5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.     

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

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

Expression of endothelial nitric oxide synthase in the postnatal developing porcine kidney

The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.     

Expression of endothelial nitric oxide synthase in the porcine oocyte and its possible function

The present study was designed to investigate the localization of endothelial nitric oxide synthase (eNOS) in porcine oocytes and its possible function during in vitro development. RT-PCR and immunoblotting analyses revealed the presence of eNOS in the oocytes prepared from small follicles, with an amplified product of 456 bp and an apparent mol wt of 130 kDa, respectively. The synthesis of oocyte NO was suppressed during a 72-h culture of cumulus-oocyte complexes in the presence of follicle-stimulating hormone (FSH), but not luteinizing hormone (LH). However, the decrease in NO synthesis did not result from the levels of eNOS mRNA and its protein, as revealed by analyses of RT-PCR and Western blot analysis, suggesting that expression of oocyte eNOS is not dependent upon gonadotropin stimulation. In proliferated cumulus cells, LH receptor mRNA expression was detected after a 48-h culture with FSH, as revealed by RT-PCR analysis. mRNA expression was inhibited by an NO-releasing agent (S-nitroso-N-acetyl-DL-penicillamine) after an additional 24-h culture. These results suggest that oocytes may release eNOS-derived NO as a signal for somatic cells to steadily suppress the development of cumulus cells, if not FSH stimulation. Conversely, the synthesis of NO is suppressed during the action of FSH on the cumulus cells with no changes in eNOS expression.     

Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin–luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.     

Docosahexaenoic acid affects endothelial nitric oxide synthase in caveolae

n-3 Polyunsaturated fatty acids are assumed to play an important role in the prevention and treatment of atherosclerosis. Endothelial nitric-oxide synthase (eNOS) is responsible for cardiovascular homeostasis involving in regulation of vascular function, and the subcellular localization is critical for its activation. Here we determined the effect of docosahexaenoic acid (DHA, 22:6 n-3) on distribution of eNOS and its activity. DHA treatment markedly altered lipid environment of caveolae microdomains, which was coincided with selective displacement of caveolin-1 and eNOS from caveolae. Akt was not detected in caveolae fractions and CaM was distributed in both of caveolin-1-enriched membranes and non-caveolar fractions, whose distribution was unaffected by DHA. These data demonstrated for the first time that DHA altered caveolae microenvironment not only by modifying membrane lipid composition, but also by changing distribution of major structural proteins. DHA-induced alterations in caveolae lipid/protein environment may be an important mechanism in the development of pathogenesis of atherosclerosis.     

L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.     

Humic acid induces the generation of nitric oxide in human umbilical vein endothelial cells: stimulation of nitric oxide synthase during cell injury

Humic acid (HA) has been implicated as an etiological factor in the peripheral vasculopathy of blackfoot disease (BFD). In this study, we examined the effects of HA upon the generation of nitric oxide (NO) during the process of lethal cell injury in cultured human umbilical vein endothelial cells (HUVECs). NO production was measured by the formation of nitrite (NO(2)(-)), the stable end-metabolite of NO. Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Treatment HUVECs with HA at a concentration of 50, 100, and 200 microg/ml concentration-dependently increased nitrite levels, reaching a peak at 12 h subsequent to HA treatment, with a maximal response of approximately 400 pmole nitrite (from 1 x 10(4) cells). HA-induced nitrite formation was blocked completely by N(G)-nitro-L-arginine methyl ester (L-NAME) and also by N(G)-methyl-L-arginine (L-NMA), both being specific inhibitors of NO synthase. The LDH released from endothelial cells was evoked at from 24 h after the addition of HA (50, 100, 200 microg/ml) in a concentration- and time-dependent manner. The HA-induced LDH release was also reduced by the presence of both L-NAME and L-NMA. The addition of Ca(2+) chelator (BAPTA) inhibited both nitrite formation and LDH release by HA. Moreover, the antioxidants (superoxide dismutase, vitamin C, vitamin E) and protein kinase inhibitor (H7) effectively suppressed HA-induced nitrite formation. These results suggest that HA treatment of endothelial cells stimulates NO production, which can elicit cell injury via the stimulation of Ca(2+)-dependent NO synthase activity by increasing cytosolic Ca(2+) levels. Because the destruction of endothelial cells has been implicated in triggering the onset of BFD, the induction of excessive levels of NO and consequent endothelial-cell injury may be important to the etiology of HA-induced vascular disorders associated with BFD for humans.     

Ultrasound induced the formation of nitric oxide and nitrosonium ions in water and aqueous solutions

Nitric oxide, nitrosonium ions, nitrites, and nitrates are formed in water saturated with air under the action of ultrasound. Nitrosonium ions react with water and hydrogen peroxide to form nitrites and nitrates in sonicated solution, correspondingly. Nitric oxide is practically completely released from sonicated water into the atmosphere and reacts with air oxygen, forming NOx compounds. The oxidation of nitric oxide in aqueous medium by hydroxyl radicals and dissolved oxygen is a minor route of the formation of nitrites and nitrates in ultrasonic field.     

Induction of inducible nitric oxide synthase by ginsenosides in cultured porcine endothelial cells

Mechanism of Nitric oxide (NO) production by ginsenosides was investigated in cultured porcine endothelial cells. Beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) staining showed that the NO production was significantly enhanced by the presence of 40 microg/ml ginsenosides with 10 microM L-arginine after 12 h incubation. NO production was suppressed by addition of 0.5 microM Nomega-Nitro-L-arginine (L-NNA), an inhibitor of NO synthases (NOSs), to the incubation medium. In addition, the immunoreactive signals of inducible NOS (iNOS) were appeared in endothelial cells after 12-h incubation of ginsenosides, whereas the signals were not observed in non-treated cells. Our findings suggest that ginsenosides can enhance NO production by induction of iNOS in addition to its direct effect on endothelial cells by increasing intracellular Ca2+ concentration.     

Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.     

Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide

Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[³H]arginine to L-[³H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.     

Podokinesis in endothelial cell migration: role of nitric oxide

Previously, we demonstrated the role of nitric oxide (NO) intransforming epithelial cells from a stationary to locomoting phenotype[E. Noiri, T. Peresleni, N. Srivastava, P. Weber, W. F. Bahou, N. Peunova, and M. S. Goligorsky. Am. J. Physiol. 270 (CellPhysiol. 39): C794-C802, 1996] and itspermissive function in endothelin-1-stimulated endothelial cellmigration (E. Noiri, Y. Hu, W. F. Bahou, C. Keese, I. Giaever, and M. S. Goligorsky. J. Biol. Chem. 272:1747-1753, 1997). In the present study, the role of functional NOsynthase in executing the vascular endothelial growth factor(VEGF)-guided program of endothelial cell migration and angiogenesiswas studied in two independent experimental settings. First, VEGF,shown to stimulate NO release from simian virus 40-immortalized microvascular endothelial cells, induced endothelial cell transwell migration, whereasN^G-nitro-L-arginine methyl ester(L-NAME) or antisenseoligonucleotides to endothelial NO synthase suppressed this effect ofVEGF. Second, in a series of experiments on endothelial cell woundhealing, the rate of VEGF-stimulated cell migration was significantlyblunted by the inhibition of NO synthesis. To gain insight into thepossible mode of NO action, we next addressed the possibility that NOmodulates cell matrix adhesion by performing impedance analysis ofendothelial cell monolayers subjected to NO. The data showed thepresence of spontaneous fluctuations of the resistance in ostensiblystationary endothelial cells. Spontaneous oscillations were induced byNO, which also inhibited cell matrix adhesion. This process we propose to term "podokinesis" to emphasize a scalar form ofmicromotion that, in the presence of guidance cues, e.g., VEGF, istransformed to a vectorial movement. In conclusion, execution of theprogram for directional endothelial cell migration requires twocoexisting messages: NO-induced podokinesis (scalar motion) andguidance cues, e.g., VEGF, which imparts a vectorial component to themovement. Such a requirement for the dual signaling may explain amismatch in the demand and supply with newly formed vessels indifferent pathological states accompanied by the inhibition of NOsynthase.

     

Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population

Porcine aortic endothelial cells (PAECs) produce glycoproteins with important biological functions, such as the control of cell adhesion, blood clotting, blood pressure, the immune system, and apoptosis. Cell surface glycoproteins play important roles in these biological activities. To understand the control of cell surface glycosylation, we elucidated biosynthetic pathways leading to N- and O-glycans in PAECs. Based on the enzyme activities, PAECs should be rich in complex biantennary N-glycans. In addition, the enzymes synthesizing complex O-glycans with core 1 and 2 structures are present in PAECs. The first enzyme of the O-glycosylation pathway, polypeptide GalNAc-transferase, was particularly active. Its specificity toward synthetic peptide substrates was found to be similar to that of purified bovine colostrum enzyme T1. A significant fraction of PAECs treated with tumour necrosis factor alpha or human serum detached from the culture plate, and most of these cells were apoptotic. The apoptotic cell population exhibited decreased core 2 beta 6-GlcNAc-transferase activity. In contrast, the activities of core 1 beta 3-Gal-transferase, which synthesizes O-glycan core 1, and of alpha 3-sialyltransferase (O), which sialylates core 1, were increased in apoptotic PAECs. Thus, apoptotic PAECs are predicted to have fewer complex O-glycans and a higher proportion of short, sialylated core 1 chains.     

Biological reduction of nitric oxide in aqueous Fe(II)EDTA solutions

The reduction of nitric oxide (NO) in aqueous solutions of Fe(II)EDTA is one of the core processes in BioDeNOx, an integrated physicochemical and biological technique for NO(x)() removal from industrial flue gases. NO reduction in aqueous solutions of Fe(II)EDTA (20-25 mM, pH 7.2 +/- 0.2) was investigated in batch experiments at 55 degrees C. Reduction of NO to N(2) was found to be biologically catalyzed with nitrous oxide (N(2)O) as an intermediate. Various sludges from full-scale denitrifying and anaerobic reactors were capable to catalyze NO reduction under thermophilic conditions. The NO reduction rate was not affected by the presence of ethanol or acetate. EDTA-chelated Fe(II) was found to be a suitable electron donor for the biological reduction of nitric oxide to N(2), with the concomitant formation of Fe(III)EDTA. In the presence of ethanol, EDTA-chelated Fe(III) was reduced to Fe(II)EDTA. This study strongly indicates that redox cycling of FeEDTA plays an important role in the biological denitrification process within the BioDeNOx concept.     

Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties

The viscoelastic deformation of porcine aortic endothelial cells grown under static culture conditions was measured using the micropipette technique. Experiments were conducted both for control cells (mechanically or trypsin detached from the substrate) and for cells in which cytoskeletal elements were disrupted by cytochalasin B or colchicine. The time course of the aspirated length into the pipette was measured after applying a stepwise increase in aspiration pressure. To analyze the data, a standard linear viscoelastic half-space model of the endothelial cell was used. The aspirated length was expressed as an exponential function of time. The actin microfilaments were found to be the major cytoskeletal component determining the viscoelastic response of endothelial cells grown in static culture.