Oxide Synthase Activity Research Articles

The Influence of Titanium Particles Size on Bone Marrow Mensenchymal Stem Cells Viability

The decrease in bone mass caused by wear debris-induced osteolysis could have been compensated through osteoblasts secreting enough new bone matrix. However, the normal osteoblastic population depends on the regular differentiation of their progenitor cells, the bone marrow mesenchymal stem cells (BMSCs). It is not possible to predict whether wear particles will affect the BMSCs’ viability, and subsequently their differentiation. Furthermore, little is known about the extent to which the sizes of the wear particles loading can impact the viability the most. This study has, therefore, concentrated on the potential mechanism for rat BMSCs’ (rBMSCs) viability influenced by different-sized titanium particle (Ti) loading in vitro.rBMSCs were harvested and loaded with circular Ti particles having three different mean diameters, 0.9, 2.7 and 6.9 .m respectively. The results showed that different-sized titanium particles all inhibited rBMSCs’ proliferation and induced rBMSCs’ apoptosis response , but this influence varied with the size of the Ti particles, their concentration and the duration of loading. The smallest Ti particles (0.9.m) exhibited the earliest and largest suppression on the proliferation and the most powerful induction on the apoptotic response of rBMSCs. qRT-PCR analysis demonstrated that those apoptotic effects were association with the abnormal accentuation of inducible nitric oxide synthase(iNOS) activity. The size of titanium particles generated through wear of a prosthetic device and the osteoblastic progenitor BMSCs may be both important considerations in the development of superior implant technology.

Abstract 601: Mechanistic Dichotomy Of Resveratrol-mediated Inhibition Of Vascular Smooth Muscle Cell Proliferation And Restenosis

Angioplasty is a widely used therapy for coronary artery stenosis, but its application is limited by restenosis occuring in 30 – 40% of patients within 3– 6 months of surgery. Restenosis is due to intimal hyperplasia resulting from migration to and proliferation of vascular smooth muscle cells (VSMC) within the intima. Recent epidemiological evidence suggests red wine exerts greater cardioprotection in women than men. Our hypothesis was that the anti-proliferative effects of resveratrol are mediated by estrogen receptor stimulation. We thus determined effects of resveratrol in VSMC isolated from female rats, compared to female VSMC treated with siRNA for estrogen receptor- (ER-)α. At low doses (0.5–25 μM) resveratrol inhibited VSMC proliferation through an ER-α dependent increase in inducible nitric oxide synthase (iNOS) activity and inhibited the cells in S phase, but at high doses (50–100 μM) the mechanism was NF-κB dependent, with inhibition of cells in G 0 /G 1 phase. At all doses, resveratrol inhibited IKK activity, an upstream kinase for NF-κB activation, but its effect at low doses was partially reversed by ER-α blockade. We also found a mechanistic link between ER-α receptors, cyclooxygenase-2 (COX-2), a known modulator of VSMC proliferation, and resveratrol. At low doses, resveratrol induced COX-2 expression, consistent with ER-α stimulation, while at high doses it inhibited COX-2 protein levels, likely due to an inhibition of NF-kB. To confirm these findings in an in vivo model for restenosis, B6.129 (n=8 per group) mice were fed with a high fat diet +/− 50 mg/kg resveratrol for 2 weeks. A carotid artery endothelial denudation procedure was conducted and after recovery, the mice were again fed a high fat diet +/−resveratrol for 2 weeks. Resveratrol increased arterial iNOS activity (p<0.05) and total NO production (p<0.05) but inhibited NF-κB activation (p<0.01). Studies in progress utilizing ER-α (−/−) mice are determining the role of ER-α in resveratrol- mediated effects and whether resveratrol prevents neointimal hyperplasia in this model. In conclusion, though resveratrol clearly inhibited NF-κB activation, it appears that it’s predominate therapeutic effect at low pharmacologically relevant concentrations is via ERα modulation.

Abstract 1420: Mitochondrial Arginase II Constrains Endothelial NOS Activity

Background: Arginase II is known as a major isoenzyme in the vascular endothelial cells of humans and other species and modulates endothelial nitric oxide synthase (eNOS) activity by regulating intracellular L-arginine bioavailability. We tested under hypothesis that subcellular localization of arginase II is confined to the mitochondria and mitochondrial arginase II deficiency reciprocally regulates the vascular endothelial NO production, contributes to improve endothelial dysfunction and decrease vascular stiffness. Methods and Results: Western blot, immunocytochem with Mitotracker and immunoEM confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly reduced, whereas NO production was significantly increased in aorta and isolated endothelial cells from arginase II knock out (ArgII−/−) compared to WT mice. Vasorelaxation to acetylcholine (ACh) was markedly enhanced and vasoconstriction to phenylephrine (PE) was impaired in ArgII−/− rings. Furthermore inhibition of eNOS by N G -nitro-L-arginine methyl ester (L-NAME) impaired ACh response and restored PE response. The baseline pulse wave velocity was significantly decreased in ArgII −/− mice (3.30 ± 0.21 vs. 3.64 ± 0.12 m/s, ArgII −/− vs. WT, *p<0.01), while 14 days of L-NAME treatment resulted in significant increase the PWV in both WT (4.23 ± 0.11 m/s, **p<0.001) and ArgII −/− mice (4.36 ± 0.20 m/s, **p<0.001). Conclusions: These data suggest that arginase II is predominantly confined to the mitochondria and this mitochondrial arginase II regulate the NO production, vascular endothelial function, and vascular stiffness by modulating eNOS activity.

Involvement of CAPON and Nitric Oxide Synthases in Rat Muscle Regeneration After Peripheral Nerve Injury

Carboxy-terminal PDZ ligand of nNOS (CAPON) protein, as an adaptor, binds to nNOS via the PDZ domain helping regulate neuronal nitric oxide synthase (nNOS) activity at post-synaptic sites in neurons (Jaffrey et al., Neuron, 20, 115-124, 1998). Recently, it has been reported that CAPON is present in mouse muscle and may be involved in mouse muscle growth, injury, and repair possibly by regulating the stability, activity, or position of nNOS (Segalat et al., Experimental Cell Research, 302, 170-179, 2005). The present study was to explore the expression patterns and roles of CAPON as well as NOS in rat muscle regeneration after nerve injury. Normal Sprague-Dawley rats were subjected to right sciatic nerve crush injury. Walking track analysis, real time polymerase chain reaction, Western blotting, in situ hybridization, immunocytochemistry, and co-immunoprecipitation techniques were used. It revealed that CAPON mRNA increased, which peaked on days 1 and 28, whereas nNOS mRNA underwent a downregulation in the ipsilateral gastrocnemius muscles after sciatic nerve injury. Their proteins approximately paralleled the mRNA expression. CAPON and nNOS were identified in the activated satellite cells or myotubes and their in vivo interaction was verified. However, eNOS and iNOS proteins suffered an upregulation and were detected in activated satellite cells or myotubes. These data suggest that CAPON and all these three isoforms of NOS might be involved in muscle regeneration after nerve injury. Further study is necessary for a better understanding of the potential functional link between CAPON, NOS, and muscle regeneration, with possible application to therapy for skeletal muscle repair from nerve injury.

Inhibition of inducible nitric oxide synthase in vitro and in vivo by a water-soluble extract of Wendita calysina leaves

Wendita calysina is a Paraguayan herbaceous plant commonly known as burrito. Our previous study indicated that burrito leaves are a very good source of phenylpropanoid glycosides, principally verbascoside. From W. calysina leaves, a standardized, water-soluble extract (WSE) rich in phenylpropanoid glycosides has been developed on an industrial scale to be used as a food supplement, cosmetic, phytomedicine, and ingredient of different formulations. In this study, we investigated the effect of the W. calysina WSE both in vitro in murine macrophage cell line J774.A1 stimulated with lipopolysaccharide (LPS) and, in vivo in an animal model of acute inflammation, carrageenan-induced pleurisy. Here we report that W. calysina WSE (0.05, 0.1, and 0.5 mg/ml) inhibited inducible nitric oxide synthase (iNOS) expression and activity in LPS-stimulated J774.A1. In vivo experiments showed that injection of carrageenan (2%) into the pleural cavity of rats elicited an acute inflammatory response characterized by iNOS expression, intercellular adhesion molecule-1 (ICAM-1) up-regulation, nitrotyrosine and poly (ADP-ribose) synthase (PARS) formation, and lung tissue damage-all parameters significantly reduced by W. calysina WSE (500 mg/kg per os). These results report, for the first time, that a treatment with W. calysina WSE exerts anti-inflammatory effects both in vitro and in vivo.

Pressure-related Increase of Asymmetric Dimethylarginine Caused by Hyperbaric Oxygen in the Rat Brain: A Possible Neuroprotective Mechanism

A decrease in nitric oxide availability in the brain tissue due to the inhibition of nitric oxide synthase (NOS) activity during the early phases of hyperbaric oxygen (HBO) exposure was found to be involved in hyperoxic vasoconstriction leading to reduced regional cerebral blood flow. We hypothesized that the concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), may be an important factor during this hyperoxic vasoconstriction state. Rats were exposed to 1, 2 and 3 atmospheres pure oxygen for two hours. A fourth group of animals served as control. Asymmetric dimethylarginine, L-Arginine and nitrite/nitrate (NOx) concentrations were measured from deproteinized rat brain cytosols. In rat brains exposed to 3 atmospheres O2, ADMA and L-Arginine levels were found to be significantly higher and NOx significantly lower than control levels. Additionally, statistically significant correlations between ADMA and L-Arginine, and ADMA and NOx concentrations were detected. In conclusion, this is the first study indicating increased ADMA levels in rat brains exposed to HBO. The simultaneously decreased NOx values suggest that ADMA elevation resulted in NOS inhibition and therefore may be responsible for the early phase hyperoxic vasoconstriction.

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.

Decreased β-adrenoceptor chronotropic response in selenium-deficient mice

With the aim to study if selenium (Se) deficiency affects the basal frequency and cardiac response to isoproterenol (ISO), mice were fed a Se-deficient diet (Se-) or the same diet supplemented with 0.2 ppm Se as sodium selenite (Se+) for 4 wk. Atria frequency, cyclic AMP (cAMP) accumulation, nitric oxide synthase (NOS) activity, and beta-adrenoceptor-binding assay were then examined. Results showed that Se-mice have both a reduction in atria frequency as well as in cAMP content but higher NOS activity levels either at basal or after ISO stimulation. These differences were suppressed by feeding Se-mice with a Se-supplemented diet for 1 wk or by inhibition of inducible nitric oxide synthase (iNOS). Alterations observed after ISO stimulation in atria of Se-mice were not related to a beta-adrenoceptor expression modification because specific radioligand-binding parameters in cardiac membranes from Se-mice and Se+ mice were similar. The reduced response on rate and cAMP in atria from Se-mice to direct adenylate cyclase (AC) stimulation by forskolin and the shifted upward levels present in 2-amino-4-methylpyridine-treated Se-mice is in agreement with a negative crosstalk between iNOS activity and AC activity in Se-mice.

Basal, but not overload-induced, myonuclear addition is attenuated by NG-nitro-l-arginine methyl ester (l-NAME) administration

The purpose of this study was to examine the effect of blocking nitric oxide synthase (NOS) activity via NG-nitro-L-arginine methyl ester (L-NAME) on myonuclear addition in skeletal muscle under basal and overloaded conditions. Female Sprague-Dawley rats (approx. 220 g) were placed into 1 of the following 4 groups (n = 7-9/group): 7-day skeletal muscle overload (O), sham operation (S), skeletal muscle overload with L-NAME treatment (OLN), and sham operation with L-NAME treatment (SLN). Plantaris muscles were overloaded via bilateral surgical ablation of the gastrocnemius muscles and L-NAME (0.75 mg/mL) was administered in the animals' daily drinking water starting 2 days prior to surgery and continued until sacrifice. Myonuclear addition was assessed as subsarcolemmal incorporation of nuclei labeled with 5-bromo-2'-deoxyuridine (approx. 25 mg.(kg body mass)-1.day-1) delivered via osmotic pump during the overload period. As expected, muscle wet mass, total protein content, fiber cross-sectional area, and myonuclear addition were significantly higher (p <or= 0.05) in O vs. S; however, only the increase in wet mass and total protein content (per body mass) were attenuated by L-NAME administration. Interestingly, L-NAME significantly reduced myonuclear addition by 75% in non overloaded muscles (SLN vs. S). Muscle hepatocyte growth factor protein content increased with overload, but was unaffected by L-NAME in either loading state. These data indicate that NOS inhibition in rat plantaris muscle attenuates myonuclear addition under basal, but not overloaded, conditions.

Differential action of ACE inhibitors on the eNOS pathway

Background: Clinical trials show that not all ACE inhibitors (ACEIs) improve outcomes in patients with CAD and preserved ventricular function. This could be related to different effects on endothelial function, i.e., endothelial nitric oxide synthase (eNOS) expression and activity. Aim: To compare the effects of various ACEIs on the eNOS pathway. Methods: Rats were orally treated with ACEIs at a dose able to cause a 25% reduction in blood pressure: perindopril 3, trandolapril 1, quinapril 10, ramipril 1 and enalapril 20 mg/kg/day. The eNOS pathway was evaluated in intact aorta and cardiomyocytes (CM). Results: All ACEIs increased eNOS protein expression and activity in the aorta (both p<0.0001 vs. vehicle, for data in table ^p<0.05,*p<0.01,°p<0.001 vs. perindopril) and in CM (both p<0.05 vs. vehicle).

A Moderately High Fat Diet Promotes Salt-Sensitive Hypertension in Obese Zucker Rats by Impairing Nitric Oxide Production

The objective of this research was to examine the contribution of a moderately high fat (MHF) diet to the development of salt-sensitive hypertension in obese Zucker rats. Lean and obese Zucker rats were fed either a MHF diet or a diet of standard rat chow (control diet) for 10 weeks. From week 4 through week 10, the drinking water was supplemented with 1% NaCl. Blood pressure was measured weekly, and urinary excretion of nitric oxide metabolites (NOx) was determined at weeks 4 and 10. At week 10, renal nitric oxide synthase (NOS) activity was assessed in kidney homogenates. Blood pressures of obese, but not lean, rats on the MHF fat diet were significantly increased by salt-supplementation, whereas blood pressures of rats on the control diet were not appreciably affected. NOx excretion was increased in response to salt-supplementation in rats on the control diet, with the effect being particularly dramatic in obese rats. After salt-supplementation, NOx excretion by rats on the MHF diet was lower than rats on the control diet. In obese rats on the MHF diet, this decrease in NO production was accompanied by a reduction in renal NOS activity. These results indicate that obese rats are more inclined than lean rats to develop diet-induced hypertension in response to a moderately high fat, salt-supplemented diet. Furthermore, they suggest that MHF diet-induced defects in NO production may promote the salt-sensitivity of blood pressure in obese Zucker rats, which appear to require more NO to maintain blood pressure during a salt challenge.

No evidence for modulation of endothelial nitric oxide synthase by the olive oil polyphenol hydroxytyrosol in human endothelial cells

Reduced nitric oxide (NO) availability is associated with the development of atherosclerosis. Upregulation of endothelial nitric oxide synthase (eNOS) activity is pursued as a strategy for the prevention of cardiovascular diseases. The polyphenol hydroxytyrosol (HT) which is present in olive oil and red wine, is regarded to be partly responsible for the beneficial effects associated with olive oil consumption and has shown antiatherogenic activity in vitro and in vivo. To elucidate the underlying molecular mechanisms, we investigated possible effects of HT on the endothelial nitric oxide synthase (eNOS). We used human endothelial cells (EA.hy926) and examined eNOS on three different levels, addressing eNOS promoter transactivation, eNOS enzyme activity and nitric oxide availability. Cells were treated with a broad range of HT concentrations (from 10 nM to 100 microM) and for different incubation times (15 min to 24 h). HT did not exert significant positive effects on eNOS in any of our assay systems. Neither did we find evidence for a possible synergism between the red wine polyphenol resveratrol and HT. We conclude that a direct modulation of eNOS is unlikely to account for the antiatherogenic properties of HT under non-inflammatory conditions.

Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity

Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P<0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P<0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.

Dietary conjugated linoleic acid enhances spleen PPAR-γ mRNA expression in broiler chicks

1. The anti-inflammatory effects of dietary conjugated linoleic acid (CLA) on broilers repeatedly challenged with lipopolysaccharide (LPS) were investigated. 2. Day-old broiler chicks were allotted into three treatment groups and fed on a control diet or diets containing 5·0 or 10·0 g CLA/kg diet. Six chicks from each treatment were injected with LPS (0·25 mg/kg body weight) at 16, 18 and 20 d of age. Splenic cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) activities, and prostaglandin E2 (PGE2) and nitric oxide (NO) production as well as peroxisome proliferator-activated receptor-γ (PPAR-γ) mRNA expression were measured at 21 d of age. 3. Chicks fed 10·0 g CLA/kg diet had lower COX activities and PGE2 production than the controls. Dietary CLA (10·0 g/kg) did not significantly diminish LPS-induced enhancement of COX-2 activity, but inhibited the subsequent increase in PGE2 production. 4. Regulation of COX-1 activity contributed to the difference in PGE2 production. 5. CLA did not markedly attenuate the increase of iNOS activity and NO production caused by LPS challenge. Chicks fed CLA had lower iNOS activity and NO production than the controls. 6. Dietary CLA activated splenic PPAR-γ mRNA expression and increased PPAR-γ mRNA expression after LPS injection. 7. These results suggest that dietary CLA has immunomodulatory effects in the spleen by restricting basal PGE2 and NO to lower levels and enhancing PPAR-γ mRNA expression. During the inflammatory response, dietary CLA did not alleviate the increase in COX-2 and iNOS activities but enhanced PPAR-γ mRNA expression.

SKIN NITRIC OXIDE AND ITS METABOLITES ARE INCREASE IN NON-BURNED SKIN AFTER THERMAL INJURIES

Local and systemic inflammation can lead to progression of burn wounds, converting second- to third-degree wounds or extending the burn to adjacent areas. Previous studies have suggested that the skin is an important site of production of nitric oxide (NO), synthesized by inducible nitric oxide synthase (iNOS) activation after injury. NO increases in burned wounds, but its formation in noninjured skin has not been investigated. We hypothesized that after severe burns, NO and cytotoxic peroxynitrite would increase in noninjured skin. We also tested the hypothesis that BBS-2, a specific inhibitor of iNOS, would impair NO formation after burn. Thirteen female sheep were randomized into burn injury and smoke inhalation (n = 5, group 1), burn and smoke treated with BBS-2 (n = 3, group 2), and sham (saline treatment, no injury) (n = 5, group 3). All the animals, including the sham-injury group, were mechanically ventilated for 48 h. Samples of nonburned skin and plasma were collected from each animal, and levels of NO and its metabolites were evaluated using a NO chemiluminescent detector. Nitrotyrosine and iNOS expression were determined in the skin by Immunoperoxidase staining, and scoring of masked slides (epidermis, hair follicles, vessels, glands, and stroma) was performed. Skin NO and metabolites significantly increased in the burn and smoke injury group, and this was inhibited by BBS-2. Nitrotyrosine expression also increased significantly in the skin of burned animals. BBS-2 prevented the increase of NOx but not the increase of nitrotyrosine expression in skin. Plasma levels of NO increased in burned animals when compared with sham, but this increase was not significant. The increase of NO and its metabolites after burn in noninjured skin is followed by a significant increase in peroxynitrite, a potent cytotoxic mediator.

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla. 2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP(1)) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure. 3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [(3)H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET(B) receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP(1) accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET(A) receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET(B) receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP(1) accumulation. 4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET(A) receptor, while the latter through the activation of ET(B) receptor. These results support the role of endothelins in the regulation of adrenal medulla function.

Chemotherapy Influences Inducible Nitric Oxide Synthase (iNOS) and Endothelial Nitric Oxide Synthase (eNOS) Activity on 3D Breast Cancer Cell Line

Multicellular tumor spheroids (MTS) are three-dimensional structural forms of tumors grown in vitro in the laboratory. In this study, the aim was to determine the regulation of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expressions on MTS in response to treatment with the commonly used anti-cancer drugs Doxorubicin and Docetaxel. The spheroids were generated using the "liquid overlay" technique. The distribution of both iNOS and eNOS was detected using indirect immunohistochemistry, while the expression of both iNOS and eNOS was measured using Western blots. Additionally, S-phase analysis using 5-bromo-2'-deoxyuridine (BrdU) was done on the MTS after treatment with doxorubicin, docetaxel, and a combination of the two. The Griess method was used to measure nitric oxide (NO) production in the cells. An increase in iNOS immunoreactivity and a decrease in eNOS immunoreactivity were observed after doxorubicin treatment, when compared with the other groups. Furthermore, upregulation of iNOS and downregulation of eNOS were detected in doxorubicin-treated cells using Western blotting. Insignificant iNOS expression was observed in all of the groups, and it was particularly low in the control and drug combination groups. NO production was also found to be significantly high after docetaxel treatment, and cell proliferation decreased after doxorubicin treatment. In conclusion, chemotherapy influences NOS activity differently with the presence of different drugs. The results with iNOS show that doxorubicin is a more effective drug than docetaxel, and a drug combination may play a helpful role in the suppression of tumorigenicity and cancer metastasis. Interestingly, eNOS expression increased after the addition of both docetaxel and the drug combination, and it was found to negatively correlate with the histological grade of the tumor. Therefore, analyzing the expression of both iNOS and eNOS might be very useful for targeting the treatment of breast carcinoma and obtaining better information on prognosis.

Tetrahydrobiopterin and Endothelial Dysfunction in Cardiovascular Diseases

Abstract Endothelial vasodilator dysfunction is a characteristic feature of patients at risk for coronary atheroscierosis. We have reported that insulin resistance may be a pathogenic factor for endothehal dysfunction through impaired endothelial nitric oxide synthase (eNOS) activity and increased oxidative breakdown of nitric oxide (NO) due to an enhanced fonnation of Superoxide anion, which arc caused by relative deficiency of tetrahydrobiopterin (BH4) in vascular endothelial cclls. Guanosine-triphosphate cyclohydrolase I, the rate-limiting cnzyme in the production of BH4, is decreased in the aorta of insulin-resistant rats and supplementation of BH4 restored the endothelial function and relieved oxidative tissue damage. The BH4 treatment may evoke these benefits not only by providing eNOS with cofactor to enhance NO synthesis, but also by acting as an indirect and/or direct antioxidant to decrease Superoxide anion derived from the endothelium. A further understanding of the physiological and pathological roles and their regulation may lead to new therapeutic avenues.

The Effect of Aminoguanidine on Blood and Tissue Lipid Peroxidation in Jaundiced Rats With Endotoxemia Induced With LPS

Obstructive jaundice (OJ) is a severe condition that leads to several complications. One of the important problems in OJ is the increased incidence of endotoxemia, which is the result of bacterial translocation (BT) and defective host immune response. Lipid peroxidation (LP) is an important problem in OJ and sepsis in which nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity are increased and antioxidative activity is decreased. Formation of peroxynitrite (ONOO−) anion leads to cellular damage and apoptosis. In this experimental study, we explore the effect of specific iNOS inhibitor aminoguanidine (AG) on blood and tissue (liver and renal) LP and iNOS levels in jaundiced rats with endotoxemia induced with lipopolysaccharide (LPS). Rats were randomized into six groups; group A, sham; group B, obstructive jaundice (OJ); group C, OJ + LPS; group D, OJ + AG; group E, OJ + LPS + AG; group F, OJ + AG + LPS. Serum malondialdehyde (MDA) and serum myeloperoxidase (MPO) activity and liver and renal tissue MDA, MPO, and Na+/K+-ATPase activity levels were detected in biochemical methods. Liver and renal tissue iNOS levels were examined immunohistopathologically. Serum and tissue MDA and MPO levels and tissue iNOS expression were increased significantly in groups B, C, and E, while tissue ATPase levels were decreased significantly in the same groups. In the group treated with AG (group D), serum and tissue MDA and MPO levels and tissue iNOS expression were decreased while tissue ATPase levels were increased significantly. In group F, if AG was administrated before LPS, we observed that serum and tissue MDA and MPO levels and tissue iNOS expression were decreased while tissue ATPase levels were increased significantly. Thus, our study showed that AG had a protective effect when it was administrated before LPS, but it failed to prevent tissue iNOS expression and LP if there was established endotoxemia in OJ.

Recurrent early pregnancy loss and endothelial nitric oxide synthase gene polymorphisms

Studies on the relation between endothelial nitric oxide synthase (eNOS) activity in implantation and maintenance of pregnancy highlights the importance of eNOS gene polymorphisms in recurrent early pregnancy loss (REPL). We investigated the relationship between idiopathic REPL and polymorphisms in eNOS among South Indian women. A case-control study comprising 145 females with REPL and 99 control females. The polymorphisms studied include a 27 bp intron 4 repeat, Glu298Asp variation of exon 7 and a novel 140 A --> G polymorphism in intron 6. A polymerase chain reaction-based di-deoxy dye terminator sequencing method was used for genotyping. A novel A --> G polymorphism was identified in intron 6. The more frequent b allele of intron 4 repeat was present at a frequency of 0.84 in cases as compared to 0.86 in controls (O.R 1.17); the G allele of exon 7 coding for the wild-type glutamate containing isoform was present at a frequency of 0.79 in cases and 0.83 in controls (O.R 1.30, CI 0.6-2.8). The intron 6 variant A allele was present at a frequency of 0.58 in cases and 0.45 in controls (O.R 0.59, CI 0.33-1.08). Overall, the polymorphism in intron 6, in homozygous condition, exhibited a significant association to the risk of REPL (O.R 0.43, CI 0.21-0.89), P: 0.021). The present study identifies and validates a novel polymorphism in the eNOS gene which was found associated with the risk of REPL.