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The Correlation between Salivary and Plasma NOx
cysteine alone can efficiently decompose the protein-bound DNICs under aerobic condition, implying that L-cysteine may have a crucial role in cellular response to NO damage.
Nitric Oxide and its Interactions 1 The Effects of Nitrogen Oxides and Sulfides on Gardos Channel Activity and Red Blood Cell (RBC) Deformability Andrea Belanger1, Tamir Kanias2, Mark Gladwin2, Daniel KimShapiro1, and Andrea Belanger1 1 Wake Forest University, USA, 2UPMC, USA Sickle cell disease is caused by a mutation in the gene encoding the beta subunit of hemoglobin (Hb) which results in Hb polymerization upon deoxygenation and the cells become rigid and misshapen (sickling). Cycles of sickling and unsickling cause an influx of calcium that leads to loss of potassium via the calcium-activated Gardos Channel (GC) which dehydrates the cells leading to increased polymerization. Some published studies have shown that various nitric oxide (NO) donors improve deformability of normal erythrocytes. Another study has shown WKDW WKH ³12 GRQRU´ VRGLXP QLWURSUXVVLGH 613 SUHYHQWV WKLV calcium-induced loss of deformability in normal erythrocytes exposed to elevated extracellular calcium and calcium ionophore A23187. We confirmed this effect of SNP when calcium and the ionophore are present, but saw no effect of any NO donors when the ionophore was absent. In addition, we tested the effects of SNP on deoxygenation-induced dehydration of RBCs from patients with sickle cell disease using laser-assisted osmotic gradient ektacytometry and saw a dramatic improvement in sRBC mechanical properties and, therefore, hydration. Nitrite was also tested, however trends did not reach significance. To test our hypothesis that this protective effect is caused by nitrosation of thiols in GC or nearby protein disulfide isomerase (PDI) that is known to influence GC activity, we tested if sulfide or nitroxyl donors also protected against calcium-induced RBC dehydration. These data suggest certain nitrogen oxides may be able to protect sickle cells from dehydration and thereby improve outcome in the disease. This research was funded by NIH grant HL058091. doi: 10.1016/j.freeradbiomed.2014.10.487
The Correlation between Salivary and Plasma NOx
William Howard Craig Clodfelter1, Swati Basu1, Patricia DosSantos1, Bruce King1, and Dany Kim-Shapiro1 1 Wake Forest University, USA There is some debate about the correlation of basal salivary and plasma NOx levels. As demonstrated by the Kelm laboratory (Lauer et al, PNAS, 2001), plasma nitrite is an indicator of eNOS activity rather than plasma NO3- and/or NOx (nitrate plus nitrite). However, despite the possible correlation between saliva and plasma NOx, no literature supports that salivary nitrite is an indicator of endothelial function. Despite this, many brands of nitric oxide saliva test strips claim that their product is indicative of circulatory NO availability. Here we investigate basal salivary and plasma NOx to determine if there is any correlation with salivary NOx and endothelial function. We found no significant correlation between basal salivary and plasma NOx in a study with 15 younger adult volunteers who had not recently consumed any high nitrate beverages or food. There was no correlation comparing salivary nitrate vs. plasma nitrate, salivary nitrite vs. plasma nitrite, salivary nitrate vs. plasma nitrite, salivary nitrite vs plasma nitrate, and salivary NOx vs plasma NOx. In a separate study, we compared the efficiency of salivary nitrate reduction with plasma nitrate and nitrite after drinking beet juice, a high nitrate drink, in 30 volunteers. Once again, no significant correlation was discovered. These results suggest that salivary nitrate, nitrite or NOx is not a good indicator of endothelial function. In addition, saliva NO test strips which only measure nitrite in saliva may not be good indicators of plasma nitrite and thus endothelial function.
4
Dynamic Metabolism of Iron-Sulfur Proteins in Response to Nitric Oxide in Rat Heart Muscle Cells Zishuo Cheng1, Maria Barnes2, and Huangen Ding1 Department of Biological Sciences, Louisiana State University, USA, 2Pennington Biomedical Research Center, USA Reactive oxygen and nitrogen species generated during ischemia/reperfusion have been shown to severely injure cardiovascular tissues. Here we have evaluated the damage of iron-sulfur proteins in isolated rat heart muscle cells after exposure to nitric oxide (NO). We found that the specific activity of the iron-sulfur enzyme aconitase was gradually decreased with concomitant increase of the protein-bound dinitrosyl iron complex (DNIC) when heart muscle cells were treated with increasing concentrations of NO. Gel filtration analyses of the cell extracts prepared from the NO-treated heart muscle cells revealed a broad distribution of the protein-bound DNICs, indicating that multiple iron-sulfur proteins are modified by NO forming the protein-bound DNICs in heart muscle cells. The NO-inactivated aconitase in the cell extracts was re-activated when the cell extracts were reincubated with L-cysteine, cysteine desulfurase and ferrous iron in the presence of dithiothreitol under aerobic conditions to reassemble new iron-sulfur clusters in the proteins. Importantly, L-
S14
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doi: 10.1016/j.freeradbiomed.2014.10.489
2
1
doi: 10.1016/j.freeradbiomed.2014.10.488
Hemoglobin Induced Vasoconstriction and Oxidative Stress are Prevented by Haptoglobin Paul Buehler1, Paul Eigenberger2, David Irwin2, Zoe Loomis2, Joanne Maltzhan2, Malcolm Anderson3, and Christina Lisk4 1 Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research (CBER), Bethesda, USA, 2Cardiovascular Pulmonary Research Group, University of Colorado, Anschutz Medical Campus, USA, 3 Department of Critical Care, Children's Hospital of Colorado, USA, 4Program of Biological Sciences, Boston University School of Medicine, USA Introduction: Cell fee hemoglobin (Hb) has been linked to the initiation and progression of systemic as well as pulmonary hypertension (PH). The primary mechanisms proposed suggest decreased NO bioavailability and Hb oxidation. However, it is not well understood if extravasation is required for Hb to exert its vaso-motor and oxidative effects. The hemoglobin-haptoglobin complex (Hb-Hp) has been shown to bind NO at the same rate as Hb alone, yet the complex may be unable to leave the plasma compartment. We therefore hypothesized that Hp binding of Hb may inhibit the vascular responses to free Hb by preventing extravasation across the vessel wall.
SFRBM 2014
Nitric Oxide and its Interactions 1 The Effects of Nitrogen Oxides and Sulfides on Gardos Channel Activity and Red Blood Cell (RBC) Deformability Andrea Belanger1, Tamir Kanias2, Mark Gladwin2, Daniel KimShapiro1, and Andrea Belanger1 1 Wake Forest University, USA, 2UPMC, USA Sickle cell disease is caused by a mutation in the gene encoding the beta subunit of hemoglobin (Hb) which results in Hb polymerization upon deoxygenation and the cells become rigid and misshapen (sickling). Cycles of sickling and unsickling cause an influx of calcium that leads to loss of potassium via the calcium-activated Gardos Channel (GC) which dehydrates the cells leading to increased polymerization. Some published studies have shown that various nitric oxide (NO) donors improve deformability of normal erythrocytes. Another study has shown WKDW WKH ³12 GRQRU´ VRGLXP QLWURSUXVVLGH 613 SUHYHQWV WKLV calcium-induced loss of deformability in normal erythrocytes exposed to elevated extracellular calcium and calcium ionophore A23187. We confirmed this effect of SNP when calcium and the ionophore are present, but saw no effect of any NO donors when the ionophore was absent. In addition, we tested the effects of SNP on deoxygenation-induced dehydration of RBCs from patients with sickle cell disease using laser-assisted osmotic gradient ektacytometry and saw a dramatic improvement in sRBC mechanical properties and, therefore, hydration. Nitrite was also tested, however trends did not reach significance. To test our hypothesis that this protective effect is caused by nitrosation of thiols in GC or nearby protein disulfide isomerase (PDI) that is known to influence GC activity, we tested if sulfide or nitroxyl donors also protected against calcium-induced RBC dehydration. These data suggest certain nitrogen oxides may be able to protect sickle cells from dehydration and thereby improve outcome in the disease. This research was funded by NIH grant HL058091. doi: 10.1016/j.freeradbiomed.2014.10.487
The Correlation between Salivary and Plasma NOx
William Howard Craig Clodfelter1, Swati Basu1, Patricia DosSantos1, Bruce King1, and Dany Kim-Shapiro1 1 Wake Forest University, USA There is some debate about the correlation of basal salivary and plasma NOx levels. As demonstrated by the Kelm laboratory (Lauer et al, PNAS, 2001), plasma nitrite is an indicator of eNOS activity rather than plasma NO3- and/or NOx (nitrate plus nitrite). However, despite the possible correlation between saliva and plasma NOx, no literature supports that salivary nitrite is an indicator of endothelial function. Despite this, many brands of nitric oxide saliva test strips claim that their product is indicative of circulatory NO availability. Here we investigate basal salivary and plasma NOx to determine if there is any correlation with salivary NOx and endothelial function. We found no significant correlation between basal salivary and plasma NOx in a study with 15 younger adult volunteers who had not recently consumed any high nitrate beverages or food. There was no correlation comparing salivary nitrate vs. plasma nitrate, salivary nitrite vs. plasma nitrite, salivary nitrate vs. plasma nitrite, salivary nitrite vs plasma nitrate, and salivary NOx vs plasma NOx. In a separate study, we compared the efficiency of salivary nitrate reduction with plasma nitrate and nitrite after drinking beet juice, a high nitrate drink, in 30 volunteers. Once again, no significant correlation was discovered. These results suggest that salivary nitrate, nitrite or NOx is not a good indicator of endothelial function. In addition, saliva NO test strips which only measure nitrite in saliva may not be good indicators of plasma nitrite and thus endothelial function.
4
Dynamic Metabolism of Iron-Sulfur Proteins in Response to Nitric Oxide in Rat Heart Muscle Cells Zishuo Cheng1, Maria Barnes2, and Huangen Ding1 Department of Biological Sciences, Louisiana State University, USA, 2Pennington Biomedical Research Center, USA Reactive oxygen and nitrogen species generated during ischemia/reperfusion have been shown to severely injure cardiovascular tissues. Here we have evaluated the damage of iron-sulfur proteins in isolated rat heart muscle cells after exposure to nitric oxide (NO). We found that the specific activity of the iron-sulfur enzyme aconitase was gradually decreased with concomitant increase of the protein-bound dinitrosyl iron complex (DNIC) when heart muscle cells were treated with increasing concentrations of NO. Gel filtration analyses of the cell extracts prepared from the NO-treated heart muscle cells revealed a broad distribution of the protein-bound DNICs, indicating that multiple iron-sulfur proteins are modified by NO forming the protein-bound DNICs in heart muscle cells. The NO-inactivated aconitase in the cell extracts was re-activated when the cell extracts were reincubated with L-cysteine, cysteine desulfurase and ferrous iron in the presence of dithiothreitol under aerobic conditions to reassemble new iron-sulfur clusters in the proteins. Importantly, L-
S14
3
doi: 10.1016/j.freeradbiomed.2014.10.489
2
1
doi: 10.1016/j.freeradbiomed.2014.10.488
Hemoglobin Induced Vasoconstriction and Oxidative Stress are Prevented by Haptoglobin Paul Buehler1, Paul Eigenberger2, David Irwin2, Zoe Loomis2, Joanne Maltzhan2, Malcolm Anderson3, and Christina Lisk4 1 Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research (CBER), Bethesda, USA, 2Cardiovascular Pulmonary Research Group, University of Colorado, Anschutz Medical Campus, USA, 3 Department of Critical Care, Children's Hospital of Colorado, USA, 4Program of Biological Sciences, Boston University School of Medicine, USA Introduction: Cell fee hemoglobin (Hb) has been linked to the initiation and progression of systemic as well as pulmonary hypertension (PH). The primary mechanisms proposed suggest decreased NO bioavailability and Hb oxidation. However, it is not well understood if extravasation is required for Hb to exert its vaso-motor and oxidative effects. The hemoglobin-haptoglobin complex (Hb-Hp) has been shown to bind NO at the same rate as Hb alone, yet the complex may be unable to leave the plasma compartment. We therefore hypothesized that Hp binding of Hb may inhibit the vascular responses to free Hb by preventing extravasation across the vessel wall.
SFRBM 2014