Antiandrogenic activity of extracts of diesel exhaust particles emitted from diesel-engine truck under different engine loads and speeds

To clarify the alteration of androgenic and antiandrogenic activities by diesel engine conditions, we collected diesel exhaust particles (DEP) samples emitted from a diesel-engine truck under different conditions of engine loads and vehicle speeds, and DEP extract (DEPE) samples were prepared from each. The androgenic and antiandrogenic activities of the DEPE samples were examined using a prostate specific antigen (PSA) promoter-luciferase reporter gene assay in PC3/AR human prostate cancer cells. While all DEPE samples did not exhibit androgenic effects, the antiandrogenic effects were enhanced by higher engine load but not by higher vehicle speed. In this study, significant correlations between antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activities were demonstrated in PC3/AR cells by 16 polycyclic aromatic compounds and beta-naphthoflavone. Yeast two-hybrid assay and cytochrome P450 (CYP) 1A1 promoter-luciferase reporter gene assay showed that the antiandrogenic constituents acting as androgen receptor (AR) antagonists and AhR agonists were increased by only the higher engine load. In conclusion, the antiandrogenic effects of DEPE samples were enhanced by a higher engine load which resulted in DEPC samples with elevated AhR agonistic and AR antagonistic activities.     

Caveolin-1 is important for nitric oxide-mediated angiogenesis in fibrin gels with human umbilical vein endothelial cells

AIM: The role of caveolin-1 (Cav-1) in angiogenesis remains poorly understood. The endothelial nitric oxide (NO) synthase (eNOS), a caveolin-interacting protein, was demonstrated to play a predominant role in vascular endothelial growth factor (VEGF) -induced angiogenesis. The purpose of our study was to examine the role of Cav-1 and the eNOS complex in NO-mediated angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in 3-D fibrin gels to form capillary-like tubules by VEGF stimulation. The expression of Cav-1 and eNOS was detected by semiquantitative RT-PCR. The HUVEC were treated with antisense oligonucleotides to downregulate Cav-1 expression. Both transduced and non-infected HUVEC were cultured in fibrin gels in the presence or absence of VEGF (20 ng/mL) and NG-nitro-L-arginine methyl ester (L-NAME; 5 mmol/L). NO was measured using a NO assay kit and capillary-like tubules were quantified by tubule formation index using the Image J program. RESULTS: RT-PCR analysis revealed that Cav-1 levels steadily increased in a time-dependent manner and reached their maximum after 5 d of incubation, but there were no obvious changes in eNOS mRNA expression in response to VEGF in the fibrin gel model. VEGF (20 ng/mL) can promote NO production and the formation of capillary-like tubules, and this promoting effect of VEGF was blocked by the addition of L-NAME (5 mmol/L). When transduced HUVEC with the antisense Cav-1 oligonucleotides were plated in the fibrin gels, the capillary-like tubules were significantly fewer than those of the non-infected cells. The capillary-like tubules formation and NO production of transduced HUVEC with the antisense Cav-1 oligonucleotides cultured in fibrin gels showed no responses to the addition of VEGF (20 ng/mL) and L-NAME (5.0 mmol/L). CONCLUSION: NO was a critical angiogenic mediator in this model. Cav-1 was essential for NO-mediated angiogenesis and may be an important target of anti-angiogenesis therapy.     

DOSE-RESPONSE ASSESSMENT AND EFFECT OF PARTICLES IN GUINEA PIGS EXPOSED CHRONICALLY TO DIESEL EXHAUST: ANALYSIS OF VARIOUS BIOLOGICAL MARKERS IN PULMONARY ALVEOLAR LAVAGE FLUID AND CIRCULATING BLOOD

To elucidate dose-response and other effects of diesel particles in guinea pigs chronically exposed to diesel exhaust, various biomarkers for chronic obstructive lung diseases were studied using bronchoalveolar lavage (BAL) fluid and blood specimens. Guinea pigs were exposed 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo to filtered air (control group, n = 8-10), a low level of diesel exhaust (L group: NO 2 = 0.22 ± 0.03 ppm; SO 2 = 0.6 ± 0.19 ppm; particles = 0.21 ± 0.07 mg/m 3, n = 8-10), medium level of diesel exhaust (M group; NO 2 = 1.07 ± 0.09 ppm; SO 2 = 2.83 ± 0.73 ppm; particles = 1.14 ± 0.26 mg/m 3, n = 8-10), and high level of diesel exhaust (H group: NO 2 = 2.88 ± 0.29 ppm; SO 2 = 6.49 ± 1.75 ppm; particles = 2.94 ± 0.69 mg/m 3, n = 8-10), or at a medium concentration of diesel exhaust without particulate matters (MG group: NO 2 = 1.01 ± 0.09 ppm; SO 2 = 2.66 ± 0.64 ppm; particles = 0.01 ± 0.01 mg/m 3, n = 8-10). Anesthetized animals were sacrificed and BAL fluid from the lung and blood from right ventricle were collected. Various biomarkers of inflammation, components of mucus and surfactant, bronchoconstrictors were determined. Changes of leukotriene C4 in plasma, eosinophil counts, biomarkers of inflammation and cytotoxicity, and mucus and surfactant components in BAL fluid were statistically different among the C, L, M, and H groups after adjustment for the exposure period and group-by-exposure period with respect to their interactions in two-way analysis of variance (ANOVA). The levels of these biomarkers in the H group were higher than those of the M group, whereas those of the L group showed no significant changes compared with those of the C group during experimental period. Onset of significant changes of these biomarkers for the M group was at 18 mo of exposure, whereas that for the H group was at 12 mo of exposure, which resulted in changes in the levels of biomarkers in BAL fluid. Although numbers of eosinophils in BAL fluid increased significantly in the M and H groups at 12 mo, only leukotrine C4 increased at 18 and 24 mo in blood and at 24 mo in BAL fluid. Animals exposed to the medium level of diesel exhaust without particulate matter showed significantly less increase of these biomarkers as compared with animals exposed to the same level of diesel exhaust with particulate matters. These findings indicate that chronic exposure to diesel exhaust induced continuous inflammation, overproduction of mucus, and phospholipids in the lung. Animals exposed to the high dose of diesel exhaust showed a plateau of biological responses at 12 mo of exposure. Particulate matter in diesel exhaust appears to play an important role in development of lung injury by chronic emission exhaust exposure.     

Dose-response assessment and effect of particles in guinea pigs exposed chronically to diesel exhaust: analysis of various biological markers in pulmonary alveolar lavage fluid and circulating blood

To elucidate dose-response and other effects of diesel particles in guinea pigs chronically exposed to diesel exhaust, various biomarkers for chronic obstructive lung diseases were studied using bronchoalveolar lavage (BAL) fluid and blood specimens. Guinea pigs were exposed 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo to filtered air (control group, n = 8-10), a low level of diesel exhaust (L group: NO(2) = 0.22 +/- 0.03 ppm; SO(2) = 0.6 +/- 0.19 ppm; particles = 0.21 +/- 0.07 mg/m(3), n = 8-10), medium level of diesel exhaust (M group; NO(2) = 1.07 +/- 0.09 ppm; SO(2) = 2.83 +/- 0.73 ppm; particles = 1.14 +/- 0.26 mg/m(3), n = 8-10), and high level of diesel exhaust (H group: NO(2) = 2.88 +/- 0.29 ppm; SO(2) = 6.49 +/- 1.75 ppm; particles = 2.94 +/- 0.69 mg/m(3), n = 8-10), or at a medium concentration of diesel exhaust without particulate matters (MG group: NO(2) = 1.01 +/- 0.09 ppm;#10; SO(2) = 2.66 +/- 0.64 ppm; particles = 0.01 +/- 0.01 mg/m(3), n = 8-10). Anesthetized animals were sacrificed and BAL fluid from the lung and blood from right ventricle were collected. Various biomarkers of inflammation, components of mucus and surfactant, bronchoconstrictors were determined. Changes of leukotriene C4 in plasma, eosinophil counts, biomarkers of inflammation and cytotoxicity, and mucus and surfactant components in BAL fluid were statistically different among the C, L, M, and H groups after adjustment for the exposure period and group-by-exposure period with respect to their interactions in two-way analysis of variance (ANOVA). The levels of these biomarkers in the H group were higher than those of the M group, whereas those of the L group showed no significant changes compared with those of the C group during experimental period. Onset of significant changes of these biomarkers for the M group was at 18 mo of exposure, whereas that for the H group was at 12 mo of exposure, which resulted in changes in the levels of biomarkers in BAL fluid. Although numbers of eosinophils in BAL fluid increased significantly in the M and H groups at 12 mo, only leukotriene C4 increased at 18 and 24 mo in blood and at 24 mo in BAL fluid. Animals exposed to the medium level of diesel exhaust without particulate matter showed significantly less increase of these biomarkers as compared with animals exposed to the same level of diesel exhaust with particulate matters. These findings indicate that chronic exposure to diesel exhaust induced continuous inflammation, overproduction of mucus, and phospholipids in the lung. Animals exposed to the high dose of diesel exhaust showed a plateau of biological responses at 12 mo of exposure. Particulate matter in diesel exhaust appears to play an important role in development of lung injury by chronic emission exhaust exposure.     

Emissions from diesel versus biodiesel fuel used in a CRDI SUV engine: PM mass and chemical composition

The diesel tailpipe emissions typically undergo substantial physical and chemical transformations while traveling through the tailpipe, which tend to modify the original characteristics of the diesel exhaust. Most of the health-related attention for diesel exhaust has focused on the carcinogenic potential of inhaled exhaust components, particularly the highly respirable diesel particulate matter (DPM). In the current study, parametric investigations were made using a modern automotive common rail direct injection (CRDI) sports utility vehicle (SUV) diesel engine operated at different loads at constant engine speed (2400?rpm), employing diesel and 20% biodiesel blends (B20) produced from karanja oil. A partial flow dilution tunnel was employed to measure the mass of the primary particulates from diesel and biodiesel blend on a 47-mm quartz substrate. This was followed by chemical analysis of the particulates collected on the substrate for benzene-soluble organic fraction (BSOF) (marker of toxicity). BSOF results showed decrease in its level with increasing engine load for both diesel and biodiesel. In addition, real-time measurements for organic carbon/elemental carbon (OC/EC), and polycyclic aromatic hydrocarbons (PAHs) (marker of toxicity) were carried out on the diluted primary exhaust coming out of the partial flow dilution tunnel. PAH concentrations were found to be the maximum at 20% rated engine load for both the fuels. The collected particulates from diesel and biodiesel-blend exhaust were also analyzed for concentration of trace metals (marker of toxicity), which revealed some interesting results.     

NADPH oxidases regulate endothelial inflammatory injury induced by PM2.5 via AKT/eNOS/NO axis

Fine particulate matter (PM_2.5)-induced detrimental cardiovascular effects have been widely concerned, especially for endothelial cells, which is the first barrier of the cardiovascular system. Among potential mechanisms involved, reactive oxidative species take up a crucial part. However, source of oxidative stress and its relationship with inflammatory response have been rarely studied in PM_2.5-induced endothelial injury. Here, as a key oxidase that catalyzes redox reactions, NADPH oxidase (NOX) was investigated. Human umbilical vein endothelial cells (EA.hy926) were exposed to Standard Reference Material 1648a of urban PM_2.5 for 24 h, which resulted in NOX-sourced oxidative stress, endothelial dysfunction, and inflammation induction. These are manifested by the up-regulation of NOX, increase of superoxide anion and hydrogen peroxide, elevated endothelin-1 (ET-1) and asymmetric dimethylarginine (ADMA) level, reduced nitric oxide (NO) production, and down-regulation of phosphorylation of endothelial NO synthase (eNOS) with increased levels of inducible NO synthase, as well as the imbalance between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1), and changes in the levels of pro-inflammatory and anti-inflammatory factors. However, administration of NOX1/4 inhibitor GKT137831 alleviated PM_2.5-induced elevated endothelial dysfunction biomarkers (NO, ET-1, ADMA, iNOS, and tPA/PAI-1), inflammatory factors (IL-1β, IL-10, and IL-18), and adhesion molecules (ICAM-1, VCAM-1, and P-selectin) and also passivated NOX-dependent AKT and eNOS phosphorylation that involved in endothelial activation. In summary, PM_2.5-induced NOX up-regulation is the source of ROS in EA.hy926, which activated AKT/eNOS/NO signal response leading to endothelial dysfunction and inflammatory damage in EA.hy926 cells.     

银杏黄酮苷对氧化损伤内皮细胞产生NO、eNOS和ICAM-1的影响

目的观察银杏黄酮苷对氧化损伤的人脐静脉内皮细胞（HUVEC）产生NO、内皮型一氧化氮合酶（eNOS）和人可溶性细胞间黏附分子（ICAM-1）的影响。方法体外培养HUVEC,传至3代后,以不同浓度的银杏黄酮苷分别作用于HUVEC,然后进行氧化损伤处理。以硝酸还原酶法测定培养液上清中的NO水平,免疫细胞化学法检测内皮细胞eNOS的表达,ELISA法测定细胞培养液中ICAM-1的含量。结果HUVEC在氧化损伤（H2O2100μmol/L,2h）后产生NO的量显著减少（P〈0.01）,eNOS表达下调,ICAM-1表达上调;银杏黄酮苷可以剂量依赖性的增加内皮细胞NO生成量,上调eNOS的表达,下调ICAM-1表达。结论银杏黄酮苷可能通过增加HUVEC eNOS的表达增加N0的释放、抑制ICAM-1的表达等机制对内皮细胞起保护作用。     

Chronic Diesel Exhaust Exposures of Rats Demonstrate Concentration and Time-Dependent Effects on Pulmonary Inflammation

Long-term, repeated exposure to particles in air pollution increases the risk for chronic respiratory diseases and cardiorespiratory mortality. The biological linkages remain poorly understood in chronic exposure to particle matter. To elucidate and verify these linkages, we investigated long-term exposure to diesel emission with respect to dose dependence and the effect of components without particles from diesel emission in rats. Wistar rats were exposed to filtered air (C group), diesel exhaust at low (L group), medium (M group), and high level (H group), or at a medium concentration diesel exhaust without particulate matter (MG group), for 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo. Anesthetized animals were sacrificed and bronchoalveolar lavage (BAL) fluid from the lung and blood from the right ventricle were collected. Various biomarkers of inflammation and components of mucus and surfactant were determined. Changes in total cell counts and cell differentiation, total protein, mucus and surfactant components, and prostaglandin E 2 in BAL fluid, but not biomarkers in plasma, showed statistical differences among the C, L, M, and H groups during the experimental period. The changes in these biomarkers in the H group were greater than those in the M group, whereas those in the L group showed no significant changes compared with those in the C group during the experimental period. The onset of significant changes in inflammatory cells and these biomarkers in BAL fluid for the M and H groups was at 6 to 12 mo of exposure. The maximum level was reached at 12 to 18 mo of exposure. Although BAL prostaglandin E 2 decreased significantly at 6 mo of exposure in the M and H groups, this trend was not observed in the C and L groups. Animals exposed to a medium level of diesel exhaust without particulate matter showed significantly less inflammatory cells and various biomarkers in BAL fluid than animals exposed to the same level of diesel exhaust with particulate matter during the experimental period. These findings suggest that biological response to inhaled particles is aggravated during chronic exposure to diesel exhaust dose-dependently. Inflammation and overproduction of mucus and surfactant components reached a plateau at 12 or 18 mo of exposure during a 24-mo experimental period. No adverse effect of particles (less than 1.0 mg particles/m 3 of diesel emission) was observed in these rats. However, our data suggest that particulate matter plays an important role during development of chronic lung injury induced by diesel emission exhaust.     

p38 MAPK通路在TNF-α诱导人脐静脉内皮细胞分泌ET-1与eNOS中的作用及通心络干预影响

目的观察p38 MAPK信号通路在肿瘤坏死因子-α(TNF-α)诱导人脐静脉内皮细胞(HUVEC)表达内皮素-1(ET-1)与内皮型一氧化氮合酶(eNOS)中的作用及通心络干预影响。方法分别采用放免法及ELISA法测定不同浓度TNF-α(0、2.5、5、10、15、20μg·L-1)在不同时间点(0、1、2、4、8、12、24h)干预后HUVEC培养上清液中ET-1和eNOS含量;分别采用Western blot和Realtime RT-PCR方法检测TNF-α干预24h后HUVEC中ET-1、eNOS蛋白及mRNA表达;采用Western blot方法检测TNF-α干预10min、30min、60min后HUVEC磷酸化p38 MAPK蛋白表达。结果不同浓度TNF-α随时间延长均明显增加HUVEC培养上清液中ET-1含量,降低eNOS含量;TNF-α升高细胞中ET-1蛋白及mRNA水平、降低eNOS蛋白及mRNA水平、在各时间点均可升高细胞p-p38 MAPK蛋白表达。通心络可降低TNF-α诱导的HUVEC培养上清ET-1含量、降低细胞中ET-1蛋白及mRNA的异常升高;增加HUVEC培养上清中eNOS的表达、增加细胞中eNOS蛋白及mRNA的表达;明显抑制TNF-α诱导的细胞p-p38 MAPK表达。结论p38 MAPK信号通路参与了TNF-α诱导HUECV细胞分泌ET-1和eNOS,通心络对内皮细胞保护作用机制与抑制该通路有关。     

Chronic diesel exhaust exposures of rats demonstrate concentration and time-dependent effects on pulmonary inflammation

Long-term, repeated exposure to particles in air pollution increases the risk for chronic respiratory diseases and cardiorespiratory mortality. The biological linkages remain poorly understood in chronic exposure to particle matter. To elucidate and verify these linkages, we investigated long-term exposure to diesel emission with respect to dose dependence and the effect of components without particles from diesel emission in rats. Wistar rats were exposed to filtered air (C group), diesel exhaust at low (L group), medium (M group), and high level (H group), or at a medium concentration diesel exhaust without particulate matter (MG group), for 16 h/day, 6 days/wk, for 6, 12, 18, or 24 mo. Anesthetized animals were sacrificed and bronchoalveolar lavage (BAL) fluid from the lung and blood from the right ventricle were collected. Various biomarkers of inflammation and components of mucus and surfactant were determined. Changes in total cell counts and cell differentiation, total protein, mucus and surfactant components, and prostaglandin E(2) in BAL fluid, but not biomarkers in plasma, showed statistical differences among the C, L, M, and H groups during the experimental period. The changes in these biomarkers in the H group were greater than those in the M group, whereas those in the L group showed no significant changes compared with those in the C group during the experimental period. The onset of significant changes in inflammatory cells and these biomarkers in BAL fluid for the M and H groups was at 6 to 12 mo of exposure. The maximum level was reached at 12 to 18 mo of exposure. Although BAL prostaglandin E(2) decreased significantly at 6 mo of exposure in the M and H groups, this trend was not observed in the C and L groups. Animals exposed to a medium level of diesel exhaust without particulate matter showed significantly less inflammatory cells and various biomarkers in BAL fluid than animals exposed to the same level of diesel exhaust with particulate matter during the experimental period. These findings suggest that biological response to inhaled particles is aggravated during chronic exposure to diesel exhaust dose-dependently. Inflammation and overproduction of mucus and surfactant components reached a plateau at 12 or 18 mo of exposure during a 24-mo experimental period. No adverse effect of particles (less than 1.0 mg particles/m(3) of diesel emission) was observed in these rats. However, our data suggest that particulate matter plays an important role during development of chronic lung injury induced by diesel emission exhaust.     

P物质对血管内皮细胞一氧化氮分泌及表达的影响

目的探讨P物质（SP）对血管内皮细胞一氧化氮（NO）分泌及表达的影响.方法采用不同浓度P物质（10-9mol/L、10-8mol/L、10-7mol/L和10-6mol/L）作用于人脐静脉血管内皮细胞（HUVEC）,分别于15 min、30 min、1 h、3 h、6 h和12 h检测培养上清液中NO含量,并应用免疫组化方法检测HUVEC内诱生型一氧化氮合成酶（iNOS）和内皮型一氧化氮合成酶（eNOS）的表达.结查10-9mol/L～10-6mol/L浓度范围的SP作用15 min,均可使HUVEC内eNOS表达增强,分泌NO水平上升,并于1 h后达峰值,与对照组比较具有显著性差异（P＜0.05）,其中10-8mol/LSP浓度组作用最为明显;而不同浓度的SP对HUVEC内iNOS的表达与对照组比较无显著性差异.结论SP可通过增强HUVEC内eNOS的表达来促进NO的分泌;SP对HUVEC内iNOS的表达则无明显影响.     

Emissions from diesel versus biodiesel fuel used in a CRDI SUV engine: PM mass and chemical composition

The diesel tailpipe emissions typically undergo substantial physical and chemical transformations while traveling through the tailpipe, which tend to modify the original characteristics of the diesel exhaust. Most of the health-related attention for diesel exhaust has focused on the carcinogenic potential of inhaled exhaust components, particularly the highly respirable diesel particulate matter (DPM). In the current study, parametric investigations were made using a modern automotive common rail direct injection (CRDI) sports utility vehicle (SUV) diesel engine operated at different loads at constant engine speed (2400 rpm), employing diesel and 20% biodiesel blends (B20) produced from karanja oil. A partial flow dilution tunnel was employed to measure the mass of the primary particulates from diesel and biodiesel blend on a 47-mm quartz substrate. This was followed by chemical analysis of the particulates collected on the substrate for benzene-soluble organic fraction (BSOF) (marker of toxicity). BSOF results showed decrease in its level with increasing engine load for both diesel and biodiesel. In addition, real-time measurements for organic carbon/elemental carbon (OC/EC), and polycyclic aromatic hydrocarbons (PAHs) (marker of toxicity) were carried out on the diluted primary exhaust coming out of the partial flow dilution tunnel. PAH concentrations were found to be the maximum at 20% rated engine load for both the fuels. The collected particulates from diesel and biodiesel-blend exhaust were also analyzed for concentration of trace metals (marker of toxicity), which revealed some interesting results.     

Responses of cultured human airway epithelial cells treated with diesel exhaust extracts will vary with the engine load

Epidemiologic evidence suggests that increased morbidity and mortality are associated with the concentrations of ambient air particulate matter (PM). Many sources contribute to the particulate fraction of ambient air pollution, including diesel exhaust particulates (DEP). Diesel exhaust also contributes gas-phase pollutants to the atmosphere, and gaseous copollutants may influence the toxicity of PM. The composition of diesel exhaust varies greatly depending on the engine load conditions as well as other factors. To determine whether different diesel exhaust composition can affect lung cell resposes, the effects of of diesel exhaust extracts derived from different engine loads were examined on normal human bronchial epithelial cells (NHBE) in vitro. Diesel exhaust was collected into chilled impingers containing phosphate-buffered saline (PBS). Cultured NHBE cells were treated with 0 to 500 microg/well extract from approximately 0% engine load (termed low load or LL) or extract from approximately 75% engine load (termed high load or HL) for 24 h. The HL extract was cytotoxic at 500 microg compared to controls as measured by (51)Cr release. Production of the neutrophil chemotaxin interleukin 8 (IL-8) was decreased 4.7-fold in cells treated with 500 microg LL extract, whereas cells treated with 500 microg HL extract showed a 2.4-fold increase in IL-8 release. Production of the inflammatory and immune system mediator prostaglandin E(2) (PGE(2)) was increased up to 2.5-fold in cells treated with HL extract, but unchanged with other treatments. Melittin stimulation of cells showed that the LL extract had an inhibitory effect on PGE(2) release at 500 microg. Differences in carbonyl content of the extracts were found by high performance liquid chromatography/mass spectroscopy HPLC/MS, with the HL extract having more intermediate size carbonyls (i.e. with six to nine carbons). The data suggest that the response of NHBE cells to treatment with diesel exhaust will vary depending on the constituent components of the exhaust.     

Evaluating the radioprotective effect of hesperidin in the liver of Swiss albino mice

Amongst calcium channel blockers, amlodipine is known to have unique cardioprotective activities likely attributable to its capacity to increase nitric oxide (NO) release from endothelial cells (EC). Because endothelial NO synthase (eNOS), the main source of NO in EC is known to be inhibited by caveolin-1 (Cav-1), the purpose of this study is to investigate the possibility that amlodipine can modulate eNOS interaction with Cav-1. Using cultured EC, we confirm that amlodipine potentiates vascular endothelial growth factor (VEGF)-induced NO release. eNOS trafficking to specialized plasma membrane microdomains, which is essential to eNOS signaling, is unaffected by amlodipine. However, glutathione s-transferase (GST) pulldown assays reveal that amlodipine can prevent binding of native, acylated eNOS complexes to the active domain of Cav-1 in a concentration-dependent fashion, suggesting that amlodipine has an antagonistic effect on the native eNOS/Cav-1 signaling complex. Moreover, experiments performed in a reconstituted cell line confirm that amlodipine's effect on NO release is highly selective for the eNOS/Cav-1 interaction. To our knowledge, these data are the first to demonstrate a direct effect of amlodipine on the eNOS/Cav-1 protein complex and support the concept of developing novel therapies specifically aimed at modulating the eNOS/Cav-1 interaction to improve endothelial function in cardiovascular diseases.     

Effects of nitrated-polycyclic aromatic hydrocarbons and diesel exhaust particle extracts on cell signalling related to apoptosis: possible implications for their mutagenic and carcinogenic effects

Nitrated-polycyclic aromatic hydrocarbons (nitro-PAHs) and diesel exhaust particle extracts (DEPE) induced apoptosis in Hepa1c1c7 cells with the following potency: 1,3-dinitropyrene (1,3-DNP)>1-nitropyrene (1-NP) > DEPE > 1,8-dinitropyrene (1,8-DNP). The compounds induced cyp1a1, and activated various intracellular signalling pathways related to apoptosis. The CYP inhibitor alpha-naphthoflavone strongly reduced 1,3-DNP-induced cell death, whereas cell death induced by 1-NP was rather increased. Toxic 1,3-DNP and 1-NP were found to induce a concentration-dependent lipid peroxidation. 1,3-DNP caused pro-apoptotic events, including increased phosphorylation and accumulation of p53 in the nucleus, cleavage of bid and of caspases 8 and 3, down-regulation of bcl-x(L) and phosphorylation of p38 and JNK MAPK. Furthermore, 1,3-DNP increased the activation of survival signals including phosphorylation of Akt and inactivation (phosphorylation) of pro-apoptotic bad. Although less potent, rather similar effects were observed following exposure to DEPE, compared to 1-NP. The most important finding was that the most mutagenic and carcinogenic compound tested, 1,8-DNP, induced little (if any) cell death, despite the fact that this compound seemed to give the most DNA damage as judged by DNA adduct formation, increased phosphorylation of p53 and accumulation of cells in S-phase. Immunocytochemical studies revealed that the p53 protein did not accumulate into the nucleus suggesting that 1,8-DNP inactivated the pro-apoptotic function of the p53 protein by a non-mutagenic event. These results suggest that after exposure to 1,8-DNP more cells may survive with DNA damage, thereby increasing its mutagenic and carcinogenic potential.     

Mechanisms underlying nebivolol-induced endothelial nitric oxide synthase activation in human umbilical vein endothelial cells

1. Nebivolol (NEB) has been shown to be a selective blocker of beta1-adrenoceptors with additional vasodilating properties that are mediated, at least in part, by an endothelial-dependent liberation of nitric oxide (NO). In the present study, we investigated the underlying mechanisms of NEB-induced vasodilation. 2. Immunohistochemical staining of endothelial nitric oxide synthase (eNOS) was performed in the absence and presence of NEB in human umbilical vein endothelial cells (HUVEC). In addition, we measured the release of nitric oxide (NO) using diaminofluorescein. Metoprolol (MET) was used for comparison. 3. Nebivolol, but not MET (each at 10 micromol/L), caused a time-dependent increase in NO release from HUVEC, as demonstrated by an increase in DAF fluorescence at 0 versus 10 min (+234 +/- 7 and 55 +/- 22% basal, respectively). Blockade of beta3-adrenoceptors by SR 59230A (1 micromol/L) partially reduced the NEB-induced increase in DAF fluorescence. Complete inhibition of NEB-induced NO liberation was achieved by the simultaneous blockade of beta3-adrenoceptors and oestrogen receptors (with 1 micromol/L ICI 182,780). 4. Application of NEB significantly increased eNOS translocation and serine 1177 phosphorylation of eNOS. However, NEB did not alter eNOS-phosphorylation at threonine 495 and at serine 114. 5. In conclusion, the endothelium-dependent NO liberation induced by NEB is due to stimulation of beta3-adrenoceptors and oestrogen receptors and coincides with eNOS translocation and a phosphorylation at eNOS-serine 1177. These characteristics of NEB may be beneficial not only when treating patients suffering from cardiovascular disease, but may also prevent further deterioration of endothelial dysfunction.