정자 운동성에 대한 Nitric Oxide와 Peroxynitrite의 효과

정헌영,서기석,임정식 大韓泌尿器科學會 1998 Korean Journal of Urology Vol.39 No.9

Involvement of Peroxynitrite in NO Donor-Induced HO-1 Expression in Rat Articular Chondrocytes

Ju Dong Song(송주동),Kang Mi Kim(김강미),Jong Min Kim(김종민),Young Hyun Yoo(유영현),Young Chul Park(박영철) 한국생명과학회 2011 생명과학회지 Vol.21 No.4

Nitric oxide (NO) donors는 heme oxygenase-1 (HO-1)의 강력한 유도제이다. 그러나 NO donors에 의한 HO-1의 발현이 NO donor에 의해 방출되는 NO에 의한 직접적인 영향인지는 불분명하다. 본 연구에서 흰쥐의 무릎으로부터 분리 배양한 관절연골세포에서 HO-1의 발현에 NO donors의 영향을 조사하였다. NO donors(SIN-1, SNAP 그리고 SNP)는 HO-1의 mRNA와 단백질의 합성을 크게 증가시켰다. 그리고 NO의 표적 분자인 guanylate cyclase와 protein kinase G의 관련성을 살펴본 결과, NO donors에 의한 Nrf2와 HO-1의 발현증가와는 무관한 것으로 보였다. 흥미롭게도, NO scavenger인 carboxy-PTIO와 SOD mimetic TEMPOL은 NO donors에 의한 HO-1의 발현을 억제하였다. 게다가, peroxynitrite scavenger인 MnTBAP에 의해서도 Nrf2와 HO-1의 발현이 완전히 억제되었다. Peroxynitrite는 NO와 superoxide의 반응에 의해 세포 내에서 자연적으로 형성되는 물질이므로 peroxynitrite가 관절연골세포에서 HO-1의 발현에 직접적인 영향을 주는지를 관찰하였다. 관절연골세포에 peroxynitrite를 처리한 결과, 시간과 농도 의존적으로 Nrf2와 HO-1의 발현을 크게 증가시켰다. 본 실험 자료는 NO donors에 의한 HO-1의 발현증가는 방출되는 NO의 직접적인 영향이라기 보다는 NO와 superoxide의 반응으로 형성되는 peroxynitrite에 의해 유도된다는 것을 시사한다. Nitric oxide (NO) donors are a potent inducer of heme oxygenase-1 (HO-1). However, it is unclear whether or not HO-1 expression induced by NO donors is a direct consequence of NO released by NO donors. Here, we investigated the effects of NO donors on the expression of HO-1 in primary rat articular chondrocytes. NO donors (SIN-1, SNAP, and SNP) significantly induced the accumulation of HO-1 protein accompanied by an increase in HO-1 mRNA. NO donor-induced HO-1 expression exerted cytoprotection against NO and/or superoxide-induced cell death. Guanylate cyclase signaling was not associated with Nrf2 and HO-1 expression in NO donor-treated chondrocytes. Interestingly, NO scavenger carboxy-PTIO and SOD mimetic TEMPOL markedly inhibited NO donor-induced HO-1 expression in chondrocytes. In addition, NO donor-induced HO-1 expression was completely abrogated by the peroxynitrite scavenger MnTBAP. Since peroxynitrite can be physiologcally formed in the cell through reaction of NO with superoxide, we analyzed whether or not peroxynitrite could directly induce HO-1 expression in chondrocytes. Peroxynitrite treatment in chondrocytes evoked dose-and time-dependent Nrf2 and HO-1 expression. These results indicate that HO-1 expression induced by NO donors in rat articular chondrocytes is due to NO-mediated peroxynitrite rather than NO.

Peroxynitrite Inactivates Carbonic Anhydrase II by Releasing Active Site Zinc Ion

Kim, Young-Mi,Han, Sang-Hwa Korean Chemical Society 2004 Bulletin of the Korean Chemical Society Vol.25 No.5

Peroxynitrite enters erythrocytes through band 3 anion exchanger and oxidizes cytosolic proteins therein. As a protein associated with band 3, carbonic anhydrase II may suffer from peroxynitrite-induced oxidative damages. Esterase activity of carbonic anhydrase II decreased as the concentration of peroxynitrite increased. Neither hydrogen peroxide nor hypochlorite affected the enzyme activity. Inactivation of the enzyme was in parallel with the release of zinc ion, which is a component of the enzyme's active site. SDS-PAGE of peroxynitrite-treated samples showed no indication of fragmentation but non-denaturing PAGE exhibited new bands with lower positive charges. Western analysis demonstrated that nitration of tyrosine residues increased with the peroxynitrite concentration but the sites of nitration could not be determined. Instead MALDI-TOF analysis identified tryptophan-245 as a site of nitration. Such modification of tryptophan residues is responsible for the decrease in tryptophan fluorescence. These results demonstrate that peroxynitrite nitrates tyrosine and tryptophan residues of carbonic anhydrase II without causing fragmentation or dimerization. The peroxynitrite-induced inactivation of the enzyme is primarily due to the release of zinc ion in the enzyme's active site.

실험적 삼출성 중이염에서 점액섬모 수송 기능에 대한 Nitric Oxide와 Peroxynitrite의 작용

전은주,박용수,채세용,여상원,장기홍,박소영 대한이비인후과학회 2003 대한이비인후과학회지 두경부외과학 Vol.46 No.9

Background and Objectives:Nitric oxide (NO)NO is an important mediator in the pathogenesis of otitis media with efusion. The harmful efect of NO is related with NO-derived intermediates such as peroxynitrite (OONO-). The author investigated the role of NO and peroxynitrite on mucociliary activity in experimental otitis media with efusion. Materials and Method:Otitis media with efusion was induced by injecting lipopolysacharide (LPS) transtympanically in guinea pigs. In the NG-nitro-L-arginine methyl ester (L-NAME) group, L-NAME was additionally injected intratympanicaly and intramuscularly. Uric acid (UA) group was treated with intraperi-toneal injection of UA 3 times. After 24 hours, dye transfer time was measured and temporal bones were taken for histopathologic examination. Expresion of peroxynitrite was determined by imunohistochemical stain for 3-nitrotyrosine (3-NT). Results:Transfer time of dye was prolonged in LPS group, whereas it was significantly reduced in L-NAME or UA not statisticaly significant. 3-NT was expresed intensely in subepithelial layer of LPS group, and decreased to mild to moderate degree in the treatment groups. Conclusion:LPS induced mucociliary dysfunction in the middle ear by NO and peroxynitrite-mediated pathways. This study sugests that inhibition of NO synthesis or scavenging of peroxynitrite may have an adjunctive role in the future treatment of otitis media with efusion. (Korean J Otolaryngol 203 ;46 :720-6)

어수리 어린 잎으로부터 Peroxynitrite 소거활성을 나타내는 플라보노이드 성분의 분리 및 함량분석

박희준,Agung Nugroho,최재수,정보람,원유화,정연주,김원배 한국자원식물학회 2010 한국자원식물학회지 Vol.23 No.5

Naturally occurring substances which potentially scavenge peroxynitrite (ONOO-) are evaluated to be beneficial for cardiovascular, diabetic, hypercholesterolemic and obese disease. To search for bioactive substances with peroxynitrite-scavenging activity from the young leaves of Heracleum moellendorffii (Umbelliferae) used for a mountainous vegetable, the experiment on peroxynitrite-scavenging assay, phytochemical isolation and HPLC analysis were undertaken. Activity-guided fractionation led to the separation of astragalin and hyperoside which were identified by physicochemical and spectroscopic data. The IC50 values of astragalin and hyperoside were shown as 5.80 ± 0.62 and 0.560 ± 0.26 μM, respectively, in the assay. The HPLC analysis led to the quantitative determination of astragalin and hyperoside by 30.0 ± 0.01 and 17.0 ± 0.02 mg/g dried weight, respectively. 식용으로 사용되는 산채의 일종인 어수리의 지상부로부터 peroxynitrite 소거활성을 나타내는 성분을 분리한 결과 플라보노이드 성분인 hyperoside와 astragalin이 분리되었다. 특히, 높은 활성을 보였던 hyperoside는 이 식물재료 중 함량도 높아 어수리 잎은 peroxynitrite 과잉생산시 유발될 수 있는 당뇨병, 비만, 고지혈증, 심장질환 등의 예방과 치료에 유익한 식품이라 할 수 있을 것이다.

Simultaneous quantification and validation of new peroxynitrite scavengers from <i>Artemisia iwayomogi</i>

Nugroho, Agung,Lim, Sang-Cheol,Karki, Subash,Choi, Jae Sue,Park, Hee-Juhn Informa Healthcare USA, Inc. 2015 PHARMACEUTICAL BIOLOGY Vol.53 No.5

<P><I>Context</I>: <I>Artemisia iwayomogi</I> Kitamura (Compositae) has been very widely used for the treatment of acute or chronic hepatitis, jaundice, and gastritis. In the course of our continuing efforts to identify and quantify peroxynitrite scavengers from Compositae plants, <I>A. iwayomogi</I> was used in this study.</P><P><I>Objective</I>: The present study was aimed to identify and quantify the peroxynitrite scavengers of <I>A. iwayomogi</I>.</P><P><I>Materials and methods</I>: Silica gel and ODS were used for column chromatography. The isolated compounds were quantified using an HPLC equipped with a Capcell Pak C18 column (5 μm, 250 mm × 4.6 mm i.d.), and the method was validated for the quality control. Peroxynitrite (ONOO<SUP>−</SUP>)-scavenging activities of the compounds and extracts were evaluated on the measurement of highly fluorescent rhodamine 123 converted from non-fluorescent dihydrorhodamine (DHR)-123 under the presence of peroxynitrite.</P><P><I>Results</I>: Based on the spectroscopic evidences, a new compound, 2″-<I>O</I>-caffeoylrutin (2″-<I>O-trans</I>-caffeic acid ester of quercetin 3-<I>O</I>-α-<SMALL>L</SMALL>-rhamnopyranosyl(1 → 6)-β-<SMALL>D</SMALL>-glucopyranoside) was isolated and determined together with patuletin 3-<I>O</I>-glucoside, scopolin, scopoletin, rutin, 3,4-dicaffeoylquinic acid, and chlorogenic acid. All of them were potent peroxynitrite scavengers (IC<SUB>50</SUB> ≤ 1.88 μg/mL).</P><P><I>Discussion and conclusion</I>: The peroxynitrite scavengers were mainly distributed in the EtOAc fraction rather than the ether and BuOH fractions. The 70% MeOH extract exhibited a high peroxynitrite-scavenging activity. Through the validation, the present HPLC method was verified to be sufficiently sensitive, accurate, precise, and stable. Therefore, this method can be used for the quality control of <I>A. iwayomogi</I>.</P>

PKC Downstream of PI3-Kinase Regulates Peroxynitrite Formation for Nrf2-Mediated GSTA2 Induction

Kim, Sang-Geon,Kim, Sun-Ok The Pharmaceutical Society of Korea 2004 Archives of Pharmacal Research Vol.27 No.7

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes including glutathione S-transferases (GSTs). NF-E2-related factor-2 (Nrf2) phosphorylation by protein kinase C (PKC) is a critical event for its nuclear translocation in response to oxidative stress. Previously, we have shown that peroxynitrite plays a role in activation of Nrf2 and Nrf2 binding to the antioxidant response element (ARE) via the pathway of phosphatidylinositol 3-kinase (PI3-kinase) and that nitric oxide synthase in hepatocytes is required for GSTA2 induction. In view of the importance of PKC and Pl3-kinase in Nrf2-mediated GST induction, we investigated the role of these kinases in peroxynitrite formation for GSTA2 induction by oxidative stress and determined the relationship between PKC and PI3-kinase. Although PKC activation by phorbol 12-myristate-13-acetate (PMA) did not increase the extents of constitutive and inducible GSTA2 expression, either PKC depletion by PMA or PKC inhibition by staurosporine significantly inhibited GSTA2 induction by tert-butylhydroquinone (t-SHa) a prooxidant chemical. Therefore, the basal PKC activity is req- uisite for GSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, induced GSTA2, which was not inhibited by PKC depletion, but slightly enhanced by PKC activation, suggesting that PKC promotes peroxynitrite formation for Nrf2-mediated GSTA2 induction. Treatment of cells with S-nitroso-N-acetyl-penicillamine (SNAP), an exogenous NO donor, in combination with t-BHQ may produce peroxynitrite. GSTA2 induction by SNAP + t-BHQ was not decreased by PKC depletion, but rather enhanced by PKC activation, showing that the activity of PKC might be required for peroxynitrite formation. LY294002 a P13-kinase inhibitor blocked GSTA2 induction by t-BHQ, which was reversed by PMA-induced PKC activation. These results provide evidence that PKC may playa role in formation of peroxynitrite that activates Nrf2 for GSTA2 induction and that PKC may serve an activator for GSTA2 induction downstream of PI3-kinase.

4-Hydroxynonenal Induces Endothelial Cell Apoptosis via ROS and Peroxynitrite Generation

Sang Woon Chung(정상운),Su Bog Yee(이수복),Ji Young Lee(이지영),Mohammad Akbar Hossain(호세인 모하메드 악바르),Dong Hwan Kim(김동환),Jeong-Hyun Yoon(윤정현),Hae Young Chung(장정윤),Nam Deuk Kim(김남득) 한국생명과학회 2011 생명과학회지 Vol.21 No.7

지질과산화로부터 생성된 aldehyde 중 4-hydroxynonenal (HNE)는 산화적 손상과 관련된 다량의 arachidonic acids, linoleic acids 등으로부터 생성될 수 있다. 그러므로 HNE는 산화적 스트레스와 관련된 세포사에서 중요한 매개인자로 작용을 할 수가 있을 것이다. 본 연구는 HNE가 세포사를 유발할 것이라 가정하고, 먼저 흰쥐 전립선 유래 내피세포인 YPEN-1 세포에서 세포독성을 측정하였다. 세포생장 저해능력은 HNE를 5~15 μM 농도로 처리하여 형태적 변화와 MTT assay를 통하여 결과를 관찰하였다. 그 결과 HNE가 이 세포에서 핵형의 변화와 세포사를 유발시키는 것을 각각의 실험을 통해 확인이 되었다. 또한 이 사실을 단백질의 변화를 통하여 확인을 할 수가 있었다. HNE를 24시간 처리한 세포에서 poly(ADP-ribose) polymerase 단백질 분절이 매개되었고 Bax의 발현량이 증가하였다. 또한 세포내의 활성 산소종들을 발생시켰다. 이에 생성된 활성 산소종과 peroxynitrite가 세포사와 관련이 있는가를 밝히기 위하여 이들의 포식자들인 N-acetylcysteine과 penicillamine을 본 연구에서 사용하였다. 이들 포식자들에 의해 HNE에 의해 유도되는 세포사가 억제가 되었기에 산화적 활성화가 HNE에 의해 유도된 세포사와 관련이 있음을 알 수 있었다. 이러한 결과들은 HNE가 내피세포에서 ROS와 peroxynitrite 생성을 통하여 세포사를 일으킨다는 사실을 뒷받침해 준다. The formation of reactive lipid aldehydes, 4-hydroxynonenal (HNE) is shown to be derived from fatty acid hydroperoxides through the oxidative process. Among its known effects in cytotoxicity, HNE has been implicated in apoptotic cell death. To delineate its putative role as a potential mediator, we investigated the mechanism by which HNE induces apoptosis of endothelial cells (ECs). The anti-proliferative effects of HNE were tested through MTT assay after exposure to various concentrations (5~15 μM) of HNE. We observed apoptotic bodies with propidium iodide staining, and measured the HNE induction of endothelial apoptosis by flow cytometry assay. We observed that cells exposed to HNE for 24 hr resulted in increased poly(ADP-ribose) polymerase cleavage and up-regulation of Bax. Data on the HNE action strongly indicated the involvement of reactive species, namely, intracellular ROS, nitrite, and peroxynitrite. To obtain evidence on the implication of ROS and peroxynitrite in HNE-induced apoptosis, a ROS scavenger, N-acetylcysteine (NAC), and a peroxynitrite scavenger, penicillamine, were tested. Results clearly indicate that the induction of apoptosis by HNE was effectively inhibited by NAC and penicillamine. Based on the present data, we conclude that the endothelial apoptosis induced by HNE involves both ROS generation and peroxynitrite activity. Our new data could lead to a redefinition of HNE action on apoptosis in ECs.

단양쑥부쟁이에 함유된 Caffeoylquinic Acid의 HPLC 분석과Peroxynitrite 소거효과

임상철,박희준 한국자원식물학회 2016 한국자원식물학회지 Vol.29 No.1

Five caffeoylquinic acids of Aster altaicus var. uchiyamae Kitamura (Compositae) leaves were identified using standard compounds by HPLC and determined as follows: 3,4-di-O-caffeoylquinic acid (4.92 ± 0.06 ㎎/g dried weight), 3,5-di-O-caffeoylquinic acid (3.95 ± 0.13 ㎎/g), 4,5-di-O-caffeoylquinic acid (1.39 ± 0.10 ㎎/g), 5-O-caffeoylquinic acid (chlorogenic acid, 8.05 ± 0.21 ㎎/g), 3-O-caffeoylquinic acid (4.97 ± 0.18 ㎎/g). The total content of five caffeoylquinic acids were calculated as 26.73 ± 0.26 ㎎/g dried weight while the percentage of the five compounds in the MeOH extract was calculated as 25.22 ± 0.25%. The IC50 value of the MeOH extract scavenging peroxynitrite (ONOO - ) was shown as 5.16 ± 0.15 ㎍/㎖. Key words - Aster altaicus, Compositae, Caffeoylquinic acid, Peroxynitrite, HPLC 적 요단양쑥부쟁이의 caffeoylquinic acid 성분을 HPLC로 분석하였다. 그 결과 3,4-DQ, 3,5-DQ, 4,5-DQ, 5-CQ, 3-CQ 5종 화합물이 검출되었다. 이 식물의 caffeoylquinic acid 총 함량은 26.73 ± 0.26 ㎎/g으로 건물중의 2.673 ± 0.026%, MeOH 추출물 중 함량은 25.22 ± 0.25%에 달하였다. 그리고 peroxynitrite 를 소거하는 IC50 값은 5.16 ± 0.15 ㎍/㎖로 나타났고, 양성대조군으로 사용된 L-penicillamine의 값은 0.89 ± 0.22 ㎍/㎖였다. 그러므로, 단양쑥부쟁이는 caffeoylquinic acid 함량에 따르는 peroxynitrite 소거효과에 기인하여 비만, 당뇨, 심혈관질환, 동맥경화 등에 유효할 것으로 예상된다.

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation

Minh Cong Nguyen,박종택,전영관,전병화,허광래,김영명,임현교,유승우 연세대학교의과대학 2016 Yonsei medical journal Vol.57 No.6

Purpose: Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-argininesupplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. Materials and Methods: Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performeda vascular tension assay. Results: SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expressionlevel of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increasedat Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistentin the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. NG-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. Conclusion: These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.