eNOS In Endothelial Cells Research Articles

Abstract 5208: S-glutathiolation Uncouples Endothelial Nitric Oxide Synthase: A Novel Mechanism of Redox Signaling and Vascular Regulation

Superoxide (·O 2 − ) and secondary oxidants are critical mediators of cellular signaling, proliferation and apoptosis. In response to oxidative stress, protein thiols undergo S-glutathiolation forming mixed disulfides and this modification plays an important role as an adaptive response to oxidative injury in cells, controlling cellular signaling in a manner similar to protein phosphorylation. We investigated the effects of S-glutathiolation on the regulation of eNOS function in vitro . We observe that S-glutathiolation greatly reduces NO synthase activity (70% ± 1%), and this loss of activity was reversed by DTT. In contrast, only a 56%±0.9% decrease in NADPH consumption was seen that was only partially inhibited by L-NAME or the calcium chelator EGTA, implicating an increase in eNOS derived ·O 2 − . Using EPR spin-trapping to directly measure eNOS derived ·O 2 − , we proved that S-glutathiolation greatly increased ·O 2 − generation from the enzyme. S-glutathiolation of human eNOS increased ·O 2 − generation >5-fold and L-NAME and EGTA only partially blocked this ·O 2 − generation. Thus, S-glutathiolation uncouples eNOS greatly increasing ·O 2 − generation and the partial or complete lack of inhibition of this by L-NAME and EGTA indicates that the observed ·O 2 − is largely derived from the reductase domain. Mass spectrometric analysis and molecular modeling revealed that two sights of S-glutathiolation in eNOS are two highly conserved cysteines found at the interface between the FMN and FAD-binding domains, C 689 and C 908 . The modification of these two cysteins appears to interrupt electron transfer to the oxygenase domain, leading to electron leakage. Furthermore, we observe that BCNU and H 2 O 2 - derived oxidative stress induces S-glutathiolation of eNOS in endothelial cells, with measured loss of NO and gain of ·O 2 − generation, and we show that this process impairs endothelium-dependent vasodilation. Additionally, we observe that in hypertensive vessels eNOS-S-glutathiolation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents. Thus, S-glutathiolation of eNOS serves as a pivotal switch that provides redox regulation of endothelial function and vascular tone. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Nitric Oxide Synthase (NOS2/3) Expression in Head and Neck Squamous Cell Carcinomas in Correlation with Clinical Patterns

Background: Increased nitric oxide synthase (NOS) expression has been demonstrated in a number of carcinomas and is discussed to play a key role in tumor progression. The aim of this immunohistochemical study was to examine the protein expression rates of endothelial (e)NOS and inducible (i)NOS in head and neck squamous cell carcinomas (HNSCCs) and oral mucosa and to correlate the results with clinicopathologic factors (TN stage). Patients and Methods: Protein expression patterns of NOS were studied immunohistochemically (score 0–7) in 58 patients with HNSCC and 7 mucosa samples, and the results were correlated with tumor stages. Results: In oral mucosa, iNOS was only expressed in the basal epithelial layers and in macrophages, eNOS in endothelial cells and lymphocytes. In contrast, both NOS isoforms were expressed in HNSCC with preference at the tumor margins. 64% of tumor specimens demonstrated a positive eNOS immunoreactivity (score ≥3), 55% a positive iNOS immunoreactivity. NOS protein expression rates reached higher scores in tumors of patients with lymph node metastasis (N > 0; iNOS protein expression rate p < 0.05). Conclusions: HNSCCs are able to express both NOS protein isoforms in relevant amounts, and we presume that synthesized NO is able to support angiogenetic patterns and facilitate tumor progression and lymphatic spread.

Role of gender and estrogen receptors in the rat aorta endothelium-dependent relaxation to red wine polyphenols

Regular intake of moderate amounts of beverages rich in polyphenols such as red wine is associated with a protective effect on the vascular system, in part, by increasing the endothelial formation of nitric oxide (NO), a major vasoprotective factor. Since estrogens are potent inducers of NO formation and polyphenols have been shown to have phytoestrogen properties, we determined whether estrogen receptors mediate the stimulatory effect of red wine polyphenols (RWPs) on the endothelial formation of NO using isolated rat aortic rings and cultured endothelial cells. RWPs caused endothelium-dependent relaxations, which were more pronounced in the aorta of female than male rats. Increased relaxations were also observed to acetylcholine but not to sodium nitroprusside. Relaxations to RWPs were abolished by nitro l-arginine and MnTMPyP, markedly reduced by polyethyleneglycol-catalase and wortmannin, and not affected by the estrogen antagonist ICI 182,780 in aortic rings from males and females. eNOS expression was higher in aortic sections of female than male rats. RWPs caused the phosphorylation of Akt and eNOS in endothelial cells, which was unaffected by ICI 182,780. Thus, RWPs cause redox-sensitive PI3-kinase/Akt-dependent NO-mediated relaxations, which are more pronounced in the aorta of female than male rats; an effect most likely due to the increased expression level of eNOS rather than activation of estrogen receptors.

C008 Endothelial NO synthase activation by a red Wine Phenolic extract: isolation of active components by bioguided fractionation

Previous studies have shown that Red Wine Phenolic extracts induce nitric oxide (NO)-mediated vasoprotective effects, mainly by causing a redox-sensitive activation of endothelial NO synthase (eNOS). However, Red Wine Phenolic extracts are complex mixtures of a several hundreds of phenolic compounds. Therefore, the aim of the present study was to isolate active phenolic compounds using multi-step bioguided fractionation of the red wine extract. Red Wine Phenolic Extract was submitted to a bioguided fractionation using chromatographic methods. The fractions obtained at each step were tested for their potential to induce the activation of eNOS in endothelial cells. Cultures of porcine coronary artery endothelial cells (P1) were used to determine the phosphorylation level of Akt and endothelial NO synthase by Western blot analysis. Identification of phenolic compounds in each active fraction was performed by MALDI-TOF and HPLC-MS techniques. The first step of fractionation on lipophilic Sephadex® yielded 9 fractions of which 4 of them significantly increased the phosphorylation level of Akt and eNOS in endothelial cells. The active fractions contained mainly procyanidins and some anthocyanins compounds. The fractionation of one of the active fractions by preparative reverse-phase HPLC yielded 11 subfractions ; all of these sub-fractions significantly increased the phosphorylation level of Akt and eNOS. The analysis of the phenolic compounds indicated that these sub-fractions contained mixtures of procyanidin dimers or conjugated anthocyanins. The red wine extract contains several types of phenolic compounds, which are able to enhance the activity of NO synthase in endothelial cells including procyanidins dimers and oligomers as well as several conjugated anthocyanins.

Caloric Restriction Stimulates Revascularization in Response to Ischemia via Adiponectin-mediated Activation of Endothelial Nitric-oxide Synthase

Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, the functional roles of CR in regulation of revascularization in response to ischemia have not been examined. Here we investigated whether CR modulates vascular response by employing a murine hindlimb ischemia model. Wild-type (WT) mice were randomly divided into two groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb ischemic surgery. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared with WT mice fed AL (AL-WT). Revascularization of ischemic hindlimb relative to the contralateral limb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow and capillary density analyses. CR-WT mice had significantly higher plasma levels of the fat-derived hormone adiponectin compared with AL-WT mice. In contrast to WT mice, CR did not affect the revascularization of ischemic limbs of adiponectin-deficient (APN-KO) mice. CR stimulated the phosphorylation of endothelial nitric-oxide synthase (eNOS) in the ischemic limbs of WT mice. CR increased plasma adiponectin levels in eNOS-KO mice but did not stimulate limb perfusion in this strain. CR-WT mice showed enhanced phosphorylation of AMP-activated protein kinase (AMPK) in ischemic muscle, and administration of AMPK inhibitor compound C abolished CR-induced increase in limb perfusion and eNOS phosphorylation in WT mice. Our observations indicate that CR can promote revascularization in response to tissue ischemia via an AMPK-eNOS-dependent mechanism that is mediated by adiponectin.

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

Inhibition of Endothelial Nitric Oxide Synthase Activity by Proline-Rich Tyrosine Kinase 2 in Response to Fluid Shear Stress and Insulin

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (approximately 50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS(-/-) mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.

Endothelial Rehabilitation: The Impact of Chronic PDE5 Inhibitors on Erectile Function and Protein Alterations in Cavernous Tissue of Diabetic Rats

BackgroundDiabetic men generally have reduced efficacy with PDE-5 inhibitors (PDE5i) for the treatment of erectile dysfunction (ED). ObjectiveTo determine whether chronic vardenafil exposure alters cavernous protein expression predicting improved erectile function in diabetes. DesignForty-two adult male Sprague Dawley rats with streptozotocin-induced (50mg/kg IP) diabetes for 4 wk, were exposed to either vehicle or vardenafil for 6 wk. Assessments compared the impact of vardenafil given at 1h and 20h to erectile function and cellular alterations and downstream translation of cavernous protein profiles were aimed. InterventionVehicle or vardenafil 0.5mg/kg/day by oral gavage for 6 wk. MeasurementsErectile function, penile tissue morphology, protein expression and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) protein profiling were determined. Results and LimitationsA significant increase of intracavernous pressure was seen in both treatment arms compared to diabetic rats not receiving vardenafil. Immunohistochemical staining showed improved endothelial and smooth muscle cell staining with chronic vardenafil use. Western blot analysis demonstrated increased endothelial cell eNOS and smooth muscle α-actin protein content. SELDI protein profiling showed enhanced proteins expression at molecular weights of 14.7, 20, 41.9, 66.2, and 83.9kDa in the chronically treated vardenafil group. ConclusionsVardenafil was effective in treating diabetic-induced ED with the greatest effect achieved through chronic dosing, with no additive effect measured with the final acute dose. Changes noted in the histology and protein expression indicate that vardenafil may have a protective effect in this disease state. This finding may serve as a basis for further work evaluating the utility of chronic vardenafil dosing in diabetic men.

Enhanced angiotensin converting enzyme 2 regulates the insulin/Akt signalling pathway by blockade of macrophage migration inhibitory factor expression

Macrophage migration inhibitory factor (MIF) is now known to be a pro-inflammatory cytokine associated with insulin resistance. Our aim was to investigate whether angiotensin converting enzyme 2 (ACE2) could modulate the expression of MIF and the insulin/Akt-endothelial nitric oxide (NO) synthase (eNOS) signalling in a human endothelial cell line (EAhy926). A recombinant plasmid encompassing human ACE2 gene was constructed and transfected into the EAhy926 cells. The mRNA, phosphorylation and protein levels of p22phox, MIF, Akt and eNOS in endothelial cells were determined by real-time PCR and Western blot analysis, respectively. Gene transfer of ACE2 suppressed the expression of p22phox and MIF induced by angiotensin (Ang) II and Ang IV, accompanied by a decreased level of malondialdehyde in cells. In addition, Ang II diminished insulin-stimulated phosphorylation of Akt (at Ser(473)) and eNOS (at Ser(1177)) and NO generation, effects which were reversed by ACE2 gene transfer and anti-MIF treatment in endothelial cells. The results reveal that gene transfer of ACE2 regulated Ang II-mediated impairment of insulin signalling and involved the Akt-eNOS phosphorylation pathway. These beneficial effects of ACE2 overexpression appear to result mainly from blocking MIF expression in endothelial cells, suggesting that the ACE2 gene may be a novel therapeutic target for diseases related to inflammation and insulin resistance.

Agonist-modulated Regulation of AMP-activated Protein Kinase (AMPK) in Endothelial Cells: EVIDENCE FOR AN AMPK → Rac1 → Akt → ENDOTHELIAL NITRIC-OXIDE SYNTHASE PATHWAY

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase beta. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKalpha1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKalpha1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKalpha1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK --> Rac1 --> Akt axis in the control of eNOS in endothelial cells.

Aldosterone

During the past decade, there has been heightened interest in aldosterone as a cardiovascular risk factor, fueled by studies documenting its effects on tissues other than epithelial cells. Of particular interest is the role of aldosterone in exacerbating vascular injury. It is likely that the mechanisms mediating the vascular effects of aldosterone differ from its effects on epithelial cells, eg, the renal collecting tubule. For more than a quarter of a century, it also had been assumed that the primary mechanism of aldosterone was mediated by activation of the mineralocorticoid receptor (MR), which in turn increased transcription of MR-responsive genes. Recently, it has been documented that aldosterone can produce rapid cellular responses—MR-mediated increases in phosphorylation of transduction factors, eg, extracellular signal regulated kinase 1 and 2 (ERK1/2), protein kinase C (PKC), and endothelial nitric oxide synthase (eNOS), and changes in intracellular calcium and hydrogen ions—in addition to its traditional genomic responses. Sometimes these rapid effects are called “nongenomic.” However, it is likely that even the “rapid” actions of aldosterone can lead to genomic effects. Thus, we will use the terms “rapid” and “genomic” to contrast 2 major mechanisms of action of aldosterone. These rapid effects may be beneficial or detrimental depending on the environment in which aldosterone is acting. For example, phosphorylation of eNOS in endothelial cells leading to increased NO production and vasodilation would likely be beneficial in an environment where there is decreased perfusion to a vital organ. Activation of vasoconstriction through increases in cytosolic calcium or phosphorylation of ERK1/2 and PKC would be beneficial in hypotension secondary to injury. …

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by L-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 microm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H(2)O(2) (assessed with H(2)O(2)-specific fluorescent dye CM-H(2)DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

Atherogenic Endothelial Cell eNOS and ET-1 Responses to Asynchronous Hemodynamics are Mitigated by Conjugated Linoleic Acid

Although local wall shear stress (WSS) induced by blood flow has been implicated in atherogenesis, another prominent and often neglected hemodynamic feature, circumferential strain (CS) driven by pressure, is induced concurrently. To investigate endothelial cell (EC) responses to pathologic hemodynamics and their possible manipulation by pharmaceuticals, we simulated complete hemodynamic conditions comprised of simultaneous WSS and CS during treatment with conjugated linoleic acid (CLA), a known PPAR (-alpha and -gamma) activator and anti-atherogenic agent, on cultured EC and examined effects on gene and metabolite expression. Two hemodynamic conditions representative of distinct regions of the circulation, coronary arteries: pro-atherogenic (asynchronous WSS and CS) and straight descending aorta: non-atherogenic (synchronous WSS and CS), were applied to cultured EC during treatment with the nutraceutical CLA. Competitive-quantitative RT-PCR showed that asynchronous hemodynamics significantly reduced ( approximately 2-fold) eNOS and PPAR-gamma mRNA levels compared to synchronous hemodynamics at 5 and 12 h. ET-1 showed an opposite trend at 12 h. CLA treatment mitigated pro-atherogenic eNOS, ET-1, PPAR-alpha and -gamma mRNA expression profiles and NO and ET-1 secretion patterns during asynchronous hemodynamics. This study demonstrates the potential for a pharmacological treatment (CLA) to normalize pro-atherogenic gene expression profiles induced by hemodynamics inherent to the circulation.

Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging

We previously reported the disruption of caveolae/rafts, dysfunction of Golgi tethers, N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptor proteins (SNAREs), and SNAPs, and inhibition of anterograde trafficking in endothelial cells in culture and rat lung exposed to monocrotaline pyrrole (MCTP) as a prelude to the development of pulmonary hypertension. We have now investigated 1) whether this trafficking block affects subcellular localization and function of endothelial nitric oxide (NO) synthase (eNOS) and 2) whether Golgi blockade and eNOS sequestration are observed after hypoxia and senescence. Immunofluorescence data revealed that MCTP-induced "megalocytosis" of pulmonary arterial endothelial cells (PAEC) was accompanied by a loss of eNOS from the plasma membrane, with increased accumulation in the cytoplasm. This cytoplasmic eNOS was sequestered in heterogeneous compartments and partially colocalized with Golgi and endoplasmic reticulum (ER) markers, caveolin-1, NOSTRIN, and ER Tracker, but not Lyso Tracker. Hypoxia and senescence also produced enlarged PAEC, with dysfunctional Golgi and loss of eNOS from the plasma membrane, with sequestration in the cytoplasm. Live-cell imaging of caveolar and cytoplasmic NO with 4,5-diaminofluorescein diacetate (DAF-2DA) as probe showed a marked loss of caveolar NO after MCTP, hypoxia, and senescence. Although ionomycin stimulated DAF-2DA fluorescence in control PAEC, this ionophore decreased DAF-2DA fluorescence in MCTP-treated and senescent PAEC, suggesting localization of eNOS in an aberrant cytoplasmic compartment that was readily discharged by Ca(2+)-induced exocytosis. Thus monocrotaline, hypoxia, and senescence produce a Golgi blockade in PAEC, leading to sequestration of eNOS away from its functional caveolar location and providing a mechanism for the often-reported reduction in pulmonary arterial NO levels in experimental pulmonary hypertension, despite sustained eNOS protein levels.

Signaling from Blood Vessels to CNS Axons through Nitric Oxide

Brain function is usually perceived as being performed by neurons with the support of glial cells, the network of blood vessels situated nearby serving simply to provide nutrient and to dispose of metabolic waste. Revising this view, we find from experiments on a rodent central white matter tract (the optic nerve) in vitro that microvascular endothelial cells signal persistently to axons using nitric oxide (NO) derived from the endothelial NO synthase (eNOS). The endogenous NO acts to stimulate guanylyl cyclase-coupled NO receptors in the axons, leading to a raised cGMP level which then causes membrane depolarization, apparently by directly engaging hyperpolarization-activated cyclic nucleotide-gated ion channels. The tonic depolarization and associated endogenous NO-dependent cGMP generation was absent in optic nerves from mice lacking eNOS, although such nerves responded to exogenous NO, with raised cGMP generation in the axons and associated depolarization. In addition to the tonic activity, exposure of optic nerves to bradykinin, a classical stimulator of eNOS in endothelial cells, elicited reversible NO- and cGMP-dependent depolarization through activation of bradykinin B2 receptors, to which eNOS is physically complexed. No contribution of other NO synthase isoforms to either the action of bradykinin or the continuous ambient NO level could be detected. The results suggest that microvascular endothelial cells participate in signal processing in the brain and can do so by generating both tonic and phasic NO signals.

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation

1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2. mPS at micromolar concentrations (1-10 microM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell-permeable version of PKCzeta pseudosubstrate fused to the HIV-TAT membrane-translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3. In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4. Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5. Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKC should be done with caution, especially in endothelial cells.

Bradykinin Mediates Phosphorylation of eNOS in Odontoblasts

While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10(-7) M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10(-6) M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.

Redox signaling in angiogenesis: Role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.

Effect of 17 β‐estradiol on the intracellular calcium concentration and eNOS protein expression in human endothelial cells

Many reports indicate nitric oxide (NO) production plays an important role in mediating the effects of estrogen on the vasculature. Here, we report the modulatory effects of 17 β-estradiol (E2) on intracellular calcium concentration ([Ca2+]i), a cofactor in the activity of endothelial NO synthase (eNOS), and eNOS expression in human endothelial cells (EA.hy926). [Ca2+]i in Fura 2 loaded cells was measured using spectrofluorometry. In the presence of extracellular Ca2+, inhibition of the endoplasmic reticulum (ER) Ca2+-ATPase with thapsigargin (TG) caused a bi-phasic increase in [Ca2+]i in the cells. However, the extent of TG-induced initial [Ca2+]i increase was significantly higher in E2 treated cells than its control counterpart. A subsequent increase in extracellular Ca2+ concentration led to a robust increase in [Ca2+]i, which was completely blocked by the non-specific calcium channel blocker Ni2+. Elevation of [Ca2+]i in response to added calcium was significantly higher in E2 treated cells relative to controls. Removal of extracellular Ca2+ changed the [Ca2+]i signal to a single [Ca2+]i transient, indicating that Ca2+ release from ER contributes to the initial [Ca2+]i peak, while the maintained plateau is due to Ca2+ entry. Using Western blotting, we demonstrated that eNOS expression in E2 treated cells was significantly higher than in controls. This action of E2 was abrogated by estrogen receptor antagonist ICI 182,780. These results suggest E2-regulated Ca2+ homeostasis may play a role in the enhancement of eNOS in endothelial cells (Supported by NHLBI).

Functional Relevance of Golgi and Plasma Membrane Localized Endothelial Nitric Oxide Synthase (eNOS) in Reconstituted Endothelial cells

Endothelial nitric oxide synthase (eNOS) is regulated by post-translational modifications which target eNOS to the plasma membrane (PM) and the perinuclear/Golgi region. It has been shown in COS-7 cells that targeting of eNOS to the Golgi or PM regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (EC). Our goal was to isolate these two pools of enzyme in EC and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels. Using an RNAi strategy, we generated stable populations of EC that had greater than 90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS “knockdown” EC with Golgi and PM targeted eNOS restored the ability of EC to produce NO. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in EC. In contrast, Golgi eNOS was less responsive to both calcium and Akt-dependent agonists. eNOS restricted to the PM was more sensitive to manipulation of membrane cholesterol levels and was significantly attenuated by modified LDL. Methyl-β-cyclodextrinβ(CD) significantly reduced and CD-cholesterol complexes significantly increased NO release from PM eNOS, but not Golgi eNOS. Similarly, modified LDL substantially reduced the activity of PM eNOS but not Golgi eNOS (HL74279).