Nyoun Soo Kwon

Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs.

The cofactor requirements of macrophage nitric oxide (NO·) synthase suggest involvement of an NADPH‐dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di‐2‐thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO· synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO· synthesis by mouse macrophages, their lysates, and partially purified macrophage NO· synthase. Inhibition of NO· synthase by these agents was potent (IC50‘s 50–150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 25‘‐ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5‐diphosphoribose, protected NO· synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO· synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine‐induced relaxation of norepinephrine‐preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine‐induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine‐induced vasoconstriction itself nor on vasorelaxation induced by the NO·‐generating agent sodium nitroprusside. These results suggest that NO∗ synthesis in both macrophages and endothelial cells depends on an NADPH‐utilizing flavoprotein. As a new class of NO· synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO·.—Stuehr, D. J.; Fasehun, O. A.; Kwon, N. S.; Gross, S. S.; Gonzalez, J. A.; Levi, R.; Nathan, C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 5: 98–103; 1991.

FAD and GSH participate in macrophage synthesis of nitric oxide

Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.

Protection from Alzheimer's-like disease in the mouse by genetic ablation of inducible nitric oxide synthase

Brains from subjects who have Alzheimers disease (AD) express inducible nitric oxide synthase (iNOS). We tested the hypothesis that iNOS contributes to AD pathogenesis. Immunoreactive iNOS was detected in brains of mice with AD-like disease resulting from transgenic expression of mutant human β-amyloid precursor protein (hAPP) and presenilin-1 (hPS1). We bred hAPP-, hPS1-double transgenic mice to be iNOS+/+ or iNOS−/−, and compared them with a congenic WT strain. Deficiency of iNOS substantially protected the AD-like mice from premature mortality, cerebral plaque formation, increased β-amyloid levels, protein tyrosine nitration, astrocytosis, and microgliosis. Thus, iNOS seems to be a major instigator of β-amyloid deposition and disease progression. Inhibition of iNOS may be a therapeutic option in AD.

Synthesis of nitrogen oxides from L-arginine by macrophage cytosol: Requirement for inducible and constitutive components

Cytosols prepared from murine peritoneal macrophages and the RAW 264 macrophage cell line catalyzed conversion of L-arginine to the labile vaso-relaxant nitric oxide and its accumulating endproducts, nitrite and nitrate. This activity required previous exposure of the cells to interferon-gamma and bacterial lipopolysaccharide. Nitrogen oxide synthetase activity was characterized further using nitrite + nitrate production as an indicator of the synthesis of all three nitrogen oxides. Nitrogen oxide synthetase activity was heat-sensitive, NADPH-dependent, and exhibited substrate stereospecificity. The nitrite + nitrate formation was proportional to time and concentration of cytosol. However, dilution decreased the specific activity, suggesting a cofactor requirement in addition to NADPH. Specific activity was restored by addition of cytosol from non-activated macrophages, which itself did not make nitric oxide. Both high and low molecular weight fractions of control macrophage cytosol were required to restore activity of cytosol from activated macrophages that had been either diluted or partially purified. Thus, the enzymatic system involved in nitric oxide synthesis by murine macrophages consists of at least one inducible and two constitutive components.

Nitric oxide generation from streptozotocin.

Streptozotocin (STZ), a diabetogenic agent, is thought to damage pancreatic β‐cells by activating immune mechanisms and by alkylating DNA. In the present study, we demonstrated that STZ can produce nitric oxide (NO), a bioregulatory and cytotoxic molecule. When STZ was dissolved in a sodium phosphate buffer (50 mM, pH 7.4) and irradiated with a 22 W circular fluorescent light, nitrite and nitrate, stable oxidation products of NO, were produced. The wavelengths of light most responsible for the photo‐decomposition were 300‐310 nm and 410‐420 nm. When a mixture of reduced hemoglobin and STZ was irradiated with UV light (280‐320 nm), hemoglobin underwent characteristic NO‐dependent spectral changes. STZ relaxed de‐endothelialized aortic strips only in the presence of light. STZ/light‐dependent relaxation was attenuated by reduced hemoglobin. These results indicated photo‐induced NO production from STZ. NO generation depended on the concentration of STZ, the duration of irradiation, and the distance between sample and light source. In acidic conditions, NO production from STZ was spontaneous even in the dark. Light‐independent NO generation was augmented by increasing acidity, and markedly diminished in a D2O‐based buffer, indicating the involvement of protons in the mechanism of STZ decomposition in acid. These results imply the usefulness of STZ as an NO‐generating reagent, and indicate that direct NO‐generation may be a mechanism of STZ toxicity in diabetogenesis.—Kwon, N. S., Lee, S. H., Choi, C. S., Kho, T., Lee, H. S. Nitric oxide generation from streptozotocin. FASEB J. 8: 529‐533; 1994.

Mutational Analysis of the Tetrahydrobiopterin-binding Site in Inducible Nitric-oxide Synthase

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.

Involvement of p38 mitogen-activated protein kinase and apoptosis signal-regulating kinase-1 in nitric oxide-induced cell death in PC12 cells

Although nitric oxide (NO) plays key signaling roles in the nervous systems, excess NO leads to cell death. In this study, the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and apoptosis signal-regulating kinase-1 (ASK1) in NO-induced cell death was investigated in PC12 cells. NO donor transiently activated p38 MAPK in the wild type parental PC12 cells, whereas the p38 MAPK activation was abolished in NO-resistant PC12 cells (PC12-NO-R). p38 MAPK inhibitors protected the cells against NO-induced death, whereas the inhibitors were not significantly protective against the cytotoxicity of reactive oxygen species. Stable transfection with dominant negative p38 MAPK mutant reduced NO-induced cell death. Stable transfection with dominant negative mutant of ASK1 attenuated NO-stimulated activation of p38 MAPK and decreased NO-induced cell death. These results suggest that p38 MAPK and its upstream regulator ASK1 are involved in NO-induced PC12 cell death.

Sphingosylphosphorylcholine inhibits melanin synthesis via pertussis toxin-sensitive MITF degradation

Objectives Sphingolipids act as structural components in cell membranes, and form lipid intermediates that have functional roles as signalling molecules in various cellular processes. Our previous findings have suggested that sphingolipid metabolites are deeply involved in the regulation of melanogenic processes. In this study we aimed to examine sphingosylphosphorylcholine‐mediated signalling pathways related to melanogenesis.

Leucine-rich glioma inactivated 3 associates with syntaxin 1.

Leucine-rich glioma inactivated 3 (LGI3) is a member of LGI/epitempin (EPTP) family. The biological function of LGI3 and its association with disease are not known. We previously reported that mouse LGI3 was highly expressed in brain in a developmentally and transcriptionally regulated manner. In this study, we identified syntaxin 1, a SNARE component in exocytosis, as a candidate functional target of LGI3. Western blot analysis of mouse brain extract with LGI3 antibodies detected multiple protein forms (75-, 60-, 35- and 25-kDa). Proteomic analysis, pull-down and coimmunoprecipitation experiments identified syntaxin 1 as an LGI3-associated protein. LGI3 colocalized with syntaxin 1 in processes of cortical neurons with punctate synaptic pattern and was enriched in synaptosomal fraction. Coimmunoprecipitation showed that LGI3-syntaxin 1 complex did not contain other SNARE components, SNAP25 and VAMP2. Recombinant LGI3 attenuated Ca(2+)-evoked glutamate release from digitonin-permeabilized synaptosomes and transfection of PC12 cells with LGI3 decreased K(+)-induced secretion of human growth hormone. Thus, LGI3 may play a regulatory role in neuronal exocytosis via its interaction with syntaxin 1.

Leucine-rich glioma inactivated 3 induces neurite outgrowth through Akt and focal adhesion kinase.

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein that belongs to LGI/epitempin family. LGI3 is highly expressed in brain in a transcriptionally and developmentally regulated manner. Here we found that LGI3 induced neurite outgrowth in Neuro-2a cells and dorsal root ganglia explants. LGI3 treatment or overexpression increased neurite outgrowth and knockdown of LGI3 by siRNA had opposite effect. LGI3 treatment increased phosphorylation of Akt and a 125-kDa protein. Immunoprecipitation identified the 125-kDa protein as focal adhesion kinase (FAK). LGI3 overexpression increased phospho-Akt, phospho-FAK and FAK protein. Inhibition of Akt activation by PI3 kinase inhibitor attenuated LGI3-induced FAK phosphorylation and neurite outgrowth. Taken together, we propose that LGI3 is a neuritogenic factor whose signaling pathway involves Akt-mediated FAK activation.