- Email: [email protected]
P4. Effect of nitric oxide donor on radiation induced MAPK signaling in mouse peritoneal macrophages
Nitric Oxide 19 (2008) S43–S72
Contents lists available at ScienceDirect
Nitric Oxide journal homepage: www.elsevier.com/locate/yniox
Poster abstracts Interaction of NO with Proteins P1. Nitrosyl iron complexes with azageterocyclic thiolyl ligands: Synthesis, structure and properties new NO-donors Nataliya Sanina RAS, Institute of Problems of Chemical Physics, Chernogolovka, Moscow Region, Russian Federation Iron nitrosyl complexes are produced in reactions of endogeneous NO with active sites of non-heme [Fe–S] proteins. It has been reliably established that iron nitrosyl complexes with thiol-containing ligands (cysteine and glutathione) play important roles in organisms, such as cellular NO storage and transport. Synthesis and study of synthetic analogs modeling nitrosyl [Fe–S] proteins reactivity are an important problem from the point of view of fundamental investigation of the bond nature of the {Fe(NO)2} fragment and practical medicine for the design prodrugs based on natural sources. The neutral mono- and dinuclear iron nitrosyl complexes of the ‘‘g = 2.03” family with analogous to natural histidines, pyridine and purine bases of nucleic acids were synthesized. The iron nitrosyl complexes different structural types were studied by X-ray diffraction, EPR, IR, mass- and 57Fe Mössbauer spectroscopy and SQUID magnetometry. Kinetics of NO formation was studied by sensor electrode amiNO700 and by registration of absorption spectra of the reaction systems containing Hb and the complex under study. The investigation of genetic, cardiotropic and antitumor activities of iron nitrosyl complexes compared to the already known clinical agents has been also studied. The results of this scientific research will become the background for development of medicines of a new generation, and they will contribute in the solution of the problem of adaptation and sensitizing to chemotherapeutic medicines. This study was financially supported by the RFBR (No. 06-03-32381) and the RAS Program ‘‘Fundamental Sciences for Medicine”. doi:10.1016/j.niox.2008.06.097
P2. Stabilization of tetranitrosyl thiosulfate iron complex by albumin Tatiana N. Roudneva, Nataliya Sanina, Denis Mischenko, Elena Frog, Raisa Kotel’nikova, Sergey Aldoshin Institute of Problems of Chemical Physics of the Russian Academy of Science, Chernogolovka, Moscow Region, Russian Federation Sulfur–nitrosyl iron complexes are synthetic analogs of active sites of nitrosyl non-heme proteins, and they can be used as NO donors for the development of cardiovascular medications of a new generation. In this work, the parameters of absorption spectra of tetranitrosyl thiosulfate iron complex (TNIC) in water solutions, in phosphatidylcholine liposomes, in micelles and proteins were determined, this being aimed at the creation of its pharmaceutical dosage form. Earlier, TNIC was proved not to keep its binuclear structure in protonic solvents, and dark decomposition of the complex due to hydrolysis was observed. TNIC stabilization in albumin solutions was found out. Kinetic dependence of the normalized values of optical density at wavelength 364 nm for TNIC [10 4 M] in albumin solution [10 4 M] in 0.05 M HEPES is presented as compared to other substrates. It was also shown, that absorption spectra of TNIC depend on polarity, viscosity and pH of the complex environment. It has been determined that TNIC is a perfect quencher of triplet excited states of eritrosine and pyrene probes. The constants of probe quenching by investigated compounds in water solutions and in phosphatidyl-
choline liposomes were determined. It has been shown that TNIC is transported through the lipid layer of liposomes, and the decrease of microviscosity of membrane hydrophobic sites has been observed. Upon intravenous introduction of TNIC in the organism of a wakeful animal (a rabbit, female of 3–4 kg), it was shown to be present in the blood flow for a long time, and in the blood plasma concentration of its paramagnetic form is much higher than in erythrocytes, thus suggesting that it binds with albumin as a predominant thiol-containing protein of the blood plasma. Acknowledgments The work was supported by the RFBR (No. 06-03-32381), the Program of the RAS ‘‘Fundamental problems of medicine” and grant of ‘‘Russian Science Support Foundation”. doi:10.1016/j.niox.2008.06.098
P3. Computer modelling of cellular reactions involving NO, superoxide and glutathione Anthony Butler a, Ridd John b, Ian Megson c Bute Medical School, University of St. Andrews b Chemistry, University College London c Physiology, Millennium Institute
a
Both NO and glutathione react with superoxide. Peroxynitrite, formed in the former reaction, is often cited as a casual factor in reduced NO activity under conditions of oxidative stress and is known to have quite different biological activity of its own. In view of the massive preponderance of glutathione in the intracellular environment (3–5 mM) over NO (pM range for constitutively generated NO), together with a vast array of other abundant intracellular components that can react with superoxide, it is difficult to believe that significant amounts of peroxynitrite form. If the reactions of superoxide with NO and with glutathione are taken in isolation this is certainly the case. However, as we increase the complexity of the reaction scheme, making it closer to the range of cellular reactions in which the three reactants are involved, this is no longer true. Computer modelling, using independently determined rate constants, leads to some unexpected conclusions. While we would not claim to have modelled completely the in vivo situation, it is clear that mere inspection of individual rate constants and concentration ranges does not give reliable insight into cellular reactions. doi:10.1016/j.niox.2008.06.099
P4. Effect of nitric oxide donor on radiation induced MAPK signaling in mouse peritoneal macrophages Himanshi Narang, Malini Krishna Bhabha Atomic Research Centre Radiation, when delivered to tumors, also affects normal cells like macrophages which are associated with tumors. Radioresistant macrophages survive radiation
S44
Poster abstracts / Nitric Oxide 19 (2008) S43–S72
and are responsible for phagocytosing the dead tumor cells after radiotherapy. They are also known to get activated by radiation and produce large amounts of nitric oxide. Some tumors are also known to produce high amounts of nitric oxide. The crucial operative pathways in macrophages, after irradiation in a nitric oxide rich environment are therefore important to study. Mitogen Activated Protein Kinase (MAPK) pathways are crucial to macrophages and play important role in their survival, activation and function. MAPK pathway components, ERK, JNK and p38 get activated after phosphorylation on tyrosine and threonine residues. Since tyrosine residues may get nitrated by nitric oxide, which could interfere with signaling, the response of MAPK in macrophages after radiation and nitric oxide was looked at. Drastic inhibition of irradiation induced phosphorylation of these MAPKs, especially ERK and JNK in presence of nitric oxide donor was observed. Increased tyrosine nitration under these conditions could be correlated to reduced phosphorylation. No change in the content of kinases was observed, ruling out the possibility of degradation. Nitration of kinases was not found to be tightly linked with their ubiquitination, one of the known fates of nitrated proteins. Despite reduced phosphorylation and increased nitration of the kinases, activation of their downstream targets could be observed. Phagocytic efficiency, viability and caspase activities were unaltered as well. This study indicates that since crucial signaling pathways in macrophages must be operative even under high nitric oxide environment, tyrosine nitration might act in cooperation with tyrosine phosphorylation so as to keep the macrophage functionally active. doi:10.1016/j.niox.2008.06.100
tathione (GSH) level decreased initially, but gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC upregulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by ZnPP IX or the siRNA knock down of HO-1 gene expression abrogated the upregulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using ZnPP IX. Furthermore, CO-RM treatment upregulated the GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC which is mediated by activation of PI3K/Akt and subsequently Nrf2 signaling. doi:10.1016/j.niox.2008.06.102
P7. Nitric oxide involved in macrophage activation and neutralization of Bacillus anthracis toxins during glycoconjugate treatment Jill Castleberry a, Ashley Scott a, Pierre Alusta b, Lee Soderberg c, Olga Tarasenko d University of Arkansas at Little Rock b Chemistry, University of Arkansas at Little Rock c University of Arkansas for Medical Sciences d Biology, University of Arkansas at Little Rock
a
P5. Differential analysis of synaptic S-nitrosome in Alzheimer disease model mice Monika Zareba-Koziol, Aleksandra Wyslouch-Cieszynska Institute of Biochemistry and Biophysics, PAS, ul. Pawinskiego 5a, 02-106 Warszawa, Poland Alzheimer’s disease (AD) is one of the most frequent neurodegenerative disorders. It is characterized by progressive deterioration of cognitive functions, learning and memory, which is in part due to dysfunction and loss of synapses. The pathogenesis of AD has been linked to imbalance in the production of reactive oxygen (ROS) and nitrogen species (RNS) [1]. The aim of the presented project is to detect the presumptive changes in synaptical protein S-nitrosome which may occur during the development of AD. The subject of the study are synaptosomes from transgenic mice with neuronal expression of human amyloid precursor protein (APP), presenilin-1 (PS1) and double mutant APP/ PS1 mice-approved models of AD [2]. Biotin Switch and SNOSID methods are used for the enrichment of the endogenously S-nitrosylated synaptosomal proteins [3,4]. Liquid chromatography (LC) coupled with electrospray mass spectrometry and new bioinformatics tools are used for the qualitative and quantitative analysis of the observed S-nitrosylated proteins. References [1] [2] [3] [4]
M.P. Mattson et al., Nature 430 (2004) 631–639. F. Van Leuven et al., Subcell. Biochem. 38 (2005) 45–63. S.R. Jaffrey, S.H. Snyder, Sci. STKE 86 (2001) PL1. S.S. Gross et al., Proc. Natl. Acad. Sci. USA 103 (2006) 1012–1017.
Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity. We have shown that glycoconjugates (GCs) stimulate binding and recognition of bacterial spores or toxins. In the present study, we studied GC (1,8) effects yielding in neutralizing B. anthracis protective antigen (PA), edema factor (EF), lethal factor (LF) single toxin (sTX) and PA-EF-LF, PA-EF, and PA-LF complexes (cTX) during phagocytosis. Neutralization of either sTX or cTX were studied under prior to, during, and following conditions. Phagocytosis, macrophage viability, and cytotoxicity assays were performed. Murine macrophages exposed to GC-treated toxins induced NO production that was correlated with high viability of macrophages and toxins neutralization. NO production during the prior to exposure condition is generally low, independent of GCs concentration or type of toxin. NO is produced in higher amounts in during and following conditions. In case of GC1, NO release declines with decreasing GC1 concentration in presence of sTX. The opposite trend is observed when cTX are present. In case of GC8, NO release raises with decreasing GC8 concentration in presence of sTX. The reverse tendency is observed when cTX are present. However, when PA is present, NO release is, above all, remarkably higher for GC8. Increased levels of inducible NO production by macrophages in presence of GCs suggested that the latter provide an activation signal directed to macrophages. GCs promote neutralization of toxins by blocking toxins-induced macrophage cell death, while increasing their activation level and NO production. The technique presented in this study may be helpful in finding GCs with antitoxic properties. Acknowledgements: National Science Foundation EPSCoR RII Grant ‘‘Arkansas AASSET Initiatives (#EPS-0701890) and the Arkansas Science & Technology Authority; SURF; American Society for Microbiology URF and UTF programs; Kathleen Thomsen Hall Charitable Trust Grants.
doi:10.1016/j.niox.2008.06.101
doi:10.1016/j.niox.2008.06.103
P6. Peroxynitrite induces heme oxygenase-1 expression via activation of Nrf2 signaling: Implications for adaptive cellular survival response to nitrosative stress
P8. Metalloproteinase-9 is secreted in an S-nitrosated, inactive form
Mei-Hua Li a, Jung-Hee Jang a, Hye-Kyung Na a, Young-Nam Cha b, Young-Joon Surh a a College of Pharmacy, Seoul National University b College of Medicine, Inha University The induction of heme oxygenase-1 (HO-1) gene expression represents the first line of cellular defence against oxidative/nitrosative stress and other noxious stimuli. Nuclear factor E2-related factor-2 (Nrf2) has been identified as a major transcription factor responsible for regulating HO-1 gene expression. Our recent studies have demonstrated that peroxynitrite induces HO-1 expression through activation of Nrf2 signaling, which confers the cellular cytoprotection. In the present study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in adaptive survival response to peroxynitriteinduced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morphoinosydonimine (SIN-1), the cellular glu-
Ana Iochabel Soares Moretti a, Marcia Cristiane Jurado a, Luiz Claúdio Godoy b, Heraldo Possolo Souza a a School of Medicine University of São Paulo b University of Massachussets Metalloproteinases (MMPs) are crucial mediators of vascular remodeling and in determining the fate of atherosclerotic plaque. Uncontrolled MMPs activity is one of the cardinal features leading to plaque rupture and triggering acute coronary syndromes. Nitric oxide (NO) may modulate MMPs activity through S-nitrosation in a tissue specific fashion. For instance, MMPs are activated by NO in neurons, while in lungs they are inhibited. Due to importance of MMPs to atherosclerosis progression and to the presence of high amounts of NO in the vessel wall, our objective was to determine the effect of S-nitrosation on the activity of MMPs produced by cells involved in atherosclerotic plaque rupture. Human umbilical vein endothelial cells (HUVECs) and cells derived from human monocytoid cell line THP-1 were co-cultured in a transwell system. MMPs 2 and 9 expression and activity were determined in culture medium and cell lysates by immunoblot and zymography, respectively.
Contents lists available at ScienceDirect
Nitric Oxide journal homepage: www.elsevier.com/locate/yniox
Poster abstracts Interaction of NO with Proteins P1. Nitrosyl iron complexes with azageterocyclic thiolyl ligands: Synthesis, structure and properties new NO-donors Nataliya Sanina RAS, Institute of Problems of Chemical Physics, Chernogolovka, Moscow Region, Russian Federation Iron nitrosyl complexes are produced in reactions of endogeneous NO with active sites of non-heme [Fe–S] proteins. It has been reliably established that iron nitrosyl complexes with thiol-containing ligands (cysteine and glutathione) play important roles in organisms, such as cellular NO storage and transport. Synthesis and study of synthetic analogs modeling nitrosyl [Fe–S] proteins reactivity are an important problem from the point of view of fundamental investigation of the bond nature of the {Fe(NO)2} fragment and practical medicine for the design prodrugs based on natural sources. The neutral mono- and dinuclear iron nitrosyl complexes of the ‘‘g = 2.03” family with analogous to natural histidines, pyridine and purine bases of nucleic acids were synthesized. The iron nitrosyl complexes different structural types were studied by X-ray diffraction, EPR, IR, mass- and 57Fe Mössbauer spectroscopy and SQUID magnetometry. Kinetics of NO formation was studied by sensor electrode amiNO700 and by registration of absorption spectra of the reaction systems containing Hb and the complex under study. The investigation of genetic, cardiotropic and antitumor activities of iron nitrosyl complexes compared to the already known clinical agents has been also studied. The results of this scientific research will become the background for development of medicines of a new generation, and they will contribute in the solution of the problem of adaptation and sensitizing to chemotherapeutic medicines. This study was financially supported by the RFBR (No. 06-03-32381) and the RAS Program ‘‘Fundamental Sciences for Medicine”. doi:10.1016/j.niox.2008.06.097
P2. Stabilization of tetranitrosyl thiosulfate iron complex by albumin Tatiana N. Roudneva, Nataliya Sanina, Denis Mischenko, Elena Frog, Raisa Kotel’nikova, Sergey Aldoshin Institute of Problems of Chemical Physics of the Russian Academy of Science, Chernogolovka, Moscow Region, Russian Federation Sulfur–nitrosyl iron complexes are synthetic analogs of active sites of nitrosyl non-heme proteins, and they can be used as NO donors for the development of cardiovascular medications of a new generation. In this work, the parameters of absorption spectra of tetranitrosyl thiosulfate iron complex (TNIC) in water solutions, in phosphatidylcholine liposomes, in micelles and proteins were determined, this being aimed at the creation of its pharmaceutical dosage form. Earlier, TNIC was proved not to keep its binuclear structure in protonic solvents, and dark decomposition of the complex due to hydrolysis was observed. TNIC stabilization in albumin solutions was found out. Kinetic dependence of the normalized values of optical density at wavelength 364 nm for TNIC [10 4 M] in albumin solution [10 4 M] in 0.05 M HEPES is presented as compared to other substrates. It was also shown, that absorption spectra of TNIC depend on polarity, viscosity and pH of the complex environment. It has been determined that TNIC is a perfect quencher of triplet excited states of eritrosine and pyrene probes. The constants of probe quenching by investigated compounds in water solutions and in phosphatidyl-
choline liposomes were determined. It has been shown that TNIC is transported through the lipid layer of liposomes, and the decrease of microviscosity of membrane hydrophobic sites has been observed. Upon intravenous introduction of TNIC in the organism of a wakeful animal (a rabbit, female of 3–4 kg), it was shown to be present in the blood flow for a long time, and in the blood plasma concentration of its paramagnetic form is much higher than in erythrocytes, thus suggesting that it binds with albumin as a predominant thiol-containing protein of the blood plasma. Acknowledgments The work was supported by the RFBR (No. 06-03-32381), the Program of the RAS ‘‘Fundamental problems of medicine” and grant of ‘‘Russian Science Support Foundation”. doi:10.1016/j.niox.2008.06.098
P3. Computer modelling of cellular reactions involving NO, superoxide and glutathione Anthony Butler a, Ridd John b, Ian Megson c Bute Medical School, University of St. Andrews b Chemistry, University College London c Physiology, Millennium Institute
a
Both NO and glutathione react with superoxide. Peroxynitrite, formed in the former reaction, is often cited as a casual factor in reduced NO activity under conditions of oxidative stress and is known to have quite different biological activity of its own. In view of the massive preponderance of glutathione in the intracellular environment (3–5 mM) over NO (pM range for constitutively generated NO), together with a vast array of other abundant intracellular components that can react with superoxide, it is difficult to believe that significant amounts of peroxynitrite form. If the reactions of superoxide with NO and with glutathione are taken in isolation this is certainly the case. However, as we increase the complexity of the reaction scheme, making it closer to the range of cellular reactions in which the three reactants are involved, this is no longer true. Computer modelling, using independently determined rate constants, leads to some unexpected conclusions. While we would not claim to have modelled completely the in vivo situation, it is clear that mere inspection of individual rate constants and concentration ranges does not give reliable insight into cellular reactions. doi:10.1016/j.niox.2008.06.099
P4. Effect of nitric oxide donor on radiation induced MAPK signaling in mouse peritoneal macrophages Himanshi Narang, Malini Krishna Bhabha Atomic Research Centre Radiation, when delivered to tumors, also affects normal cells like macrophages which are associated with tumors. Radioresistant macrophages survive radiation
S44
Poster abstracts / Nitric Oxide 19 (2008) S43–S72
and are responsible for phagocytosing the dead tumor cells after radiotherapy. They are also known to get activated by radiation and produce large amounts of nitric oxide. Some tumors are also known to produce high amounts of nitric oxide. The crucial operative pathways in macrophages, after irradiation in a nitric oxide rich environment are therefore important to study. Mitogen Activated Protein Kinase (MAPK) pathways are crucial to macrophages and play important role in their survival, activation and function. MAPK pathway components, ERK, JNK and p38 get activated after phosphorylation on tyrosine and threonine residues. Since tyrosine residues may get nitrated by nitric oxide, which could interfere with signaling, the response of MAPK in macrophages after radiation and nitric oxide was looked at. Drastic inhibition of irradiation induced phosphorylation of these MAPKs, especially ERK and JNK in presence of nitric oxide donor was observed. Increased tyrosine nitration under these conditions could be correlated to reduced phosphorylation. No change in the content of kinases was observed, ruling out the possibility of degradation. Nitration of kinases was not found to be tightly linked with their ubiquitination, one of the known fates of nitrated proteins. Despite reduced phosphorylation and increased nitration of the kinases, activation of their downstream targets could be observed. Phagocytic efficiency, viability and caspase activities were unaltered as well. This study indicates that since crucial signaling pathways in macrophages must be operative even under high nitric oxide environment, tyrosine nitration might act in cooperation with tyrosine phosphorylation so as to keep the macrophage functionally active. doi:10.1016/j.niox.2008.06.100
tathione (GSH) level decreased initially, but gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC upregulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by ZnPP IX or the siRNA knock down of HO-1 gene expression abrogated the upregulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using ZnPP IX. Furthermore, CO-RM treatment upregulated the GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC which is mediated by activation of PI3K/Akt and subsequently Nrf2 signaling. doi:10.1016/j.niox.2008.06.102
P7. Nitric oxide involved in macrophage activation and neutralization of Bacillus anthracis toxins during glycoconjugate treatment Jill Castleberry a, Ashley Scott a, Pierre Alusta b, Lee Soderberg c, Olga Tarasenko d University of Arkansas at Little Rock b Chemistry, University of Arkansas at Little Rock c University of Arkansas for Medical Sciences d Biology, University of Arkansas at Little Rock
a
P5. Differential analysis of synaptic S-nitrosome in Alzheimer disease model mice Monika Zareba-Koziol, Aleksandra Wyslouch-Cieszynska Institute of Biochemistry and Biophysics, PAS, ul. Pawinskiego 5a, 02-106 Warszawa, Poland Alzheimer’s disease (AD) is one of the most frequent neurodegenerative disorders. It is characterized by progressive deterioration of cognitive functions, learning and memory, which is in part due to dysfunction and loss of synapses. The pathogenesis of AD has been linked to imbalance in the production of reactive oxygen (ROS) and nitrogen species (RNS) [1]. The aim of the presented project is to detect the presumptive changes in synaptical protein S-nitrosome which may occur during the development of AD. The subject of the study are synaptosomes from transgenic mice with neuronal expression of human amyloid precursor protein (APP), presenilin-1 (PS1) and double mutant APP/ PS1 mice-approved models of AD [2]. Biotin Switch and SNOSID methods are used for the enrichment of the endogenously S-nitrosylated synaptosomal proteins [3,4]. Liquid chromatography (LC) coupled with electrospray mass spectrometry and new bioinformatics tools are used for the qualitative and quantitative analysis of the observed S-nitrosylated proteins. References [1] [2] [3] [4]
M.P. Mattson et al., Nature 430 (2004) 631–639. F. Van Leuven et al., Subcell. Biochem. 38 (2005) 45–63. S.R. Jaffrey, S.H. Snyder, Sci. STKE 86 (2001) PL1. S.S. Gross et al., Proc. Natl. Acad. Sci. USA 103 (2006) 1012–1017.
Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity. We have shown that glycoconjugates (GCs) stimulate binding and recognition of bacterial spores or toxins. In the present study, we studied GC (1,8) effects yielding in neutralizing B. anthracis protective antigen (PA), edema factor (EF), lethal factor (LF) single toxin (sTX) and PA-EF-LF, PA-EF, and PA-LF complexes (cTX) during phagocytosis. Neutralization of either sTX or cTX were studied under prior to, during, and following conditions. Phagocytosis, macrophage viability, and cytotoxicity assays were performed. Murine macrophages exposed to GC-treated toxins induced NO production that was correlated with high viability of macrophages and toxins neutralization. NO production during the prior to exposure condition is generally low, independent of GCs concentration or type of toxin. NO is produced in higher amounts in during and following conditions. In case of GC1, NO release declines with decreasing GC1 concentration in presence of sTX. The opposite trend is observed when cTX are present. In case of GC8, NO release raises with decreasing GC8 concentration in presence of sTX. The reverse tendency is observed when cTX are present. However, when PA is present, NO release is, above all, remarkably higher for GC8. Increased levels of inducible NO production by macrophages in presence of GCs suggested that the latter provide an activation signal directed to macrophages. GCs promote neutralization of toxins by blocking toxins-induced macrophage cell death, while increasing their activation level and NO production. The technique presented in this study may be helpful in finding GCs with antitoxic properties. Acknowledgements: National Science Foundation EPSCoR RII Grant ‘‘Arkansas AASSET Initiatives (#EPS-0701890) and the Arkansas Science & Technology Authority; SURF; American Society for Microbiology URF and UTF programs; Kathleen Thomsen Hall Charitable Trust Grants.
doi:10.1016/j.niox.2008.06.101
doi:10.1016/j.niox.2008.06.103
P6. Peroxynitrite induces heme oxygenase-1 expression via activation of Nrf2 signaling: Implications for adaptive cellular survival response to nitrosative stress
P8. Metalloproteinase-9 is secreted in an S-nitrosated, inactive form
Mei-Hua Li a, Jung-Hee Jang a, Hye-Kyung Na a, Young-Nam Cha b, Young-Joon Surh a a College of Pharmacy, Seoul National University b College of Medicine, Inha University The induction of heme oxygenase-1 (HO-1) gene expression represents the first line of cellular defence against oxidative/nitrosative stress and other noxious stimuli. Nuclear factor E2-related factor-2 (Nrf2) has been identified as a major transcription factor responsible for regulating HO-1 gene expression. Our recent studies have demonstrated that peroxynitrite induces HO-1 expression through activation of Nrf2 signaling, which confers the cellular cytoprotection. In the present study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in adaptive survival response to peroxynitriteinduced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morphoinosydonimine (SIN-1), the cellular glu-
Ana Iochabel Soares Moretti a, Marcia Cristiane Jurado a, Luiz Claúdio Godoy b, Heraldo Possolo Souza a a School of Medicine University of São Paulo b University of Massachussets Metalloproteinases (MMPs) are crucial mediators of vascular remodeling and in determining the fate of atherosclerotic plaque. Uncontrolled MMPs activity is one of the cardinal features leading to plaque rupture and triggering acute coronary syndromes. Nitric oxide (NO) may modulate MMPs activity through S-nitrosation in a tissue specific fashion. For instance, MMPs are activated by NO in neurons, while in lungs they are inhibited. Due to importance of MMPs to atherosclerosis progression and to the presence of high amounts of NO in the vessel wall, our objective was to determine the effect of S-nitrosation on the activity of MMPs produced by cells involved in atherosclerotic plaque rupture. Human umbilical vein endothelial cells (HUVECs) and cells derived from human monocytoid cell line THP-1 were co-cultured in a transwell system. MMPs 2 and 9 expression and activity were determined in culture medium and cell lysates by immunoblot and zymography, respectively.