iNOS Inhibitor 1400W Research Articles

Abstract A45: Signaling Events Associated with Cytoprotective Induction of Nitric Oxide Synthase in a Photodynamic Therapy Model

Abstract Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and photosensitizer-exciting light to produce reactive oxygen species that lead to tumor eradication. Nitric oxide (NO) produced by tumor or tumor vasculature cells could be pro-carcinogenic by inhibiting apoptosis and/or promoting angiogenesis and tumor growth. We recently showed that photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX in mitochondria of COH-BR1 breast tumor cells strongly upregulated inducible nitric oxide synthase (iNOS) and steady state NO level in these cells. Including the iNOS inhibitor 1400W during photochallenge dramatically enhanced caspase-9 activation and Annexin-assessed apoptotic cell killing while reducing NO production assessed by the intracellular NO probe DAF-2DA. Short hairpin RNA (ShRNA)-based iNOS knockdown (kd) produced similar results, confirming iNOS involvement. An apoptosis-promoting effect of iNOS inhibition was also observed when breast MDA-MB231 and prostate PC-3 cancer cells were subjected to ALA/light stress. We have now examined the signaling events associated with iNOS-mediated hyperresistance of COH-BR1 cells using a combination of immunocytochemistry, western blotting, immunoprecipitaion, and iNOS-kd methods. A soluble guanylyl cyclase inhibitor failed to stimulate ALA/light-provoked apoptosis, ruling out cGMP involvement in stress resistance. ALA/light activated PI3-kinase-dependent signaling via phosphorylation-inactivation of the tumor suppressor PTEN, leading to phosphorylation-activation of pro-survival Akt. Inhibition of PI3K by Wortmannin prevented ALA/light-induced Akt activation as well as iNOS upregulation while enhancing apoptotic photokilling. Moreover, photostress activation of PI3K/Akt was accompanied by a cytosol-to-nucleus translocation of the iNOS transcription factor NF-κB, and an inhibitor of NF-kB activation prevented iNOS induction while stimulating apoptosis. Furthermore, iNOS-kd as well as 1400W treatment resulted in intensified and more prolonged activation of MAP kinases JNK and p38α by ALA/light stress. In addition, photostressed iNOS-kd cells exhibited p53 upregulation and Survivin inactivation/downregulation. The following general mechanism of NO-based cytoprotection is suggested from these and related findings: Photostress activation of PI3K/Akt suppresses pro-apoptotic MAP kinases and p53 while upregulating/activating Survivin via NF-κB-mediated induction of iNOS/NO. This study provides important new insights into photostress-elicited pro-survival signaling that could reduce PDT effectiveness, and suggests iNOS inhibitor-based interventions for counteracting this. (Supported by NIH Grant CA70823 and a grant from the MCW Cancer Center.)

Is preconditioning by oxytocin administration mediated by iNOS and/or mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart?

AimsOxytocin (OXT) pretreatment protects the heart during ischemia–reperfusion injury by activating ATP-dependent potassium (KATP) channels. The aim of the current study was to elucidate the roles of nitric oxide synthaseNOS and myocardial biochemistry in the cardioprotective effects of OXT and ischemic preconditioning (IPC). Main methodsMale New Zealand White anesthetized rabbits (13 groups) were subjected to 30min of occlusion of the left coronary artery and 120min of reperfusion with or without IPC. Key findingsIPC (1cycle), OXT (0.03μg/kg, i.p.) or IPC+OXT yield significant infarct size reductions (21.8±1.5%, 20.5±1.2% and 19.4±1.4%, respectively, versus 38.9±3.5% in the S-CONT group; P<0.01) and antiarrhythmic effects, including VF (0%, 0% and 0%, versus 50% in S-CONT group; P<0.05) sustained VT (13%, 13% and 13%, versus 100% in S-CONT group; P<0.005) and other arrhythmias (25%, 13% and 25%, versus 100% in S-CONT group; P<0.005, P<0.01 and P<0.005, respectively). Atosiban (ATO, a selective OXT receptor antagonist), 5-HD and l-NAME (a nonspecific NOS inhibitor) abolished the beneficial effects of IPC and OXT, suggesting that the benefits are achieved via selective activation of OXT receptors, mitochondrial KATP channels and NO. An iNOS inhibitor (1400W) blocked the beneficial effects of IPC but not OXT. The IPC, OXT, IPC+OXT and 1400W+OXT interventions significantly preserved ATP levels in the heart. SignificanceThis study demonstrates similarities between acute OXT pretreatment and IPC in terms of infarct size reduction, antiarrhythmic activity, and metabolic status.

Interplay between NO, prostaglandins and KATP channels in the lymphatic pumping dysfunction observed during experimental intestinal inflammation

Lymphatic vessels rhythmically contract to actively propel lymph. This function, critical to tissue fluid homeostasis and immune cell trafficking is impaired in Crohn's disease and in the guinea‐pig model of TNBS ileitis, suggesting compromised lymph flow and lymphatic vessels potential players in the disease. Nitric oxide (NO) and prostaglandins (PGs), abundantly produced during intestinal inflammation, are known to activate ATP‐sensitive potassium (KATP) channels expressed in lymphatic muscle. Our aim was to assess the role of KATP channels, NO and PGs in inflammation‐induced lymphatic pumping dysfunction.After induction of intestinal inflammation, mesenteric lymphatics were collected for qPCR analysis, or isolated to assess their contractile and electrophysiological properties.Expression of mRNA for KATP channel subunits Kir6.1 and SUR2B, nitric oxide synthase iNOS and cyclooxygenase COX2 was significantly upregulated in lymphatics from TNBS‐compared to sham‐treated animals. Vessels from TNBS‐treated animals were dilated, quiescent, with strongly hyperpolarized muscle. Administration of the KATP blocker glibenclamide, iNOS inhibitor 1400W and COX inhibitor indomethacin restored pumping and depolarized lymphatic muscle.Our findings suggest that NO and PGs mediate lymphatic contractile dysfunction seen in TNBS‐treated animals via KATP channel opening.Supported by CIHR

Lipopolysaccharide inhibits ghrelin-excited neurons of the arcuate nucleus and reduces food intake via central nitric oxide signaling

Lipopolysaccharide (LPS) induces anorexia and expression of inducible nitric oxide synthase (iNOS) in the hypothalamic arcuate nucleus (Arc). Peripheral administration of the iNOS inhibitor 1400 W counteracts the anorectic effects of LPS. Here we investigated the role of central NO signaling in LPS anorexia. In electrophysiological studies we tested whether 1400 W counteracts the iNOS-dependent inhibition of Arc neurons triggered by in vivo or in vitro stimulation with LPS. We used the hormone ghrelin as a functional reference stimulus because ghrelin is known to activate orexigenic Arc neurons. Further, we investigated whether in vitro LPS stimulation induces an iNOS-mediated formation of the second messenger cGMP. Since the STAT1 pathway contributes to the regulation of iNOS expression we investigated whether LPS treatment induces STAT1 phosphorylation in the Arc. Finally we tested the effect of intracerebroventricular injection of 1400 W on LPS-induced anorexia. Superfusion with 1400 W (10(-4) M) increased neuronal activity in 37% of neurons in Arc slices from LPS treated (100 μg/kg ip) but not from saline treated rats. Similarly, 1400 W excited 45% of Arc neurons after in vitro stimulation with LPS (100 ng/ml). In both approaches, a considerable percentage of 1400 W sensitive neurons were excited by ghrelin (10(-8)M; 50% and 75%, respectively). In vitro stimulation with LPS induced cGMP formation in the Arc, which was blocked by co-incubation with 1400 W. LPS treatment elicited a pSTAT1 response in the Arc of mice. Central 1400 W injection (4 μg/rat) attenuated LPS-induced anorexia and counteracted the LPS-dependent decrease in respiratory quotient and energy expenditure. In conclusion, the current findings substantiate a role of central iNOS dependent NO formation in LPS-induced effects on eating and energy homeostasis. A pharmacological blockade of NO formation might be a therapeutic approach to ameliorate disease-related anorexia.

Ouabain increases iNOS-dependent nitric oxide generation which contributes to the hypertrophic effect of the glycoside: possible role of peroxynitrite formation

In addition to inotropic effects, cardiac glycosides exert deleterious effects on the heart which limit their use for cardiac therapeutics. In this study, we determined the possible contribution of ouabain-induced iNOS stimulation to the resultant hypertrophic as well as cytotoxic effects of the glycoside on cultured adult rat ventricular myocytes. Myocytes were treated with ouabain (50 μM) for up to 24 h. Ouabain significantly increased gene and protein levels of inducible nitric oxide synthase (iNOS) which was associated with significantly increased release of NO from myocytes as well as increased total release of reactive oxygen species (ROS), superoxide anion (O(2) (-)), and increased peroxynitrite formation as assessed by protein tyrosine nitration. Administration of ouabain was also associated with increased levels of myocyte toxicity as determined by myocyte morphology, trypan blue staining and lactate dehydrogenase (LDH) efflux. The nonspecific NOS inhibitor Nω-nitro-L: -arginine methyl ester and the more selective iNOS inhibitor 1400W both abrogated the increase in LDH release but had no significant effect on either morphology or trypan blue staining. Ouabain also significantly increased both myocyte surface area and expression of atrial natriuretic peptide indicating a hypertrophic response with both parameters being completely prevented by NOS inhibition. The effects of iNOS inhibitors were associated with diminished ouabain tyrosine nitration as well as abrogation of ouabain-induced p38 and ERK phosphorylation. Our study shows that ouabain is a potent inducer of NO formation, iNOS upregulation, and increased production of ROS. Inhibition of ouabain-dependent peroxynitrite formation may contribute to the antihypertrophic effect of iNOS inhibition possibly by preventing downstream MAPK activation.

Pulmonary arterial dysfunction in insulin resistant obese Zucker rats

BackgroundInsulin resistance and obesity are strongly associated with systemic cardiovascular diseases. Recent reports have also suggested a link between insulin resistance with pulmonary arterial hypertension. The aim of this study was to analyze pulmonary vascular function in the insulin resistant obese Zucker rat.MethodsLarge and small pulmonary arteries from obese Zucker rat and their lean counterparts were mounted for isometric tension recording. mRNA and protein expression was measured by RT-PCR or Western blot, respectively. KV currents were recorded in isolated pulmonary artery smooth muscle cells using the patch clamp technique.ResultsRight ventricular wall thickness was similar in obese and lean Zucker rats. Lung BMPR2, KV1.5 and 5-HT2A receptor mRNA and protein expression and KV current density were also similar in the two rat strains. In conductance and resistance pulmonary arteries, the similar relaxant responses to acetylcholine and nitroprusside and unchanged lung eNOS expression revealed a preserved endothelial function. However, in resistance (but not in conductance) pulmonary arteries from obese rats a reduced response to several vasoconstrictor agents (hypoxia, phenylephrine and 5-HT) was observed. The hyporesponsiveness to vasoconstrictors was reversed by L-NAME and prevented by the iNOS inhibitor 1400W.ConclusionsIn contrast to rat models of type 1 diabetes or other mice models of insulin resistance, the obese Zucker rats did not show any of the characteristic features of pulmonary hypertension but rather a reduced vasoconstrictor response which could be prevented by inhibition of iNOS.

NPM-ALK Oncogene Upregulates the Expression of Inducible Nitric Oxide Synthase in T-Cell Lymphoma Through STAT3/MicroRNA-26a-Dependent Mechanism

Abstract 1383The oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma tumors. NPM-ALK promotes cellular survival and transformation, and initiates lymphomagenesis. To induce its effects, NPM-ALK interacts with a comprehensive network of oncogenes and tumor suppressor genes. Previous studies have identified upstream and downstream members of this network that modulate the effects of NPM-ALK. However, the exact mechanisms by which NPM-ALK induces its effects are not completely identified.Nitric oxide (NO) is a gaseous molecule and a highly active free radical. It plays critical, yet versatile, roles in physiological cellular functions including survival, adhesion, migration, and angiogenesis. It also has important contributions to tumor progression and metastasis. NO is generated from L-arginine via 3 distinct isoforms of the enzyme NO synthase including the inducible form (iNOS). The expression and role of iNOS in NPM-ALK-expressing T-cell lymphoma is not known.We found that iNOS mRNA and protein are expressed in the NPM-ALK-expressing T-cell lymphoma cell lines Karpas 299, DEL, and SR-786. In agreement with a functional role of iNOS in these cells, the selective iNOS inhibitor 1400W and the NO scavenger CPITO decreased the proliferation of the NPM-ALK-expressing T-cell lymphoma cell lines (p < 0.05). In addition, the NO donor SNAP recovered the decrease in the proliferation of these cell lines after treatment with the ALK inhibitors TAE684 and PF-2341066 (p < 0.05). Interestingly, iNOS protein was totally absent in normal human T lymphocytes in spite of a notable increase in iNOS mRNA. These observations suggested that the downregulation of iNOS protein in T lymphocytes vs. its high levels in the NPM-ALK-expressing T-cell lymphoma cell lines might occur posttranscriptionally. Because microRNA are major posttranscriptional regulators of protein expression, we set to analyze possible aberrancies in microRNA. First, we performed an array study comparing the expression of microRNA in T lymphocytes vs. NPM-ALK-expressing T cell lymphoma cell lines. Three web-based algorithms [TargetScan (http://genes.mit.edu/targetscan/), miRanda (http://www.microrna.org/microrna/home.do), and PicTar (http://pictar.mdc-berlin.de/] identified microRNA-26a (miR-26a) to potentially bind with iNOS 3’-UTR. Importantly, statistical analysis of the array data showed that the expression of miR-26a is much more pronounced in T lymphocytes in comparison with the lymphoma cell lines (p < 0.00001), and real-time qPCR further confirmed these results. To examine the functional interactions between miR-26a and iNOS 3’-UTR, we performed a luciferase assay in 293T cells after transfection with iNOS 3’-UTR reporter gene. Wild type miR-26a, and not mutated miR-26a, induced a marked decrease in the luciferase activity of iNOS 3’-UTR reporter gene (p < 0.05). We then questioned whether NPM-ALK underlies the aberrancies in the expression of miR-26a and iNOS. Specific targeting of NPM-ALK by siRNA increased miR-26a and simultaneously decreased iNOS protein levels in the lymphoma cell lines. Next, we reasoned to examine how NPM-ALK induces its effects on this system. A transcription factor screening showed that miR-26a gene promoter, which is located within the promoter of a host gene, CTDSPL, contains 2 consensus sequences where it can potentially bind with STAT3, a major downstream target of NPM-ALK. Chromatin immunoprecipitation studies illustrated the binding between STAT3 and CTDSPL promoter. To analyze the functional interactions between STAT3 and miR-26a, a luciferase assay performed in NPM-ALK-expressing T-cell lymphoma cell lines showed that the downregulation of STAT3 by siRNA increased CTDSPL promoter activity. In addition, downregulation of STAT3 protein or the decrease in STAT3 phosphorylation by STAT3 or NPM-ALK siRNA, respectively, increased miR-26a levels and decreased iNOS protein expression in NPM-ALK-expressing T-cell lymphoma cell lines.Our study proposes novel mechanisms by which NPM-ALK contributes to the survival of T-cell lymphoma. We are currently analyzing the expression of miR-26a and iNOS in primary human lymphoma tumors, and examining the effects of transfecting NPM-ALK-expressing T-cell lymphoma cell lines with miR-26a on cell proliferation, adhesion, and migration. Disclosures:No relevant conflicts of interest to declare.

Modulation of the myogenic response in renal blood flow autoregulation by NO depends on endothelial nitric oxide synthase (eNOS), but not neuronal or inducible NOS

Nitric oxide (NO) blunts the myogenic response (MR) in renal blood flow (RBF) autoregulation. We sought to clarify the roles of NO synthase (NOS) isoforms, i.e. neuronal NOS (nNOS) from macula densa, endothelial NOS (eNOS) from the endothelium, and inducible NOS (iNOS) from smooth muscle or mesangium. RBF autoregulation was studied in rats and knockout (ko) mice in response to a rapid rise in renal artery pressure (RAP). The autoregulatory rise in renal vascular resistance within the first 6 s was interpreted as MR, from ∼6 to ∼30 s as tubuloglomerular feedback (TGF), and ∼30 to ∼100 s as the third regulatory mechanism. In rats, the nNOS inhibitor SMTC did not significantly affect MR (67 ± 4 vs. 57 ± 4 units). Inhibition of all NOS isoforms by l-NAME in the same animals markedly augmented MR to 78 ± 4 units. The same was found when SMTC was combined with angiotensin II to reproduce the hypertension and vasoconstriction seen with l-NAME (58 ± 3 vs. 54 ± 7 units, l-NAME 81 ± 2 units), or when SMTC was replaced by the nNOS inhibitor NPA (57 ± 5 vs. 56 ± 7 units, l-NAME 79 ± 4 units) or by the iNOS inhibitor 1400W (50 ± 1 vs. 55 ± 4 units, l-NAME 81 ± 3 units). nNOS-ko mice showed the same autoregulation as wild-types (MR 36 ± 4 vs. 38 ± 3 units) and the same response to l-NAME (111 ± 9 vs. 114 ± 10 units). eNOS-ko had similar autoregulation as wild-types (44 ± 8 vs. 33 ± 4 units), but failed to respond to l-NAME (37 ± 7 vs. 78 ± 16 units). We conclude that the attenuating effect of NO on MR depends on eNOS, but not on nNOS or iNOS. In eNOS-ko mice MR is depressed by NO-independent means.

Akt2 knockout mitigates chronic iNOS inhibition-induced cardiomyocyte atrophy and contractile dysfunction despite persistent insulin resistance

Increased levels of inducible nitric oxide synthase (iNOS) during cardiac stress such as ischemia–reperfusion, sepsis and hypertension may display both beneficial and detrimental roles in cardiac contractile performance. However, the precise role of iNOS in the maintenance of cardiac contractile function remains elusive. This study was designed to determine the impact of chronic iNOS inhibition on cardiac contractile function and the underlying mechanism involved with a special focus on the NO downstream signaling molecule Akt. Male C57 or Akt2 knockout [Akt2(−/−)] mice were injected with the specific iNOS inhibitor 1400W (2mg/kg/d) or saline for 7 days. Both 1400W and Akt2 knockout dampened glucose and insulin tolerance without additive effects. Treatment of 1400W decreased heart and liver weights as well as cardiomyocyte cross-sectional area in C57 but not Akt2 knockout mice. 1400W but not Akt2 knockout compromised cardiomyocyte mechanical properties including decreased peak shortening and maximal velocity of shortening/relengthening, prolonged relengthening duration, reduced intracellular Ca2+ release and decay rate, the effects of which were ablated or attenuated by Akt2 knockout. Akt2 knockout but not 1400W increased the levels of intracellular Ca2+ regulatory proteins including SERCA2a and phospholamban phosphorylation. 1400W reduced the level of anti-apoptotic protein Bcl-2, the effect of which was unaffected by Akt2 knockout. Neither 1400W nor Akt2 knockout significantly affected ER stress, autophagy, the post-insulin receptor signaling Akt, GSK3β and AMPK, as well as the stress signaling IκB, JNK, ERK and p38 with the exception of elevated IκB phosphorylation with jointed effect of 1400W and Akt2 knockout. Taken together, these data indicated that an essential role of iNOS in the maintenance of cardiac morphology and function possibly through an Akt2-dependent mechanism.

Atorvastatin Prevents Vascular Hyporeactivity to Norepinephrine in Sepsis

Hyporeactivity to vasoconstrictors is one of the clinical manifestations of sepsis in man and experimental animals. The objective of the investigation was to examine whether atorvastatin can prevent hyporeactivity to norepinephrine (NE) in mouse aorta in sepsis, and if so, what are the mechanisms involved. Sepsis in mice was induced by cecal ligation and puncture. The aorta was harvested for tension experiment, nitric oxide (NO) and cyclic guanosine monophosphate measurements, and inducible NO synthase (iNOS) and α(1D)-adrenoceptor mRNA expression studies. In comparison with sham-operated controls, sepsis significantly decreased the contractile response to NE in the mouse aorta. Pretreatment with atorvastatin of septic animals completely restored NE-induced contractions to levels similar to those of sham-operated controls and significantly increased survival time and mean arterial pressure. Atorvastatin also attenuated iNOS-induced overproduction of NO, as well as iNOS mRNA expression. Accordingly, hyporeactivity to NE was not evident in tissues pretreated with selective iNOS inhibitor 1400W in sepsis. Although basal cyclic guanosine monophosphate accumulation in the aorta was reduced in sepsis, pretreatment of the tissues with soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) partially restored the reactivity to NE. Interestingly, hyporeactivity to NE in sepsis was associated with a decreased α(1D)-adrenoceptor mRNA expression in the mouse aorta. Atorvastatin pretreatment, however, prevented the decrease in α(1D)-adrenoceptor mRNA expression in septic animals. In conclusion, atorvastatin seems to prevent hyporeactivity to vasoconstrictor NE in the aorta from septic mice through attenuation of overproduction of NO as well as improved α(1D)-adrenoceptor mRNA expression. The findings of the present study may explain the beneficial effects of atorvastatin on improved hemodynamic functions in sepsis.

Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1–iNOS–CD36 pathway

Obesity due to nutrient excess leads to chronic pathologies including type 2 diabetes and cardiovascular disease. Related to nutrient excess, FoxO1 has a role in regulating fatty acid uptake and oxidation and triglyceride (TG) storage by mechanisms that are largely unresolved. We examined the mechanism behind palmitate (PA)-induced TG accumulation in cardiomyocytes. To mimic lipid excess, rat ventricular myocytes were incubated with albumin-bound PA (1mM) or rats were administered Intralipid (20%). PA-treated cardiomyocytes showed a substantial increase in TG accumulation, accompanied by amplification of nuclear migration of phospho-p38 and FoxO1, iNOS induction, and translocation of CD36 to the plasma membrane. PA also increased Cdc42 protein and its tyrosine nitration, thereby rearranging the cytoskeleton and facilitating CD36 translocation. These effects were duplicated by TNF-α and reversed by the iNOS inhibitor 1400W. PA increased the nuclear interaction between FoxO1 and NF-κB, reduced the nuclear presence of PGC-1α, and downregulated expression of oxidative phosphorylation proteins. In vivo a robust increase in cardiac TGs after Intralipid administration was also associated with augmentation of nuclear FoxO1 and iNOS expression. Impeding this FoxO1–iNOS–CD36 pathway could decrease cardiac lipid accumulation and oxidative/nitrosative stress and help ameliorate the cardiovascular complications associated with obesity and diabetes.

Toll-like Receptor 4 Signaling Confers Cardiac Protection against Ischemic Injury via Inducible Nitric Oxide Synthase- and Soluble Guanylate Cyclase-dependent Mechanisms

Prior administration of a small dose of lipopolysaccharide confers a cardiac protection against ischemia-reperfusion injury. However, the signaling mechanisms that control the protection are incompletely understood. We tested the hypothesis that Toll-like receptor 4 (TLR4) mediates the ability of lipopolysaccharide to protect against cardiac ischemia-reperfusion injury through distinct intracellular pathways involving myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein-inducing interferon-β-mediated transcription factor (Trif), inducible nitric oxide synthase (iNOS), and soluble guanylate cyclase (sGC). Wild-type mice and genetically modified mice, that is TLR4-deficient (TLR4(-def)), TLR2 knockout (TLR2(-/-)), MyD88(-/-), Trif(-/-), iNOS(-/-), and sGCα1(-/-), were treated with normal saline or 0.1 mg/kg lipopolysaccharide intraperitoneally. Twenty-four hours later, isolated hearts were perfused in a Langendorff apparatus and subsequently subjected to 30 min global ischemia and reperfusion for as long as 60 min. Left ventricular function and myocardial infarction sizes were examined. Compared with saline-treated mice, lipopolysaccharide-treated mice had markedly improved left ventricular developed pressure and dP/dt(max) (P < 0.01) and reduced myocardial infarction sizes (37.2 ± 3.4% vs. 19.8 ± 4.9%, P < 0.01) after ischemia-reperfusion. The cardiac protective effect of lipopolysaccharide was abolished in the TLR4(-def) and MyD88(-/-) mice but remained intact in TLR2(-/-) or Trif(-/-) mice. iNOS(-/-) mice or wild-type mice treated with the iNOS inhibitor 1400W failed to respond to the TLR4-induced nitric oxide production and were not protected by the lipopolysaccharide preconditioning. Although sGCα(1)(-/-) mice had robust nitric oxide production in response to lipopolysaccharide, they were not protected by the TLR4-elicited cardiac protection. TLR4 activation confers a potent cardiac protection against ischemia-reperfusion injury via a MyD88-dependent, but Trif-independent, mechanism. iNOS/sGC are essential for the TLR4-induced cardiac protection.

Insulin resistance and liver microcirculation in a rat model of early NAFLD

Insulin contributes to vascular homeostasis in peripheral circulation, but the effects of insulin in liver microvasculature have never been explored. The aim of this study was to assess the vascular effects of insulin in the healthy and fatty liver. Wistar rats were fed a control or a high fat diet (HFD) for 3days, while treated with a placebo, the insulin-sensitizer metformin, or the iNOS inhibitor 1400W. Vascular responses to insulin were evaluated in the isolated liver perfusion model. Insulin sensitivity at the sinusoidal endothelium was tested by endothelium-dependent vasodilation in response to acetylcholine in the presence or absence of insulin and by the level of liver P-eNOS after an insulin injection. Rats from the HFD groups developed liver steatosis. Livers from the control group showed a dose-dependent hepatic vasodilation in response to insulin, which was blunted in livers from HFD groups. Metformin restored liver vascular insulin-sensitivity. Pre-treatment with insulin enhanced endothelium-dependent vasodilation of the hepatic vasculature and induced hepatic eNOS phosphorylation in control rats but not in HFD rats. Treatment with metformin or 1400W restored the capacity of insulin to enhance endothelium dependent vasodilation and insulin induced eNOS phosphorylation in HFD rats. The administration of a HFD induces insulin resistance in the liver sinusoidal endothelium, which is mediated, at least in part, through iNOS upregulation and can be prevented by the administration of metformin. Insulin resistance at the hepatic vasculature can be detected earlier than inflammation or any other sign of advanced NALFD.

The protective effect of enteric glial cells on intestinal epithelial barrier function is enhanced by inhibiting inducible nitric oxide synthase activity under lipopolysaccharide stimulation

Enteric glial cells (EGC) play an essential role in maintaining the integrity of intestinal epithelial barrier (IEB). However, the mechanism of EGCs in the regulation of IEB functions under lipopolysaccharide (LPS) stimulation is unknown. To investigate the barrier-related role of EGCs in response to the LPS challenge, the coculture model of EGCs and intestinal epithelial cells (IEC) IEC-6 was established in vitro. Transepithelial resistance (TER) measurements showed that, LPS treatment significantly increased barrier permeability of IEC monolayer from the basolateral side (35.4±6.3Ω/cm2, p<0.05) but not the apical side (69.7±6.3Ω/cm2) when compared with the control group (81.8±10.9Ω/cm2). The assessment of intestinal epithelial integrity by TER reading and by measuring expression of tight junction protein revealed that, incubation with EGCs or EGC conditioned media significantly increased the TER of IEC monolayers under normal condition as well as the LPS stimulation, accompanied with upregulating zonula occludens-1 and occludin expression at mRNA and protein levels. Real-time quantitative polymerase chain reaction and nitric production assay demonstrated that LPS exposure elicited a maximally 13-fold increase of inducible nitric oxide synthase (iNOS) mRNA expression and 10-fold increase of nitric oxide production of EGCs. After being pretreated with the selective iNOS inhibitor 1400W, EGCs significantly increased the TER of IEC monolayers against the disruption effect of LPS (p<0.05). These findings suggest that EGCs play an important role in maintaining the IEB function in response to the LPS stimulation. The protective effect of EGCs on IEB functions could be enhanced by inhibiting the increase of iNOS activity induced by LPS.

Postconditioning attenuates myocardial injury by reducing nitro-oxidative stress in vivo in rats and in humans

In the present study, we hypothesized that postcon (postconditioning) confers cardioprotection in vivo by reducing the production of ONOO- (peroxynitrite) and nitro-oxidative stress subsequent to the inhibition of the iNOS (inducible NO synthase). Patients with AMI (acute myocardial infarct) were randomly assigned to undergo percutaneous coronary intervention without (control) or with ischaemic postcon by three episodes of 30-s inflation and 30-s deflation of the angioplasty balloon. Animal models of MI/R (myocardial ischaemia/reperfusion) injury were induced in rats by occluding the left coronary artery for 40min followed by 4-h reperfusion. Rats were randomized to receive vehicle, postcon (three cycles of 10-s reperfusion and 10-s coronary re-occlusion preceding full reperfusion), the selective iNOS inhibitor 1400W or postcon plus 3-morpholinosydnonimine (an ONOO- donor). Postcon in patients reduced iNOS activity in white blood cells, decreased plasma nitrotyrosine, a fingerprint of ONOO- and an index of nitro-oxidative stress, and improved cardiac function (P<0.01 compared with control). In rats, postcon reduced post-ischaemic myocardial iNOS activity and nitrotyrosine formation, reduced myocardial infarct size (all P<0.05 compared with control) and improved cardiac function. Administration of 1400W resembled, whereas 3-morpholinosydnonimine abolished, the effects of postcon. In conclusion, reduction in ONOO--induced nitro-oxidative stress subsequent to the inhibition of iNOS represents a major mechanism whereby postcon confers cardioprotection in vivo.

Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung

Nitric oxide (NO•) from inducible NO• synthase (iNOS) has been reported to either protect against, or contribute to, hypoxia/re-oxygenation lung injury. The present work aimed to clarify this double role in the hypoxic lung. With this objective, a follow-up study was made in Wistar rats submitted to hypoxia/re-oxygenation (hypoxia for 30 min; re-oxygenation of 0 h, 48 h, and 5 days), with or without prior treatment with the selective iNOS inhibitor 1400W (10 mg/kg). NO• levels (NOx), lipid peroxidation, apoptosis, and protein nitration were analysed. This is the first time-course study which investigates the effects of 1400W during hypoxia/re-oxygenation in the rat lung. The results showed that the administration of 1400W lowered NOx levels in all the experimental groups. In addition, lipid peroxidation, the percentage of apoptotic cells, and nitrated protein expression fell in the late post-hypoxia period (48 h and 5 days). Our results reveal that the inhibition of iNOS in the hypoxic lung reduced the damage observed before the treatment with 1400W, suggesting that iNOS-derived NO• may exert a negative effect on this organ during hypoxia/re-oxygenation. These findings are notable, since they indicate that any therapeutic strategy aimed at controlling excess generation of NO• from iNOS may be useful in alleviating NO•-mediated adverse effects in hypoxic lungs.

Resveratrol improves left ventricular diastolic relaxation in type 2 diabetes by inhibiting oxidative/nitrative stress: in vivo demonstration with magnetic resonance imaging

Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control (m-Lepr(db)) mice and type 2 diabetic (Lepr(db)) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Lepr(db) mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O(2)(·-) production by inhibiting NAD(P)H oxidase activity and gp91(phox) mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Lepr(db) mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Lepr(db) mice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Lepr(db) mice. Both Lepr(db) mice null for TNF-α (db(TNF-)/db(TNF-) mice) and Lepr(db) mice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Lepr(db) mice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes.

Systemic use of selective iNOS inhibitor 1400W or non-selective NOS inhibitor l-NAME differently affects systemic nitric oxide formation after oral Porphyromonas gingivalis inoculation in mice

Objective Nitric oxide synthase (NOS) inhibitors are reported to protect against the local tissue damage in gingivitis and periodontal disease by reducing nitroxidative stress during inflammation, but their systemic effects are not well investigated. Design NOS inhibitors systemic effects were investigated in a murine chronic oral inoculation model using live Porphyromonas gingivalis ATCC 33277 (0.3 ml; 10 9 cfu/ml) or sterile broth (0.3 ml). Organ nitric oxide (NO) and plasma nitrite/nitrate (NOx) were determined in mice treated with non-selective NOS inhibitor l-NAME (50 mg/kg/24 h i.p.) or selective iNOS inhibitor 1400W (10 mg/kg/6 h i.p.) for the last 5 days of the experiment. Differences between groups were evaluated by nonparametric Wilcoxon's rank-sum one-sided two-sample test and the results compared to those obtained from sham-treated (sterile broth) sham-inoculated animals (water for injection i.p./6 h). Results Repeated ingestion of P. gingivalis resulted in generalized production of NO in organs and NOx in plasma, the levels of both typically being reduced in P. gingivalis-inoculated-1400W-treated mice, whilst the use of l-NAME was largerly ineffective, even promoting NO/NOx formation. Application of either inhibitor to sham-inoculated animals enhanced NO/NOx formation, due only in part to the repeated i.p. injections. Conclusions The systemic use of 1400W or l-NAME differently affects systemic nitric oxide formation in mice orally challenged with P. gingivalis, but the sequelae of such an intervention should be evaluated further.

Abstract 4105: Nitric-oxide-mediated inhibition of Na+/H+ exchange (NHE) is a novel mechanism of bile acid induced damage: Relevance to esophageal carcinogenesis

Abstract Background: Barrett's esophagus (BE) is a premalignant condition that is associated with gastroesophageal reflux disease (GERD). Gastric acid and bile acids are two components of refluxate that induce proliferation, DNA damage and oxidative stress. In combination, the effects of gastric acid and bile acids are amplified and synergistic. The major goal of this study was to evaluate the effect of bile acids alone or in combination with acid on intracellular pH (pHi). A mechanism responsible for bile acid-induced pHi alterations was examined. Methods: CP-A cells (non-dysplastic Barrett's esophagus cells) were treated with medium acidified to pH 5.5 and/or 0.5mM bile acid cocktail in the presence/absence of inhibitors of nitric oxide synthase (NOS) or NHE (sodium-hydrogen exchanger). pHi was measured by BCECF microfluorimetry in perfused cells, nitric oxide by DAF-FM fluorescence and DNA damage by the comet assay. Western blot analysis was used to detect the expression of phosphorylated (activated) eNOS, iNOS and NHE1. Results: A dose-dependent decrease in pHi was observed in CP-A cells exposed to bile acids. Bile acid-induced intracellular acidification was abolished when NO production was inhibited by a nonspecific NOS inhibitor L-NAME or by thea specific iNOS inhibitor 1400W. In contrast the NHE inhibitor DMA, significantly decreased the bile acid-mediated pHi response. Exposure of the cells to acidified medium (pH5.5) containing a 0.5mM bile acid cocktail, markedly decreased pHi pHi to a value below extracellular pH. Importantly, medium acidified to pH5.5 containing 0.5mM bile acids induced a similar decrease in pHi as medium acidified to pH 4.5 without bile acids. This The same treatment resulteds in a 250% increase in acid-mediated DNA damage compared to medium acidified to pH5.5 in CP-A cells. Conclusion: In summary, a new mechanism of bile acid induced damage was identified. Bile acids alone or in combination with acidic pH significantly decrease intracellular pH in esophageal cells. The results suggest the effect ofresponse tpo bile acids is the consequence of NOS activation and that leads to NHE inhibition. The mMarked increase in DNA damage after suggests exposure to bile acids in combination with acid may play a crucial role in esophageal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4105.

The release of microparticles by RAW 264.7 macrophage cells stimulated with TLR ligands

MPs are small membrane-bound particles that originate from activated and dying cells and mediate intercellular communication. Once released from cells, MPs can serve as novel signaling elements in innate immunity, with levels elevated in immune-mediated diseases. This study tested the hypothesis that TLR stimulation can induce MP release by macrophages. In these experiments, using the RAW 264.7 murine macrophage cell line as a model, LPS, a TLR4 ligand, and poly(I:C), a TLR3 ligand, induced MP release effectively, as measured by flow cytometry; in contrast, a CpG oligonucleotide, which can stimulate TLR9, induced much lower levels of particle release. To determine the role of other mediators in this response, the effects of NO were tested. Thus, MP release from RAW 264.7 cells stimulated by LPS or poly(I:C) correlated with NO production, and treatment with the iNOS inhibitor 1400W decreased particle release and NO production. Furthermore, treatment of RAW 264.7 cells with NO donors induced MP production. As TLR ligands can induce apoptosis, the effect of caspase inhibition on MP release by stimulated cells was assessed. These experiments showed that the pan-caspase inhibitor, ZVAD, although decreasing NO production, increased MP release by stimulated cells. Together, these experiments demonstrate that TLR stimulation of macrophages can lead to MP release, and NO plays a key role in this response.