Involvement Of Inducible Nitric Oxide Synthase Research Articles

Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells

Hydroxyurea (hydroxycarbamide, HU) arrests cells in the S-phase by inhibiting ribonucleotide reductase and DNA synthesis, significantly contributing to the release of nitric oxide (NO). We investigated the involvement of inducible NO synthase (NOS2) in the cytostatic effect of HU using in vitro shRNA-induced knockdown of the NOS2 transcript (NOS2kd) or a specific NOS2 inhibitor (1400W) in human erythroleukemic HEL92.1.7 cells, as well as murine erythroid progenitors (mERPs) from HU-treated wild-type (WT) and Nos2 knockout (Nos2–/–) mice. Over the long-term, HU increased NOS2 expression in HEL92.1.7 cells (via nuclear factor kappa B [NFκB] signaling) and in mERP. In the short-term, HU increased the activity of human recombinant and erythroleukemic cell-derived NOS2, as confirmed by NO metabolite nitrite/citrulline production. In silico molecular docking predicted that HU binds to the NOS2 active site and substrate L-arginine via hydrogen bonds. Molecular dynamics simulations showed reduced rigidity of the NOS2 active site upon interaction with HU, indicating stabilization of the enzyme-substrate complex. Both 1400W and NOS2kd prevented the in vitro reduction in proliferation and induction of apoptosis in HEL92.1.7 cells by HU. NOS2kd preferentially blocked early apoptosis and HU-induced S-phase arrest in HEL92.1.7 cells. The HU-induced decrease in proliferation and stimulation of early apoptosis in mERP were prevented in Nos2–/– mice and by 1400W in WT mice. This study demonstrated that HU induces NOS2 activity through direct interaction and increased protein expression via NFκB signaling. Moreover, NOS2 mediates the HU-induced inhibition of proliferation and stimulation of apoptosis in erythroid cells.

Poly I:C and R848 facilitate nitric oxide production via inducible nitric oxide synthase in chicks

Nitric oxide (NO) is a gaseous bioactive molecule associated with many physiological functions including vasodilation and neurotransmission. NO also plays an important role in immune responses during viral infections in mammals. However, there is a paucity of knowledge regarding the involvement of NO in viral infections in birds. Therefore, the purpose of the present study was to determine if intraperitoneal (IP) injection of poly I:C and R848 (resiquimod), which are analogues of virus component, affects NO production in chicks (Gallus gallus) as a bird model. The involvement of inducible NO synthase (iNOS) in poly I:C- and R848-induced anorexia and corticosterone release was also investigated. These virus analogues significantly increased plasma NO metabolites (NOx) concentrations. IP injection of poly I:C and R848 significantly increased iNOS mRNA expression in several organs including the liver. On the other hand, poly I:C and R848 significantly decreased mRNA expressions of endothelial NOS and neural NOS in several organs, indicating that induction of iNOS might be responsible for increased NOx levels in plasma. This finding was further confirmed by using a selective iNOS inhibitor, S-methylisothiourea sulfate (SMT), which abolished the poly I:C- and R848-induced increase in plasma NOx concentration. In addition, SMT partly attenuated the poly I:C- and R848-induced increase in plasma corticosterone concentration, suggesting that corticosterone release induced by these virus analogues may be partly mediated by iNOS. Collectively, the present results suggest that viral infections facilitate NO production by inducing iNOS. The liver would play an important role in the NO production because the response in iNOS mRNA expression to poly I:C and R848 was remarkable. The present results also suggest that NO is associated with corticosterone release in birds under viral infection.

Abstract 948: Silibinin selectively inhibits growth and progression of urethane-induced lung tumors in wild-type but not inducible nitric oxide synthase (-/-) mice: potential usefulness of micro-computed tomography scanning in lung cancer chemoprevention studies

Abstract Lung cancer is the second most common cancer but accounts for highest mortality in both men and women in the United States and Europe. The overall 5 year survival rate is still 16% underscoring the importance of new approaches to prevention and treatment of this deadly malignancy. Since sustained and excessive nitric oxide (NO) generation is positively correlated with lung cancer development and progression, recent studies have also focused on the involvement of inducible NO synthase (iNOS) and its inhibition by natural and synthetic agents for both prevention and therapy of lung cancer. Here we evaluated the role of iNOS in lung tumor occurrence and progression as well as the angiopreventive efficacy of silibinin employing B6/129J wild-type (WT) and iNOS (−/−) mice. Mice were injected with 1 mg/g body weight (i.p.) urethane once weekly for seven consecutive weeks, and thereafter were divided into two groups and gavaged with vehicle (control) or silibinin (742 mg/kg body weight) 5 days/week for 18 weeks. Using micro-computed tomography (micro-CT), we monitored the growth and progression of lung tumors from 8 through 20 weeks in WT mice. At 25 weeks, mice were sacrificed and their lung tumor multiplicity and diameters recorded. Quantification of micro-CT data showed that silibinin treatment significantly decreases urethane-induced tumor number and size in WT mice. This correlated with the decreases in tumor number and size directly measured at the termination of the study. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 89% (P<0.001) compared to WT mice, but unlike its strong chemopreventive efficacy in WT mice, silibinin treatment failed to decrease tumor number or size in iNOS (-/-) mice. Tumors in WT control mice expressed more iNOS than those in silibinin-treated mice. Further analyses of tumors from WT mice showed that compared to controls silibinin treatment significantly decreased newly formed microvessels as measured by nestin immunostaining, activation of STAT3 and NF-κB as determined by pSTAT3 (ser727) and p65NF-κB (ser276) levels, and proliferation as measured by PCNA expression. Whereas a decrease in lung tumor number in iNOS (-/-) mice compared to WT confirms previous studies that iNOS is a critical regulator of lung tumorigenesis; this study identified an essential role of iNOS in lung tumor angiogenesis. The lack of effect of silibinin in iNOS null mice suggests that silibinin exerts its angiopreventive effects at least in part through its inhibition of iNOS expression in lung tumors. Together, our results suggest iNOS as a potential target for treating lung cancer and support the value of micro-CT as a non-invasive imaging modality for evaluating the efficacy of lung cancer chemopreventive agents. (RO1 CA113876) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 948.

Activation of Cytosolic Phospholipase A2α through Nitric Oxide-induced S-Nitrosylation: INVOLVEMENT OF INDUCIBLE NITRIC-OXIDE SYNTHASE AND CYCLOOXYGENASE-2

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E(2) (PGE(2)), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA(2)alpha protein is S-nitrosylated, and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric-oxide synthase (iNOS) in human epithelial cells induced cPLA(2)alpha S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA(2)alpha activation is blocked by the specific iNOS inhibitor, 1400W. The addition of the NO donor, S-nitrosoglutathione, to isolated cell lysates or purified recombinant human cPLA(2)alpha protein induced S-nitrosylation of cPLA(2)alpha in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA(2)alpha activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA(2)alpha in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA(2)alpha is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA(2)alpha S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE(2) production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2-fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA(2)alpha S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA(2)alpha, and COX-2. Since COX-2 enhances the formation of cPLA(2)alpha-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA(2)alpha S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA(2)alpha. These findings disclose a novel link among cPLA(2)alpha, iNOS, and COX-2, which form a multiprotein complex leading to cPLA(2)alpha S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE(2) production and prevent inflammation and carcinogenesis.

Effects of dexamethasone andl-canavanine on the intracellular calcium-contraction relation of the rat tail artery during septic shock

The intracellular mechanism by which sepsis lowers vascular reactivity and the subsequent reversal by dexamethasone or nitric oxide synthase (NOS) inhibitors remain unclear. We measured the sensitivity of contraction of the rat tail artery to intracellular Ca2+ in a model of polymicrobial septic shock. At 22 h after cecal ligation and puncture (CLP), rats were treated with an anti-inflammatory glucocorticoid (dexamethasone, 1 mg/kg ip), an inducible NOS inhibitor (L-canavanine, 100 mg/kg ip), or saline. At 24 h after CLP, endothelium-denuded, perfused segments of tail artery were loaded with the intracellular Ca2+-sensitive dye fura 2 in vitro. Intracellular Ca2+ concentration and perfusion pressure were measured simultaneously. The rightward shift of the perfusion pressure-intracellular Ca2+ mobilization curve after norepinephrine stimulation subsequent to CLP indicates decreased intracellular Ca2+ sensitivity of contraction. The relation was restored by dexamethasone (which also restored in vivo blood pressure and flow), but not by L-canavanine (which restored perfusion pressure by further mobilization of intracellular Ca2+). We conclude that CLP lowers vasomotion by lowering intracellular Ca2+ sensitivity, which can be restored with glucocorticoid treatment. The involvement of inducible NOS does not solely account for the sepsis-induced reduction in Ca2+ sensitivity of contraction.

Nitric oxide metabolites in the cisternal cerebral spinal fluid of patients with subarachnoid hemorrhage.

To investigate nitric oxide (NO) metabolism after subarachnoid hemorrhage (SAH). We measured the concentrations of the NO metabolites, nitrite and nitrate, in cerebrospinal fluid (CSF) obtained from the cisternal drainage of patients with SAH. Studies were performed for 31 patients who had undergone surgical obliteration of bleeding aneurysms within 3 days of their hemorrhage. The concentrations of nitrite and nitrate in the CSF were measured for 14 days using a nitrate/nitrite kit and samples that were obtained on a daily basis from the cisternal drainage. Compared with the control values in the CSF (2.6 +/- 0.4 mumol/L, n = 14) obtained from patients with hemifacial spasm, trigeminal neuralgia, or nonruptured aneurysms, the concentrations of nitrite and nitrate in the CSF were significantly elevated in the acute stage of SAH and remained elevated. The concentration of NO metabolites may correlate with the amount of bleeding, inasmuch as the values in patients in Fisher Group 3 (n = 25) were higher than those in patients in Fisher Group 2 (n = 6). The concentration of nitrate was higher than that of nitrite, suggesting that NO in the subarachnoid space is mainly absorbed by hemoglobin and degraded to nitrate. No differences were demonstrated in patients treated with high doses of methylprednisolone (n = 17) compared with those treated with usual-dose steroids (n = 14). Steroids are known to prevent the formation of inducible NO synthase mediated by inflammatory cytokines. NO metabolism in the brain is stimulated after SAH. Nitrate is the dominant NO metabolite in CSF after SAH. The involvement of inducible NO synthase in the pathophysiology of NO metabolism after SAH was not clearly suggested based on the present data.