Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.     

Endothelial nitric oxide production is tightly coupled to the citrulline–NO cycle

Nitric oxide (NO) is an important vasorelaxant produced along with L-citrulline from L-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of L-citrulline to L-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of L-citrulline to L-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of L-citrulline formed due to the efficiency of L-citrulline recycling via the citrulline-NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-L-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-L-citrulline formation were simultaneously determined. NO production exceeded apparent L-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, alpha-methyl-DL-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline-NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of L-citrulline recycling, via the citrulline-NO cycle, to endothelial NO production.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells

Although cellular levels of arginine greatly exceed the apparent K(m) for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.     

胰岛素促进血管内皮细胞产生一氧化氮的实验研究

目的：探讨胰岛素对血管内皮细胞增殖、NO产生和NOS基因表达的影响。方法：培养牛主动脉内皮细胞,测定培养上清液中NO氧化产物NO2^－的水平并应用定量RT－PCR技术检测内皮细胞NOS mRNA的表达水平。结果：①胰岛素对大血管内皮细胞无细胞毒作用,也不影响细胞增殖;②在1－15μg/ml浓度范围内,胰岛素加强内皮细胞释放NO,且呈剂量依赖的方式,NOS特异性抑制剂L－NAME可阻抑之;③胰岛素轻度增加NOS mRNA表达水平,但无统计学意义。结论：胰岛素既不影响大血管内皮细胞增殖,也不影响内皮细胞NOS mRNA表达水平,但以剂量依赖的方式加强内皮细胞产生NO,推测其诱导NO产生的机制可能是通过酶活性的诱导,加速NO的合成。     

Regulation of endothelial argininosuccinate synthase expression and NO production by an upstream open reading frame

Argininosuccinate synthase (AS) catalyzes the rate-limiting step in the recycling of citrulline to arginine, which in endothelial cells, is tightly coupled to the production of nitric oxide (NO). In previous work, we established that endothelial AS mRNA can be initiated from multiple start sites, generating co-expressed mRNA variants with different 5'-untranslated regions (5'-UTRs). One of the 5'-UTRs, the shortest form, represents greater than 90% of the total AS mRNA. Two other extended 5'-UTR forms of AS mRNA, resulting from upstream initiations, contain an out-of-frame, upstream open reading frame (uORF). In this study, the function of the extended 5'-UTRs of AS mRNA was investigated. Single base insertions to place the uORF in-frame, and mutations to extend the uORF, demonstrated functionality, both in vitro with AS constructs and in vivo with luciferase constructs. Overexpression of the uORF suppressed endothelial AS protein expression, whereas specific silencing of the uORF AS mRNAs resulted in the coordinate up-regulation of AS protein and NO production. Expression of the full-length of the uORF was necessary to mediate a trans-suppressive effect on endothelial AS expression, demonstrating that the translation product itself affects regulation. In conclusion, the uORF found in the extended, overlapping 5'-UTR AS mRNA species suppresses endothelial AS expression, providing a novel mechanism for regulating endothelial NO production by limiting the availability of arginine.     

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.     

Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10⁻³, 10⁻⁵, 10⁻⁷ and 10⁻⁹ M sodium nitroprusside (SNP). PFs were further cultured with 10⁻⁵ M SNP + 1.0 mM N^ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10⁻³, 10⁻⁵, 10⁻⁷ M) inhibited (P < 0.05) while, lower dose of SNP (10⁻⁹ M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

Grape seed extract enhances eNOS expression and NO production through regulating calcium‐mediated AKT phosphorylation in H2O2‐treated endothelium

GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H₂O₂ (hydrogen peroxide)‐treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H₂O₂‐treated cells in a dose‐dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose‐dependent manner in H₂O₂‐treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 μM GSE, 2.0 μM TG (thapsigargin) and 20.0 μM 2‐APB (2‐aminoethoxydiphenyl borate) instead of 0.25 μM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p‐AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up‐regulation of eNOS and NO expression via inhibiting InsP₃Rs (inositol 1,4,5‐trisphosphate receptors)‐mediated intracellular excessive calcium release and by activating p‐AKT in endothelial cells.     

Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.     

Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

Background  

The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.     

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.