Continuous exposure to low amplitude extremely low frequency electrical fields characterizing the vascular streaming potential alters elastin accumulation in vascular smooth muscle cells

In normal development and pathology, the vascular system depends on complex interactions between cellular elements, biochemical molecules, and physical forces. The electrokinetic vascular streaming potential (EVSP) is an endogenous extremely low frequency (ELF) electrical field resulting from blood flowing past the vessel wall. While generally unrecognized, it is a ubiquitous electrical biophysical force to which the vascular tree is exposed. Extracellular matrix elastin plays a central role in normal blood vessel function and in the development of atherosclerosis. It was hypothesized that ELF fields of low amplitude would alter elastin accumulation, supporting a link between the EVSP and the biology of vascular smooth muscle cells. Neonatal rat aortic smooth muscle cell cultures were exposed chronically to electrical fields characteristic of the EVSP. Extracellular protein accumulation, DNA content, and electron microscopic (EM) evaluation were performed after 2 weeks of exposure. Stimulated cultures showed no significant change in cellular proliferation as measured by the DNA concentration. The per‐DNA normalized protein in the extracellular matrix was unchanged while extracellular elastin accumulation decreased 38% on average. EM analysis showed that the stimulated cells had a 2.85‐fold increase in mitochondrial number. These results support the formulation that ELF fields are a potential factor in both normal vessel biology and in the pathogenesis of atherosclerotic diseases including heart disease, stroke, and peripheral vascular disease. Bioelectromagnetics 34:358–365, 2013. © 2012 Wiley Periodicals, Inc.     

Extremely low‐frequency electromagnetic fields affect the immune response of monocyte‐derived macrophages to pathogens

This study aimed to determine the effect of extremely low‐frequency electromagnetic fields (ELF‐EMF) on the physiological response of phagocytes to an infectious agent. THP‐1 cells (human monocytic leukemia cell line) were cultured and 50 Hz, 1 mT EMF was applied for 4–6 h to cells induced with Staphylococcus aureus or interferon gamma/lipopolysaccharide (IFγ/LPS). Alterations in nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels, heat shock protein 70 levels (hsp70), cGMP levels, caspase‐9 activation, and the growth rate of S. aureus were determined. The growth curve of exposed bacteria was lower than the control. Field application increased NO levels. The increase was more prominent for S. aureus‐induced cells and appeared earlier than the increase in cells without field application. However, a slight decrease was observed in iNOS levels. Increased cGMP levels in response to field application were closely correlated with increased NO levels. ELF‐EMF alone caused increased hsp70 levels in a time‐dependent manner. When cells were induced with S. aureus or IFγ/LPS, field application produced higher levels of hsp70. ELF‐EMF suppressed caspase‐9 activation by a small extent. These data confirm that ELF‐EMF affects bacterial growth and the response of the immune system to bacterial challenges, suggesting that ELF‐EMF could be exploited for beneficial uses. Bioelectromagnetics 31:603–612, 2010. © 2010 Wiley‐Liss, Inc.     

Extracellular currents alter gap junction intercellular communication in synovial fibroblasts

We studied the effect of extremely low frequency (ELF) currents on gap junction intercellular communication (GJIC) mediated by connexin43 protein. Confluent monolayers of synovial fibroblasts (HIG-82) and neuroblastoma cells (5Y) were exposed in bath solution to 0-75 mA/m(2) (0-56 mV/m), 60 Hz. Single channel conductance, cell membrane current-voltage (I-V) curves, and Ca(2+) influx were measured using the nystatin single and double patch methods. The conductances of the closed and open states of the gap junction channel in HIG-82 cells were each significantly reduced (by 0.76 and 0.39 pA, respectively) in cells exposed to 20 mA/m(2). Current densities as low as 10 mA/m(2) significantly increased Ca(2+) influx in HIG-82 cells. No effects were seen in 5Y cells. The I-V curves of the plasma membranes of both types of cells were independent of 60 Hz electric fields and current densities, 0-75 mA/m(2), indicating that the effect of the 60 Hz fields on GJIC in HIG-82 cells was not mediated by a change in membrane potential. We conclude that ELF electric fields can alter GJIC in synovial cells via a mechanism that does not depend on changes in membrane potential, but may depend on Ca(2+) influx. The results open the possibility that GJIC mediated responses in synovial cells, such as for example, their secretory responses to proinflammatory cytokines, could be antagonized by the application of ELF electric fields.     

Vascular endothelial growth factor-dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitination. Consequences on nitric oxide production from endothelial cells

Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.     

Extremely low-frequency magnetic fields modulate nitric oxide signaling in rat brain

Our previous study has shown that an extremely low‐frequency magnetic field (ELF‐MF) induces nitric oxide (NO) synthesis by Ca²⁺‐dependent NO synthase (NOS) in rat brain. The present study was designed to confirm that ELF‐MF affects neuronal NOS (nNOS) in several brain regions and to investigate the correlation between NO and nNOS activation. The exposure of rats to a 2 mT, 60 Hz ELF‐MF for 5 days resulted in increases of NO levels in parallel with cGMP elevations in the cerebral cortex, striatum, and hippocampus. Cresyl violet staining and electron microscopic evaluation revealed that there were no significant differences in the morphology and number of neurons in the cerebral cortex, striatum, and hippocampus. Differently, the numbers of nNOS‐immunoreactive (IR) neurons were significantly increased in those cerebral areas in ELF‐MF‐exposed rats. These data suggest that the increase in NO could be due to the increased expression and activation of nNOS in cells. Based on NO signaling in physiological and pathological states, ELF‐MF created by electric power systems may induce various physiological changes in modern life. Bioelectromagnetics 33:568–574, 2012. © 2012 Wiley Periodicals, Inc.     

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system

The region of elongation in Cucumis sativus and Cucurbita maxima roots was marked at increasing distances from the apex to provide an analog of increasing cell size. These roots were exposed/sham-exposed to 60 Hz electric fields and the growth rates of the root segments measured. The growth rate effect magnitude varied with increasing distance from the root tip at constant field strength, and with increasing applied field strength. These results provide strong, qualitative support for the postulate that ELF transmembrane potential induction is involved in the stimulation of ELF electric field effects in the plant root model system.     

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.     

Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF‐A independent mechanism

The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF‐PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF‐PEMF on angiogenesis. The hypothesis of this study is that ELF‐PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)‐A‐based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF‐PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF‐PEMF increased endothelial proliferation 54‐fold, whereas media from endothelial cells stimulated with ELF‐PEMF did not affect osteoblast proliferation. We examined the role of the pro‐angiogenic mediator VEGF‐A in the mitogenic effect of ELF‐PEMF‐stimulated osteoblast media on endothelial cells. The production of VEGF‐A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF‐PEMF‐induced osteoblast‐derived endothelial mitogen observed in these studies was not VEGF‐A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189–197, 2009. © 2008 Wiley‐Liss, Inc.     

高密度脂蛋白促进内皮衍生的一氧化氮抑制血小板聚集的作用

用培养的小牛主动脉内皮细胞与兔水洗血小板直接相互作用的模型 ,探讨高密度脂蛋白对内皮衍生的一氧化氮抗血小板聚集作用的影响。培养的小牛主动脉内皮细胞预先用 10 0 μmol/L阿斯匹林处理 ,抑制细胞内的环氧化物酶活性。凝血酶 ( 0 1U/ml)可诱导兔血小板 ( 2× 10 8/ml) 67 3 3± 7 5 2 %的聚集反应。内皮细胞 ( 1× 10 5～ 1× 10 6 /ml)能抑制凝血酶诱导的血小板聚集 ,抑制强度与内皮细胞的数目正相关。且此作用可被 1mmol/L硝基精氨酸完全取消。表明内皮细胞对凝血酶诱导血小板聚集的抑制作用都是由内皮衍生的一氧化氮所致。在加凝血酶之前加入高密度脂蛋白 ( 1mg/ml)可增强内皮细胞 ( 1× 10 5/ml)的这种作用。高密度脂蛋白 ( 1mg/ml)与内皮细胞共同孵育 1h后 ,将高密度脂蛋白离心弃去 ,内皮细胞对凝血酶诱导血小板聚集的抑制作用不受影响。高密度脂蛋白及内皮细胞对静息血小板均无直接作用。结果表明 ,高密度脂蛋白增强内皮细胞抗凝血酶诱导的血小板聚集反应的作用是通过直接作用于内皮衍生的一氧化氮而产生的     

血管内皮细胞的Cl-通道--电特性和有关细胞功能

内皮细胞在心血管系统具有重要功能,除通过分泌内皮舒张因子－－一氧化氮（ＮＯ）及收缩性物质内皮素等控制血管平滑肌张力外,并能调节血管通透性。近年来发现内皮细胞上的Ｃ１＾－通道能调节细胞体积和细胞膜电位的稳定性。通过离子通道调控膜电位一机理,能较好理解血管内皮的功能,并可望由此开拓新型血管药物。本文综述了内皮细胞的Ｃ１＾－通道的电生理特性、类别,并探讨该通道调控细胞体积,ＮＯ的分泌及调控细胞膜电位的可     

ELF in vitro exposure systems for inducing uniform electric and magnetic fields in cell culture media.

Many in vitro experiments on the biological effects of extremely low frequency (ELF) electromagnetic fields utilize a uniform external magnetic flux density (B) to expose biological materials. A significant number of researchers do not measure or estimate the resulting electric field strength (E) or current density (J) in the sample medium. The magnitude and spatial distribution of the induced E field are highly dependent on the sample geometry and its relative orientation with respect to the magnetic field. We have studied the E fields induced in several of the most frequently used laboratory culture dishes and flasks under various exposure conditions. Measurements and calculations of the E field distributions in the aqueous sample volume in the containers were performed, and a set of simple, quantitative tables was developed. These tables allow a biological researcher to determine, in a straightforward fashion, the magnitudes and distributions of the electric fields that are induced in the aqueous sample when it is subjected to a uniform, sinusoidal magnetic field of known strength and frequency. In addition, we present a novel exposure technique based on a standard organ culture dish containing two circular, concentric annular rings. Exposure of the organ culture dish to a uniform magnetic field induces different average electric fields in the liquid medium in the inner and outer rings. Results of experiments with this system, which were reported in a separate paper, have shown the dominant role of the magnetically induced E field in producing specific biological effects on cells, in vitro. These results emphasize the need to report data about the induced E field in ELF in-vitro studies, involving magnetic field exposures. Our data tables on E and J in standard containers provide simple means to enable determination of these parameters.     

Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.     

Induction of heat shock gene expression in RAT1 primary fibroblast cells by ELF electric fields

Recent studies have demonstrated that the Ku70 gene fragment can be placed in the anti‐sense orientation under the control of a heat‐inducible heat shock protein 70 (HSP70) promoter and activated through heat shock exposure. This results in attenuation of the Ku70 protein expression, inhibiting cellular repair processes, and sensitizing the transfected cells to exposures such as the ionizing radiation exposures used clinically. However, achieving the tissue temperatures necessary to thermally induce the HSP70 response presents significant limitations to the clinical application of this strategy. Previous findings suggest an alternative approach to inducing a heat shock response, specifically through the use of extremely low frequency (ELF) electrical field stimulation. To further pursue this approach, we investigated HSP70 responses in transfected rat primary fibroblast (RAT1) cells exposed to 10 Hz electric fields at intensities of 20–500 V/m. We confirmed that low frequency electric fields can induce HSP70 heat shock expression, with peak responses obtained at 8 h following a 2 h field exposure. However, the approximate threefold increase in expression is substantially lower than that obtained using thermal stimulation, raising questions of the clinical utility of the response. Bioelectromagnetics 34:405–413, 2013. © 2012 Wiley Periodicals, Inc.     

Real-time control of neutrophil metabolism by very weak ultra-low frequency pulsed magnetic fields

In adherent and motile neutrophils NAD(P)H concentration, flavoprotein redox potential, and production of reactive oxygen species and nitric oxide, are all periodic and exhibit defined phase relationships to an underlying metabolic oscillation of approximately 20 s. Utilizing fluorescence microscopy, we have shown in real-time, on the single cell level, that the system is sensitive to externally applied periodically pulsed weak magnetic fields matched in frequency to the metabolic oscillation. Depending upon the phase relationship of the magnetic pulses to the metabolic oscillation, the magnetic pulses serve to either increase the amplitude of the NAD(P)H and flavoprotein oscillations, and the rate of production of reactive oxygen species and nitric oxide or, alternatively, collapse the metabolic oscillations and curtail production of reactive oxygen species and nitric oxide. Significantly, we demonstrate that the cells do not directly respond to the magnetic fields, but instead are sensitive to the electric fields which the pulsed magnetic fields induce. These weak electric fields likely tap into an endogenous signaling pathway involving calcium channels in the plasma membrane. We estimate that the threshold which induced electric fields must attain to influence cell metabolism is of the order of 10(-4) V/m.     

The role of sex steroids on cellular events involved in vascular disease

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.     

Shear stress enhances glutathione peroxidase expression in endothelial cells

Hemodynamic forces have profound effects on vasculature. Laminar shear stress upregulates superoxide dismutase (SOD) expression in endothelial cells. SOD converts superoxide anion to H(2)O(2), which, however, promotes atherosclerosis. Therefore, defense against H(2)O(2) may be crucial in reducing oxidative stress. Since glutathione peroxidase (GPx-1) reduces H(2)O(2) to H(2)O, the regulation of GPx-1 expression by mechanical stress was examined. Cultured bovine aortic endothelial cells (BAECs) were subjected to laminar shear stress and stretch force. Shear stress upregulated GPx-1 mRNA expression in a time- and force-dependent manner in BAECs, whereas stretch force was without effect. Furthermore, shear stress increased GPx activity. L-NAME, an inhibitor of nitric oxide synthase, did not affect shear stress-induced GPx-1 mRNA expression. The ability of laminar shear stress to induce GPx-1 expression in endothelial cells may be an important mechanism whereby shear stress protects vascular cells against oxidative stress.     

Bioelectric potential gradients may initiate cell cycling: ELF and zeta potential gradients may mimic this effect

When a number of experimental studies in bioelectromagnetics were reviewed, those in which weak, exogenous extremely low frequency (ELF) fields were applied in fixed juxtaposition to their target tissues, were found to initiate mitogenesis or mitogenesis-related signals more successfully than when the target tissue moved freely during the irradiation. It is suggested that ELF fields in fixed juxtaposition to their target tissue and implanted foreign bodies or endogenous tissues with a significant zeta potential, mimic bioelectric fields generated at wounds. When the potential is high enough, they assist healing by moving cells into the wound and stimulating quiescent cells at the wound margin to cycle. Electrophoresis may help the initial migration of cells into the wound to form a clot, and migration of fibroblasts and epithelial cells from the wound margin. When exposed for a long time in a fixed juxtaposition to a potential gradient too weak to show in situ microelectrophoresis along the cell membrane surface, surface particles may coalesce to form microclusters, where like-charged surface particles are in close proximity, and growth factor receptor oligomerization and other cycle-initiating reactions are facilitated. Bioelectromagnetics 18:341–348, 1997. © 1997 Wiley-Liss, Inc.     

Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.     

Inhibition of dynamin‐2 confers endothelial barrier dysfunctions by attenuating nitric oxide production

Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia‐mediated barrier dysfunctions. Dyn‐2 (dynamin‐2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn‐2 in hypoxia‐mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn‐2 by transfecting the cells with K44A, a dominant negative construct of dyn‐2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia‐mediated leakiness of K44A‐overexpressed endothelial monolayer. Our study confirms that inhibition of dyn‐2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia.     

3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.