Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Bacterial nitric oxide synthesis.

The structure-function relationships in nitrite reductases, key enzymes in the dissimilatory denitrification pathway which reduce nitrite to nitric oxide (NO), are reviewed in this paper. The mechanisms of NO production are discussed in detail and special attention is paid to new structural information, such as the high resolution structure of the copper- and heme-containing enzymes from different sources. Finally, some implications relevant to regulation of the steady state levels of NO in denitrifiers are presented.     

Inhibition of protein synthesis by nitric oxide correlates with cytostatic activity: nitric oxide induces phosphorylation of initiation factor eIF-2 alpha.

BACKGROUND: Nitric oxide (NO) is cytostatic for proliferating cells, inhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correlates with established cytostatic activities of NO. MATERIALS AND METHODS: In in vitro experiments, various cell types were exposed to NO using either donors or expression of inducible NO synthase (iNOS). The capacity of NO to suppress total protein synthesis, measured by incorporation of 35S-methionine into protein, was correlated with the capacity of NO to suppress cell proliferation, viral replication, or iNOS expression. Phosphorylation of eIF-2 alpha was examined as a possible mechanism for the suppressed protein synthesis by NO. RESULTS: Both NO donors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decreased cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine peritoneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expression in pancreatic islets or RAW264.7 cells almost completely inhibited total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synthesis of specific proteins such as insulin or iNOS itself. This possibility was confirmed in RAW264.7 cells where the inhibition of total protein synthesis correlated with the decreased iNOS protein. The decrease in protein levels occurred without changes in iNOS mRNA levels, implicating an inhibition of translation. Mechanistic studies revealed that iNOS expression in RAW264.7 cells resulted in the phosphorylation of eIF-2 alpha and inhibition of the 80S ribosomal complex formation. CONCLUSIONS: These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2 alpha. Furthermore, our observations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of NO.     

Inhibition of inducible nitric oxide synthase attenuates platelet adhesion in subpleural arterioles caused by lung ischemia-reperfusion in rabbits.

Oxidative stress, induced by lung ischemia-reperfusion, leads to platelet and leukocyte activation and may contribute to decreased alveolar perfusion by platelet adhesion to the arteriolar wall. We investigated the hypothesis that ischemia-reperfusion injury increases inducible nitric oxide synthase (iNOS) activity and subsequent generation of reactive nitrogen species with P-selectin-dependent platelet-endothelial interactions and vasoconstriction during lung reperfusion. Subpleural arterioles, labeled platelets, and leukocytes were examined in anesthetized, open-chest rabbits by intravital fluorescence microscopy. Ischemia was caused by reversible occlusion of the right pulmonary artery for 1 or 2 h (1IR and 2IR groups). During 2 h of reperfusion, postischemic platelet rolling and adhesion were independent from leukocyte-arteriolar wall interactions and correlated with pulmonary arteriolar constriction in proportion to the length of ischemia. In rabbits treated with an iNOS inhibitor (1400W) before occlusion (2IR + 1400W group), platelet-arteriolar wall interactions and vasoconstriction were prevented. iNOS expression and activity in ischemic lung tissue were markedly greater than control and also were proportional to ischemia duration. NOS activity, immunochemically detected P-selectin, and nitrotyrosine expression in ischemic lung tissue from animals subjected to ischemia-reperfusion, as well as the plasma level of soluble P-selectin, were significantly higher than in nonischemic lungs and were inhibited by pretreatment with 1400W. These results show that platelet adhesion and arteriolar constriction during early reperfusion in the ventilated lung can result from increased iNOS activity and is highly correlated with reactive nitrogen species and P-selectin expression.     

Inhibition of nitric oxide synthesis in primary cultured mouse hepatocytes by alpha-lipoic acid

Recent work shows that septic or endotoxic shock is associated with lipopolysaccharide and cytokine mixture-induced nitric oxide (NO) synthesis in liver. Here we found that DL-alpha-lipoic acid inhibited but other thiol-containing antioxidants such as glutathione and N-acetylcysteine enhanced lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis in hepatocytes. The inhibitory action of alpha-lipoic acid on hepatocyte NO synthesis was as potent as that of NG-monomethyl-L-arginine without obvious cytotoxicity. Deletion by diethylmaleate or inhibition by buthionine sulfoximine of intracellular glutathione caused a significant decrease in hepatocyte NO synthesis, implying that increased intracellular reduced glutathione levels could not be the reason for alpha-lipoic acid inhibited NO synthesis. alpha-Lipoic acid inhibition of NO synthesis seems to be from alpha-lipoic acid improved carbohydrate metabolism in hepatocytes. Since alpha-lipoic acid is an essential compound existing naturally in physiological systems, it may serve as both a research and therapeutic agent for sepsis.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Role of exhaled nitric oxide in asthma

Nitric oxide (NO), an evanescent atmospheric gas, has recently been discovered to be an important biological mediator in animals and humans. Nitric oxide plays a key role within the lung in the modulation of a wide variety of functions including pulmonary vascular tone, nonadrenergic non-cholinergic (NANC) transmission and modification of the inflammatory response. Asthma is characterized by chronic airway inflammation and increased synthesis of NO and other highly reactive and toxic substances (reactive oxygen species). Pro- inflammatory cytokines such as TNFalpha and IL-1beta are secreted in asthma and result in inflammatory cell recruitment, but also induce calcium- and calmodulin-independent nitric oxide synthases (iNOS) and perpetuate the inflammatory response within the airways. Nitric oxide is released by several pulmonary cells including epithelial cells, eosinophils and macrophages, and NO has been shown to be increased in conditions associated with airway inflammation, such as asthma and viral infections. Nitric oxide can be measured in the expired air of several species, and exhaled NO can now be rapidly and easily measured by the use of chemiluminescence analysers in humans. Exhaled NO is increased in steroid-naive asthmatic subjects and during an asthma exacerbation, although it returns to baseline levels with appropriate anti-inflammatory treatment, and such measurements have been proposed as a simple non-invasive method of measuring airway inflammation in asthma. Here the chemical and biological properties of NO are briefly discussed, followed by a summary of the methodological considerations relevant to the measurement of exhaled NO and its role in lung diseases including asthma. The origin of exhaled NO is considered, and brief mention made of other potential markers of airway inflammation or oxidant stress in exhaled breath.     

Inhibition of neuronal nitric oxide synthase by N-phenacyl imidazoles.

Nitric oxide (NO) mediates a series of physiological processes, including regulation of vascular tone, macrofage-mediated neurotoxicity, platelet aggregation, learning and long-term potentiation, and neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in several pathological diseases. Accordingly, more potent inhibitors, more selective for neuronal nitric oxide synthase (nNOS) than endothelial NOS (eNOS) or inducible NOS (iNOS), could be useful in the treatment of cerebral ischemia and other neurodegenerative diseases. We recently described the synthesis of a series of imidazole derivatives. Among them N-(4-nitrophenacyl) imidazole (A) and N-(4-nitrophenacyl)-2-methyl-imidazole (B) were considered selective nNOS inhibitors. In the present study the action mechanism of compounds A and B was analyzed. Spectral changes observed in the presence of compound A indicate that this inhibitor exerts its effect without interaction with heme iron. Moreover compounds A and B, inhibit nNOS "noncompetitively" versus arginine, but "competitively" versus BH(4).     

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.     

Non-enzymatic nitric oxide synthesis in biological systems.

Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, NO can also be generated in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia. This NO formation is not blocked by NOS inhibitors and with long periods of ischemia progressing to necrosis, this mechanism of NO formation predominates. In postischemic tissues, NOS-independent NO generation has been observed to result in cellular injury with a loss of organ function. The kinetics and magnitude of nitrite disproportionation have been recently characterized and the corresponding rate law of NO formation derived. It was observed that the generation and accumulation of NO from typical nitrite concentrations found in biological tissues increases 100-fold when the pH falls from 7.4 to 5.5. It was also observed that ischemic cardiac tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions, the magnitude of enzyme-independent NO generation exceeds that which can be generated by tissue concentrations of NOS. The existence of this enzyme-independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury.     

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole attenuates acute lung injury in an ovine model

Nitric oxide (NO) has been shown to play a major role in acute lung injury (ALI) after smoke inhalation. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that mimics human sepsis and pneumonia. We hypothesized that the inhibition of neuronal NO synthase (nNOS) might be beneficial in treating ALI associated with this model. Female sheep (n = 26) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa [5 x 10(11) colony forming units (CFU)] into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of 7-nitroindazole (7-NI), an nNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), a nonspecific NOS inhibitor, or aminoguanidine (AG), an inducible NOS inhibitor, was started 1 h after insult. The administration of 7-NI improved pulmonary gas exchange (PaO2/FiO2; where PaO2 is arterial PO2 and FiO2 is fractional inspired oxygen concentration) and pulmonary shunt fraction and attenuated the increase in lung wet-to-dry weight ratio seen in the nontreated sheep. Histologically, 7-NI prevented airway obstruction. The increase in airway blood flow after injury in the nontreated group was significantly inhibited by 7-NI. The increase in plasma concentration of nitrate and nitrite (NOx) was inhibited by 7-NI as well. Posttreatment with l-NMMA improved the pulmonary gas exchange, but AG did not. The results of the present study show that nNOS may be involved in the pathogenesis of ALI after smoke inhalation injury followed by bacterial instillation in the airway.     

Catalpol attenuates nitric oxide increase via ERK signaling pathways induced by rotenone in mesencephalic neurons

Catalpol has been shown to rescue neurons from kinds of damage in vitro and in vivo in previous reports. However, the effect of catalpol on the nitric oxide (NO) system via MAPKs signaling pathway of mesencephalic neurons largely remains to be verified. The current study examined that whether catalpol modulated NO and iNOS increase by rotenone in primary mesencephalic neurons and investigated its underlying signaling pathways. Present results indicated that catalpol inhibited primary mesencephalic neurons from apoptosis by morphological assay, immunocytochemistry and flow cytometric evaluation. Moreover, the ERK signaling pathway plays an important role in NO-mediated degeneration of neuron. The current results suggest that catalpol is a potential agent for the prevention of neurons apoptosis by regulating NO and iNOS increase in ERK-mediated neurodegenerative disorders.     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

Desiccation and hypertonicity of the airway surface fluid and thermally induced asthma.

To determine whether drying and hypertonicity of the airway surface fluid (ASF) are involved in thermally induced asthma, nine subjects performed isocapnic hyperventilation (HV) (minute ventilation 62.2 +/- 8.3 l/min) of frigid air (-8.9 +/- 3.3 degrees C) while periciliary fluid was collected endoscopically from the trachea. Osmolality was measured by freezing-point depression. The baseline 1-s forced expiratory volume was 73 +/- 4% of predicted and fell 26.4% 10 min postchallenge (P > 0.0001). The volume of ASF collected was 11.0 +/- 2.2 microl at rest and remained constant during and after HV as the airways narrowed (HV 10.6 +/- 1.9, recovery 6.5 +/- 1.7 microl; P = 0.18). The osmolality also remained stable throughout (rest 336 +/- 16, HV 339 +/- 16, and recovery 352 +/- 19 mosmol/kgH(2)O, P = 0.76). These data demonstrate that airway desiccation and hypertonicity of the ASF do not develop during hyperpnea in asthma; therefore, other mechanisms must cause exercise- and hyperventilation-induced airflow limitation.     

Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury

Previous studies indicate that deficiency of endothelial nitric oxide (NO) synthase (eNOS)-derived NO exacerbates myocardial reperfusion injury. We hypothesized that overexpression of eNOS would reduce the extent of myocardial ischemia-reperfusion (MI/R) injury. We investigated two distinct strains of transgenic (TG) mice overexpressing the eNOS gene (eNOS TG). Bovine eNOS was overexpressed in one strain (eNOS TG-Kobe), whereas the human eNOS gene was overexpressed in the other strain (eNOS TG-RT). Non-TG (NTG) and eNOS TG mice were subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion, and the extent of myocardial infarction was determined. Myocardial infarct size was reduced by 33% in the eNOS TG-Kobe strain (P < 0.05 vs. NTG) and by 32% in the eNOS TG-RT strain (P < 0.05 vs. NTG). However, postischemic cardiac function (cardiac output, fractional shortening) was not improved in the eNOS TG-Kobe mouse at 24 h of reperfusion [P = not significant (NS) vs. NTG]. In additional studies, eNOS TG-Kobe mice were subjected to 30 min of myocardial infarction and 7 days of reperfusion. Fractional shortening and the first derivative of left ventricular pressure were measured in eNOS TG-Kobe and NTG mice, and no significant differences in contractility were observed (P = NS) between the eNOS TG mice and NTG controls. Left ventricular end-diastolic pressure was significantly (P < 0.05 vs. NTG) reduced in the eNOS TG-Kobe strain at 7 days of reperfusion. The cardioprotective effects of eNOS overexpression on myocardial infarct size were ablated by Nomega-nitro-l-arginine methyl ester (300 mg/kg) pretreatment. Thus genetic overexpression of eNOS in mice attenuates myocardial infarction after MI/R but fails to significantly protect against postischemic myocardial contractile dysfunction in mice.     

Airway diffusing capacity of nitric oxide and steroid therapy in asthma.

Exhaled nitric oxide (NO) concentration is a noninvasive index for monitoring lung inflammation in diseases such as asthma. The plateau concentration at constant flow is highly dependent on the exhalation flow rate and the use of corticosteroids and cannot distinguish airway and alveolar sources. In subjects with steroid-naive asthma (n = 8) or steroid-treated asthma (n = 12) and in healthy controls (n = 24), we measured flow-independent NO exchange parameters that partition exhaled NO into airway and alveolar regions and correlated these with symptoms and lung function. The mean (+/-SD) maximum airway flux (pl/s) and airway tissue concentration [parts/billion (ppb)] of NO were lower in steroid-treated asthmatic subjects compared with steroid-naive asthmatic subjects (1,195 +/- 836 pl/s and 143 +/- 66 ppb compared with 2,693 +/- 1,687 pl/s and 438 +/- 312 ppb, respectively). In contrast, the airway diffusing capacity for NO (pl.s-1.ppb-1) was elevated in both asthmatic groups compared with healthy controls, independent of steroid therapy (11.8 +/- 11.7, 8.71 +/- 5.74, and 3.13 +/- 1.57 pl.s-1.ppb-1 for steroid treated, steroid naive, and healthy controls, respectively). In addition, the airway diffusing capacity was inversely correlated with both forced expired volume in 1 s and forced vital capacity (%predicted), whereas the airway tissue concentration was positively correlated with forced vital capacity. Consistent with previously reported results from Silkoff et al. (Silkoff PE, Sylvester JT, Zamel N, and Permutt S, Am J Respir Crit Med 161: 1218-1228, 2000) that used an alternate technique, we conclude that the airway diffusing capacity for NO is elevated in asthma independent of steroid therapy and may reflect clinically relevant changes in airways.     

Dimethylarginine dimethylaminohydrolase-2 overexpression improves impaired nitric oxide synthesis of endothelial cells induced by glycated protein.

Nitric oxide (NO) synthesis is modulated by dimethylarginine dimethylaminohydrolase (DDAH) via metabolizing asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor. This study investigated whether glycosylated bovine serum albumin (GBSA) could impair NO synthesis by inhibition of DDAH expression and activity, and whether DDAH2 overexpression could reverse the impaired NO synthesis induced by GBSA in endothelial cells. Overexpression of DDAH2 gene was established by liposome-mediated gene transfection in ECV304 endothelial cell line. Cells were incubated with 1.70 mmol/L GBSA for 48h. And the expressions of DDAH1 and DDAH2, gene activities of DDAH and NOS in cells, as well as concentrations of ADMA and NO in media were assayed. The activity of DDAH and expression of DDAH2 gene but not DDAH1 gene were inhibited in endothelial cells after exposure to GBSA, whereas the concentrations of ADMA were increased concomitantly with the decrease of NOS activity in cells and NO production in media. Overexpression of DDAH2 gene could prevent the inhibition of DDAH activity induced by GBSA (0.55+/-0.02 vs 0.42+/-0.02U/g pro; n=3; P<0.05), decrease ADMA concentration (0.59+/-0.04 vs 1.13+/-0.11 micromol/L; n=3; P<0.01), and increase NOS activity and NO production (53.77+/-3.40 vs 34.59+/-2.57 micromol/L; P<0.05) compared with untransfected cells treated with GBSA. These results suggest that decreased DDAH activity and subsequent elevated endogenous ADMA are implicated in the inhibition of NO synthesis induced by GBSA, and overexpression of DDAH2 gene can prevent these changes in DDAH/ADMA/NOS/NO pathway of endothelial cells exposed to GBSA.