Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells.

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes.

Aging alters the production of iNOS, arginase and cytokines in murine macrophages

The limited amount of information on the primary age-related deficiencies in the innate immune system led us to study the production of inducible nitric oxide synthase (iNOS), arginase, and cytokines in macrophages of young (8 weeks old) and old (72 weeks old) female BALB/c mice. We first evaluated iNOS and arginase inducers on peritoneal (PMΦ) and bone marrow-derived (BMMΦ) macrophages of young BALB/c and C57BL/6 mice, and then investigated their effects on macrophages of old mice. Upon stimulation with lipopolysaccharide (LPS), resident and thioglycolate-elicited PMΦ from young mice presented higher iNOS activity than those from old mice (54.4%). However, LPS-stimulated BMMΦ from old mice showed the highest NO levels (50.1%). Identical NO levels were produced by PMΦ and BMMΦ of both young and old mice stimulated with interferon-γ. Arginase activity was higher in resident and elicited PMΦ of young mice stimulated with LPS (48.8 and 32.7%, respectively) and in resident PMΦ stimulated with interleukin (IL)-4 (64%). BMMΦ of old mice, however, showed higher arginase activity after treatment with IL-4 (46.5%). In response to LPS, PMΦ from old mice showed the highest levels of IL-1α (772.3 ± 51.9 pg/mL), whereas, those from young mice produced the highest amounts of tumor necrosis factor (TNF)-α (937.2 ± 132.1 pg/mL). Only TNF-α was expressed in LPS-treated BMMΦ, and cells from old mice showed the highest levels of this cytokine (994.1 ± 49.42 pg/mL). Overall, these results suggest that macrophages from young and old mice respond differently to inflammatory stimuli, depending on the source and maturity of the cell donors.

Nitric oxide modulates lipopolysaccharide-induced endothelial platelet endothelial cell adhesion molecule expression via interleukin-10

We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 mu g/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions.

Human visceral leishmaniasis expresses Th1 pattern in situ liver lesions

The architectural and infiltrate pattern of liver human visceral leishmaniasis (HVL) have been systematically classified as typical, fibrogenic or nodular. Despite this histopathological classification, the immune response based on cytokines and cellular phenotypes have never been performed. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of the nodular involvement of the Liver in HVL. We evaluated nine cases of the nodular form of HVL. In situ immune response was studied through cytokine analysis and immunohistochemical study for phenotype markers: IL-1, IL-4, IL-1 0, TNF-alpha, IFN-gamma, CD4+ T cells, CD8+ T cells, CD20, CD68, CD57 and macrophage activation was determined by evaluation of iNOS activity. HVL seems to be related to a better immune response. Amastigotes were rarely found on liver sections. Leishmania antigen expression was also rare and located in the inflammatory nodules. The lower expression of IL-4 and IL-10, moderate expression of TNF-alpha and IFN-gamma demonstrate a panorama of Th1 phenotype. The increased expression of NK cells could help in sustaining this model of response. This pattern of immune response is probably responsible for improvement in the parasite`s clearance from liver tissue and it is a prognostic marker of human visceral leishmaniasis. (C) 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Régulation du processus métastatique tumoral par l'oxyde nitrique (NO), le TGF-β el les cellules de l'hôte

RESUME Nous avons étudié le rôle de deux molécules, le Transfon-ning Growth Factor (TGF-β) et l'oxyde nitrique (NO), dans le processus métastatique. Deux clones tumoraux ont été sélectionnés à partir d'un carcinome du côlon pour leur différence de potentiel tumorigénique dans des rats syngéniques. La croissance tumorale du clone progressif PROb a été corrélée à sa capacité à sécréter le TGF-β actif Cependant, la transfection du clone régressif REGb, sécrétant du TGF-β latent, par une vecteur codant pour le TGF-β bio-actif n'a pas permis d'induire le développement tumoral. Les deux clones tumoraux présentent des activités des protéases MMP-2, APN et DPPIV identiques et qui ne semblent pas modifiées par le TGF-β. L'interaction des cellules tumorales avec l'endothélium et l'activité de la NO synthase (iNOS) responsable de la synthèse de NO sont impliqués dans la progression de nombreux cancers. Le clone PROb, mais pas le clone REGb, inhibe l'activation de la iNOS des cellules endothéliales par sa sécrétion de TGF-β actif Les deux clones montrent cependant des propriétés d'adhésion identiques aux cellules endothéliales et sont capables d'inhiber par contact cellulaire direct l'activation de la iNOS endothéliale. Ceci suggère que ces contacts directs pourraient créer un micro-environnement favorable à la conversion du TGF-β latent en TGF-β actif ou à d'autres interactions moléculaires pouvant réguler l'activation endothéliale. Par ailleurs, les deux clones activent des macrophages du système nerveux central, organe où ils ne forment pas de métastases, mais pas les macrophages circulants, illustrant des mécanismes différentiels et spécifiques dans l'activation de différents types de cellules immunitaires. Afin de mieux comprendre le rôle du NO dans la dissémination métastatique, deux clones cellulaires différant par le taux d'activité de la iNOS ont été sélectionnés à partir de la lignée murine parentale de carcinome du sein EMT-6. Bien que le NO soit un inhibiteur potentiel de la prolifération cellulaire, les deux clones montrent des propriétés prolifératives identiques in vitro. Les cellules EMT-6H qui produisent peu de NO in vitro forment de nombreux nodules tumoraux pulmonaires in vivo corrélés à une mortalité significative des souris syngéniques injectées. Les cellules EMT-6J qui présentent une expression élevée de iNOS et de NO induisent de rares nodules tumoraux pulmonaires et peu de mortalité. Dans ce modèle, l'expression tumorale de NO semble donc défavoriser la croissance tumorale. Les deux clones cellulaires ont des propriétés identiques d'adhésion et de prolifération mesurées in vitro sur des cellules endothéliales primaires isolées de différents organes et in vivo par une colocalisation identique dans les poumons de souris syngéniques 48h après leur injection. Les cellules EMT-6H présentent une activité MMP-2 plus élevée alors que les activités des protéases APN et DPPIV sont identiques dans les deux clones cellulaires. Le TGF-β soluble ainsi que les fibroblastes primaires bloquent la prolifération des deux clones cellulaires. Cependant, l'activation préalable des fibroblastes par du TGF-β restaure partiellement la prolifération du clone EMT-6H mais pas celle du clone EMT-6J. Ces résultats montrent que le rôle de molécules telles que le TGF-β et le NO tumoral dans la progression tumorale doit être considéré dans un contexte d'interactions des cellules tumorales avec les différentes types cellulaires de l'hôte: en particulier, notre travail souligne que les macrophages et les fibroblastes sont déterminants dans la progression métastatique des carcinomes du côlon ou du sein. RESUME DESTINE A UN LARGE PUBLIC Les métastases tumorales, disséminées et intraitables par chirurgie, représentent un problème majeur dans le traitement clinique du cancer. Elles sont dues à des cellules tumorales qui ont migré de leur site tumoral primaire, circulé et survécu dans le système vasculaire de l'hôte, échappé au système immunitaire, adhéré à et survécu sur l'endothélium des vaisseaux, et envahi le tissu sous-jacent où elles ont proliféré. Cette capacité à former des métastases implique de nombreux facteurs dont certains ont été identifiés mais dont le rôle reste controversé dans les différentes études. Nous nous sommes intéressés au rôle de l'oxyde nitrique (NO) et du facteur de croissance et de transformation cellulaire TGF-β. Dans les carcinomes du sein, l'expression des enzymes responsables de la synthèse de NO a été corrélée avec l'invasion tumorale mais aussi avec un pronostic favorable selon les études. Deux clones cellulaires ont été isolés à partir de la tumeur mammaire EMT-6 chez la souris. Le clone EMT-6H sécrète peu de NO et forme de nombreuses tumeurs dans les poumons des souris *entraînant leur décès. Le clone EMT-6J sécrète beaucoup de NO et ne se développe que peu dans les poumons. Dans ce modèle expérimental, le NO semble donc défavoriser la croissance tumorale. L'analyse des interactions avec les cellules de l'hôte rencontrées lors de la formation de métastases pulmonaires a montré que les deux clones cellulaires adhérent et prolifèrent de manière similaire sur les cellules endothéliales tapissant l'intérieur des vaisseaux sanguins. L'arrêt des cellules tumorales dans les poumons ne permet donc pas d'expliquer la différence de croissance tumorale. Cependant, le clone agressif EMT-6H présente une activité élevée d'une protéase (MMP-2) qui lui permettrait par la suite d'envahir le tissu pulmonaire. Par ailleurs, l'activation des fibroblastes du tissu pulmonaire par le TGF-β, une molécule observée dans des conditions inflammatoires, permet au clone agressif EMT-6H de proliférer mais inhibe la croissance du clone EMT-6J. Dans un modèle expérimental de carcinome du côlon, le TGF-β est considéré favorable à la croissance tumorale. Isolées à partir de la même tumeur initiale, deux lignées de cellules ont des comportements opposés lorsqu'elles sont injectées sous la peau des rats. La capacité de la lignée PROb à former des tumeurs a été corrélée à la sécrétion de TGF-β actif L'introduction du gène codant pour le TGF-β actif dans la lignée REGb, qui ne sécrète pas de TGF-β actif et ne forme pas de tumeurs chez le rat, ne restaure pas leur potentiel tumorigénique. Dans ce modèle, l'expression de TGF-β actif ne semble donc pas suffisante à la croissance tumorale. Les interactions avec différents types cellulaires de l'hôte ont été étudiées. Les deux lignées tumorales adhérent de manière similaire sur les cellules endothéliales et sont capables d'inhiber leur activation, un mécanisme qui pourrait participer à la destruction. Les deux lignées activent les cellules immunitaires du système nerveux central, un organe où elles ne forment pas de métastase. Ces résultats suggèrent que la sélection des cellules métastatiques ne s'effectue pas sur l'endothélium des vaisseaux sanguins mais à des étapes ultérieures dans le micro- environnement cellulaire du nouvel organe colonisé. SUMMARY Metastasis results from the migration of tumor cells from their primary tumor, circulation through the bloodstream, attachment to the endothelium, and invasion of the surrounding tissue where they create a microenvironnement favoring their growth. This multistep process implies various cellular interactions and molecules. Among those, we were interested in the role of the Transforming Growth Factor beta (TGF-β) and the nitric oxide (NO). Two cell lines were isolated from a rat colon tumor and assessed for their metastatic potential in vivo. The PROb cell line that expresses active TGF-β formed subcutaneous tumors in rats while the REGb cell line that expresses only latent TGF-β did not. Transfection of REGb cells with a plasmid encoding for the active form of TGF-β failed to restore their metastatic ability. Thus TGF-β secretion is not sufficient to induce colon carcinoma progression. Activities of various proteases such as APN, DPPIV and MMP were similar in both cell lines and were not regulated by TGF-β. Interactions with the endothelium as well as NO synthase activity (iNOS) and local NO concentrations are believed to be crucial steps in cancer metastasis. Coculture of the two clones with endothelial cells inhibited the cytokine-triggered activation of the iNOS enzyme in primary rat endothelial cells but only PROb cells were capable of increasing the expression of IL-6, a protumoral interleukin that may participate in the impairment of the anti-tumoral immune response of the host. Both cell lines exhibited potential to activate microglial cells but not bone marrow-derived macrophages, pointing to a differential regulation of specialized immune cells. To better understand the conflicting role of NO in breast cancer progression, two cell clones were selected from the murine tumorigenic cell line EMT-6 based on their iNOS activity and NO secretion. Although NO has been shown to inhibit cell proliferation, the two cell clones exhibited similar proliferation rates in vitro. The EMT-6H cells expressed little NO and grew actively in the lungs of syngenic mice, leading to their death. Opposite results were observed with the EMT-6J cells. In these in vivo conditions, NO seems to impair tumor growth. Both clones exhibited similar in vitro adhesive properties to primary endothelial cells isolated from various mouse organs and similar localization in the lungs of mice 48 hours after injection. Sustained metalloproteinase MMP-2 activity was detected in the tumorigenic EMT-6H clone, but not in the EMT-6J cells while other proteases such as APN and DPPIV showed no difference. These results suggested that the two clones differed in invasion steps following adhesion to the endothelium and that NO did not participate in previous steps. Consistent with this, both soluble TGF-β and supernatants of cultures of mouse primary lung fibroblasts inhibited the growth of the two clones. However, previous activation of these fibroblasts with TGF-β restored the growth of the tumorigenic EMT-6H cells, but not of EMT-6J cells. Altogether, these results indicate that the role of a given molecule, such as NO or TGF-β, must be considered in a context of interaction of tumor cells with host cells. They further imply that interaction of tumor cells with specialized immune cells and with stromal cells of the colonized organ, rather than with the endothelium, are critical in regulating metastasis.

Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells.

Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3'-5'-monophosphate (NO-cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of alpha2 and beta1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription-polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Caveolin-1-mediated post-transcriptional regulation of inducible nitric oxide synthase in human colon carcinoma cells.

Reactive oxygen species are now widely recognized as important players contributing both to cell homeostasis and the development of disease. In this respect nitric oxide (NO) is no exception. The discussion here will center on regulation of the inducible form of nitric oxide synthase (iNOS) for two reasons. First, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca2(+)-controlled NO production by endothelial eNOS or neuronal nNOS. Second, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to endothelial eNOS or neuronal nNOS, is widely considered to occur at the transcriptional level only. In particular, we were interested in the possibility that caveolin-1, a protein that functions as a tumor suppressor in colon carcinoma cells (Bender et al., 2002; this issue), might regulate iNOS activity. Our results provide evidence for the existence of a post-transcriptional mechanism controlling iNOS protein levels that involves caveolin-1-dependent sequestration of iNOS within a detergent-insoluble compartment. Interestingly, despite the high degree of conservation of the caveolin-1 scaffolding domain binding motif within all NOS enzymes, the interaction detected between caveolin-1 and iNOS in vitro is crucially dependent on presence of a caveolin-1 sequence element immediately adjacent to the scaffolding domain. A model is presented summarizing the salient aspects of these results. These observations are important in the context of tumor biology, since down-regulation of caveolin-1 is predicted to promote uncontrolled iNOS activity, genotoxic damage and thereby facilitate tumor development in humans.

Heme oxygenase-1 induction by endogenous nitric oxide: influence of intracellular glutathione.

To investigate the influence of glutathione (GSH) on cellular effects of nitric oxide (NO) formation, human colon adenocarcinoma cells were transfected with a vector allowing controlled expression of inducible nitric oxide synthase (iNOS). Protein levels of oxidative stress-sensitive heme oxygenase-1 (HO-1) were analyzed in the presence or absence of GSH depletion using L-buthionine-[S,R]-sulfoximine and iNOS induction. While no effect was observed in the presence of iNOS activity alone, a synergistic effect on HO-1 expression was observed in the presence of iNOS expression and GSH depletion. This effect was prevented by addition of N-methyl-L-arginine. Therefore, targeting of endogenous NO may be modulated by intracellular GSH.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35% (N = 10), and from patients with CAD and LVEF >60% (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H³]-arginine to L-[H³]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 ± 0.18 vs 1.51 ± 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 ± 0.08 vs 0.78 ± 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 ± 0.90 vs 1.54 ± 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 ± 0.27 vs 1.43 ± 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

In vivo and in vitro anti-inflammatory and anti-nociceptive activities of lovastatin in rodents

Statins are among the most prescribed drugs in recent clinical practice. They are also known for their pleiotropic actions, which are independent of their lipid-lowering properties. The effect of lovastatin was investigated against carrageenan-induced paw edema in male Wistar rats (200-250 g) and on leukocyte migration, as measured by carrageenan-induced peritonitis in male Swiss mice (20-25 g), which are models of acute inflammation. Lovastatin (administered 1 h prior to carrageenan), at oral doses of 2, 5, and 10 mg/kg, markedly attenuated paw edema formation in rats at the 4th hour after carrageenan injection (25, 43, and 37% inhibition, respectively). Inhibitions of 20, 45 and 80% were observed in the leukocyte migration, as evaluated by carrageenan-induced peritonitis in mice with lovastatin doses of 0.5, 1 and 5 mg/kg, as compared to controls. Furthermore, lovastatin (administered 1 h before initiation) reduced the nociceptive effect of the formalin test in mice, at both phases, at doses of 2, 5, and 10 mg/kg: first phase (51, 65, and 70%, respectively) and second phase (73, 57, and 66% inhibition of licking time, respectively). The anti-nociceptive activity of lovastatin was inhibited by naloxone (3 mg/kg, sc). Lovastatin (0.01, 0.1, and 1 µg/mL) inhibited by 23, 79, and 86%, respectively, the release of myeloperoxidase from human neutrophils. Leukocyte (predominantly neutrophils) infiltration was almost completely reduced by lovastatin treatment, as observed in the model of acute paw edema with hematoxylin and eosin staining. In addition, lovastatin decreased the number of cells expressing tumor necrosis factor-α (TNF-α) and the inducible form of nitric oxide synthase (iNOS) activity. Therefore, the alterations in leukocyte activity and cytokine release could contribute to the anti-inflammatory activity of lovastatin.

Cardiac cell fate control by the imidazoline I1 receptor/nischarin : application in cardiac pathology

La moxonidine, un médicament antihypertenseur sympatholytique de type imidazolinique, agit au niveau de la médulla du tronc cérébral pour diminuer la pression artérielle, suite à l’activation sélective du récepteur aux imidazolines I1 (récepteur I1, aussi nommé nischarine). Traitement avec de la moxonidine prévient le développement de l’hypertrophie du ventricule gauche chez des rats hypertendus (SHR), associé à une diminution de la synthèse et une élévation transitoire de la fragmentation d’ADN, des effets antiprolifératifs et apoptotiques. Ces effets se présentent probablement chez les fibroblastes, car l’apoptose des cardiomyocytes pourrait détériorer la fonction cardiaque. Ces effets apparaissent aussi avec des doses non hypotensives de moxonidine, suggérant l’existence d’effets cardiaques directes. Le récepteur I1 se trouvé aussi dans les tissus cardiaques; son activation ex vivo par la moxonidine stimule la libération de l’ANP, ce qui montre que les récepteurs I1 cardiaques sont fonctionnels malgré l’absence de stimulation centrale. Sur la base de ces informations, en plus du i) rôle des peptides natriurétiques comme inhibiteurs de l’apoptose cardiaque et ii) des études qui lient le récepteur I1 avec la maintenance de la matrix extracellulaire, on propose que, à part les effets sympatholytiques centrales, les récepteurs I1 cardiaques peuvent contrôler la croissance-mort cellulaire. L’activation du récepteur I1 peut retarder la progression des cardiopathies vers la défaillance cardiaque, en inhibant des signaux mal adaptatifs de prolifération et apoptose. Des études ont été effectuées pour : 1. Explorer les effets in vivo sur la structure et la fonction cardiaque suite au traitement avec moxonidine chez le SHR et le hamster cardiomyopathique. 2. Définir les voies de signalisation impliquées dans les changements secondaires au traitement avec moxonidine, spécifiquement sur les marqueurs inflammatoires et les voies de signalisation régulant la croissance et la survie cellulaire (MAPK et Akt). 3. Explorer les effets in vitro de la surexpression et l’activation du récepteur I1 sur la survie cellulaire dans des cellules HEK293. 4. Rechercher la localisation, régulation et implication dans la croissance-mort cellulaire du récepteur I1 in vitro (cardiomyocytes et fibroblastes), en réponse aux stimuli associés au remodelage cardiaque : norépinephrine, cytokines (IL-1β, TNF-α) et oxydants (H2O2). Nos études démontrent que la moxonidine, en doses hypotensives et non-hypotensives, améliore la structure et la performance cardiaque chez le SHR par des mécanismes impliquant l’inhibition des cytokines et des voies de signalisation p38 MAPK et Akt. Chez le hamster cardiomyopathique, la moxonidine améliore la fonction cardiaque, module la réponse inflammatoire/anti-inflammatoire et atténue la mort cellulaire et la fibrose cardiaque. Les cellules HEK293 surexprimant la nischarine survivent et prolifèrent plus en réponse à la moxonidine; cet effet est associé à l’inhibition des voies ERK, JNK et p38 MAPK. La surexpression de la nischarine protège aussi de la mort cellulaire induite par le TNF-α, l’IL-1β et le H2O2. En outre, le récepteur I1 s’exprime dans les cardiomyocytes et fibroblastes, son activation inhibe la mort des cardiomyocytes et la prolifération des fibroblastes induite par la norépinephrine, par des effets différentiels sur les MAPK et l’Akt. Dans des conditions inflammatoires, la moxonidine/récepteur aux imidazolines I1 protège les cardiomyocytes et facilite l’élimination des myofibroblastes par des effets contraires sur JNK, p38 MAPK et iNOS. Ces études démontrent le potentiel du récepteur I1/nischarine comme cible anti-hypertrophique et anti-fibrose à niveau cardiaque. L’identification des mécanismes cardioprotecteurs de la nischarine peut amener au développement des traitements basés sur la surexpression de la nischarine chez des patients avec hypertrophie ventriculaire. Finalement, même si l’effet antihypertenseur des agonistes du récepteur I1 centraux est salutaire, le développement de nouveaux agonistes cardiosélectifs du récepteur I1 pourrait donner des bénéfices additionnels chez des patients non hypertendus.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose: Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee. Experimental approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (dl-propargylglycine) or an H(2)S donor [Lawesson`s reagent (LR)]. Key results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Conclusions and implications: Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B(1) receptor gene

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ(10 mu M). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 mu M), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice. (C) 2009 Elsevier Inc. All rights reserved.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NO(x) levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite N(omega)-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the N(omega)-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with L-NAME yields low serum N(omega)-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO. (c) 2008 Elsevier Ltd. All rights reserved.