经性一氧化氮合酶的活性调控中也起到重要的调节作用。     

Nitric oxide mediates lung injury induced by ischemia-reperfusion in rats

Nitric oxide (NO) has been reported to play a role in lung injury (LI) induced by ischemia-reperfusion (I/R). However, controversy exists as to the potential beneficial or detrimental effect of NO. In the present study, an in situ, perfused rat lung model was used to study the possible role of NO in the LI induced by I/R. The filtration coefficient (Kfc), lung weight gain (LWG), protein concentration in the bronchoalveolar lavage (PCBAL), and pulmonary arterial pressure (PAP) were measured to evaluate the degree of pulmonary hypertension and LI. I/R resulted in increased Kfc, LWG, and PCBAL. These changes were exacerbated by inhalation of NO (20-30 ppm) or 4 mM L-arginine, an NO precursor. The permeability increase and LI caused by I/R could be blocked by exposure to 5 mM N omega-nitro-L-arginine methyl ester (L-NAME; a nonspecific NO synthase inhibitor), and this protective effect of L-NAME was reversed with NO inhalation. Inhaled NO prevented the increase in PAP caused by I/R, while L-arginine had no such effect. L-NAME tended to diminish the I/R-induced elevation in PAP, but the suppression was not statistically significant when compared to the values in the I/R group. These results indicate that I/R increases Kfc and promotes alveolar edema by stimulating endogenous NO synthesis. Exogenous NO, either generated from L-arginine or delivered into the airway, is apparently also injurious to the lung following I/R.     

膜雌激素受体介导一氧化氮合酶活性增高的快速非基因效应

实验利用新生小牛胸主动脉内皮细胞(BAECs)作为模型,观察17β-雌二醇(E2)、E2BSA对BAECs中内皮型一氧化氯合酶(eNOS)的快速激活作用,并探讨了丝裂素活化蛋白激酶(MAPK)信号通路在其中的作用。结果显示,不同浓度的E2(0．001—1lμmol/L)作用于BAECs l5 min均能快速激活eNOS;0．01μmol/L浓度的E2作用于BAECs,5min即能激活eNOS,15min达到最大效应,随后eNOS快速失活;E2BSA(17．5ng／m1)作用于BAECs,15min同样可激活eNOS。E2、E2BSA激活eNOS的作用均能被雌激素受体(ER)拮抗剂tamoxifen(0．1μmol/L)或MAPK激酶特异抑制剂PD98059(50μmol/L)所阻断。放线菌素D(25μg/ml)不能阻断E2、E2BSA对eNOS的激活作用。E2(0．01μmol/L)、E2BSA(17．5ng/ml)作用于BAECs l5 min后可明显促进p42／p44磷酸化MAPK蛋白表达,而对p42／p44 MAPK总蛋白表达无影响。Tamoxifen可部分阻断E2;E2BSA激活p42／p44磷酸化MAPK的作用。这些结果提示,BAECs膜上可能存在膜雌激素受体(membrane estrogen receptor,mER),E2、E2BSA作用于mER后可通过MAPK信号途径快速激活eNOS。     

Nitric oxide modulates the responses of osteoclast formation to static magnetic fields

Nitric oxide (NO) is involved in osteoclast differentiation. Our previous studies showed that static magnetic fields (SMFs) could affect osteoclast differentiation. The inhibitory effects of 16 T of high SMF (HiMF) on osteoclast differentiation was correlated with increased production of NO. We raised the hypothesis that NO mediated the regulatory role of SMFs on osteoclast formation. In this study, 500 nT of hypomagnetic field (HyMF), 0.2 T of moderate SMF (MMF) and 16 T of high SMF (HiMF) were utilized as SMF treatment. Under 16 T, osteoclast formation was markedly decreased with enhanced NO synthase (NOS) activity, thus producing a high level of NO. When treated with NOS inhibitor N-Nitro-L-Arginine Methyl Ester (L-NAME), NO production could be inhibited, and osteoclast formation was restored to control group level in a concentration-dependent manner. However, 500 nT and 0.2 T increased osteoclast formation with decreased NOS activity and NO production. When treated with NOS substrate L-Arginine (L-Arg) or NO donor sodium nitroprusside (SNP), the NO level in the culture medium was obviously elevated, thus inhibiting osteoclast differentiation in a concentration-dependent manner under 500 nT or 0.2 T. Therefore, these findings indicate that NO mediates the regulatory role of SMF on osteoclast formation.