共查询到20条相似文献,搜索用时 15 毫秒
1.
C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling 总被引:3,自引:0,他引:3
Singh U Devaraj S Vasquez-Vivar J Jialal I 《Journal of molecular and cellular cardiology》2007,43(6):780-791
C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O(2)(-)) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH(4) levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH(4), were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O(2)(-) production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O(2)(-) production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcgamma receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation. 相似文献
2.
Bevers LM Braam B Post JA van Zonneveld AJ Rabelink TJ Koomans HA Verhaar MC Joles JA 《Hypertension》2006,47(1):87-94
Endothelial NO synthase (eNOS) produces superoxide when depleted of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-arginine by uncoupling the electron flow from NO production. High expression of eNOS has been reported to have beneficial effects in atherosclerotic arteries after relatively short periods of time. However, sustained high expression of eNOS may have disadvantageous vascular effects because of uncoupling. We investigated NO and reactive oxygen species (ROS) production in a microvascular endothelial cell line (bEnd.3) with sustained high eNOS expression and absent inducible NOS and neuronal NOS expression using 4,5-diaminofluorescein diacetate and diacetyldichlorofluorescein as probes, respectively. Unstimulated cells produced both NO and ROS. After stimulation with vascular endothelial growth factor (VEGF), NO and ROS production increased. VEGF-induced ROS production was even further increased by the addition of extra L-arginine. Nomega-nitro-L-arginine methyl ester decreased ROS production. These findings strongly suggest that eNOS is a source of ROS in these cells. Although BH4 levels were increased as compared with another endothelial cell line, eNOS levels were >2 orders of magnitude higher. The addition of BH4 resulted in increased NO production and decreased generation of ROS, indicating that bEnd.3 cells produce ROS through eNOS uncoupling because of relative BH4 deficiency. Nevertheless, eNOS-dependent ROS production was not completely abolished by the addition of BH4, suggesting intrinsic superoxide production by eNOS. This study indicates that potentially beneficial sustained increases in eNOS expression and activity could lead to eNOS uncoupling and superoxide production as a consequence. Therefore, sustained increases of eNOS or VEGF activity should be accompanied by concomitant supplementation of BH4. 相似文献
3.
《Journal of molecular and cellular cardiology》2008,44(6):780-791
C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O2−) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH4 levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH4, were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O2− production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O2− production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcγ receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation. 相似文献
4.
Augmented BH4 by gene transfer restores nitric oxide synthase function in hyperglycemic human endothelial cells 总被引:9,自引:0,他引:9
OBJECTIVE: Endothelial dysfunction in diabetes is characterized by decreased nitric oxide (NO) bioactivity and increased superoxide (SO) production. Reduced levels of tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase (eNOS), appear to be associated with eNOS enzymatic uncoupling. We sought to investigate whether augmented BH4 biosynthesis in hyperglycemic human aortic endothelial cells (HAEC) by adenovirus-mediated gene transfer of GTP cyclohydrolase I (GTPCH, the rate-limiting enzyme for the de novo BH4 synthesis), would be sufficient to rescue eNOS activity and dimerization. HAEC were cultured in media with low glucose (5 mM) or high glucose (30 mM). METHODS: After 5 days, the cells with/without GTPCH gene transfer (AdeGFP as a control) were prepared for assays of (1) NO with electron paramagnetic resonance (EPR); (2) SO with cytochrome c reduction and dihydroethidine (DHE) fluorescence; (3) BH4 with high-performance liquid chromatography (HPLC); (4) eNOS expression and dimerization with immunoblotting. RESULTS: We found that high glucose decreased HAEC NO and increased SO production, in association with reductions in both total biopterin and BH4 levels. High glucose increased total eNOS protein levels in HAEC 1.5-fold, but this was present principally in the monomeric form. GTPCH gene transfer increased cellular biopterin levels and NO production but decreased SO production. Furthermore, augmenting BH4 increased the eNOS dimer:monomer ratio 2.6-fold. CONCLUSION: This study demonstrates a critical role for BH4 in regulating eNOS function, suggesting that GTPCH is a rational target to augment endothelial BH4 and recover eNOS activity in hyperglycemic endothelial dysfunction states. 相似文献
5.
Craige SM Chen K Pei Y Li C Huang X Chen C Shibata R Sato K Walsh K Keaney JF 《Circulation》2011,124(6):731-740
BACKGROUND- Reactive oxygen species serve signaling functions in the vasculature, and hypoxia has been associated with increased reactive oxygen species production. NADPH oxidase 4 (Nox4) is a reactive oxygen species-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia. METHODS AND RESULTS: Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the vascular endothelial cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hindlimb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by reactive oxygen species, we probed the effect of Nox4 on eNOS. In cultured endothelial cells overexpressing Nox4, we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4, we crossed our transgenic Nox4 mice with eNOS(-/-) mice. Aortas from these mice did not demonstrate enhanced aortic sprouting, and VECad-Nox4 mice on the eNOS(-/-) background did not demonstrate enhanced recovery from hindlimb ischemia. CONCLUSIONS: Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner. 相似文献
6.
BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension. 相似文献
7.
Kitamura T Kimura K Makondo K Furuya DT Suzuki M Yoshida T Saito M 《Diabetologia》2003,46(12):1698-1705
8.
9.
Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive rats 总被引:6,自引:0,他引:6
OBJECTIVE: Nitric oxide synthase (NOS)-derived nitric oxide (NO) production is regulated posttranslationally through enzyme's inhibitory interaction with the caveolar coat protein, caveolin and stimulatory interaction with the chaperone heat shock protein, Hsp90. However, changes in the expression of these regulators with the development of hypertrophic cardiomyopathy are unknown. METHODS: Histochemical and immunoblotted signals for the NOS isoforms, caveolin and Hsp90 were compared in left ventricle (LV) and aortic or mesenteric vessels between spontaneously hypertensive rats (SHR; 18 and 63 weeks old) and age-matched normotensive Wistar-Kyoto (WKY) rats. To assess functional impacts on downstream NO signaling, superoxide anions (O(2)(-)) and cGMP contents were measured in the same tissues by oxidative fluorescent hydroethidine staining and enzyme immunoassay, respectively. RESULTS: Compared with levels in age-matched WKY rats, endothelial NOS (eNOS) proteins were increased in aorta of SHR at 18 weeks. Conversely, aortic caveolin-1 and -3 were decreased in SHR, whereas Hsp90 remained unchanged. In LV tissue of SHR at 18 weeks, caveolin-1 and -3 were similarly decreased, but Hsp90 upregulated, together with a downregulation of eNOS. However, at 63 weeks, both eNOS and neuronal NOS (nNOS) were markedly upregulated in the LV of SHR, together with an upregulation of Hsp90. No difference in cardiac and aortic cGMP contents was found between the two strains. In LV sections, O(2)(-) generation was higher in older compared with younger rats from both strains and highest in 63 weeks SHR. CONCLUSIONS: Changes in NOS protein abundance in SHR rats compared with WKY controls are differentially regulated according to the age of hypertension and the tissue examined and are not necessarily correlated with cGMP contents. The coordinate expressional changes in NOS isoforms and their allosteric regulators, such as caveolin and Hsp90, may act as a compensatory mechanism to maintain the production of bioactive NO in the face of increased oxidant stress. 相似文献
10.
Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca2+/calmodulin, eNOS has several regulatory sites. Ser1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca2+ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca2+ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC) and PKC or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser1179 phosphorylation-dependent eNOS activation through a Ca2+-dependent, PKC-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway. 相似文献
11.
Pomace olive oil improves endothelial function in spontaneously hypertensive rats by increasing endothelial nitric oxide synthase expression 总被引:1,自引:0,他引:1
Rodriguez-Rodriguez R Herrera MD de Sotomayor MA Ruiz-Gutierrez V 《American journal of hypertension》2007,20(7):728-734
BACKGROUND: The effect of dietary pomace olive oil, which has the same concentration of oleic acid but a higher proportion of oleanolic acid (OA) than olive oil, was examined on animal models of hypertension for the first time. METHODS: During 12 weeks, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were fed with either a control 2% corn oil diet (BD), or high-fat diets containing 15% of refined olive oil (OL), pomace olive oil (POM), or pomace olive oil supplemented in OA (up to 800 ppm) (POMO). Then, vascular reactivity and endothelial nitric oxide (NO) synthase (eNOS) expression were studied in aortic rings. Plasma nitrite + nitrate levels were also determined. RESULTS: Diets had no effects on blood pressure (BP). In contrast to the BD and OL dietary groups, POM intake improved relaxation evoked by acetylcholine in SHR aorta. The POMO intake increased vasodilatation to acetylcholine and attenuated phenylephrine-induced contractions in both strains of rats associated with a major NO participation revealed by inhibition of NOS. The enhanced relaxation shown in POM and POMO SHR aorta was attributed to an increased eNOS protein expression. Plasma nitrite levels were also increased in these groups. Although olive and pomace oils used in diets contained similar fatty acid composition, beneficial effects on endothelial function were absent in the OL group. Therefore, these effects must be associated with some minor components from pomace olive oil such as OA. CONCLUSIONS: Chronic intake of diets rich in pomace olive oil improves endothelial dysfunction in SHR aorta by mechanisms associated with enhanced eNOS expression. Important evidence is provided regarding the effects of pomace olive oil and OA on endothelial function in hypertensive animals. 相似文献
12.
Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP 总被引:2,自引:0,他引:2
Alexander MY Brosnan MJ Hamilton CA Fennell JP Beattie EC Jardine E Heistad DD Dominiczak AF 《Cardiovascular research》2000,47(3):609-617
OBJECTIVE: Previous studies from our group have shown a deficit in nitric oxide (NO) bioavailability and an excess production of the superoxide anion (O(2)(-)) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the NO/O(2)(-) balance, thereby improving endothelial function. METHODS: A recombinant adenovirus, Ad/Hu/eNOS, containing the human eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into SHRSP carotid arteries in vivo, using a titre of 2x10(9)-2x10(10) plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. RESULTS: Western blotting confirmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L-NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insignificant increase in NO bioavailability. CONCLUSIONS: Our results support our previous findings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a significant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function. 相似文献
13.
Stroes ES van Faassen EE Yo M Martasek P Boer P Govers R Rabelink TJ 《Circulation research》2000,86(11):1129-1134
5-methyltetrahydrofolate (MTHF), the active form of folic acid, has been reported to restore NO status in hypercholesterolemic patients. The mechanism of this effect remains to be established. We assessed the effects of L- and D-MTHF on tetrahydrobiopterin (BH(4))-free and partially BH(4)-repleted endothelial NO synthase (eNOS). Superoxide production of eNOS and the rate constants for trapping of superoxide by MTHF were determined with electron paramagnetic resonance using 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) as spin trap for superoxide. NO production was measured with [(3)H]arginine-citrulline conversion or nitrite assay. The rate constants for scavenging of superoxide by L- and D-MTHF were similar, 1.4 x 10(4) ms(-1). In BH(4)-free eNOS, L- and D-MTHF have no effect on enzymatic activity. In contrast, in partially BH(4)-repleted eNOS, we observe a 2-fold effect of MTHF on the enzymatic activity. First, superoxide production is reduced. Second, NO production is enhanced. In cultured endothelial cells, a similar enhancement of NO production is induced by MTHF. In the present study, we show direct effects of MTHF on the enzymatic activity of NO synthase both in recombinant eNOS as well as in cultured endothelial cells, which provides a plausible explanation for the previously reported positive effects of MTHF on NO status in vivo. 相似文献
14.
Maffei A Poulet R Vecchione C Colella S Fratta L Frati G Trimarco V Trimarco B Lembo G 《Journal of hypertension》2002,20(6):1135-1142
INTRODUCTION : Although in hypertension a defect in stimulated nitric oxide (NO) is well established, little is known about basal NO levels. Thus, we measured directly in vessels from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHR) both basal and stimulated NO production using a novel technique [4,5-diaminofluorescein (DAF-2) fluorescence]. METHODS : Isolated vessels were exposed to the fluorescent probe DAF-2. After the technique was validated with increasing doses of acetylcholine in the presence and absence of NG-nitro-L-arginine methyl ester (l-NAME), we measured NO production in vessels from WKY rats and SHR in the same experimental setting. Finally, to explore the impact of reactive oxygen species (ROS) on NO release, we analysed the effect of an antioxidant, such as ascorbic acid, on basal and stimulated NO in aortic rings of WKY rats and SHR. RESULTS : Aortic rings from SHR exhibited a higher basal NO production and a lower responsiveness to agonist-induced NO release as compared with those observed in WKY rats. Also in resistance vessels such as mesenteric arteries, basal NO production was higher in hypertension. In hypertensive rats, ascorbic acid was able to further increase basal NO release and recovered the impaired stimulated NO production, whereas no effect was detected in normotensive rats. CONCLUSIONS : Our data reveal an increased basal NO availability in hypertension despite the increased production of ROS, suggesting a greater complexity in hypertensive endothelial dysfunction when the analysis is focused on direct NO measurement. 相似文献
15.
Aims/hypothesis Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice.Methods Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting.Results In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged.Conclusions/interpretation Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes. 相似文献
16.
Earlier studies have demonstrated that nitric oxide (NO) exerts a fast-acting inhibitory influence on endothelial NO synthase (eNOS) enzymatic activity in isolated vascular tissue preparations. The present study was designed to examine the possible effect of NO on eNOS protein expression in cultured endothelial cells and intact animals. Human coronary endothelial cells were incubated with S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor), oxyhemoglobin (HGB, an NO trapping agent), SNAP plus HGB, or inactive vehicle (control). In other experiments, cells were treated with 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), 1H-[1,2, 4]oxadiazolo-[4,3-2]quinoxalin-1-one (ODQ, a guanylate cyclase inhibitor), SNAP plus ODQ, 8-bromo-cGMP (8-Br-cGMP, a cell-permeable cGMP compound), 8-Br-cGMP plus HGB, or inactive vehicle in order to discern the effect of cGMP. The incubations were conducted for 24 hours, and total nitrate plus nitrite production and eNOS protein abundance (Western analysis) were measured. To determine the effect of NO on eNOS expression in vivo, rats were treated with either the NO donor isosorbide dinitrate or placebo by gastric gavage for 48 hours, and aortic eNOS protein expression was examined. The NO donor SNAP markedly depressed, whereas the NO scavenger HGB significantly raised, eNOS protein expression. The downregulatory action of SNAP was completely abrogated by HGB. Phosphodiesterase inhibitor and 8-Br-cGMP downregulated, whereas the guanylate cyclase inhibitor ODQ upregulated eNOS protein expression. The downregulatory action of SNAP was completely overcome by the guanylate cyclase inhibitor ODQ, and the upregulatory action of the NO scavenger HGB was abrogated by 8-Br-cGMP. Administration of NO donor resulted in a marked downregulation of aortic eNOS protein expression in intact animals, thus confirming the in vitro findings. NO serves as a negative-feedback regulator of eNOS expression via a cGMP-mediated process. 相似文献
17.
目的探讨非诺贝特能否对脂多糖(LPS)诱导的血管内皮一氧化氮合酶(eNOS)脱偶联发挥保护作用。方法体外培养人脐静脉内皮细胞(HUVECs),用非诺贝特预处理HUVECs 2 h,再与LPS共孵育24 h,采用高效液相色谱法检测细胞四氢生物蝶呤(BH4)的表达水平,ELISA检测细胞eNOS表达水平和细胞上清一氧化氮(NO)浓度,利用Confocal方法检测细胞内活性氧(ROS)产生水平。结果与对照组比较,单纯LPS刺激组内皮细胞BH4表达水平降低,伴有eNOS表达下调和NO水平降低,而内皮细胞内ROS产生增加(P均〈0.05)。与单纯LPS刺激组比较,非诺贝特预处理组内皮细胞BH4表达水平升高,同时伴有eNOS表达上调和NO水平增加,而内皮细胞内ROS产生降低(P均〈0.05)。结论非诺贝特通过上调BH4水平,对LPS诱导的血管内皮细胞eNOS脱偶联有逆转作用,这可能是其发挥血管内皮保护作用的机制之一。 相似文献
18.
Bendall JK Alp NJ Warrick N Cai S Adlam D Rockett K Yokoyama M Kawashima S Channon KM 《Circulation research》2005,97(9):864-871
Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2*-) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2*- rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O2*- production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2*- production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2*- production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOS-BH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo. 相似文献
19.
Erythropoietin protects cardiomyocytes from apoptosis via up-regulation of endothelial nitric oxide synthase 总被引:15,自引:0,他引:15
Burger D Lei M Geoghegan-Morphet N Lu X Xenocostas A Feng Q 《Cardiovascular research》2006,72(1):51-59
OBJECTIVE: Erythropoietin (EPO), a cytokine best known for its ability to increase red blood cell mass, has recently been shown to protect cardiomyocytes from apoptotic cell death. The objective of the present study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the anti-apoptotic effects of EPO in cardiomyocytes. METHODS AND RESULTS: Neonatal mouse ventricular cardiomyocytes were isolated and cultured from wild-type and eNOS(-/-) mice. Treatment with EPO significantly reduced apoptosis induced by norepinephrine (NE) in the wild-type cardiomyocytes. The reduction of apoptosis was associated with significant increases in eNOS expression, phosphorylation and NO production. However, the anti-apoptotic effects of EPO were significantly decreased in wild-type cardiomyocytes treated with L-NAME, which inhibits nitric oxide synthase activity. The results were further confirmed using eNOS(-/-) cardiomyocytes. To investigate the in vivo significance of eNOS in mediating the anti-apoptotic effects of EPO, wild-type and eNOS(-/-) mice were subjected to myocardial ischemia and reperfusion. EPO decreased myocardial apoptosis and infarct size in wild-type mice. However, the protective effects of EPO were significantly diminished in eNOS(-/-) mice. CONCLUSIONS: EPO increases eNOS expression and NO production in cardiomyocytes. The anti-apoptotic effects of EPO in cardiomyocytes are mediated by eNOS-derived NO production. 相似文献
20.
Atherosclerosis is associated with an impairment of endothelium-dependent relaxations, which represents the reduced bioavailability of nitric oxide (NO) produced from endothelial NO synthase (eNOS). Among various mechanisms implicated in the impaired EDR in atherosclerosis, superoxide generated from dysfunctional eNOS has attracted attention. Under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a cofactor for NOS, are deficient or lacking, eNOS becomes dysfunctional and produces superoxide rather than NO. Experimental studies in vitro have revealed that NO from eNOS constitutes an anti-atherogenic molecule. A deficiency of eNOS was demonstrated to accelerate atherosclerotic lesion formation in eNOS knockout mice. In contrast, eNOS overexpression with hypercholesterolemia may promote atherogenesis via increased superoxide generation from dysfunctional eNOS. Thus, eNOS may have 2 faces in the pathophysiology of atherosclerosis depending on tissue BH4 metabolisms. An improved understanding of tissue BH4 metabolisms in atherosclerotic vessels is needed, which would help in developing new strategies for the inhibition and treatment of atherosclerosis. 相似文献