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Detection of Nitric Oxide from Metabolism of Exogenous Snitrosothiols
RBC as measured by staining the cells with the NO fluorescence probe DAF-FM and laser scanning microscopy, fluorimetry and flow cytometry. By comparing the intracellular NO production among blood cells by flow cytometry, we found that monocytes produce the highest levels of NO, followed by neutrophiles, lymphocytes, RBC, and platelets. Thus, due to their number RBC are the major NO producing compartment in blood. In summary, we here show that human RBC express a classical isoform of NOS3, which is active in vitro and produces NO within the cells. Thus RBC might directly contribute to NOS-mediated effect within the circulation doi: 10.1016/j.freeradbiomed.2010.10.301
294 Extracellular Matrix Proteoglycans are a Major Target for Peroxynitrite in the Artery Wall Eleanor Kennett1, Christine Y Chuang1,2, Georg Degendorfer1, Martin D Rees1, Astrid Hammer3, Ernst Malle3, John M Whitelock2, and Michael J. Davies1 1 2 Heart Research Institute, Sydney, Australia, University of New 3 South Wales, Sydney, Australia, Medical University of Graz, Graz, Austria The extracellular matrix (ECM) of the vascular wall plays a key role in the functional, mechanical and elastic properties of arteries. Matrix molecules such as laminin, perlecan and type XVIII collagen, interact with many growth factors and enzymes to regulate endothelial cell adhesion, proliferation and migration. Atherosclerotic lesions are characterised by chronic inflammation and accumulation of activated monocytes and macrophages, that generate oxidants that may alter the composition and function of the ECM. This may contribute to endothelial cell loss, dysfunction and weakening of ECM structure, thereby enhancing lesion rupture. This study examined the effects of peroxynitrite (ONOOH), a putative lesion oxidant, on both isolated and intact ECM materials synthesized by human coronary artery endothelial cells (HCAECs). ONOOH damaged both the perlecan protein core and attached heparan sulfate chains, and resulted in decreased HCAEC adhesion and diminished FGF-2 mediated proliferation. This was accompanied by dose-dependent 3nitrotyrosine formation and proteoglycan aggregation. These effects were modulated by both bicarbonate and pH. Laminin exposed to ONOOH was fragmented and aggregated, resulting in decreased HCAEC adhesion. Exposure of HCAEC-derived ECM to ONOOH, resulted in decreased antibody recognition of the incorporated laminin, perlecan and type XVIII collagen, which also led to a decreased level of HCAEC adhesion. These effects were dependent on the oxidant dose; decomposed ONOOH gave less damage. Immunofluorescence studies of advanced human atherosclerotic lesions provided evidence for the co-localisation of both perlecan and 3-nitrotyrosine epitopes in the intimal region. These data indicate that peroxynitrite induces major structural and functional changes to molecules in the ECM, and that this occurs within human atherosclerotic lesions. doi: 10.1016/j.freeradbiomed.2010.10.302
295
modification inhibits their activity. Here, we examine the effects of S-nitrosation on integrated metabolism in endothelial cells using extracellular flux technology. The intracellular nitrosating agent Snitroso-L-cysteine (L-CysNO) is transported into cells, initiates Snitrosation in vitro, and has been used as a model for nitrosative stress. Extracellular acidification (ECAR), a surrogate marker of glycolytic flux, is stimulated by low concentrations of L-CysNO (25-50 μM), whereas high concentrations (100-200 μM) inhibit this parameter. Inhibition of ECAR is diminished by L-leucine, a competitive inhibitor of L-CysNO uptake, and cannot be recapitulated with the D isomer of CysNO (D-CysNO) which is not transported into cells. In addition, the activity of the glycolytic enzyme GAPDH is concentration-dependently inhibited by LCysNO. Mitochondrial function was also examined, and L-CysNO inhibits basal respiration, respiration linked to ATP production, and reserve respiratory capacity. Surprisingly, D-CysNO also impaired mitochondrial function suggesting impairment results not from intracellular S-nitrosation, but from extracellular mediated mechanisms. In support of this, L-leucine did not restore mitochondrial function after L-CysNO treatment. Taken together, these results indicate that nitrosative stress differentially regulates metabolic pathways through both intracellular S-nitrosation and extracellular events and suggests exofacial thiol modification at the cell surface may be involved. Moreover, these data provide insight into the role of nitric oxide and related compounds in vascular (patho)physiology. doi: 10.1016/j.freeradbiomed.2010.10.303
296 Cellular Repair Mechanism for ProteinBound Dinitrosyl Iron Complexes Juanjuan Yang1, Aaron Landry1, and Huangen Ding1 1 Louisiana State University Increasing evidence suggests that iron-sulfur proteins are the primary targets of nitric oxide (NO). Exposure of Escherichia coli cells to NO readily converts iron-sulfur proteins to the proteinbound dinitrosyl iron complexes (DNICs). While the proteinbound DNICs are stable in vitro under aerobic or anaerobic conditions, they are efficiently repaired in aerobically growing E. coli cells even without new protein synthesis. The cellular repair mechanism for the NO-modified iron-sulfur proteins remains largely elusive. Here we report that unlike aerobically growing E. coli cells, the starved E. coli cells fail to re-activate the NOmodified iron-sulfur proteins. Nevertheless, addition of Lcysteine, but not other related biological thiols, results in decomposition of the protein-bound DNICs in the starved E. coli cells and in the cell extracts under aerobic conditions. However, L-cysteine has little or no effect on the protein-bound DNICs in the starved E. coli cells and in vitro under anaerobic conditions, suggesting that oxygen is required for the L-cysteine-mediated decomposition of the protein-bound DNICs. Additional studies reveal that L-cysteine is able to exchange the DNIC with the protein-bound DNICs to form the L-cysteine-bound DNIC which is rapidly disrupted by oxygen, resulting in eventual decomposition of the protein-bound DNICs under aerobic conditions. A model for cellular repair of the NO-modified iron-sulfur proteins will be discussed. doi: 10.1016/j.freeradbiomed.2010.10.304
Differential Regulation of Metabolic Pathways by Snitrosation in Endothelial Cells Anne R. Diers1, Katarzyna A. Broniowska1, and Neil Hogg1 1 Department of Biophysics, Redox Biology Program, Medical College of Wisconsin S-nitrosation of protein thiols is thought to be an important element of nitric oxide-dependent signaling mechanisms in vascular physiology and pathology. Multiple metabolic enzymes are targets of S-nitrosation (e.g. GAPDH and Complex I), and
297 Detection of Nitric Oxide from Metabolism of Exogenous Snitrosothiols Zhen Ding1, and Neil Hogg1 1 DepartmentMedical College of Wisconsin S-nitrosothiols have the generic structure R-SNO are important
SFRBM/SFRRI 2010
S113
intermediates of nitric oxide (NO) bioactivity. The formation and metabolism of RSNO have been implicated as a mechanism of NO storage, transport and signaling. RSNO have been used as exogenous NO donors and have potent antiplatelet and vasodilation effects. We have previously demonstrated that RSNO are able to activate soluble guanylyl cyclase (sGC) after they are transported into the cells (Riego, JA et al. (2009) Free Radic Biol Med 47(3):269-274) indicating that cells contain a specific RSNO metabolic mechanism to form NO. In this study, mouse leukemic monocyte macrophage cells (RAW 264.7) have been exposed to exogenous S-nitrosothiols. Intracellular NO release from the metabolism of RSNO reacted with DAF-2 to yield highly fluorescent trizazolofluorescein DAF-2T which was detected by HPLC and microscopy. Headspace NO analysis was also performed by chemiluminescence detection after RSNO metabolism by cell lysate. The results demonstrated that 1. The majority NO formed from exogenous RSNO is from intracellular metabolism rather than extracellular spontaneous decomposition. 2. Different RSNO have different rates of metabolic NO release. 3. Physiological metabolic NO release is thiol dependent and heat susceptible which may indicate a role of thiol containing proteins in this process. doi: 10.1016/j.freeradbiomed.2010.10.305
298 Snitrosothiols Formation during the Reductive Nitrosylation of Methemoglobin Nagababu Enika1, and Joseph M Rifkind1 1 National Institute on Aging Methemoglobin (metHb) undergoes S-nitrosation when nitric oxide (NO) binds to the metal center. One electron oxidation of NO by transferring an electron to ferric iron of the heme forms a + nitrosonium (NO ) ion, which presumably reacts with β-93 cysteine to form S-nitrosohemoglobin (SNOHb). However, the + short life-time of NO (less than 1ns ) raises doubt about the + required diffusion of NO 13 Å from the iron to react with cysteine. An alternate mechanism proposed for a bolus dose of NO involves the reaction of nitrite, a common contaminant in NO + solutions, with NO producing the nitrosation species N2O3, which reacts with cysteine to generate SNOHb. To avoid nitrite contamination, we have used a purified iron nitrosylhemoglobin (Hb(II)NO) without any nitrite contamination as a source for metHb and NO. Hb(II)NO or NEM treated Hb(II)NO (NEMHb(II)NO) were slowly oxidized to form metHb and NO with potassium ferricyanide over a period of 2 hours in the presence and the absence of GSH under anaerobic conditions. The oxidation of Hb(II)NO was monitored spectrophotometrically, while SNOHb and S-nitrosoglutathione (GSNO) were measured by chemiluminiscence method. The involvement of a reaction of NO with metHb is indicated by the 80% inhibition of the SNO signal with cyanide. To rule out any contribution from nitrite or cysteine radicals during this reaction, we added nitrite or the spin trap (DMPO) and observed no additional affect on the SNO signals detected. Oxidation of Hb(II)NO, but not NEMHb(II)NO resulted in the conversion of 29% of the NO released during Hb oxidation to SNOHb. Oxidation of Hb(II)NO in the presence of GSH resulted in 32% of the released NO in formation of SNOHb (12%) as well as GSNO (20%). The reduction of the SNOHb from 29% to 12% with GSH suggests a direct interaction of the GSH with the Fe(II)NO+ species, which inhibits the transfer of NO+ from the heme to the thiol. For NEMHb(II)NO, where SNOHb is not formed, the reaction in the presence of GSH resulted in 45% of the released NO producing GSNO. The formation of GSNO can + result from an interaction of GSH with Hb(II)NO and/or the + release of NO from the heme pocket. However, the formation of + SNOHb suggests that NO is stable enough in the hydrophobic globin heme pocket to be able to be transferred intra-molecularly from the heme iron to the β-93 cysteine to form SNOHb. doi: 10.1016/j.freeradbiomed.2010.10.306
S114
299 The Nitrosoproteome of Skeletal and Cardiac Myofibrils William H Guilford1, and Ashley R Filo1 1 University of Virginia Nitric oxide (NO) regulates a number of cellular responses including contraction in both smooth and striated muscles. While some of these responses are mediated by guanylyl cyclase, it is increasingly accepted that protein S-nitrosylation is an important regulatory mechanism. NO donors in demembranated skeletal 2+ muscle fibers lead to reduced Ca sensitivity, velocity of shortening, and isometric force (Galler et al., 1997; Perkins et al., 1997; Andrade et al., 1998). Similar effects have been observed in demembranated cardiac muscle (Takahashi et al., 1999). These data suggest a direct effect of NO on the myofibrillar proteins of striated muscles. In fact, we previously showed that NO and small nitrosothiols nitrosylate and directly regulate muscle myosins (Evangelista et al., 2010). The current study focuses on identifying other rat skeletal and cardiac myofibrillar proteins that undergo S-nitrosylation and therefore may be candidates for regulation by NO. Using a modification of the biotin switch assay (Jaffrey et al., 2001) we generated a myofibrillar nitrosoproteome in response to trans-nitrosylation by nitroso-L-cysteine (SNO-Lcys). In rat cardiac myofibrils, α-myosin heavy chain, myosin binding protein-C, actin, troponin-I, and the slow isoform of the myosin essential light chain were significantly and consistently nitrosylated by SNO-L-cys. In rat skeletal myofibrils, myosin heavy chain, α-actinin, actin, troponin-I, the slow and fast isoforms of the essential light chain, and the myosin regulatory light chain were all significantly nitrosylated. These data reveal possible protein targets for regulation of muscle contraction and cardiac function by NO and nitrosothiols. doi: 10.1016/j.freeradbiomed.2010.10.307
300 Cellular Quantification and Kinetic Analysis of Dinitrosyliron Complexes Jason Hickok1, Sumit Sahani1, Hong Shen1, and Douglas D Thomas1 1 UIC • While it is well established that nitric oxide ( NO) reacts with cellular iron and thiols to form dinitrosyl iron complexes (DNIC),rigorous characterization of this paramagnetic species under controlled, biologically-relevant, experimental conditions has never been undertaken. As there are physiologic properties • of NO that cannot be explained mechanistically by the formation • s-nitrosothiols or NO-heme interactions, delineating the intrinsic nature of DNIC is potentially of great importance. Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous • and exogenous sources of NO in relation to iron and O2 • concentrations. Low physiologic NO concentrations (50-500 nM) resulted in the rapid (sec) quantitative conversion of the chelatable iron pool (CIP) into DNIC with a stoichiometry of 1:1. • Treating cells with pathologic NO concentrations (>1 μM) increased the amount of DNIC beyond the basal concentrations of • chelatable iron, indicating NO-mediated iron mobilization. Temporal measurements revealed that DNIC disappear by firstorder kinetics (t½ = 80 min) in an oxygen independent manner • and remain detectable long after the NO source had been removed (>24h). As predicted, changing the O2 concentration • (21→1%) reduced the rate of O2-dependent enzymatic NO synthesis in endotoxin-stimulated RAW cells. Unexpectedly, however, greater amounts of intracellular DNIC were detected at 1% as compared to 21% O2 (2,200 vs. 900 pmol/mg protein). • Just as NO will effect iron homeostasis, which has far-reaching ramifications for cellular physiology, iron will influence the • biological actions of NO. These results demonstrate that DNIC
SFRBM/SFRRI 2010
294 Extracellular Matrix Proteoglycans are a Major Target for Peroxynitrite in the Artery Wall Eleanor Kennett1, Christine Y Chuang1,2, Georg Degendorfer1, Martin D Rees1, Astrid Hammer3, Ernst Malle3, John M Whitelock2, and Michael J. Davies1 1 2 Heart Research Institute, Sydney, Australia, University of New 3 South Wales, Sydney, Australia, Medical University of Graz, Graz, Austria The extracellular matrix (ECM) of the vascular wall plays a key role in the functional, mechanical and elastic properties of arteries. Matrix molecules such as laminin, perlecan and type XVIII collagen, interact with many growth factors and enzymes to regulate endothelial cell adhesion, proliferation and migration. Atherosclerotic lesions are characterised by chronic inflammation and accumulation of activated monocytes and macrophages, that generate oxidants that may alter the composition and function of the ECM. This may contribute to endothelial cell loss, dysfunction and weakening of ECM structure, thereby enhancing lesion rupture. This study examined the effects of peroxynitrite (ONOOH), a putative lesion oxidant, on both isolated and intact ECM materials synthesized by human coronary artery endothelial cells (HCAECs). ONOOH damaged both the perlecan protein core and attached heparan sulfate chains, and resulted in decreased HCAEC adhesion and diminished FGF-2 mediated proliferation. This was accompanied by dose-dependent 3nitrotyrosine formation and proteoglycan aggregation. These effects were modulated by both bicarbonate and pH. Laminin exposed to ONOOH was fragmented and aggregated, resulting in decreased HCAEC adhesion. Exposure of HCAEC-derived ECM to ONOOH, resulted in decreased antibody recognition of the incorporated laminin, perlecan and type XVIII collagen, which also led to a decreased level of HCAEC adhesion. These effects were dependent on the oxidant dose; decomposed ONOOH gave less damage. Immunofluorescence studies of advanced human atherosclerotic lesions provided evidence for the co-localisation of both perlecan and 3-nitrotyrosine epitopes in the intimal region. These data indicate that peroxynitrite induces major structural and functional changes to molecules in the ECM, and that this occurs within human atherosclerotic lesions. doi: 10.1016/j.freeradbiomed.2010.10.302
295
modification inhibits their activity. Here, we examine the effects of S-nitrosation on integrated metabolism in endothelial cells using extracellular flux technology. The intracellular nitrosating agent Snitroso-L-cysteine (L-CysNO) is transported into cells, initiates Snitrosation in vitro, and has been used as a model for nitrosative stress. Extracellular acidification (ECAR), a surrogate marker of glycolytic flux, is stimulated by low concentrations of L-CysNO (25-50 μM), whereas high concentrations (100-200 μM) inhibit this parameter. Inhibition of ECAR is diminished by L-leucine, a competitive inhibitor of L-CysNO uptake, and cannot be recapitulated with the D isomer of CysNO (D-CysNO) which is not transported into cells. In addition, the activity of the glycolytic enzyme GAPDH is concentration-dependently inhibited by LCysNO. Mitochondrial function was also examined, and L-CysNO inhibits basal respiration, respiration linked to ATP production, and reserve respiratory capacity. Surprisingly, D-CysNO also impaired mitochondrial function suggesting impairment results not from intracellular S-nitrosation, but from extracellular mediated mechanisms. In support of this, L-leucine did not restore mitochondrial function after L-CysNO treatment. Taken together, these results indicate that nitrosative stress differentially regulates metabolic pathways through both intracellular S-nitrosation and extracellular events and suggests exofacial thiol modification at the cell surface may be involved. Moreover, these data provide insight into the role of nitric oxide and related compounds in vascular (patho)physiology. doi: 10.1016/j.freeradbiomed.2010.10.303
296 Cellular Repair Mechanism for ProteinBound Dinitrosyl Iron Complexes Juanjuan Yang1, Aaron Landry1, and Huangen Ding1 1 Louisiana State University Increasing evidence suggests that iron-sulfur proteins are the primary targets of nitric oxide (NO). Exposure of Escherichia coli cells to NO readily converts iron-sulfur proteins to the proteinbound dinitrosyl iron complexes (DNICs). While the proteinbound DNICs are stable in vitro under aerobic or anaerobic conditions, they are efficiently repaired in aerobically growing E. coli cells even without new protein synthesis. The cellular repair mechanism for the NO-modified iron-sulfur proteins remains largely elusive. Here we report that unlike aerobically growing E. coli cells, the starved E. coli cells fail to re-activate the NOmodified iron-sulfur proteins. Nevertheless, addition of Lcysteine, but not other related biological thiols, results in decomposition of the protein-bound DNICs in the starved E. coli cells and in the cell extracts under aerobic conditions. However, L-cysteine has little or no effect on the protein-bound DNICs in the starved E. coli cells and in vitro under anaerobic conditions, suggesting that oxygen is required for the L-cysteine-mediated decomposition of the protein-bound DNICs. Additional studies reveal that L-cysteine is able to exchange the DNIC with the protein-bound DNICs to form the L-cysteine-bound DNIC which is rapidly disrupted by oxygen, resulting in eventual decomposition of the protein-bound DNICs under aerobic conditions. A model for cellular repair of the NO-modified iron-sulfur proteins will be discussed. doi: 10.1016/j.freeradbiomed.2010.10.304
Differential Regulation of Metabolic Pathways by Snitrosation in Endothelial Cells Anne R. Diers1, Katarzyna A. Broniowska1, and Neil Hogg1 1 Department of Biophysics, Redox Biology Program, Medical College of Wisconsin S-nitrosation of protein thiols is thought to be an important element of nitric oxide-dependent signaling mechanisms in vascular physiology and pathology. Multiple metabolic enzymes are targets of S-nitrosation (e.g. GAPDH and Complex I), and
297 Detection of Nitric Oxide from Metabolism of Exogenous Snitrosothiols Zhen Ding1, and Neil Hogg1 1 DepartmentMedical College of Wisconsin S-nitrosothiols have the generic structure R-SNO are important
SFRBM/SFRRI 2010
S113
intermediates of nitric oxide (NO) bioactivity. The formation and metabolism of RSNO have been implicated as a mechanism of NO storage, transport and signaling. RSNO have been used as exogenous NO donors and have potent antiplatelet and vasodilation effects. We have previously demonstrated that RSNO are able to activate soluble guanylyl cyclase (sGC) after they are transported into the cells (Riego, JA et al. (2009) Free Radic Biol Med 47(3):269-274) indicating that cells contain a specific RSNO metabolic mechanism to form NO. In this study, mouse leukemic monocyte macrophage cells (RAW 264.7) have been exposed to exogenous S-nitrosothiols. Intracellular NO release from the metabolism of RSNO reacted with DAF-2 to yield highly fluorescent trizazolofluorescein DAF-2T which was detected by HPLC and microscopy. Headspace NO analysis was also performed by chemiluminescence detection after RSNO metabolism by cell lysate. The results demonstrated that 1. The majority NO formed from exogenous RSNO is from intracellular metabolism rather than extracellular spontaneous decomposition. 2. Different RSNO have different rates of metabolic NO release. 3. Physiological metabolic NO release is thiol dependent and heat susceptible which may indicate a role of thiol containing proteins in this process. doi: 10.1016/j.freeradbiomed.2010.10.305
298 Snitrosothiols Formation during the Reductive Nitrosylation of Methemoglobin Nagababu Enika1, and Joseph M Rifkind1 1 National Institute on Aging Methemoglobin (metHb) undergoes S-nitrosation when nitric oxide (NO) binds to the metal center. One electron oxidation of NO by transferring an electron to ferric iron of the heme forms a + nitrosonium (NO ) ion, which presumably reacts with β-93 cysteine to form S-nitrosohemoglobin (SNOHb). However, the + short life-time of NO (less than 1ns ) raises doubt about the + required diffusion of NO 13 Å from the iron to react with cysteine. An alternate mechanism proposed for a bolus dose of NO involves the reaction of nitrite, a common contaminant in NO + solutions, with NO producing the nitrosation species N2O3, which reacts with cysteine to generate SNOHb. To avoid nitrite contamination, we have used a purified iron nitrosylhemoglobin (Hb(II)NO) without any nitrite contamination as a source for metHb and NO. Hb(II)NO or NEM treated Hb(II)NO (NEMHb(II)NO) were slowly oxidized to form metHb and NO with potassium ferricyanide over a period of 2 hours in the presence and the absence of GSH under anaerobic conditions. The oxidation of Hb(II)NO was monitored spectrophotometrically, while SNOHb and S-nitrosoglutathione (GSNO) were measured by chemiluminiscence method. The involvement of a reaction of NO with metHb is indicated by the 80% inhibition of the SNO signal with cyanide. To rule out any contribution from nitrite or cysteine radicals during this reaction, we added nitrite or the spin trap (DMPO) and observed no additional affect on the SNO signals detected. Oxidation of Hb(II)NO, but not NEMHb(II)NO resulted in the conversion of 29% of the NO released during Hb oxidation to SNOHb. Oxidation of Hb(II)NO in the presence of GSH resulted in 32% of the released NO in formation of SNOHb (12%) as well as GSNO (20%). The reduction of the SNOHb from 29% to 12% with GSH suggests a direct interaction of the GSH with the Fe(II)NO+ species, which inhibits the transfer of NO+ from the heme to the thiol. For NEMHb(II)NO, where SNOHb is not formed, the reaction in the presence of GSH resulted in 45% of the released NO producing GSNO. The formation of GSNO can + result from an interaction of GSH with Hb(II)NO and/or the + release of NO from the heme pocket. However, the formation of + SNOHb suggests that NO is stable enough in the hydrophobic globin heme pocket to be able to be transferred intra-molecularly from the heme iron to the β-93 cysteine to form SNOHb. doi: 10.1016/j.freeradbiomed.2010.10.306
S114
299 The Nitrosoproteome of Skeletal and Cardiac Myofibrils William H Guilford1, and Ashley R Filo1 1 University of Virginia Nitric oxide (NO) regulates a number of cellular responses including contraction in both smooth and striated muscles. While some of these responses are mediated by guanylyl cyclase, it is increasingly accepted that protein S-nitrosylation is an important regulatory mechanism. NO donors in demembranated skeletal 2+ muscle fibers lead to reduced Ca sensitivity, velocity of shortening, and isometric force (Galler et al., 1997; Perkins et al., 1997; Andrade et al., 1998). Similar effects have been observed in demembranated cardiac muscle (Takahashi et al., 1999). These data suggest a direct effect of NO on the myofibrillar proteins of striated muscles. In fact, we previously showed that NO and small nitrosothiols nitrosylate and directly regulate muscle myosins (Evangelista et al., 2010). The current study focuses on identifying other rat skeletal and cardiac myofibrillar proteins that undergo S-nitrosylation and therefore may be candidates for regulation by NO. Using a modification of the biotin switch assay (Jaffrey et al., 2001) we generated a myofibrillar nitrosoproteome in response to trans-nitrosylation by nitroso-L-cysteine (SNO-Lcys). In rat cardiac myofibrils, α-myosin heavy chain, myosin binding protein-C, actin, troponin-I, and the slow isoform of the myosin essential light chain were significantly and consistently nitrosylated by SNO-L-cys. In rat skeletal myofibrils, myosin heavy chain, α-actinin, actin, troponin-I, the slow and fast isoforms of the essential light chain, and the myosin regulatory light chain were all significantly nitrosylated. These data reveal possible protein targets for regulation of muscle contraction and cardiac function by NO and nitrosothiols. doi: 10.1016/j.freeradbiomed.2010.10.307
300 Cellular Quantification and Kinetic Analysis of Dinitrosyliron Complexes Jason Hickok1, Sumit Sahani1, Hong Shen1, and Douglas D Thomas1 1 UIC • While it is well established that nitric oxide ( NO) reacts with cellular iron and thiols to form dinitrosyl iron complexes (DNIC),rigorous characterization of this paramagnetic species under controlled, biologically-relevant, experimental conditions has never been undertaken. As there are physiologic properties • of NO that cannot be explained mechanistically by the formation • s-nitrosothiols or NO-heme interactions, delineating the intrinsic nature of DNIC is potentially of great importance. Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous • and exogenous sources of NO in relation to iron and O2 • concentrations. Low physiologic NO concentrations (50-500 nM) resulted in the rapid (sec) quantitative conversion of the chelatable iron pool (CIP) into DNIC with a stoichiometry of 1:1. • Treating cells with pathologic NO concentrations (>1 μM) increased the amount of DNIC beyond the basal concentrations of • chelatable iron, indicating NO-mediated iron mobilization. Temporal measurements revealed that DNIC disappear by firstorder kinetics (t½ = 80 min) in an oxygen independent manner • and remain detectable long after the NO source had been removed (>24h). As predicted, changing the O2 concentration • (21→1%) reduced the rate of O2-dependent enzymatic NO synthesis in endotoxin-stimulated RAW cells. Unexpectedly, however, greater amounts of intracellular DNIC were detected at 1% as compared to 21% O2 (2,200 vs. 900 pmol/mg protein). • Just as NO will effect iron homeostasis, which has far-reaching ramifications for cellular physiology, iron will influence the • biological actions of NO. These results demonstrate that DNIC
SFRBM/SFRRI 2010