Inhibition of inducible nitric oxide synthase-derived nitric oxide as a therapeutical target for acute pancreatitis induced by secretory phospholipase A(2)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

Involvement of nitric oxide on the pathogenesis of irinotecan-induced intestinal mucositis: role of cytokines on inducible nitric oxide synthase activation

Purpose Intestinal mucositis and the closely associated diarrhea are common costly side effects of irinotecan. Cytokine modulators, such as thalidomide and pentoxifylline, are found capable of attenuating intestinal mucositis progression. Nitric oxide (NO) seems to be a key mediator of the antineoplastic drug toxicity. The aim of this study was to investigate the role of NO on the pathogenesis of intestinal mucositis, as well as the participation of cytokines upon inducible nitric oxide synthase (iNOS) expression in irinotecan-induced intestinal mucositis. Methods iNOS-knockout (iNOS(-/-)) and C57BL/6 (WT, wild type) animals (n = 5-6) were given either saline or irinotecan (60 mg/kg i.p for 4 days), with or without pretreatment with aminoguanidine (50 mg/kg s.c.), thalidomide (60 mg/kg s.c), infliximab (5 mg/kg i.v.), or pentoxifylline (1.7 mg/kg s.c). On day 5, diarrhea was assessed, and following euthanasia, proximal intestinal samples were obtained for myeloperoxidase (MPO) and iNOS activity, morphometric analysis, western blot and immunohistochemistry to iNOS, cytokine dosage, and for in vitro evaluation of gut contractility. Results Irinotecan induced severe diarrhea and intestinal smooth muscle over-contractility, accompanied with histopathological changes. Additionally, increased MPO and iNOS activity and iNOS immunoexpression were found in WT animals treated with irinotecan. The rise in MPO, smooth muscle over-contractility, and diarrhea were abrogated in aminoguanidine-treated and iNOS(-/-) mice. Moreover, through western blot, we verified that infliximab and pentoxifylline significantly inhibited irinotecan-induced iNOS expression. In addition, cytokine concentration was found only partially decreased in irinotecan-treated iNOS(-/-) mice when compared with wild-type animals that were given irinotecan. Conclusions This study suggests a role of nitric oxide in the pathogenesis of irinotecan-induced intestinal mucositis and also provides evidence for the participation of cytokines on iNOS induction.

Synthesis, biological evaluation and molecular docking studies of 3-(triazolyl)coumarin derivatives: Effect on inducible nitric oxide synthase

A series of 3-(triazolyl)-coumarins were synthesized and tested as anti-inflammatory agents. It was possible to infer that these compounds do not alter the interaction of LPS with TLR-4 or TLR-2, as the intracellular pathways involved in the TNF-alpha secretion and COX-2 activity were not affected. Nevertheless, the compounds inhibited iNOS-derived NO production, without affecting the eNOS activity. The outcome of the docking studies showed that it pi center dot center dot center dot pi interactions with the heme group are important for the iNOS inhibition, thus making compound 3c a promising lead. Moreover, the efficacy of this compound was visualized by the reduced number of neutrophils in the LPS-inflamed subcutaneous tissue. Together, biological and docking data show that triazolyl-substituted coumarins, that can act on iNOS, are a good scaffold to be explored. (C) 2012 Elsevier Masson SAS. All rights reserved.

A mechanism of paraquat toxicity involving nitric oxide synthase

Paraquat (PQ) is a well described pneumotoxicant that produces toxicity by redox cycling with cellular diaphorases, thereby elevating intracellular levels of superoxide (O2⨪). NO synthase (NOS) has been shown to participate in PQ-induced lung injury. Current theory holds that NO reacts with O2⨪ generated by PQ to produce the toxin peroxynitrite. We asked whether NOS might alternatively function as a PQ diaphorase and reexamined the question of whether NO/O2⨪ reactions were toxic or protective. Here, we show that: (i) neuronal NOS has PQ diaphorase activity that inversely correlates with NO formation; (ii) PQ-induced endothelial cell toxicity is attenuated by inhibitors of NOS that prevent NADPH oxidation, but is not attenuated by those that do not; (iii) PQ inhibits endothelium-derived, but not NO-induced, relaxations of aortic rings; and (iv) PQ-induced cytotoxicity is potentiated in cytokine-activated macrophages in a manner that correlates with its ability to block NO formation. These data indicate that NOS is a PQ diaphorase and that toxicity of such redox-active compounds involves a loss of NO-related activity.

Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries

Nitric oxide (NO), synthesized from l-arginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or β-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal fluid (CSF) via the cisterna magna (final viral titer in CSF, 109 pfu/ml). Adventitial transgene expression was demonstrated 24 h later by β-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels (P < 0.05). The increased receptor-mediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood flow.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of alpha-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and alpha-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon gamma. alpha-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. alpha-MSH increased cAMP accumulation in RAW cells, characteristic of alpha-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary alpha-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecular of alpha-MSH, was expressed in RAW cells, and tumor necrosis factor alpha increased production and release of alpha-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor alpha can induce macrophages to increase production of alpha-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that alpha-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Endothelial nitric oxide synthase polymorphisms and adaptation of parasympathetic modulation to exercise training

SILVA, B. M., F. J. NEVES, M. V. NEGRÃO, C. R. ALVES, R. G. DIAS, G. B. ALVES, A. C. PEREIRA, M. Urbana A. RONDON, J. E. KRIEGER, C. E. NEGRÃO, and A. C. DA NOBREGA. Endothelial Nitric Oxide Synthase Polymorphisms and Adaptation of Parasympathetic Modulation to Exercise Training. Med. Sci. Sports Exerc., Vol. 43, No. 9, pp. 1611-1618, 2011. Purpose: There is a large interindividual variation in the parasympathetic adaptation induced by aerobic exercise training, which may be partially attributed to genetic polymorphisms. Therefore, we investigated the association among three polymorphisms in the endothelial nitric oxide gene (-786T>C, 4b4a, and 894G>T), analyzed individually and as haplotypes, and the parasympathetic adaptation induced by exercise training. Methods: Eighty healthy males, age 20-35 yr, were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis, and haplotypes were inferred using the software PHASE 2.1. Autonomic modulation (i.e., HR variability and spontaneous baroreflex sensitivity) and peak oxygen consumption ((V) over dotO(2peak)) were measured before and after training (running, moderate to severe intensity, three times per week, 60 min.day(-1), during 18 wk). Results: Training increased (V) over dotO(2peak) (P < 0.05) and decreased mean arterial pressure (P < 0.05) in the whole sample. Subjects with the -786C polymorphic allele had a significant reduction in baroreflex sensitivity after training (change: wild type (-786TT) = 2% +/- 89% vs polymorphic (-786TC/CC) = -28% +/- 60%, median +/- quartile range, P = 0.03), and parasympathetic modulation was marginally reduced in subjects with the 894T polymorphic allele (change: wild type (894GG) = 8% +/- 67% vs polymorphic (894GT/TT) = -18% +/- 59%, median +/- quartile range, P = 0.06). Furthermore, parasympathetic modulation percent change was different between the haplotypes containing wild-type alleles(-786T/4b/894G) and polymorphic alleles at positions -786 and 894 (-786C/4b/894T) (-6% +/- 56% vs -41% +/- 50%, median T quartile range, P = 0.04). Conclusions: The polymorphic allele at position -786 and the haplotype containing polymorphic alleles at positions -786 and 894 in the endothelial nitric oxide gene were associated with decreased parasympathetic modulation after exercise training.

Exercise training improves muscle vasodilatation in individuals with T786C polymorphism of endothelial nitric oxide synthase gene

Negrão M.V, Alves CR, Alves G.B, Pereira A.C, Dias R.G, Laterza M.C, Mota G.F, Oliveira E.M, Bassaneze V, Krieger J.E, Negrão C.E, Rondon M.U.P. Exercise training improves muscle vasodilatation in individuals with T786C polymorphism of endothelial nitric oxide synthase gene. Physiol Genomics 42A: 71-77, 2010. First published July 6, 2010; doi:10.1152/physiolgenomics.00145.2009.-Allele T at promoter region of the eNOS gene has been associated with an increase in coronary disease mortality, suggesting that this allele increases susceptibility for endothelial dysfunction. In contrast, exercise training improves endothelial function. Thus, we hypothesized that: 1) Muscle vasodilatation during exercise is attenuated in individuals homozygous for allele T, and 2) Exercise training improves muscle vasodilatation in response to exercise for TT genotype individuals. From 133 preselected healthy individuals genotyped for the T786C polymorphism, 72 participated in the study: TT (n = 37; age 27 +/- 1 yr) and CT + CC (n = 35; age 26 +/- 1 yr). Forearm blood flow (venous occlusion plethysmography) and blood pressure (oscillometric automatic cuff) were evaluated at rest and during 30% handgrip exercise. Exercise training consisted of three sessions per week for 18 wk, with intensity between anaerobic threshold and respiratory compensation point. Resting forearm vascular conductance (FVC, P = 0.17) and mean blood pressure (P = 0.70) were similar between groups. However, FVC responses during handgrip exercise were significantly lower in TT individuals compared with CT + CC individuals (0.39 +/- 0.12 vs. 1.08 +/- 0.27 units, P = 0.01). Exercise training significantly increased peak VO(2) in both groups, but resting FVC remained unchanged. This intervention significantly increased FVC response to handgrip exercise in TT individuals (P = 0.03), but not in CT + CC individuals (P = 0.49), leading to an equivalent FVC response between TT and CT + CC individuals (1.05 +/- 0.18 vs. 1.59 +/- 0.27 units, P = 0.27). In conclusion, exercise training improves muscle vasodilatation in response to exercise in TT genotype individuals, demonstrating that genetic variants influence the effects of interventions such as exercise training.

Gender-specific vascular effects elicited by chronic ethanol consumption in rats: a role for inducible nitric oxide synthase

Background and purpose: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. Experimental approach: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. Key results: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. Conclusions and implications: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS.

Induction of smooth muscle cell nitric oxide synthase by human leukaemia inhibitory factor: Effects in vitro and in vivo

We have previously shown that human leukaemia inhibitory factor (hLIF) inhibits perivascular cuff-induced neointimal formation in the rabbit carotid artery. Since nitric oxide (NO) is a known inhibitor of smooth muscle growth, NO synthase (NOS) activity in the presence of hLIF was examined in vivo and in vitro. In rabbit aortic smooth muscle cell (SMC) culture, significant NOS activity was observed at 50 pg/ml hLIF, with maximal activity at 5 ng/ml. In the presence of the NOS inhibitor L-NAME, hLIF-induced activation of NOS was greatly decreased, however it was still 63-fold higher than in control (p < 0.05). SMC-DNA synthesis was significantly reduced (-47%) following incubation with hLIF plus L-arginine, the substrate required for NO production (p < 0.05), with no effect observed in the absence of L-arginine. Silastic cuff placement over the right carotid artery of rabbits resulted in a neointima 19.3 +/- 5.4% of total wall cross-sectional area, which was increased in the presence of L-NAME (27.0 +/- 2.0%; p < 0.05) and reduced in the presence of L-arginine (11.3 +/- 2.0%; p < 0.05). The effect of L-arginine was ameliorated by co-administration of L-NAME (16.4 +/- 1.5%). However, administration of L-NAME with hLIF had no effect on the potent inhibition of neointimal formation by hLIF (3.2 +/- 2.5 vs. 2.1 +/- 5.4%, respectively). Similarly, with hLIF administration, NOS activity in the cuffed carotid increased to 269.0 +/- 14.0% of saline-treated controls and remained significantly higher with coadministration of L-NAME (188.5 +/- 14.7%). These results indicate that hLIF causes superinduction of NO by SMC, and that it is, either partially or wholly, through this mechanism that hLIF is a potent inhibitor of neointimal formation in vivo and of smooth muscle proliferation in vitro.

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p < 0.05), eosinophils counting (p < 0.001), iNOS-positive cells (p < 0.001), collagen and elastic fiber deposition (p < 0.05), actin density (p < 0.05) and 8-iso-PGF2 alpha expression (p < 0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p < 0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway. (C) 2008 Elsevier B.V. All rights reserved.