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Ca2+/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell line ME-180.

ABSTRACT: We show here that the human cervix carcinoma cell line ME-180 expresses a constitutive nitric oxide (NO) synthase, as demonstrated by formation of [3H]citrulline and nitrite. The enzyme is dependent on tetrahydrobiopterin, NADPH, flavins and Ca2+/calmodulin. Enzyme activity is located in the cytosol rather than in the membrane fraction and can be inhibited by NG-monomethyl-L-arginine (NMMA). An antiserum to NO synthase purified from porcine cerebellum inhibited the enzyme activity. ME-180 cells released NO, as was shown by stimulation of guanylate cyclase (EC 4.6.1.2) in RFL-6 detector cells; this release was stimulated 8-fold by the Ca2+ ionophore A23187 and 2-fold by increasing the intracellular tetrahydrobiopterin levels with cytokines. This is the first characterization of a Ca2+/calmodulin-dependent NO synthase activity in human epithelial-type tumour cells.

SUBMITTER: Werner-Felmayer G

PROVIDER: S-EPMC1132175 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase.

Project description:L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with half-maximal effects at 11 microM and 0.3 microM respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. N omega-Nitro-L-arginine, its methyl ester and N omega-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 microM) with half-maximal effects at 0.74 microM, 2.8 microM and 15 microM respectively. The N omega-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N omega-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2+ at subphysiological levels of intracellular L-arginine or H4biopterin may result in the formation of reactive oxygen species instead of NO, and N omega-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.

| S-EPMC1130735 | biostudies-other

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Project description:Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.

| S-EPMC40926 | biostudies-other

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I.

Project description:The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

| S-EPMC2198825 | biostudies-literature

Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase.

Project description:Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr-calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.

| S-EPMC7039552 | biostudies-literature

Calmodulin-induced structural changes in endothelial nitric oxide synthase.

Project description:We have derived structures of intact calmodulin (CaM)-free and CaM-bound endothelial nitric oxide synthase (eNOS) by reconstruction from cryo-electron micrographs. The CaM-free reconstruction is well fitted by the oxygenase domain dimer, but the reductase domains are not visible, suggesting they are mobile and thus delocalized. Additional protein is visible in the CaM-bound reconstruction, concentrated in volumes near two basic patches on each oxygenase domain. One of these corresponds with a presumptive docking site for the reductase domain FMN-binding module. The other is proposed to correspond with a docking site for CaM. A model is suggested in which CaM binding and docking position the reductase domains near the oxygenase domains and promote docking of the FMN-binding modules required for electron transfer.

| S-EPMC3569036 | biostudies-literature

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis.

Project description:The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [(32)P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC. Phosphorylation of CaM by purified CK2 in vitro reduces the V(max) of immunopurified eNOS by a factor of 2 but has no effect on the K(A) for CaM or calcium. Additionally, [(32)P]orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM ("phosphonull" S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the "phosphomimetic" Ser-81 to aspartate (S81D) mutant is unchanged. Further studies using Escherichia coli-expressed and phenyl-Sepharose-purified CaM mutants revealed that the S81A mutation abrogates in vitro CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved. Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS. Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue. We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses.

| S-EPMC337024 | biostudies-literature

Correction: Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase.

Project description:[This corrects the article DOI: 10.1371/journal.pone.0121782.].

| S-EPMC7546496 | biostudies-literature

Structural basis for endothelial nitric oxide synthase binding to calmodulin.

Project description: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.

| S-EPMC145438 | biostudies-literature

Nitric oxide synthase-dependent SNOome identification

Project description:This study aimed to identify proteins whose S-nitrosylation is mediated by each of the three human NOS isoforms.

2023-06-01 | PXD040633 | Pride

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation.

Project description:Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several high-resolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogen-deuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen-deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.

| S-EPMC3780838 | biostudies-literature

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