Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

A comparative study of neuronal and inducible nitric oxide synthases: generation of nitric oxide, superoxide, and hydrogen peroxide

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (.NO), superoxide (O2.-), and hydrogen peroxide (H2O2). Since .NO has been implicated in many physiological processes, the importance of O2.- and H2O2 in regulating cell signaling by .NO cannot be overlooked. Before addressing these questions, we investigated the production of .NO, O2.-, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of .NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of .NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2.- and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production of .NO, O2.-, and H2O2 may define the specific physiologic function of each isozyme.     

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.     

Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants

Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.     

Contribution of inducible and neuronal nitric oxide synthases to mitochondrial damage and melatonin rescue in LPS-treated mice

NOS isoform activation is related to liver failure during sepsis, but the mechanisms driving mitochondrial impairment remain unclear. We induced sepsis by LPS administration to inducible nitric oxide synthase (iNOS^?/?) and neuronal nitric oxide synthase (nNOS^?/?) mice and their respective wild-type controls to examine the contribution of iNOS to mitochondrial failure in the absence of nNOS. To achieve this goal, the determination of messenger RNA (mRNA) expression and protein content of iNOS in cytosol and mitochondria, the mitochondrial respiratory complex content, and the levels of nitrosative and oxidative stress (by measuring 3-nitrotyrosine residues and carbonyl groups, respectively) were examined in the liver of control and septic mice. We detected strongly elevated iNOS mRNA expression and protein levels in liver cytosol and mitochondria of septic mice, which were related to enhanced oxidative and nitrosative stress, and with fewer changes in respiratory complexes. The absence of the iNOS, but not nNOS, gene absolutely prevented mitochondrial impairment during sepsis. Moreover, the nNOS gene did not modify the expression and the effects of iNOS here shown. Melatonin administration counteracted iNOS activation and mitochondrial damage and enhanced the expression of the respiratory complexes above the control values. These effects were unrelated to the presence or absence of nNOS. iNOS is a main target to prevent liver mitochondrial impairment during sepsis, and melatonin represents an efficient antagonist of these iNOS-dependent effects whereas it may boost mitochondrial respiration to enhance liver survival.     

Constitutive and inducible nitric oxide synthases incorporate molecular oxygen into both nitric oxide and citrulline.

Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO.     

Influenza virus-induced sleep responses in mice with targeted disruptions in neuronal or inducible nitric oxide synthases.

Influenza viral infection induces increases in non-rapid eye movement sleep and decreases in rapid eye movement sleep in normal mice. An array of cytokines is produced during the infection, and some of them, such as IL-1beta and TNF-alpha, are well-defined somnogenic substances. It is suggested that nitric oxide (NO) may mediate the sleep-promoting effects of these cytokines. In this study, we use mice with targeted disruptions of either the neuronal NO synthase (nNOS) or the inducible NO synthase (iNOS) gene, commonly referred to as nNOS or iNOS knockouts (KOs), to investigate sleep changes after influenza viral challenge. We report that the magnitude of viral-induced non-rapid eye movement sleep responses in both nNOS KOs and iNOS KOs was less than that of their respective controls. In addition, the duration of rapid eye movement sleep in nNOS KO mice did not decrease compared with baseline values. All strains of mice had similar viral titers and cytokine gene expression profiles in the lungs. Virus was not isolated from the brains of any strain. However, gene expression in the brain stem differed between nNOS KOs and their controls: mRNA for the interferon-induced gene 2',5'-oligoadenylate synthase 1a was elevated in nNOS KOs relative to their controls at 15 h, and IL-1beta mRNA was elevated in nNOS KOs relative to their controls at 48 h. Our results suggest that NO synthesized by both nNOS and iNOS plays a role in virus-induced sleep changes and that nNOS may modulate cytokine expression in the brain.     

Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase

Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation.     

Multimodality imaging of nitric oxide and nitric oxide synthases

Nitric oxide (NO) and NO synthases (NOSs) are crucial factors in many pathophysiological processes such as inflammation, vascular/neurological function, and many types of cancer. Noninvasive imaging of NO or NOS can provide new insights in understanding these diseases and facilitate the development of novel therapeutic strategies. In this review, we will summarize the current state-of-the-art multimodality imaging in detecting NO and NOSs, including optical (fluorescence, chemiluminescence, and bioluminescence), electron paramagnetic resonance (EPR), magnetic resonance (MR), and positron emission tomography (PET). With continued effort over the last several years, these noninvasive imaging techniques can now reveal the biodistribution of NO or NOS in living subjects with high fidelity which will greatly facilitate scientists/clinicians in the development of new drugs and/or patient management. Lastly, we will also discuss future directions/applications of NO/NOS imaging. Successful development of novel NO/NOS imaging agents with optimal in vivo stability and desirable pharmacokinetics for clinical translation will enable the maximum benefit in patient management.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

Mammalian nitric oxide synthases.

The nitric oxide (NO) synthase family of enzymes generate NO from L-arginine, which acts as a biologic effector molecule in a broad number of settings. This report summarizes some of the current information regarding NO synthase structure-function, reaction mechanism, control of catalysis, and protein interactions.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Regulation of mammalian nitric oxide synthases by electrostatic interactions in the linker region of calmodulin

Calmodulin (CaM), the ubiquitous Ca(2+)-sensing protein, consists of two globular domains separated by a flexible central linker that properly orients CaM's globular domains to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. In the present study we determined that the charge and length of the central linker of CaM has an effect on the binding and activation of the NOS isozymes by using a variety of charge CaM mutants (T79D, S81D, T79D/S81D, S101D and E84R/E87K) and CaM mutants with residues removed (Delta84, Delta83-84, and Delta81-84). Our kinetic and spectropolarimetry results demonstrate that the NOS enzymes are not adversely affected by the CaM mutants with the exceptions of S101D, E84R/E87K and the deletion of residue 84. Electrostatic interactions in the central linker between residues 82-87 in combination with hydrophobic interactions in the globular domains of CaM are important for its tight association to inducible NOS.     

Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.     

Radical reactions of nitric oxide synthases

NOSs (nitric oxide synthases) are flavohaem enzymes that function broadly in human health and disease. We are combining mutagenesis, crystallographic and rapid kinetic methods to understand their mechanism and regulation. The NOSs create a transient tetrahydrobiopterin radical within the enzyme to generate their free radical product (NO). Recent work is revealing how critically important this process is at all levels of catalysis. This article will synthesize four seemingly disparate but related aspects of NOS tetrahydrobiopterin radical formation: (i) how it enables productive O2 activation by providing an electron to the enzyme haem, (ii) what structural features help to regulate this electron transfer, (iii) how it enables NOS to synthesize NO from its diamagnetic substrate and (iv) how it allows NOS to release NO after each catalytic cycle instead of other nitorgen oxide-containing products.     

Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-kB activation

Artemisinin is a natural product used as an alternative drug in the treatment of severe and multidrug-resistant malaria. In the present work we show that artemisinin shares with other sesquiterpene lactones the ability to inhibit the activation of the nuclear factor NF-kB: by this mechanism, artemisinin, as well as parthenolide, inhibits nitric oxide synthesis in cytokine-stimulated human astrocytoma T67 cells. These results suggest that artemisinin, in addition to its antiparasitic properties, could also exert a therapeutic effect on neurological complications of malaria.     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。