Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.     

Nitric oxide enhances PGI(2)production by human pulmonary artery smooth muscle cells

To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI(2)) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1(beta)(IL-1(beta)), tumor necrosis factor alpha (TNF(alpha)) or interferon gamma (IFN(gamma)), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, N(G)-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI(2)by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS, IL-1(beta)or TNF(alpha)for 24 h caused a significant increase in nitrite release and PGI(2)production. However, IFN(gamma)slightly increased the release of nitrite and had little effect on PGI(2)production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI(2)production, the incubation of HPASMC with SNP and 10 microg/ml LPS, or with SNP and 100 U/ml IL-1(beta)further increase PGI(2)production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI(2)production was not found in TNF(alpha)- and IFN(gamma)- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1(beta)reduced nitrite release and attenuated the stimulatory effect of those agents on PGI(2)production. MeB significantly suppressed the production of PGI(2)by HPASMC treated with or without LPS or IL-1(beta). The addition of SNP partly reversed the inhibitory effect of MeB on PGI(2)production by HPASMC. These experimental results suggest that NO might stimulate PGI(2)production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI(2)production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome.     

Requirement for Nitric Oxide in Retinal Neuronal Cell Death Induced by Activated Müller Glial Cells

Retinal Müller glial cells express the inducible isoform (-2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS-2 [NOS-2 (-/-)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN-gamma-stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS-2 (-/-) mice and because inhibition of NOS-2 induction by transforming growth factor-beta or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP-ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells is responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.     

Endogenous peroxynitrite modulates PGHS-1-dependent thromboxane A2 formation and aggregation in human platelets

Aggregation of activated platelets is considerably mediated by the autocrine action of thromboxane A2 (TxA2) which is formed in a prostaglandin endoperoxide H2 synthase-1 (PGHS-1 or COX-1)-dependent manner. The activity of PGHS-1 can be stimulated by peroxides, an effect termed "peroxide tone", that renders PGHS-1 the key regulatory enzyme in the formation of TxA2. Activated platelets release nitric oxide (*NO) and superoxide (O*2) but their interactions with the prostanoid pathway have been controversially discussed in platelet physiology and pathophysiology. The current study demonstrates that endogenously formed peroxynitrite at nanomolar concentrations, originating from the interaction of *NO and *O2, potently activated PGHS-1, which parallels TxA2 formation and aggregation in human platelets. Inhibition of the endogenous formation of either *NO or O*2 resulted in a concentration-dependent decline of PGHS-1 activity, TxA2 release, and aggregation. The concept of peroxynitrite as modulator of TxA2 formation and aggregation explains the interaction of *NO and O*2 with the PGHS pathway and suggests a mechanism by which antioxidants can regulate PGHS-1-dependent platelet aggregation. This may provide a molecular explanation for the clinically observed hyperreactivity of platelets in high-risk patients and serve as a basis for novel therapeutic interventions.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.     

Differential effects of nitric oxide on the activity of prostaglandin endoperoxide H synthase-1 and -2 in vascular endothelial cells

A number of studies have demonstrated that prostacyclin and nitric oxide (NO) regulate blood pressure, blood flow and platelet aggregation. In this paper, we have examined the possible relationship between NO and prostaglandin endoperoxide H synthase (PGHS)-1 and -2 activities in cultured bovine aortic endothelial cells. In the non-activated condition endothelial cells expressed PGHS-1 activity alone. When these cells were pretreated with aspirin to inactivate their PGHS-1 and then activated by serum and phorbol ester (TPA) for 6 h, the cells expressed PGHS-2 activity alone. The PGHS activity was assessed by the generation of 6-ketoprostaglandin F1alpha (6-ketoPGF1alpha), a stable metabolite of prostacyclin, after the treatment of these cells with arachidonic acid. The simultaneous addition of NOC-7, a NO donor, with arachidonic acid did not affect the production of 6-ketoPGF1alpha in PGHS-1 expressed cells, but attenuated it in PGHS-2-expressed cells. The inhibitory effect of NOC-7 on PGHS-2 activity was dose dependent, and the different effects of NOC-7 on the activities of PGHS isozymes were also observed in other NO donors. To confirm the different effect of NO on PGHS isozymes demonstrated in the cultured endothelial cells, we carried out an ex vivo perfusion assay in aorta isolated from normal and lipopolysaccharide (LPS)-treated rats. In the aortae isolated from normal rats, where dominant expression of PGHS-1 was expected, the NO donor did not affect the PGHS activity, while in aortae isolated from LPS-treated rats, where PGHS-2 was dominantly expressed, the NO donor dramatically inhibited the PGHS activity, suggesting that NO suppressed PGHS-2 activity alone. The inhibitory effect of NO on PGHS-2 activity was not mediated by cyclic GMP (cGMP), since (a) methylene blue, an inhibitor of soluble guanylate cyclase did not abolish the inhibitory effect of the NO donor on PGHS-2 activity, and (b) 8-Br-cGMP, a permeable cGMP analogue, failed to mimic the effect of NO donors. These data suggest that the effect of NO on prostacyclin production in endothelial cells was dependent on the expression rate of PGHS-1 and PGHS-2 in the cells.     

Porphyromonas gingivalis lipopolysaccharide interferes with salivary mucin synthesis through inducible nitric oxide synthase activation by ERK and p38 kinase

Porphyromonas gingivalis is a Gram-negative periodontopathic bacterium colonizing the oral cavity and its lipopolysaccharide (LPS) is a key factor in the development of periodontitis. We investigated the effect of P. gingivalis LPS on the cellular responses associated with mucin synthesis in sublingual salivary gland acinar cells. Exposure of the acinar cells to the LPS led to a dose-dependent decrease in mucin synthesis and was accompanied by a massive induction in inducible nitric oxide synthase (NOS-2) activity and the increase in NO production, caspase-3 activity and apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) with PD98059 accelerated the LPS-induced decrease in the glycoprotein synthesis and caused further increase in apoptosis and NOS-2 activity, while the blockade of p38 mitogen-activated kinase (MAPK) with SB203580 countered the LPS-induced reduction in the glycoprotein synthesis and obviated the induced increases in NOS-2 and apoptosis. Introduction of NOS-2 inhibitor, L-NAME, not only countered the LPS-induced increase in NO generation, caspase-3 activity and apoptosis, but caused the impedance of the LPS inhibition on mucin synthesis. The findings point to the upregulation in NOS-2 expression by P. gingivalis LPS as a key detrimental culprit affecting salivary mucin synthesis.     

TGF beta 1 and TNF alpha potentiate nitric oxide production in astrocyte cultures by recruiting distinct subpopulations of cells to express NOS-2

Nitric oxide (NO) synthase-2 (NOS-2), a key source of NO at sites of neuroinflammation, is induced in astrocyte cultures treated with lipopolysaccharide (LPS) plus interferon-gamma (IFN gamma). A recent study examining the regulation of astrocytic NOS-2 expression demonstrated that transforming growth factor-beta1 (TGF beta 1) potentiated LPS plus IFN gamma-induced NOS-2 expression via expansion of the pool of astrocytes that express NOS-2. Results in the current report indicate that this population-based mechanism of increasing NOS-2 expression is not restricted to TGF beta 1, since it also accounts for the potentiation of NO production in astrocyte cultures by tumor necrosis factor-alpha (TNFalpha). In contrast to TGF beta 1, which required 24h preincubation for optimal potentiation of NO production, TNF alpha was maximally effective when added concurrently with LPS plus IFN gamma. Nevertheless, under conditions that optimally potentiated NO production, both cytokines recruited similar numbers of astrocytes to express NOS-2 (% NOS-2-positive cells after LPS plus IFN gamma alone or with TNFalpha or TGF beta 1 was 9.5+/-1.2, 25.3+/-2.9, and 32.4+/-3.0, respectively). Interestingly, stimulation of astrocytes in the presence of both TGF beta 1 and TNFalpha additively increased the number of astrocytes that expressed NOS-2 protein (% NOS-2-positive cells was 61.0+/-4.2) relative to each cytokine alone. Potentiation of NO production by either TNF alpha or TGF beta 1 was not ablated by neutralizing antibodies to TGF beta 1 or TNFalpha, respectively. Thus, the two cytokines act independently to recruit separate pools of astrocytes to express NOS-2. These results are consistent with the notion that astrocytes possess an innate heterogeneity with respect to responsiveness to these cytokines.     

Inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats

Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-beta (IL-1beta) are postulated to play a key pathophysiologic role during sepsis. In this study, we examined the time course of inducible NO synthase (iNOS) mRNA expression and the plasma TNF-alpha and IL-1beta in lipopolysaccharide (LPS)-treated conscious rats. The hemodynamic pattern in septic shock is more similar to clinical conditions without anesthesia. The data showed that a significant increase in iNOS mRNA levels was found in the spleen, lung, liver, with slight elevation in the heart and kidney at 3 h after LPS administration. However, iNOS mRNA levels were not elevated significantly in all tissues examined at 24 h. In the plasma, TNF-alpha and IL-1beta culminated within 1 h, and reduced gradually to baseline levels in a relatively short period (within 9 h). The results suggest that local NO production by activation of iNOS mRNA expression and cytokine release may contribute to LPS-induced organ dysfunction at various time points.     

Lipopolysaccharide (LPS) induction of nitric oxide synthase-2 and cyclooxygenase-2 is impaired in fructose overloaded rats

AimsFructose (F) overload in rats induces metabolic dysfunctions that resemble the human metabolic syndrome. In this paper, we aimed to investigate the response of F overload rats to lipopolysaccharide (LPS) challenge in terms of nitric oxide (NO) production and prostanoids (PR) release.Main methodsNO blood steady-state concentration was monitored through the detection of nitrosyl–hemoglobin complexes (NO–Hb) by electronic spin resonance. Production of 6-keto PGF₁α, PGE₂, PGF₂α and TXB₂ was measured in aorta and mesenteric beds by HPLC. Western blot analysis was used to examine the changes in the expression levels of NOS-2 and COX-2 in aorta.Key findingsOur results showed that increases in NO circulating steady-state concentration and PR production by aorta and mesenteric beds 6 h after LPS administration were significantly attenuated in F overload rats with respect to control animals. Oxidative stress parameters were equally affected in the presence or absence of the F treatment. Aorta protein levels of NOS-2 and COX-2, two enzymes inducible by LPS, were significantly lower in F overload rats with respect to control rats at the end of the treatment (?39% and ?61% for NOS-2 and COX-2 respectively).SignificanceThese results suggest that the metabolic alterations established by 15 weeks of F overload should affect the response to LPS challenge due to an attenuation in the induction of NOS-2 and COX-2. This effect would be one of the components contributing to abnormalities in the course of the inflammatory response in other conditions associated to insulin resistance, such as diabetes.     

Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences

The role of adventitial cells in bacterial lipopolysaccharide (LPS)-induced vascular nitric oxide (NO) overproduction has been largely ignored. In rat aortas exposed to LPS in vitro or in vivo, it was found that adventitia contained the major part of NO synthase (NOS)-2 protein (Western blot and immunohistochemistry) and generated the largest amount of NO (electron paramagnetic resonance spin trapping). NOS-2 immunoreactive cells were mainly resident macrophages at an early stage (5 h, in vitro or in vivo) and fibroblasts at a later stage (20 h, in vitro). Adventitial NOS-2 activity largely accounted for 1) the relaxing effect of L-arginine in rings exposed to LPS in vivo, 2) generation of an "NO store" revealed by N-acetylcysteine-induced relaxation, and 3) formation of protein-bound dinitrosyl iron complexes in the medial layer of aortic rings exposed to LPS in vitro. In conclusion, the adventitia is a powerful source of NO triggered by LPS in the rat aorta. This novel source of NO has an important impact on smooth muscle function and might be implicated in various inflammatory diseases.     

Co-induction of nitric oxide and tetrahydrobiopterin synthesis in the myocardium in vivo

Induction of the inducible isoform of nitric oxide (NO) synthase (iNOS) in the myocardium is implicated as a mechanism in the development of cardiac depression in immune activated states associated with an enhanced release of cytokines, such as septic shock. We evaluated the in vivo synthesis of NO and tetrahydrobiopterin (BH4), a cofactor of NOS, in the heart tissue using a model of LPS injection in rats (LPS: 10 mg/kg, i.v.). In control rats, iNOS activity or iNOS mRNA in the heart was negligible. Three hours after LPS administration, a marked induction of iNOS mRNA and activity was observed in the heart. A significant increase in BH4 content and GTP cyclohydrolase mRNA abundance was also observed in the heart from LPS-treated rats. Our results demonstrate induction of NO synthesis and parallel increase in BH4 concentration in the heart of rats after LPS treatment in vivo and may provide molecular evidence responsible for the increased production of BH4 which may up-regulate iNOS activity in the heart in vivo. (Mol Cell Biochem 166: 177-181, 1997)     

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.     

Acute exercise stimulates macrophage function: possible role of NF-kappaB pathways

Moderate physical activity when performed on a regular basis presents a number of benefits to the whole organism, especially regarding immune system function, such as augmenting resistance to infections and to cancer growth. Although glutamine production by active muscle cells as well as neuroendocrine alterations mediated by the chronic adaptation to exercise may play a role, the entire mechanism by which exercise makes the immune system aware of challenges remains mostly uncovered. This is particularly true for the effects of an acute exercise session on immune function. In this work, circulating monocytes/macrophages from sedentary rats submitted to an acute (1 h) swimming session were tested for the ability of phagocytosing zymosan particles, phorbol myristate acetate (PMA)-induced hydrogen peroxide production, nitric oxide (NO) release (assessed by nitrate and nitrite production) and the expression of NO synthases (NOS-1, NOS-2 and NOS-3). The results showed that an exercise bout induced a 2.4-fold rise in macrophage phagocytic capacity (p = 0.0041), a 9.6-fold elevation in PMA-induced hydrogen peroxide release into the incubation media (1-h, p = 0.0022) and a 95.5%-augmentation in nitrite basal production (1-h incubation; p = 0.0220), which was associated with a marked expression of NOS-2 (the inducible NOS isoform; p = 0.0319), but not in other NOS gene products. Although NOS-2 expression is nuclear factor-kappaB (NF-kappaB)-dependent, no systemic oxidative stress was found, as inferred from the data of plasma TBARS and glutathione disulphide (GSSG) to glutathione (GSH) ratio in circulating blood erythrocytes which remained constant after the acute exercise. Also, no stressful situation seemed to be faced by monocytes/macrophages, since the expression of the 70-kDa heat shock protein (HSP70) remained unchanged. We conclude that NF-kappaB-dependent induction of NOS-2 and macrophage activation must be related to local factor(s) produced in the surroundings of monocytes/macrophages.     

Inhibition of NOS-2 induction in LPS-stimulated J774.2 cells by 1, 5-isoquinolinediol, an inhibitor of PARP.

Activation of both poly (ADP-ribose) polymerase (PARP) and inducible nitric oxide synthase (NOS-2) have been implicated in the pathogenesis of various forms of inflammation, therefore compounds which may simultaneously inhibit both pathways are of potential therapeutic interest. We tested the influence of potent inhibitor of PARP, 1, 5-isoquinolinediol (ISO), on NOS-2 induction in model of mouse macrophages (cell line J774.2) stimulated with lipopolysaccharide (1 microg/ml). Pretreatment with ISO (1-300 microM) resulted in dose-dependent inhibition of accumulation of NOS-2-derived nitrite in culture medium (IC(50) = 9,3 microM) as well as inhibition of NOS-2 protein induction in cultured J774.2 cells; ISO given 10 hours after LPS did not influence activity of NOS-2. Interestingly, another PARP inhibitor, 3-aminobenzamide (3-AB, 10-3000 microM), did not influence 24-hr nitrite accumulation in J774.2 cell culture, either administered 15 minutes prior to LPS or 10 hrs after LPS. Scavenging of reactive oxygen species by use of mixture of SOD and catalase (SOD/Cat, 100/300 - 1000/3000 U/ml) as well as cell permeable SOD-mimetic [Mn(III)TBAP, 1- 100 microM], did not influence NOS-2 induction in J774.2 cells. In summary, we identified 1, 5-isoquinoline as potent inhibitor of induction of NOS-2 in LPS-treated mouse macrophages. The exact mechanism of inhibitory action of this compound on NOS-2 induction requires further investigation.     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.