Co-induction of nitric oxide and tetrahydrobiopterin synthesis in the myocardium in vivo

Induction of the inducible isoform of nitric oxide (NO) synthase (iNOS) in the myocardium is implicated as a mechanism in the development of cardiac depression in immune activated states associated with an enhanced release of cytokines, such as septic shock. We evaluated the in vivo synthesis of NO and tetrahydrobiopterin (BH4), a cofactor of NOS, in the heart tissue using a model of LPS injection in rats (LPS: 10 mg/kg, i.v.). In control rats, iNOS activity or iNOS mRNA in the heart was negligible. Three hours after LPS administration, a marked induction of iNOS mRNA and activity was observed in the heart. A significant increase in BH4 content and GTP cyclohydrolase mRNA abundance was also observed in the heart from LPS-treated rats. Our results demonstrate induction of NO synthesis and parallel increase in BH4 concentration in the heart of rats after LPS treatment in vivo and may provide molecular evidence responsible for the increased production of BH4 which may up-regulate iNOS activity in the heart in vivo. (Mol Cell Biochem 166: 177-181, 1997)     

Inducible nitric oxide synthase expression is inhibited by myeloperoxidase.

Nitric oxide (NO) plays key roles in vasodilation and host defense, yet the overproduction of NO by inducible nitric oxide synthase (iNOS) at inflammatory sites can also be pathogenic. Here, we investigate the role of MPO in modulating the induction of iNOS by IFNgamma/LPS (IL). In monocyte-macrophages (Mvarphi) treated with IL, MPO gene expression was found to be downregulated as iNOS was upregulated. In Mvarphi from MPO-knockout (KO) mice, the induction of iNOS by IL was earlier and higher than in MPO-positive cells, suggesting MPO is inhibitory. Consistent with that interpretation, the addition of purified MPO enzyme to cultured macrophages inhibited iNOS induction by IL. In addition, an inhibitor of MPO enzyme, 4-aminobenzohydrazide, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. Similarly, taurine, a scavenger of MPO-generated HOCl, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. MPO affects an early event, suppressing iNOS induction when added within 2h of IL, but not when added several hours after IL. The suppression by MPO was alleviated by NO donor, sodium nitroprusside, suggesting the suppression results from scavenging of NO by MPO. This interpretation is consistent with earlier reports that MPO consumes NO, and that low levels of NO donor augment induction of iNOS by IFNgamma/LPS. The implication of these findings is that MPO acts as gatekeeper, suppressing the deleterious induction of iNOS at inflammatory sites by illegitimate signals. The combined signaling of IFNgamma/LPS overrides the gatekeeper function by suppressing MPO gene expression.     

Up-regulation of rat adrenomedullin gene expression by endotoxin: Relation to nitric oxide synthesis

The effect of bacterial endotoxin (LPS) on adrenomedullin (AM) gene expression was investigated in cultured rat aortic vascular smooth muscle (VSM) cells and in tissues from anesthetized rats. The addition of LPS together with interferon-y to VSM cells resulted in a marked increase in the abundance of AM mRNA as well as the appearance of mRNA for the inducible isoform of nitric oxide (NO) synthase (iNOS). Intravenous injection of LPS into rats also increased AM mRNA abundance and induced iNOS mRNA in lung, heart, liver, and kidney. AM significantly enhanced NO synthesis evoked by LPS and interferon-v in cultured VSM cells. These data suggest that AM may contribute to circulatory failure during endotoxin shock, in part, by modulating NO synthesis.     

Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.     

Antifibrotic role of inducible nitric oxide synthase.

Long-term treatment in rats with l-NAME, an isoform-non-specific inhibitor of nitric oxide synthase (NOS), leads to fibrosis of the heart and kidney, suggesting that nitric oxide (NO) may play a role in preventing tissue fibrosis. In this process, a likely target of NO is the quenching of reactive oxygen species (ROS) through peroxynitrite formation, and one possible source for this NO is inducible NOS (iNOS). Using Peyronie's disease (PD) tissue from both human specimens and from a rat model of PD as the source of fibrotic tissue, we investigated if NO derived from iNOS could act as such an antifibrogenic defense mechanism by determining whether: (a) tunical ROS and iNOS are increased in PD; and (b) the long-term inhibition of iNOS activity decreases the NO/ROS balance in the tunica albuginea thereby promoting collagen deposition. It was determined that in the human PD plaque, iNOS mRNA and protein, ROS, collagen, and the peroxynitrite marker, nitrotyrosine, were all increased in comparison to the normal tunica. In the rat model of PD, the fibrotic plaque also showed significant increases in iNOS mRNA and protein, nitrotyrosine, ROS as measured by heme oxygenase-1, and collagen when compared with the normal control tunica. When a selective inhibitor of iNOS, L-NIL, was given to rats with the PD-like plaque, this resulted in a decrease in nitrotyrosine levels but intensified ROS levels and collagen deposition. These data demonstrate that: (a) iNOS induction occurs in both the human and rat PD fibrotic plaque; and (b) that the NO derived from iNOS appears to counteract ROS formation and collagen deposition. Because the inhibition of iNOS activity leads to a decrease in the NO/ROS ratio, thereby favoring the development of fibrosis, it is proposed that iNOS induction in this tissue may be a protective mechanism against fibrosis and abnormal wound healing.     

autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.     

Urotensin-II activates L-arginine/nitric oxide pathway in isolated rat aortic adventitia

Urotensin-II (U-II), a cyclic peptide widely expressed in blood vessels, has diverse vascular actions that range from potent vasoconstriction to vasodilation. Although, U-II-induced vasodilation has been shown to be partially dependent on nitric oxide (NO), the involvement of vascular adventitia-derived NO, remains unknown. The present study aimed to elucidate the activation of U-II on L-arginine/NO pathway in isolated rat aortic adventitia. In adventitia of thoracic and abdominal aortas, the l-arginine/NO pathway was similarly characterized: the uptake of l-[(3)H]arginine was Na(+)-independent, with the peak occurring over around 40 min incubation; the total NO synthase (NOS) activity was mostly calcium-independent (>90%), and significantly inhibited by a specific iNOS inhibitor AMT; the production of NO metabolites nitrate and nitrite (NO(x)) was stimulated by L-arginine but not by D-arginine. In aortic adventitia exposed to rat U-II (10(-9) and 10(-8)M) for 6 h, the V(max) of l-[(3)H]arginine uptake over 40 min incubation was significantly increased, while the K(m) of l-[(3)H]arginine uptake showed no significant change. Besides, the iNOS mRNA level was up-regulated, the total NOS activity, largely calcium-independent, was significantly induced, and the NO(x) production was significantly stimulated by U-II. According to the same protocol as U-II, the positive control lipopolysaccharide (LPS, 10 microg/ml), which had been established to activate adventitial L-arginine/NO pathway, increased l-[(3)H]arginine uptake, iNOS activity and NO(x) production to a greater extent than U-II. In addition, the total NOS activities induced by 3 and 6h incubation of U-II and LPS were significantly inhibited by a specific inhibitor of protein synthesis, actinomycin D. In conclusion, the results showed that rat U-II activated L-arginine/NOS/NO pathway in rat aortic adventitia, suggesting a potential contributive role of adventitia-derived NO in the vasodilator response of U-II.     

Ascorbate enhances iNOS activity by increasing tetrahydrobiopterin in RAW 264.7 cells

Studies on the effect of ascorbic acid on inducible nitric oxide synthase (iNOS) activity are few and diverse, likely to be dependent on the species of cells. We investigated a role of ascorbic acid in iNOS induction and nitric oxide (NO) generation in mouse macrophage cell line RAW 264.7. Although interferon- (IFN-) gamma alone produced NO end products, ascorbic acid enhanced NO production only when cells were synergistically stimulated with IFN-gamma plus Escherichia coli lipopolysaccharide (LPS). Ascorbate neither enhanced nor decreased the expression of iNOS protein in RAW 264.7 cells, in contrast to the reports that ascorbic acid augments iNOS induction in a mouse macrophage-like cell line J774.1 and that ascorbate suppresses iNOS induction in rat skeletal muscle endothelial cells. Intracellular levels of tetrahydrobiopterin (BH4), a cofactor for iNOS, were increased by ascorbate in RAW 264.7 cells. However, ascorbate did not increase GTP cyclohydrolase I mRNA, the main enzyme at the critical steps in the BH4 synthetic pathway, expression levels and activity. Sepiapterin, which supplies BH4 via salvage pathway, more efficiently enhanced NO production if ascorbate was added. These data suggest that enhanced activation of iNOS by ascorbic acid is mediated by increasing the stability of BH4 in RAW 264.7 cells.     

Interferon-gamma plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus

Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into the rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N(G)-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUNEL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS. Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.     

Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment

In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.     

Cytokine- and Endotoxin-Induced Nitric Oxide Synthase in Rat Astroglial Cultures: Differential Modulation by Angiotensin II

Abstract: Recent studies have shown that the stimulatory effects of bacterial endotoxin [lipopolysaccharide (LPS)] on inducible nitric oxide (NO) synthase (iNOS) in astroglia are significantly reduced by the peptide angiotensin II (Ang II). In the present study we have compared the modulatory actions of Ang II on cytokine- and LPS-stimulated iNOS in astroglia cultured from adult rat brain. Incubation of astroglia with LPS (100 ng/ml; 24 h) and/or combinations of interleukin-1β (IL-1β; 10 ng/ml, 24 h), interferon-γ (IFN-γ; 100 U/ml, 24 h), or tumor necrosis factor-α (TNF-α; 100 ng/ml, 24 h) resulted in significant increases of iNOS mRNA, iNOS protein, and NO production, with the latter indicated by increased nitrite accumulation. The effects of LPS, IL-1β, and TNF-α were significantly decreased by coincubation with Ang II (100 ng/ml, 24 h). In contrast, Ang II did not alter the stimulation of iNOS mRNA levels and NO production elicited by IFN-γ. Therefore, Ang II differentially modulates the stimulatory actions of LPS and cytokines on iNOS, and subsequently NO production, in astroglia. These data suggest that Ang II may have an important modulatory role in intracerebral immune responses that involve production of NO by astroglia.