Free Radical Production and Oxidative Stress in Lung Tissue of Patients Exposed to Sulfur Mustard: An Overview of Cellular and Molecular Mechanisms.

Sulfur mustard (SM) is a chemical alkylating compound that primary targets lung tissue. It causes a wide variety of pathological effects in respiratory system such as chronic bronchitis, bronchiolitis obliterans, necrosis of the mucosa and inflammation, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. However, molecular and cellular mechanisms for these pathologies are still unclear. Oxidative stress (OS) induced by reactive oxygen species (ROS) is likely a significant mechanism by which SM leads to cell death and tissues injury. SM can trigger various molecular and cellular pathways that are linked to ROS generation, OS, and inflammation. Hypoxia-induced oxidative stress, reduced activity of enzymatic antioxidants, depletion of intercellular glutathione (GSH), decreased productivity of GSH-dependent antioxidants, mitochondrial dysfunction, accumulation of leukocytes and proinflammatory cytokines, and increased expression of ROS producing-related enzymes and inflammatory mediators are the major events in which SM leads to massive production of ROS and OS in pulmonary system. Therefore, understanding of these molecules and signaling pathways gives us valuable information about toxicological effects of SM on injured tissues and the way for developing a suitable clinical treatment. In this review, we aim to discuss the possible mechanisms by which SM induces excessive production of ROS, OS, and antioxidants depletion in lung tissue of exposed patients.

Transplantation of enteric nervous system stem cells rescues nitric oxide synthase deficient mouse colon.

Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase (nNOS-/-) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS+ neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS-/- colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.

Decreased Bronchial Eosinophilic Inflammation and Mucus Hypersecretion in Asthmatic Mice Lacking All Nitric Oxide Synthase Isoforms.

BACKGROUND: Asthma is characterized by airflow limitation with chronic airway inflammation, hyperresponsiveness and mucus hypersecretion. NO is generated by three nitric oxide synthase (i/n/eNOSs) isoforms, but conflicting results have been reported using asthmatic mice treated with NOSs inhibitors and NOS-knockout mice. To elucidate the authentic role of NO/NOSs in asthma, we used asthmatic mice lacking all NOSs (n/i/eNOS(-/-)). METHODS: Wild-type and n/i/eNOS(-/-) mice were sensitized and challenged with ovalbumin. Pathological findings and expressions of interferon (IFN)-γ, interleukin (IL)-4, -5, -10, -13 and chemokines in the lung were evaluated. RESULTS: Decreased eosinophilic inflammation, bronchial thickening and mucus secretion, IL-4, -5 and -13, monocyte chemoattractant protein-1, eotaxin-1 and thymus and activation-regulated chemokine expressions were observed in n/i/eNOS(-/-) mice compared to wild-type, but expressions of IFN-γ and IL-10 were similar. CONCLUSION: Using asthmatic n/i/eNOS(-/-) mice, NO plays important roles in accelerating bronchial eosinophilic inflammation and mucus hypersecretion in the pathophysiology of asthma.

Untargeted metabolite profiling reveals that nitric oxide bioynthesis is an endogenous modulator of carotenoid biosynthesis in Deinococcus radiodurans and is required for extreme ionizing radiation resistance.

Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult.

Genetic characterization of Toxoplasma gondii isolates from eared doves (Zenaida auriculata) in Brazil / Caracterização genética de isolados de Toxoplasma gondii de pombos (Zenaida auriculata) no Brasil

Eared doves (Zenaida auriculata), which are common in urban, rural and wild areas in many regions of Brazil, are frequently prey for domestic cats. Therefore Toxoplasma gondii isolates obtained from doves may reflect greater environmental diversity than those from other hosts. The aim of the present study was to evaluate T. gondii seroprevalence, isolate and genotype strains from Z. auriculata. Serum and tissue samples were collected from 206 doves for use in the modified agglutination test (MAT) and mouse bioassay. The prevalence of T. gondii antibodies in the doves was 22.3% (46/206), with titers ranging from 16 to 4096, and T. gondii strains were isolated from 12 of these doves. Five genotypes were detected by means of PCR-RFLP, including ToxoDB genotypes #1, #6, #17 and #65, and one genotype that had not previously been described (ToxoDB#182). This was the first report on isolation of T. gondii from Z. auriculata. This study confirmed the genetic diversity of T. gondii isolates and the existence of clonal type II (ToxoDB genotype #1) in Brazil.

Pombos silvestres (Zenaida auriculata), comuns em áreas urbanas, rurais e selvagens em muitas regiões do Brasil, são frequentemente predados por gatos domésticos. Sendo assim, os isolados de T. gondii obtidos de pombos podem refletir uma maior diversidade ambiental do que os outros hospedeiros. O objetivo do presente estudo foi avaliar a soroprevalência, isolar e genotipar T. gondii de Z. auriculata. Amostras de soro e tecido foram coletadas de 206 pombos para o teste de aglutinação modificado (MAT) e o bioensaio em camundongos. A prevalência de anticorpos contra T. gondii em pombos foi 22,3% (46/206), com títulos variando de 16 a 4096, e T. gondii foi isolado de 12 pombos. Cinco genótipos foram detectados por PCR-RFLP, incluindo os genótipos ToxoDB #1, #6, #17, #65 e um genótipo não descrito anteriormente (ToxoDB#182). Esse é o primeiro relato de isolamento de T. gondii de Z. auriculata. Este estudo também confirmou a diversidade dos isolados de T. gondii e a presença de tipo clonal II (ToxoDB #1) no Brasil.

Perturbed neural activity disrupts cerebral angiogenesis during a postnatal critical period.

During the neonatal period, activity-dependent neural-circuit remodelling coincides with growth and refinement of the cerebral microvasculature. Whether neural activity also influences the patterning of the vascular bed is not known. Here we show in neonatal mice, that neither reduction of sensory input through whisker trimming nor moderately increased activity by environmental enrichment affects cortical microvascular development. Unexpectedly, chronic stimulation by repetitive sounds, whisker deflection or motor activity led to a near arrest of angiogenesis in barrel, auditory and motor cortices, respectively. Chemically induced seizures also caused robust reductions in microvascular density. However, altering neural activity in adult mice did not affect the vasculature. Histological analysis and time-lapse in vivo two-photon microscopy revealed that hyperactivity did not lead to cell death or pruning of existing vessels but rather to reduced endothelial proliferation and vessel sprouting. This anti-angiogenic effect was prevented by administration of the nitric oxide synthase (NOS) inhibitor L-NAME and in mice with neuronal and inducible NOS deficiency, suggesting that excessive nitric oxide released from hyperactive interneurons and glia inhibited vessel growth. Vascular deficits persisted long after cessation of hyperstimulation, providing evidence for a critical period after which proper microvascular patterning cannot be re-established. Reduced microvascular density diminished the ability of the brain to compensate for hypoxic challenges, leading to dendritic spine loss in regions distant from capillaries. Therefore, excessive sensorimotor stimulation and repetitive neural activation during early childhood may cause lifelong deficits in microvascular reserve, which could have important consequences for brain development, function and pathology.

Reactive oxygen and nitrogen species in sepsis-induced hepatic microvascular dysfunction.

OBJECTIVE AND DESIGN: Hepatic microvascular dysfunction is a critical event in the development of liver failure during sepsis. Activated blood cells and reactive oxygen and nitrogen species (RONS) have been implicated in the pathogenesis of sepsis. METHODS: Intravital-videomicroscopy was used to determine whether RONS contribute to the recruitment of leukocytes/platelets in the hepatic microvasculature during sepsis. Six hours following cecal-ligation and puncture (CLP), disturbances of the hepatic microvasculature were assessed in WT-mice (C57Bl/6 J; n = 8), in mice lacking gp91(phox)(n = 5), overexpressing superoxide-dismutase (SOD, n = 8), in WT-mice treated with a NOS-inhibitor (L-NAME, n = 5), lacking nNOS, eNOS or iNOS (n = 5 each), treated with the NO-donor DetaNO (n = 5), in WT-mice treated with gadolinium-chloride (GdCl(2), n = 5) and compared to a group of WT-mice following a sham operation (n = 8). Six hours post-CLP, the adhesion of leukocytes and platelets in terminal hepatic venules (THV) and sinusoids was quantified. RESULTS: In WT-mice, CLP elicited increases in the number of adherent leukocytes and platelets. Similar responses to CLP were noted in mice overexpressing SOD or lacking either eNOS or gp91(phox). The blood-cell recruitment was significantly blunted in septic iNOS-knockout mice and this response was reversed by pre-treatment with DetaNO. CONCLUSION: These findings suggest that iNOS-derived NO is a determinant of the pro-inflammatory phenotype assumed by the hepatic microvasculature during sepsis.

Analysis of cardiac and renal endothelin receptors by in situ hybridization in mice.

BACKGROUND: Endothelin-1 (ET-1) is a multifunctional peptide, which is implicated in the renal and cardiac physicology as well as in many pathologies of these systems. ET-1's actions take place after the activation of two receptors: ET(A) and ET(B). The expression of these receptors may be modulated during the pathologic process. The analysis of the distribution and level of expression of the receptors in animal models is therefore crucial. METHODS: We developed a protocol for non-radioactive in situ hybridization for the mRNA of the two endothelin receptors on paraffin-embedded tissue using digoxigenin-labeled RNA probes. RESULTS: In heart and kidney, the staining was reliable and specific. In a mouse model for endothelin/nitric oxide imbalance, cardiac ET(B) expression was reduced. The distribution of the receptors was in accordance with the actual knowledge. Differences in cell specific expression are discussed. CONCLUSIONS: We developed a protocol for the in situ hybridization of the endothelin receptors in mice. Given that the endothelin system is implicated in the development of many diseases, we believe that this protocol may be useful for a number of future preclinical studies..

Compensatory lung growth in NOS3 knockout mice suggests synthase isoform redundancy.

Nitric oxide synthase 3 (NOS3) produces nitric oxide (NO) in endothelial cells, which stimulates cyclic guanosine monophosphate (cGMP) production and thereby mediates pulmonary vasodilation. Inhibition of cGMP enzymatic cleavage by sildenafil might be involved in lung growth stimulating processes in pulmonary hypoplasia. The aim of this study was to discover insights into the transcriptional regulation of NOS3 in a mouse model of compensatory lung growth (CLG). CLG was studied in wild type animals (WT) and NOS3 knockout mice (NOS3-/-) by dry weight, DNA, and protein quantification as well as relative quantification of NOS mRNA. All assessments were done on adult female mice, 10 days after left pneumonectomy (PNX) or sham thoracotomy. Weight ratios of right NOS3-/- lungs were no different than controls. There was a compensatory increase in DNA and a noncompensating increase in protein ratios in NOS3-/- mice compared with controls. Pharmacological knockdown with the pan-NOS inhibitor l-NAME (nitro-arginine methyl ester) reduced CLG by only 8% compared with the d-NAME treated control mice. Relative quantification of lung mRNA revealed no up-regulation of NOS3 expression in WT lungs after PNX, but NOS3-/- lungs showed a 2.6-fold higher inducible NOS2 expression compared with shams. These data suggest that NOS3 loss of function alone does not impair CLG in mice, possibly because of redundancy mechanisms involving NOS2.

Contributions of nitric oxide synthases, dietary nitrite/nitrate, and other sources to the formation of NO signaling products.

UNLABELLED: Mice lacking all three nitric oxide synthase (NOS) genes remain viable even though deletion of the major downstream target of NO, soluble guanylyl cyclase, is associated with a dramatically shortened life expectancy. Moreover, findings of relatively normal flow responses in eNOS knockouts are generally attributed to compensatory mechanisms including upregulation of remaining NOS isoforms, but the alternative possibility that dietary nitrite/nitrate (NOx) may contribute to basal levels of NO signaling has never been investigated. AIM: The aim of the present study was to examine how NO signaling products (nitrosated and nitrosylated proteins) and NO metabolites (nitrite, nitrate) are affected by single NOS deletions and whether dietary NOx plays a compensatory role in any deficiency. Specifically, we sought to ascertain whether profound alterations of these products arise upon genetic deletion of either NOS isoform, inhibition of all NOS activity, NOx restriction, or all of the above. RESULTS: Our results indicate that while some significant changes do indeed occur, they are surprisingly moderate and compartmentalized to specific tissues. Unexpectedly, even after pharmacological inhibition of all NOSs and restriction of dietary NOx intake in eNOS knockout mice significant levels of NO-related products remain. Innovation/Conclusion: These findings suggest that a yet unidentified source of NO, unrelated to NOSs or dietary NOx, may be sustaining basal NO signaling in tissues. Given the significance of NO for redox regulation in health and disease, it would seem to be important to identify the nature of this additional source of NO products as it may offer new therapeutic avenues for correcting NO deficiencies.

Crucial vasculoprotective role of the whole nitric oxide synthase system in vascular lesion formation in mice: Involvement of bone marrow-derived cells.

Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS(-/-) mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS(-/-) and iNOS(-/-) genotypes, it was most accelerated in the n/i/eNOS(-/-) genotype. While neointimal formation was evident in the eNOS(-/-) and nNOS(-/-) genotypes, it was also most aggravated in the n/i/eNOS(-/-) genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS(-/-) mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS(-/-) bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism.

Reduced spinal microglial activation and neuropathic pain after nerve injury in mice lacking all three nitric oxide synthases.

BACKGROUND: Several studies have investigated the involvement of nitric oxide (NO) in acute and chronic pain using mice lacking a single NO synthase (NOS) gene among the three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). However, the precise role of NOS/NO in pain states remains to be determined owing to the substantial compensatory interactions among the NOS isoforms. Therefore, in this study, we used mice lacking all three NOS genes (n/i/eNOS-/-mice) and investigated the behavioral phenotypes in a series of acute and chronic pain assays. RESULTS: In a model of tissue injury-induced pain, evoked by intraplantar injection of formalin, both iNOS-/-and n/i/eNOS-/-mice exhibited attenuations of pain behaviors in the second phase compared with that in wild-type mice. In a model of neuropathic pain, nerve injury-induced behavioral and cellular responses (tactile allodynia, spinal microglial activation and Src-family kinase phosphorylation) were reduced in n/i/eNOS-/-but not iNOS-/-mice. Tactile allodynia after nerve injury was improved by acute pharmacological inhibition of all NOSs and nNOS. Furthermore, in MG-5 cells (a microglial cell-line), interferon-γ enhanced NOSs and Mac-1 mRNA expression, and the Mac-1 mRNA increase was suppressed by L-NAME co-treatment. Conversely, the NO donor, sodium nitroprusside, markedly increased mRNA expression of Mac-1, interleukin-6, toll-like receptor 4 and P2X4 receptor. CONCLUSIONS: Our results provide evidence that the NOS/NO pathway contributes to behavioral pain responses evoked by tissue injury and nerve injury. In particular, nNOS may be important for spinal microglial activation and tactile allodynia after nerve injury.

Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in mice.

The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS(-/-) mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS(-/-) mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS(-/-) mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved.

Protection against age-dependent renal injury in the F344xBrown Norway male rat is associated with maintained nitric oxide synthase.

Age-dependent renal damage is influenced by genetic background and the Fisher344xBrown Norway (F344xBN) rat is resistant to glomerular injury. In vulnerable strains, a fall in renal nitric oxide synthase (NOS) contributes to age-dependent renal damage. Here, we investigated renal NOS in young (3 months) and old (30 months) male F344xBN to test the hypothesis that renal NOS is maintained in "protected" strains. We also examined if 6 months of renin-angiotensin system (RAS) blockade using angiotensin converting enzyme inhibition (ACEI) and angiotensin receptor blockade (ARB) provides further benefit in these "protected" old rats. Aging increased tubulointerstitial injury but glomerular sclerosis was minimal and NOS and superoxide dismutase abundance increased. There was no change in the NOS inhibitor, ADMA (asymmetric dimethylarginine) or its regulatory enzymes. RAS blockade with ARB protected against tubulointerstitial injury and increased nNOSα, but ACEI, which also increased nNOSα, had no protective effect on the tubulointerstitium. We conclude that the glomerular sclerosis-resistant aged male F344xBN rat maintains renal NOS, thus reinforcing our hypothesis that progressive glomerular injury is related to renal NOS deficiency. The tubulointerstitial injury seen with aging is reversed with 6 months of ARB but not ACEI and is not associated with renal NOS.

Complete disruption of all nitric oxide synthase genes causes markedly accelerated renal lesion formation following unilateral ureteral obstruction in mice in vivo.

The role of nitric oxide (NO) derived from all three NO synthases (NOSs) in renal lesion formation remains to be fully elucidated. We addressed this point in mice lacking all NOSs. Renal injury was induced by unilateral ureteral obstruction (UUO). UUO caused significant renal lesion formation (tubular apoptosis, interstitial fibrosis, and glomerulosclerosis) in wild-type, singly, and triply NOS(-/-) mice. However, the extents of renal lesion formation were markedly and most accelerated in the triply NOS(-/-) genotype. UUO also elicited the infiltration of inflammatory macrophages, up-regulation of transforming growth factor (TGF)-ß1, and induction of epithelial mesenchymal transition (EMT) in all of the genotypes; however, the extents were again largest by far in the triply NOS(-/-) genotype. Importantly, long-term treatment with the angiotensin II type 1 (AT(1))-receptor blocker olmesartan significantly prevented the exacerbation of those renal structural changes after UUO in the triply NOS(-/-) genotype, along with amelioration of the macrophage infiltration, TGF-ß1 levels, and EMT. These results provide the first evidence that the complete disruption of all NOS genes results in markedly accelerated renal lesion formation in response to UUO in mice in vivo through the AT(1)-receptor pathway, demonstrating the critical renoprotective role of all NOSs-derived NO against pathological renal remodeling.

Pathophysiological relevance of NO signaling in the cardiovascular system: novel insight from mice lacking all NO synthases.

Nitric oxide (NO) exerts a variety of biological actions under both physiological and pathological conditions. NO is synthesized by three distinct NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS), all of which are expressed in the human cardiovascular system. The roles of endogenous NO in the cardiovascular system have been investigated in pharmacological studies with NOS inhibitors and in studies with mice that lack each NOS isoform. However, in the pharmacological studies, the specificity of the NOS inhibitors continues to be an issue of debate, while in each of the NOS isoform-deficient mice, a compensatory mechanism by other NOSs that are not genetically deleted is apparently involved. Thus, the authentic roles of endogenous NO are still poorly understood. To address this issue, genetically engineered mice in which all three NOS genes are completely disrupted have been developed. In the triply n/i/eNOS(-/-) mice, but not in singly eNOS(-/-) mice, several cardiovascular phenotypes, including arteriosclerosis/atherosclerosis, myocardial infarction, and dyslipidemia, have been described. Furthermore, by using the triply NOS(-/-) mice, the roles of the NOS system in endothelium-dependent hyperpolarization and stain-induced NO production have been elucidated. These results provide novel insight into the cardiovascular role of the endogenous NO/NOS system at the molecular level. This review, based on the research outcomes obtained from the triply NOS(-/-) genetic model, summarizes the latest knowledge of the pathophysiological relevance of NO signaling in the cardiovascular system.

High-protein-induced glomerular hyperfiltration is independent of the tubuloglomerular feedback mechanism and nitric oxide synthases.

A high protein intake is associated with increased glomerular filtration rate (GFR), which has been suggested to be mediated by reduced signaling of the tubuloglomerular feedback (TGF) mechanism. Nitric oxide (NO) has been shown to contribute to high protein-induced glomerular hyperfiltration, but the specific NO synthase (NOS) isoform responsible is not clear. In this study, a model for high-protein-induced hyperfiltration in conscious mice was developed. Using this model, we investigated the role of TGF using adenosine A(1)-receptor knockout mice lacking the TGF mechanism. Furthermore, the role of the different NOS isoforms was studied using neuronal-, inducible-, and endothelial-NOS knockout mice, and furthermore, wild-type mice acutely administered with the unspecific NOS inhibitor N(ω)-nitro-l-arginine methyl ester (100 mg/kg). GFR was measured consecutively in mice given a low-protein diet (8% casein) for 10 days, followed by a high-protein diet (50% casein) for 10 days. All mice developed high protein-induced hyperfiltration to a similar degree. These results demonstrate that high protein-induced glomerular hyperfiltration is independent of the TGF mechanism and NOS isoforms.

Polyamine transport is mediated by both endocytic and solute carrier transport mechanisms in the gastrointestinal tract.

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.

Severe dyslipidaemia, atherosclerosis, and sudden cardiac death in mice lacking all NO synthases fed a high-fat diet.

AIMS: The precise role of the nitric oxide synthase (NOS) system in lipid metabolism remains to be elucidated. We addressed this point in mice that we have recently developed and that lack all three NOS isoforms. METHODS AND RESULTS: Wild-type (WT), singly, doubly, and triply NOS(-/-) mice were fed either a regular or high-cholesterol diet for 3-5 months. The high-cholesterol diet significantly increased serum low-density lipoprotein (LDL) cholesterol levels in all the genotypes when compared with the regular diet. Importantly, when compared with the WT genotype, the serum LDL cholesterol levels in the high-cholesterol diet were significantly and markedly elevated only in the triply NOS(-/-) genotype, but not in any singly or doubly NOS(-/-) genotypes, and this was associated with remarkable atherosclerosis and sudden cardiac death, which occurred mainly in the 4-5 months after the high-cholesterol diet. Finally, hepatic LDL receptor expression was markedly reduced only in the triply NOS(-/-) genotype, accounting for the diet-induced dyslipidaemia in the genotype. CONCLUSION: These results provide the first direct evidence that complete disruption of all NOS genes causes severe dyslipidaemia, atherosclerosis, and sudden cardiac death in response to a high-fat diet in mice in vivo through the down-regulation of the hepatic LDL receptor, demonstrating the critical role of the whole endogenous NOS system in maintaining lipid homeostasis.

Roles of CYP2C29 and RXR gamma in vascular EET synthesis of female mice.

We aimed to identify which cytochrome P-450 (CYP) family/subfamily, as well as related transcription factor(s), is responsible for the estrogen-dependent synthesis of epoxyeicosatrienoic acids (EETs) to initiate shear stress-induced vasodilation. Microarray analysis indicated a significant upregulation of CYP2C29 and retinoid X receptor gamma (RXRgamma) in isolated mesenteric arteries/arterioles of female endothelial nitric oxide synthase-knockout mice, a result that was validated by real-time RT-PCR. The cannulated vessels were then perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress dose-dependently stimulated the release of EETs into the perfusate, associated with an EET-mediated vasodilation, in which predominantly 14,15-EET and 11,12-EET contributed to the responses ( approximately 87.4% of total EETs). Transfection of vessels with CYP2C29 siRNA eliminated the release of EETs into the perfusate, which was evidenced by an abolished vasodilation, and confirmed by RT-PCR and Western blot analyses. Knockdown of RXRgamma in these vessels significantly inhibited the production of EETs, parallel to a reduced vasodilation. RXRgamma siRNA not only silenced the vascular RXRgamma expression, but synchronously downregulated CYP2C29 expression, leading to a reduced EET synthesis. In conclusion, our data provide the first evidence for a specific signaling cascade, by which estrogen potentially activates the CYP2C29 gene in the absence of nitric oxide, to synthesize EETs in response to shear stress, via an RXRgamma-related regulatory mechanism.