eNOS Nitric Oxide Research Articles

Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells

Objectives Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells. Methods We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods. Results L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N G-nitro- l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium. Conclusion NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS–NO–sGC β–cGMP–PKG I pathway.

Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro leishmanicidal activity of an Agaricus blazei Murill mushroom extract as compared to different Leishmania species and stages. The water extract proved to be effective against promastigote and amastigote-like stages of Leishmania amazonensis, L. chagasi, and L. major, with IC 50 (50% inhibitory concentration) values of 67.5, 65.8, and 56.8 μg/mL for promastigotes, and 115.4, 112.3, and 108.4 μg/mL for amastigotes-like respectively. The infectivity of the three Leishmania species before and after treatment with the water extract was analyzed, and it could be observed that 82%, 57%, and 73% of the macrophages were infected with L. amazonensis, L. major, and L. chagasi, respectively. However, when parasites were pre-incubated with the water extract, and later used to infect macrophages, they were able to infect only 12.7%, 24.5%, and 19.7% of the phagocytic cells for L. amazonensis, L. chagasi, and L. major, respectively. In other experiments, macrophages were infected with L. amazonensis, L. chagasi, or L. major, and later treated with the aforementioned extract, presented reductions of 84.4%, 79.6%, and 85.3% in the parasite burden after treatment. A confocal microscopy revealed the loss of the viability of the parasites within the infected macrophages after treatment with the water extract. The applied extract presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O + human red blood cells. No nitric oxide (NO) production, nor inducible nitric oxide syntase expression, could be observed in macrophages after stimulation with the water extract, suggesting that biological activity may be due to direct mechanisms other than macrophage activation by means of NO production. In conclusion, the results demonstrate that the A. blazei Murill water extract can potentially be used as a therapeutic alternative on its own, or in association with other drugs, to treat Visceral and Cutaneous Leishmaniasis.

Sulfur dioxide upregulates the aortic nitric oxide pathway in rats

Sulfur dioxide (SO 2) is a common gaseous pollutant. It is also, however, endogenously generated from sulfur-containing amino acids. Recent studies have demonstrated that rat blood pressure can be lowered by SO 2-exposure in vivo and that vasodilation caused by SO 2 at low concentrations (< 450 μM) is endothelium-dependent in rat aorta. However, effects of SO 2 on nitric oxide synthase (NOS) and nitric oxide (NO) production have not been previously studied in rat aorta. The objective of the present study is to assess the effects of acute (10 min) and prolonged (2 h) stimulation with different concentrations of SO 2 on NO/cGMP pathway in isolated rat aorta. The results show that: (1) the acute and prolonged pretreatments with SO 2 produced an inhibition of vasoconstrictions induced by norepinephrine. (2) SO 2 potentiated activity of endothelial nitric oxide synthase (eNOS), but not of induced NOS (iNOS). (3) SO 2 could increase expression of eNOS gene on the transcription and translation levels in rat aorta. (4) SO 2 enhanced NO formation in aortic tissue. (5) The level of cGMP in rat aorta was increased by SO 2 and no change of cAMP. These findings led to the conclusion: there were acute and prolonged effects of SO 2 on the NO/cGMP signalling pathway; and SO 2 could upregulate the eNOS–NO–cGMP pathway and at least partly by which the SO 2 might cause vasodilation and inhibition to vasoconstriction.

Molecular comparison of WT and F92A Caveolin‐1: Direction towards increasing Nitric Oxide bioavailability

INTRODUCTIONEndothelial dysfunction is characterized by the loss of endothelial cell (EC)‐derived Nitric Oxide (NO) release and increased superoxide anion (O2−). Endothelial NO synthase (eNOS) is the enzyme that synthesizes NO and O2− in physiological and pathological settings, respectively. Caveolin‐1 (Cav‐1) is the major coat protein of caveolae that binds to and inhibits eNOS NO release, and we have shown that Cav‐1 F92 is the amino acid that inhibits NO release (Bernatchez, PNAS 2005). Interestingly, our unpublished observations show that F92A Cav‐1 mutant can INCREASE NO release.OBJECTIVETo determine how F92A Cav‐1 increases NO releaseMETHODSWe performed a biochemical comparison of WT vs F92A Cav‐1 to determine if the F92A mutation affects its activity. We also looked at Cav‐1 F92A substitution on eNOS O2− release.RESULTSWT Cav‐1 (25kDa) targets to caveolae and oligomerizes into high MW homo‐oligomers (350–600 kDa). Interestingly, F92A Cav‐1 enrichment in caveolae and homo‐oligomers are identical to WT Cav‐1, and by GST pulldown assays we show that WT and F92A Cav‐1 bind identically to eNOS. However, we have observed that F92A Cav‐1 can decrease O2− release as compared to WT Cav‐1.CONCLUSIONThese data show that F92A Cav‐1 binds eNOS, prevents WT Cav‐1 inhibition of eNOS and decreases O2− release, and suggest that eNOS‐Cav‐1 interaction is a therapeutic target for improving NO bioavailability.

A kinetic model of endothelial dysfunction from the NOS NO synthesis biochemical pathway

Endothelial nitric oxide synthase (enos) under the influence of shear stress due to blood flow and agonist produces the vasorelaxant nitric oxide (NO). In hyperglycemia, hypertension and hypercholesterolemia, enos uncouples and produces superoxide instead of NO, leading to endothelial dysfunction. One of the key events in the process of endothelial dysfunction involves reduction of enos co‐factor tetrahydrobiopterin (BH4) with respect to dihydrobiopterin (BH2). However, it is not known how this reduction affects NO/superoxide synthesis by enos. In this study, a mathematical kinetic model for biochemical pathways of enos was developed that accounts for both the normal and endothelial dysfunctional state. The effect of the biopterin ratio (BH2:BH4) and kinetic parameters on NO/superoxide synthesis was analyzed. The results predicted that an increase in BH2:BH4 ratio leads to a significant decrease in production of NO and increase in production of superoxide and peroxynitrite. The model also provides a range of kinetic parameters for NOS biochemical pathways over which significant superoxide formation occurs. In addition, superoxide dismutase significantly affects the concentrations of NO, superoxide and peroxynitrite. These results have implications for understanding NOS uncoupling related oxidative stress. Supported by NIH R01 HL084337.

Involvement of Ca2+ in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca2+ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca2+ ([Ca2+]i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca2+]i occurred 30min after gAd treatment, after which [Ca2+]i increased continually and exceeded the initial level. The mitochondrial Ca2+ ([Ca2+]m) detected by Rhod-2 fluorescence started to increase at 6h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca2+]i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca2+]m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.

Significance of pulmonary artery size and blood flow as a predictor of outcome in congenital diaphragmatic hernia

To determine if pulmonary artery size and blood flow have prognostic value in congenital diaphragmatic hernia (CDH). Twenty-eight consecutive left-sided CDH patients treated according to a standard protocol with high frequency oscillatory ventilation (HFOV) + nitric oxide (NO) had right and left pulmonary artery (RPA, LPA) diameters, LPA/RPA diameter (L/R) ratios, and PA blood flows examined by echocardiography (EC) on days 0, 2, and 5 after birth and compared prospectively. Twenty-two patients (78.6%) survived. Of these, 15 required NO (NO-s), and seven did not (non-NO-s). All six patients that died required NO (NO-d). RPA in the NO-d group was significantly smaller than in the NO-s or non-NO-s groups on day 0 (2.90 +/- 0.41 vs. 3.40 +/- 0.49 or 4.01 +/- 0.43; P < 0.01, respectively). LPA in the NO-d group was significantly smaller than in the non-NO-s on day 0 (2.13 +/- 0.45 vs. 3.39 +/- 0.34; P < 0.01). L/R ratios in NO subjects were significantly smaller (NO-s 0.74 +/- 0.11; NO-d 0.73 +/- 0.11) than in non-NO-s subjects (0.84 +/- 0.03) on day 0 (P < 0.01). PA diameters and L/R ratios did not change significantly from day 0 to day 5 in all three groups. There was LPA flow on day 0 in all non-NO-s subjects, but none in all NO subjects. In the NO-s group, LPA flow was confirmed in 87% (13/15) on day 2 and in 100% on day 5, however, there was no LPA flow from day 0 to day 5 in any of the NO-d group. Our data indicate that PA diameters on day 0 and LPA flow are strongly prognostic in left-sided CDH and L/R ratio would appear to be a simple highly reliable indicator of the necessity for NO therapy.

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

PROTECTIVE EFFECT OF FR183998, A Na+/H+ EXCHANGER INHIBITOR, AND ITS INHIBITION OF iNOS INDUCTION IN HEPATIC ISCHEMIA-REPERFUSION INJURY IN RATS

Recent evidence indicates that inhibition of the Na+/H+ exchanger improves heart and brain injuries induced by I/R. Studies were performed to investigate whether FR183998, a Na/H exchanger inhibitor, has protective effects on hepatic I/R injury in rats. Male Sprague-Dawley rats were subjected to 70% hepatic ischemia by occluding the hepatic artery, portal vein, and bile duct associated with the left and median liver lobes with a microvascular clip for 2 h. FR183998 (1 mg/kg) was administered i.v. 10 min before the hepatic ischemia. Hepatic I/R increased the serum levels of aspartate transaminase, alanine transaminase, and lactate dehydrogenase, which peaked at 9 h after reperfusion. FR183998 reduced these injury markers and recovered liver functions. Histopathologic analysis revealed that FR183998 prevented the incidences of hepatic necrosis, apoptosis, and neutrophil infiltration at 6 and 9 h (P < 0.05). FR183998 reduced the increases in proinflammatory cytokines such as TNF-alpha (1-6 h), IL-6 (1-12 h), interferon-gamma (6-12 h), IL-1beta (1-3 h), and cytokine-induced neutrophil chemoattractant 1 (1-3 h), but enhanced the anti-inflammatory cytokine IL-10 (1 h). FR183998 inhibited the hepatic I/R-induced activation of the transcription factor nuclear factor-kappaB at 1 to 6 h and reduced the induction of iNOS at 6 to 12 h, followed by inhibition of nitric oxide production. Furthermore, FR183998 decreased the expression of the iNOS gene antisense transcript, which is involved in the stability of iNOS messenger RNA, at 9 to 12 h in the liver of hepatic I/R rats. These results demonstrate that FR183998 reduces the induction of proinflammatory cytokines and iNOS at least in part through inhibition of nuclear factor-kappaB activation and iNOS antisense transcript expression, thereby preventing hepatic I/R injury.

Nitric oxide, antioxidants, and mitochondria activity for preimplantation development

OBJECTIVE: Nitric oxide (NO) is an important regulator of apoptosis for early embryo development and a documented modulator of mitochondrial activity for several types of somatic cells. This study attempts to elucidate the effect of NO on mitochondrial activity for early cleavage stage embryos. Furthermore, the capability of antioxidants to against the apoptotic effect of NO for preimplantation embryos is also investigated. DESIGN: Prospective, controlled study. MATERIALS AND METHODS: Zygotes from superovulated B6CBF1 female mice were retrieved from excised oviducts and cultured within modified human tubal fluid (mHTF) medium. Sodium nitroprusside (SNP) and NG-nitro-L-arginine (L-NA) were supplemented into the culture medium and functioned as NO donor and NOS inhibitor, respectively. Mitochondrial membrane potential and amount of ATP were measured for monitoring mitochondrial activity. Mn(III) tetrakis(4-benzoic acid) porphyrin choloride (TBAP), ebselen (EBS), and glutathione methyl ester (GSH) were applied into the culture medium to reduce oxidative stress. Apoptosis assay for blastomeres was performed with terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay. The blastocyst formation rate and number of apoptotic cells were recorded as an index of embryo development. RESULTS: Supplementation of SNP impaired embryo development and induced disruption of mitochondrial membrane potential in a dose-dependent manner. Although L-NA also arrested embryo development, L-NA did not break the integrity of mitochondrial membrane potential. The degenerated embryos after NO exposure exhibited apoptosis demonstrated by TUNEL stain. Subsequent to NO exposure, the addition of TBAP (superoxide scavenger) or GSH was able to rescue the embryos. However, the supplementation of TBAP was associated with a decline of blastocyst formation rate. Furthermore, EBS (peroxynitrite scavenger) was not able to rescue the embryos from NO toxicity. CONCLUSIONS: The NO donor exerted deleterious effect on embryo development, which might through disruption of mitochondrial membrane potential and induced mitochondria-dependent apoptosis. In order to counteract this adverse effect, GSH might be a required addictive of culture medium.

Quantitative nitric oxide production by rat, bovine and porcine macrophages

The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-γ) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-γ together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.

Nitric oxide synthesis in neutrophil precursor cells and effect of nitric oxide modulators on their maturation

Neutrophil maturation in bone marrow and their release in circulation have been shown to be modulated by nitric oxide (NO). Present study was undertaken to further assess the role of NO in neutrophil maturation in vitro. Neutrophil precursor cells were isolated from Sprague Dawley rat (200–250 g) bone marrow by Percoll density gradient in myeloblasts/promyelocytes rich band 3, myelocytes/metamyelocytes rich band 2 and band cells/segmented Neutrophils rich band 1, purity was confirmed by Giemsa staining/CD markers, NADPH oxidase and myeloperoxidase activity.Intracellular NO level (assessed by DAF‐2DA), total nitrite (measured by Griess reagent) content, NOS activity (using 3H‐L‐arginine) and nNOS protein (Western blotting) were highest in band 1 and lowest in band 3. We also investigated effect of NO donor (SNP 1μM‐1mM) and NOS inhibitor (L‐NAME 1mM) on cell cycle by using Propidium iodide and BrdU staining during their culture for 24 hrs. SNP treated cells exhibited an increase in apoptosis, while viable cells showed a marginal increase in the S phase in band 2 and band 3. NOS inhibitor (L‐NAME) however increased the survival of cells without any modulation of cell cycle. Results obtained indicate that NO content augment with neutrophil maturation and seems to play an important role in the maturation of neutrophils.The work was supported by CSIR and DBT India.

Similarities Between Ozone Oxidative Preconditioning and Ischemic Preconditioning in Renal Ischemia/Reperfusion Injury

Many studies indicate that the production of reactive oxygen species (ROS) after renal ischemia/reperfusion (I/R) may initiate the cascade of cellular injury. It has been demonstrated that ozone oxidative preconditioning (OzoneOP) may prevent the damage induced by ROS and attenuate renal I/R injury. On the basis of those results, we postulated that OzoneOP was similar to the ischemic preconditioning (IP). The aim of our present work was to assess whether the combination of OzoneOP and IP provided synergistic protection. Seven groups of rats were classified as follows: 1) sham-operated control; 2) I/R; 3) OzoneOP+I/R; 4) IP+I/R; 5) OzoneOP+IP+I/R; 6) O2+I/R; 7) sham-operated control+OzoneOP. Rats were sacrificed at 24 h after I/R injury. Serum and tissue were taken to determine urea nitrogen (BUN), creatinine (Cr), nitric oxide (NO), histological examination, and NO synthase (endothelial, eNOS and inducible, iNOS) expression. Malondialdehyde (MDA) content, glutathione (GSH) content, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity were determined in renal tissue. Renal dysfunction, histological damage, and renal oxidative stress were significantly improved by OzoneOP or IP alone. OzoneOP+IP could not further relieve severe renal damage. Either IP or OzoneOP treatment alone increased NO release and NO synthase (endothelial, eNOS and inducible, iNOS) expression. The combination of OzoneOP and IP could not further enhance NO levels and NOS expression. These findings indicate that both of the preconditioning settings shared similar mechanisms of protection in the parameters measured. However, OzoneOP combined with IP had no synergistic effect. IP and OzoneOP appeared to share a common mediator: NO. These findings suggested the potential role of OzoneOP against renal failure during surgery or transplantation.

FEBB: Fuel Efficiency Buy-Back Program

This Efficiency Program is design to encourage the skilled truck drivers in a way to save precious fuel during their work shift. The main objectives of FEBB are: 1st The transportation and forwarding companies offer an incentive to its drivers that called FEBB program: It means all fuel savings will transformed to driver bonus monthly payment additional to its basic salary. How it is calculate? All engines have technical specification by producer state the average urban or highway fuel consumption. All below those specifications are fuel savings. You simply multiply the fuel saving in litters or gallons by the fuel price at the middle of the month (to monthly eliminate standard deviation of fuel price). How the drivers do savings? - The skilled driver would adjust the vehicle speed not only accelerating the engine but most with switching the gear while the engine is working at its optimum of rotational force (torque); 2nd It is announced as incentives to SKILLED DRIVERS, which predefined that only drivers saving fuel are skilled. That will change driver mentality to more economic and environmental safety manner. Benefits: 1. You can lower fuel consumption in average vary from 1 litter/100kms for engines below 2000cc to 5 litters/100kms for heavy engines over 12,000cc; 2. Environmental more safety; 3. Encourage the drivers not to exceed speed limits because fast driving means higher fuel consumption. Conforming with speeds limits will decrease the road incidents; 4. Engine's and suspension system physical depreciation is lower. The lower depreciation the lower costs of maintenance; 5. More cost-effective routing is chosen by the driver in order to save fuel; 6. In some countries it will reduce the fuel pilferage. Common phenomenon in south-east Europe; 7. Fuel save = Bonus Payment = Motivation. Application: Effective: In countries where the highway network is well developed; Ineffective: In urban environment where a lot of maneuvers are required during transportation. Broad application: Automotive industry: this theory leads to the term Economic Drive Gear System (EDGS). In general it is 12-18 gear automatic transmission system that is working on maximum of moment (torque) in terms of RPM. How it works? - The transmission changes the gear up when the RPM moves up with more than 10% vs. ORPM and the transmission changes the gear down when RPM drops down with more than 10% vs. ORPM. * ORPM = optimum of revolutions per minutes (see Prerequisites for the theory). Prerequisites for the theory (the following facts are well known but fundamental for FEBB theory and could be defined as Axioms): 1. Newton metre is the unit of moment (torque**) of self propelling internal combustion engines. The maximum of moment (torque) for diesel engines is achievable with the range of 1000-2000 rpm that could differ from one producer to another. The best engines are those that have wider range of revolutions per minutes (rpm) while keeping the maximum of the moment (torque). The driver can adjust the speed of the vehicle switching to higher Gear for higher speed through engine acceleration in a range of ORPM and vice versa. Herein we will call this maximum of torque in terms of rpm as ORPM Optimum of Revolutions Per Minutes. ** Torque = rotational force (Nm). 2. While keeping the maximum of moment (torque) in terms of RPM it results in lower depreciation of the engine and better combustion and lower emission of CO and (HC NOx) where NO-Nitric Oxide is toxic air pollutant.Example table of correlation between RPM (r/min), Torque (Nm) and Engine power (kW) is given below.

Arginine vasopressin increases iNOS–NO system activity in cardiac fibroblasts through NF-κB activation and its relation with myocardial fibrosis

Previous studies have shown that arginine vasopressin (AVP) promotes myocardial fibrosis (MF), whereas nitric oxide (NO) inhibits MF. Cardiac fibroblasts (CFs) are the main target cells of MF. However, the modulatory effect of AVP on NO production in CFs and the role of this effect in MF are still unknown. In the present study, CFs obtained from Sprague–Dawley rats were stimulated with or without AVP and pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor kappa-B (NF-κB). NO production and NOS activity were detected with absorption spectrometry, inducible nitric oxide synthase (iNOS) protein with Western blot analysis, iNOS mRNA with real-time PCR, CF collagen synthesis with [ 3H]proline incorporation, and NF-κB activation with immunofluorescence staining and Western blot analysis. The results showed that AVP increased NO production in a dose- and time-dependent manner, with maximal effects at 10 − 7 mol/l after 24-h stimulation. AVP also increased NOS activity, protein and mRNA levels of iNOS in a coincident manner. Furthermore, AVP also increased CF collagen synthesis in a dose- and time-dependent manner. In addition, it was found that NF-κB was activated by AVP, and that PDTC could inhibit NO production, NOS activity, protein and mRNA levels of iNOS stimulated by AVP in a dose-dependent manner. The inhibitory effects of PDTC on NF-κB translocation were coincident with the effects of PDTC on iNOS–NO system activity. It is suggested that AVP increases NO production via the regulation of iNOS gene expression, and the upregulation of iNOS gene expression stimulated by AVP is mediated through NF-κB activation. NO production induced by AVP may counteract the profibrotic effects of AVP, thus the development of MF perhaps depends on the balance between profibrotic AVP and antifibrotic NO effects on MF.

Ethanol Metabolism and Effects: Nitric Oxide and its Interaction

Ethanol (EtOH) in alcoholic beverages is consumed by a large number of individuals and its elimination is primarily by oxidation. The role of nitric oxide (NO) in EtOH's effects is important since NO is one of the most prominent biological factors in mammals. NO is constantly formed endogenously from L-arginine. Dose and length of EtOH exposure, and cell type are the main factors affecting EtOH effects on NO production. Either acute or chronic EtOH ingestion affects inducible NO synthase (iNOS) activity. However it seems that EtOH suppresses induced-NO production by inhibition of iNOS in different cells. On the other hand, it is clear that acute low doses of EtOH increase both the release of NO and endothelial NOS (eNOS) expression, and augment endothelium-mediated vasodilatation, whereas higher doses impair endothelial functions. EtOH selectively affects neuronal NOS (nNOS) activity in different brain cells, which may relate to various behavioral interactions. Therefore, there is an excellent chance for EtOH and NO to react with each other. Effects of EtOH on NO production and NOS activity may be important to EtOH modification of cell or organ function. Nitrosated compounds (alkyl nitrites) are often found as the interaction products, which might be one of the minor pathways of EtOH metabolism. NO also inhibits EtOH metabolizing enzymes. Furthermore, NO is involved in EtOH induced liver damage and has a role in fetal development during EtOH exposure in pregnancy. The mechanisms underlying these effects are only partially understood. Hence, the current discussion of the interaction of EtOH and NO is presented.

THE ROLE OF INITIAL TRAUMA IN THE HOST'S RESPONSE TO INJURY AND HEMORRHAGE

Trauma and hemorrhagic shock (HS) elicit severe physiological disturbances that predispose the victims to subsequent organ dysfunction and death. The general lack of effective therapeutic options for these patients is mainly due to the complex interplay of interacting inflammatory and physiological elements working at multiple levels. Systems biology has emerged as a new paradigm that allows the study of large portions of physiological networks simultaneously. Seeking a better understanding of the interplay among known inflammatory pathways, we constructed a mathematical model encompassing the dynamics of the acute inflammatory response that incorporates the intertwined effects of inflammation and global tissue damage. The model was calibrated using data from C57Bl/6 mice subjected to endotoxemia, sham operation (i.e., surgical trauma induced by cannulation [ST]) or ST + HS+ resuscitation (ST-HS-R). An in silico simulation, made at whole-organism level, suggested that similar pathways of different magnitudes were operant as the degree of total body damage increased. We sought to validate this hypothesis by subjecting mice to HS and comparing the models predictions to circulating markers of inflammation and tissue injury as well as the global transcriptomic response of the liver. C57Bl/6 mice were subjected to ST or ST-HS (without resuscitation). Liver gene expression was assessed using an Affymetrix DNA microarray (GeneChip Mouse Expression Set 430A, Affymetrix, Santa Clara, CA), which contains 22,621 probe sets and effectively interrogates 12,341 mouse genes. The microarray data sets were subjected to hierarchical clustering and pathway analysis. In agreement with model predictions, circulating levels of inflammation/tissue injury markers and the microarray analysis both demonstrated that ST alone accounts for a substantial proportion of the observed phenotypic and genetic/molecular changes versus untreated animals. The addition of HS further increased the magnitude of gene expression, but relatively few additional genes were recruited. Mathematical simulations and DNA microarrays, both systems biology tools, may provide valuable insight into the complex global physiological interactions that occur in response to trauma and hemorrhagic shock.

THE ROLE OF NITRIC OXIDE IN CAERULEIN-INDUCED ACUTE PANCREATITIS IN MICE

Introduction: Nitric oxide (NO) is involved in the physiology of circulation and in pathophysiological conditions such as inflammation. However, the role of NO in experimental acute pancreatitis still remains controversial. The present study investigated the role of nitric oxide in caerulein induced acute pancreatitis in mice. Methods: Acute pancreatitis was induced in adult swiss mice by hourly intraperitoneal injections of caerulein (50 μg/kg/hr) for 10 hours while control mice were injected with saline. The mice were sacrificed at 3, 6 and 10 hours after the first injection to assay for NO production and NOS expression. Nitrate and nitrite (NOx) were detected in plasma, pancreas and lungs as an indicator of nitric oxide level by Griess colorimetric assay. The expression of three NOS isoforms; nNOS or NOS-1, eNOS or NOS-3 and iNOS or NOS-2 were determined by western blot analysis. Results: The plasma NOx level showed a significant increase at 3 hrs (1.6 fold) when compared to the control. The NOx level in pancreas showed a significant increase at 3, 6 and 10 hrs (2.3, 2 and 1.75 fold) respectively, whereas, the NOx level in lungs showed significant increase only at 10 hrs (1.85 fold) when compared to control. The protein expression of iNOS in pancreas was significantly higher at all time points with a peak of four fold increase at 6 hrs. The nNOS expression showed a significant increase at 3 hrs followed by a decline at 6 hrs and 10 hrs respectively. The eNOS expression showed a decreasing trend although it was significant only at 10 hrs when compared to control. Conclusion: These results suggest the differential expression of the various NOS isoforms, with a decrease in eNOS expression and iNOS possibly being the major sustainable contributor to the NO production in the pancreas while nNOS appears to have an early transient effect.

Peroxynitrite production and NOS expression in astrocytes U373MG incubated with lipoproteins from Alzheimer patients

Apolipoprotein E (apo E), a plasma protein involved both in the metabolism of cholesterol and triglycerides, particularly in nervous tissue, has been associated with a higher risk of Alzheimer's disease. It has been shown that apo E increased the production of nitric oxide (NO) from human monocyte-derived macrophages (MDM); this effect could represent an important link between tissue redox balance and inflammation, since inflammation and oxidative stress are involved in chronic neurodegenerative disorders. Moreover, it has been evidenced that an overproduction of NO in the central nervous system (CNS) may play a key role in aging and that the glial cells (microglials cells and probably astrocytes) are able to form consistent amounts of NO through the induction of a nitric oxide synthase (iNOS) isoform so-called inducible or inflammatory. This report was performed in order to elucidate the effects produced by lipoproteins from control subjects, AD patients and first degree relatives (offspring) on human astrocyte cells after a short incubation. Peroxynitrite and NO production and NOS expression in cultured astrocytes were measured. We observed a decreased NO production after incubation with both LDL and HDL and an increased peroxynitrite production. As it concerns NOS expression, densitometric analysis of bands indicated that iNOS protein levels were significantly higher in the cells incubated with both AD lipoproteins and offspring lipoproteins compared to cells incubated with control lipoproteins. These findings suggest the possibility to identify in NO pathway a precocious marker of AD.

Role of tumor necrosis factor-α and nitric oxide in luteolysis in cattle

Although prostaglandin (PG) F 2α is known to be a principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intra-ovarian factors. Tumor necrosis factor-α (TNFα) and its specific receptors are present in the bovine CL with the highest expressions at luteolysis. TNFα in combination with interferon-γ reduced progesterone (P4) secretion, increased PGF 2α and leukotriene C 4 (LTC 4) production, and induced apoptosis of the luteal cells in vitro. Low concentrations of TNFα caused luteolysis, which resulted in a decreased level of P4, and increased levels of PGF 2α, LTC 4 and nitrite/nitrate (stable metabolites of nitric oxide-NO) in the blood. Inhibition of local NO production counteracts spontaneous and PGF 2α-induced luteolysis. Therefore, NO is a likely candidate for the molecule that mediates PGF 2α and TNFα actions during luteolysis. Both PGF 2α and TNFα increase NO concentrations in blood, and stimulate NO synthase expression on protein level in the bovine CL cells. NO stimulates PGF 2α and LTC 4 secretion, inhibits P4 production and reduces the number of viable luteal cells. TNFα and NO induce apoptotic death of the CL by modulating expression of bcl-2 family genes and by stimulating expression and activity of caspase-3. The above findings indicate that TNFα and NO play crucial roles in functional and structural luteolysis in cattle.