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Nitric oxide metabolism in erythrocytes of breast cancer patients
122
Abstracts/Nitric Oxide 42 (2014) 99–153
Results and conclusions: Characterization of NO-nps revealed that size and release rates could be altered with facile manipulation of incorporated ingredients. NO-nps were nontoxic in all assays assessed using broad ranges of concentrations. The mechanism of production of NO from incorporated nitrite appears to occur via a dinitrogen trioxide intermediate capable of S-nitrosation under anaerobic conditions. The NO-nps demonstrated significant impacts in all in vivo, preclinical topical and systemic application models, which will be reviewed. Together, these data highlight NO-nps tremendous potential for a broad range of medical applications. Keywords: Nitric oxide; Nanoparticles; Erectile dysfunction; Hemorrhagic shock; Wound healing; Antimicrobial.
P123. 8-Nitro-cGMP-mediated autophagy in host defense and its regulation by hydrogen sulfide produced by bacteria http://dx.doi.org/10.1016/j.niox.2014.09.070 Shigemoto Fujii a, Shahzada Khan b, Tomoaki Ida a, Tomohiro Sawa a, Tetsuro Matsunaga a, Takaaki Akaike a a Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine b Gladstone Institutes, University of California, San Francisco, CA, USA
A nitrated nucleotide, 8-nitro-cGMP, is formed depending on nitric oxide and reactive oxygen species in cells, and functions as a signaling molecule via a unique posttranslational modification of cysteine thiol in proteins, which is called protein S-guanylation. Interestingly, we recently found that hydrogen sulfide-related compounds react with and degrade 8-nitro-cGMP and regulate its signaling functions. Emerging evidence shows an important role of autophagy in host defense against bacterial infections. However, the mechanism of autophagy induction during bacterial infection is not fully understood. Here, we investigated the molecular mechanism of 8-nitro-cGMP-mediated autophagy induction working on host defense against bacterial infection, and its regulation by hydrogen sulfide produced by bacteria. In mouse macrophages (RAW 264.7 cells and peritoneal macrophages), Salmonella Typhimurium LT2 infection markedly increased in cellular formation of 8-nitro-cGMP and induced autophagy. Direct treatment with 8-nitro-cGMP of macrophages caused autophagy induction, which suggests that 8-nitrocGMP would mediate autophagy induction in bacterial infection. Confocal microscopic analysis revealed that Salmonella cells incorporated in autophagosomes in macrophages were heavily S-guanylated, and autophagosome-related proteins, p62 and LC3, were colocalized with bacterial S-guanylated proteins. We also identified bacterial proteins targeted for S-guanylation (fimbial protein FimA, molecular chaperone DnaK, etc.) by means of 2Dgel electrophoresis and mass spectrometric analysis, suggesting that S-guanylation of bacterial proteins may be involved in the induction of autophagy and subsequent intracellular bacterial killing. Compared with wild-type LT2 strain, Salmonella strains knocked-out of hydrogen sulfide-producing enzymes (sulfite reductase PhsABC and thiosulfate reductase AsrABC) induced autophagy in infected macrophages more extensively, and intracellular survival was significantly suppressed in PhsABC/ AsrABC knockout strains. These results suggest that 8-nitro-cGMP formed during bacterial infection induces autophagy via protein S-guanylation and contributes to antibacterial host defense, and that hydrogen sulfide produced by bacteria may contribute to its intracellular survival by inhibition of 8-nitro-cGMP-mediated autophagy. Keywords: 8-nitro-cGMP; Host defense; Bacterial infection; Autophagy; Hydrogen sulfide.
P124. Nitrite reduction by heme and cobalamin enzymes involved in homocysteine metabolism http://dx.doi.org/10.1016/j.niox.2014.09.071 Carmen Gherasim, Ruma Banerjee Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
Inorganic nitrite is a potent vasodilator and the largest source of bioavailable nitric oxide (NO) that has been recognized as a signaling molecule and modulator of gene expression. Biochemical and physiological evidence exists suggesting that nitrite, the one electron oxidation product of NO, represents a circulating pool of NO that can be accessed under hypoxic conditions. In addition to non-enzymatic nitrite reduction, conversion of nitrite to NO has been shown to be mediated by a range of metalloproteins, including hemoglobins and molybdoflavoproteins. In this study, we have identified human cystathionine ß-synthase (CBS) and a vitamin B12 processing chaperone as new players in nitrite reduction with implications for the nitrite-dependent control of H2S and homocysteine metabolism. This novel activity of CBS exposes the catalytic property of its unusual regulatory heme cofactor to reduce nitrite and generate Fe(II)-NO, in the presence of either dithionite (a chemical reducing system to generate Fe(II)-CBS) and NADPH and human methionine reductase (a diflavin oxidoreductase) as a physiological reducing system. Formation of Fe(II)-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H2S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Additionally, we also show that a novel metal center, the superreduced form of vitamin B12, i.e. cob(I)alamin bound to the trafficking protein, CblC, can also reduce nitrite to generate nitrosylcobalamin (NOCbl). Release of NO as a function of nitrite concentration was monitored using an NO analyzer. Reduction of nitrite by cob(I)alamin expands the range of metal centers with NO releasing abilities and provides additional molecular mechanisms that might regulate nitrite metabolism. P125. Nitric oxide metabolism in erythrocytes of breast cancer patients http://dx.doi.org/10.1016/j.niox.2014.09.072 Marijke Grau, Eileen Geffroy, Wolfram Malter, Freerk Baumann, Wilhelm Bloch Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
Breast cancer is associated with an increased nitric oxide synthase (NOS) activity and also high rate of metastasis has been related to high NO levels and NOS activity. High NO levels have been shown to increase blood flow to the tumor facilitating tumor growth. Both NOS expression and NO production thus correlate with the tumor grade with grad III tumors showing higher NO synthesis rate than grade II tumors. Investigated NOS isoforms include inducible and endothelial NOS while the role of red blood cell (RBC) NOS remains unclear. Therefore, this study aimed to investigate RBC-NOS activation, NO production and deformability of RBC in breast cancer patients. A total of 15 (10 breast cancer (BC) and 5 age matched health controls (HC)) persons were included in this pilot study. Blood from BC were taken before neo-adjuvant therapy (surgery, chemotherapy, radiotherapy). RBC-NOS activation was measured immunohistochemically, nitrite levels were determined using chemiluminescence detection and deformability was measured by ektacytometry. Data showed that activation of RBC-NOS enzyme was significantly higher in BC compared to HC. Also, nitrite levels in
Abstracts/Nitric Oxide 42 (2014) 99–153
RBC, as marker for NO synthesis, were higher in BC than in HC and consequently RBC deformability was higher in BC. The present data revealed that NO production is increased in breast cancer due to higher RBC-NOS activation thus leading to higher deformability. Higher NO production may facilitate tumor growth but it remains to be investigated whether NO levels decrease upon tumor resection and whether NOS inhibition might interfere with tumor growth. Keywords: Breast cancer; NO; NOS; RBC; Deformability. P126. Influence of chronic endurance exercise on human red blood cell deformability http://dx.doi.org/10.1016/j.niox.2014.09.073 Marijke Grau, John-Maxwell Cremer, Wilhelm Bloch Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
In recent years, red blood cell (RBC) deformability was extensively examined as this important parameter mainly influences the microcirculation. It is known that physical activity increases shear stress, which activates RBC nitric oxide synthase (NOS) leading to increased NO production and consequently increases RBC deformability. But the influence of chronic endurance training (>12 weeks) on RBC deformability and especially NO production has not been investigated so far. Therefore, the aim of the present study was to monitor RBC deformability, nitrite levels as marker for NO production and nitrosated/nitrosylated NO species (RxNO) in male and female athletes and non-athletes during a fivemonth investigation period. Thereby, the female groups were further divided depending on whether they used hormonal contraceptives (HC) or not. Twenty-five participants (12M, mean age 24.5 years /13F, mean age 26.5 years) were included in this study and persons with an endurance training content >4 h per week were classified as athletes. Deformability was measured weekly by ektacytometry while NO parameters were determined on a total of 7 investigation days by chemiluminescence detection. Data revealed significant higher deformability values in male athletes compared to male non-athletes as well higher values in female athletes than non-athletes. Also, deformability was higher in female athletes without HC than in athletes with HC. Additional investigations confirmed that estrogen positively influenced deformability. The results also showed that nitrite and RxNO levels in both, plasma and RBC, were higher in athletes than in nonathletes and that again, female athletes without HC showed higher values than athletes with HC. In conclusion, the data revealed a positive correlation between estrogen and RBC deformability in females. Further, data showed that chronic endurance training consequently increases RBC deformability due to an increase in NO production which possibly improves oxygen transport to the working muscle and thus sheds new light on physiological alterations in RBC deformability in female and male subjects due to exercise interventions. Keywords: RBC; Deformability; NO; NOS; Endurance exercise. P127. Influence of hypoxia on RBC–NOS activation, NO production and deformability of human red blood cells http://dx.doi.org/10.1016/j.niox.2014.09.074 Marijke Grau, Alexander Lauten, Steffen Hoeppener, Bjoern Goebel, Julian Brenig, Christian Jung, Wilhelm Bloch, Frank Suhr Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
The present study addressed the question whether hypoxia affects nitric oxide synthase activation (NOS) in red blood cells (RBC), NO generation and deformability of RBC. For this purpose,
123
oxygen saturation of the ambient air of fourteen healthy volunteers was reduced from 21% to 16.21%, 12.35% and 10% O2 every two hours before normal oxygen concentration was restored. Vital- and hypoxia associated parameters were determined at 21% and 10% O2. RBC–NOS phosphorylation sites and deformability were measured after each concentration. In a subsequent in vitro study, blood from ten healthy volunteers was adjusted to a hematocrit of 40% and gas-flushed with oxygen concentrations of 21%, 16.21%, 12.35% and 10% O2 for two hours each. At the end, samples were flushed for 45 min with 21% O2. RBC and plasma nitrite content as well as RBC deformability were measured after each concentration. Volunteers adapted to hypoxia with decreasing blood pressure and increases in heart rate and respiratory frequency. Blood oxygen saturation decreased while hypoxic parameters FCOHb and FMetHb increased from 21% O2 to 10% O2. Phosphorylation of RBC–NOS at its serine 1177 residue, indicating NOS activation, decreased with increasing hypoxia, while phosphorylation of serine 116 and threonine 495 residues, indicating decrease in NOS activation, increased. RBC deformability decreased until 12.35% O2, but significantly increased upon 10% O2 both in vivo and in vitro. In vitro experiments showed that plasma nitrite content was not affected by hypoxia while nitrite levels in RBC decrease with deceasing oxygen saturation suggesting a reduction of nitrite to NO in RBC under hypoxia. Both, in vivo and in vitro data first time provide clear evidence that hypoxia reduces RBC–NOS activation, and thus NO production, and RBC deformability to a certain extent. The results moreover suggest a compensatory mechanism by reducing RBC nitrite to NO, allowing the maintenance of RBC deformability and therefore sustaining microcirculation under high hypoxic conditions. The data gain new mechanistic understanding of hypoxic regulatory pathways and hypoxic vasodilatation, with a special emphasis on RBCs. Keywords: Hypoxia; RBC; Deformability; NO; NOS; Nitrite. P128. Production of nitric oxide by red blood cells in sickle cell anemia http://dx.doi.org/10.1016/j.niox.2014.09.075 Marijke Grau, Anais Mozar, Yann Lamarre, Frank Suhr, Keyn Charlot, Linda Weyel, Bianca Collins, Marc Romana, Marie-Dominique Hardy-Dessources, Nathalie Lemonne, Maryse Etienne-Julan, Wilhelm Bloch, Philippe Connes Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
Sickle cell anemia (SCA) is an inherited blood disorder caused by single nucleotide substitution in the b-globin gene leading to the synthesis of an abnormal hemoglobin (HbS) much less soluble than normal hemoglobin (HbA) when deoxygenated. The polymerization of HbS upon deoxygenation is the basic pathophysiologic event leading to red blood cell (RBC) sickling and stiffening, hemolysis, vasoocclusion and, ultimately, chronic organ damage. Sickle cell hemoglobin C disease (SC) is another sickle cell syndrome characterized by the presence of both HbS and HbC, with HbC being able to crystallize when oxygenated. Both SCA and SC disease are marked by anemia and the occurrence of vaso-occlusive events. Complications of these hemoglobinopathies, more particularly in SCA, have been associated with a reduction of nitric oxide (NO) bioavailability caused by the free hemoglobin and free arginase released into the plasma during red blood cell lysis. NO is enzymatically produced by NO synthases (NOS). RBC–NOS produced NO has been shown to regulate and maintain RBC deformability through direct S-nitrosylation of α- and β-spectrins in normal RBCs. SCA patients show reduced RBC deformability but so far RBC–NOS
Abstracts/Nitric Oxide 42 (2014) 99–153
Results and conclusions: Characterization of NO-nps revealed that size and release rates could be altered with facile manipulation of incorporated ingredients. NO-nps were nontoxic in all assays assessed using broad ranges of concentrations. The mechanism of production of NO from incorporated nitrite appears to occur via a dinitrogen trioxide intermediate capable of S-nitrosation under anaerobic conditions. The NO-nps demonstrated significant impacts in all in vivo, preclinical topical and systemic application models, which will be reviewed. Together, these data highlight NO-nps tremendous potential for a broad range of medical applications. Keywords: Nitric oxide; Nanoparticles; Erectile dysfunction; Hemorrhagic shock; Wound healing; Antimicrobial.
P123. 8-Nitro-cGMP-mediated autophagy in host defense and its regulation by hydrogen sulfide produced by bacteria http://dx.doi.org/10.1016/j.niox.2014.09.070 Shigemoto Fujii a, Shahzada Khan b, Tomoaki Ida a, Tomohiro Sawa a, Tetsuro Matsunaga a, Takaaki Akaike a a Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine b Gladstone Institutes, University of California, San Francisco, CA, USA
A nitrated nucleotide, 8-nitro-cGMP, is formed depending on nitric oxide and reactive oxygen species in cells, and functions as a signaling molecule via a unique posttranslational modification of cysteine thiol in proteins, which is called protein S-guanylation. Interestingly, we recently found that hydrogen sulfide-related compounds react with and degrade 8-nitro-cGMP and regulate its signaling functions. Emerging evidence shows an important role of autophagy in host defense against bacterial infections. However, the mechanism of autophagy induction during bacterial infection is not fully understood. Here, we investigated the molecular mechanism of 8-nitro-cGMP-mediated autophagy induction working on host defense against bacterial infection, and its regulation by hydrogen sulfide produced by bacteria. In mouse macrophages (RAW 264.7 cells and peritoneal macrophages), Salmonella Typhimurium LT2 infection markedly increased in cellular formation of 8-nitro-cGMP and induced autophagy. Direct treatment with 8-nitro-cGMP of macrophages caused autophagy induction, which suggests that 8-nitrocGMP would mediate autophagy induction in bacterial infection. Confocal microscopic analysis revealed that Salmonella cells incorporated in autophagosomes in macrophages were heavily S-guanylated, and autophagosome-related proteins, p62 and LC3, were colocalized with bacterial S-guanylated proteins. We also identified bacterial proteins targeted for S-guanylation (fimbial protein FimA, molecular chaperone DnaK, etc.) by means of 2Dgel electrophoresis and mass spectrometric analysis, suggesting that S-guanylation of bacterial proteins may be involved in the induction of autophagy and subsequent intracellular bacterial killing. Compared with wild-type LT2 strain, Salmonella strains knocked-out of hydrogen sulfide-producing enzymes (sulfite reductase PhsABC and thiosulfate reductase AsrABC) induced autophagy in infected macrophages more extensively, and intracellular survival was significantly suppressed in PhsABC/ AsrABC knockout strains. These results suggest that 8-nitro-cGMP formed during bacterial infection induces autophagy via protein S-guanylation and contributes to antibacterial host defense, and that hydrogen sulfide produced by bacteria may contribute to its intracellular survival by inhibition of 8-nitro-cGMP-mediated autophagy. Keywords: 8-nitro-cGMP; Host defense; Bacterial infection; Autophagy; Hydrogen sulfide.
P124. Nitrite reduction by heme and cobalamin enzymes involved in homocysteine metabolism http://dx.doi.org/10.1016/j.niox.2014.09.071 Carmen Gherasim, Ruma Banerjee Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
Inorganic nitrite is a potent vasodilator and the largest source of bioavailable nitric oxide (NO) that has been recognized as a signaling molecule and modulator of gene expression. Biochemical and physiological evidence exists suggesting that nitrite, the one electron oxidation product of NO, represents a circulating pool of NO that can be accessed under hypoxic conditions. In addition to non-enzymatic nitrite reduction, conversion of nitrite to NO has been shown to be mediated by a range of metalloproteins, including hemoglobins and molybdoflavoproteins. In this study, we have identified human cystathionine ß-synthase (CBS) and a vitamin B12 processing chaperone as new players in nitrite reduction with implications for the nitrite-dependent control of H2S and homocysteine metabolism. This novel activity of CBS exposes the catalytic property of its unusual regulatory heme cofactor to reduce nitrite and generate Fe(II)-NO, in the presence of either dithionite (a chemical reducing system to generate Fe(II)-CBS) and NADPH and human methionine reductase (a diflavin oxidoreductase) as a physiological reducing system. Formation of Fe(II)-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H2S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Additionally, we also show that a novel metal center, the superreduced form of vitamin B12, i.e. cob(I)alamin bound to the trafficking protein, CblC, can also reduce nitrite to generate nitrosylcobalamin (NOCbl). Release of NO as a function of nitrite concentration was monitored using an NO analyzer. Reduction of nitrite by cob(I)alamin expands the range of metal centers with NO releasing abilities and provides additional molecular mechanisms that might regulate nitrite metabolism. P125. Nitric oxide metabolism in erythrocytes of breast cancer patients http://dx.doi.org/10.1016/j.niox.2014.09.072 Marijke Grau, Eileen Geffroy, Wolfram Malter, Freerk Baumann, Wilhelm Bloch Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
Breast cancer is associated with an increased nitric oxide synthase (NOS) activity and also high rate of metastasis has been related to high NO levels and NOS activity. High NO levels have been shown to increase blood flow to the tumor facilitating tumor growth. Both NOS expression and NO production thus correlate with the tumor grade with grad III tumors showing higher NO synthesis rate than grade II tumors. Investigated NOS isoforms include inducible and endothelial NOS while the role of red blood cell (RBC) NOS remains unclear. Therefore, this study aimed to investigate RBC-NOS activation, NO production and deformability of RBC in breast cancer patients. A total of 15 (10 breast cancer (BC) and 5 age matched health controls (HC)) persons were included in this pilot study. Blood from BC were taken before neo-adjuvant therapy (surgery, chemotherapy, radiotherapy). RBC-NOS activation was measured immunohistochemically, nitrite levels were determined using chemiluminescence detection and deformability was measured by ektacytometry. Data showed that activation of RBC-NOS enzyme was significantly higher in BC compared to HC. Also, nitrite levels in
Abstracts/Nitric Oxide 42 (2014) 99–153
RBC, as marker for NO synthesis, were higher in BC than in HC and consequently RBC deformability was higher in BC. The present data revealed that NO production is increased in breast cancer due to higher RBC-NOS activation thus leading to higher deformability. Higher NO production may facilitate tumor growth but it remains to be investigated whether NO levels decrease upon tumor resection and whether NOS inhibition might interfere with tumor growth. Keywords: Breast cancer; NO; NOS; RBC; Deformability. P126. Influence of chronic endurance exercise on human red blood cell deformability http://dx.doi.org/10.1016/j.niox.2014.09.073 Marijke Grau, John-Maxwell Cremer, Wilhelm Bloch Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
In recent years, red blood cell (RBC) deformability was extensively examined as this important parameter mainly influences the microcirculation. It is known that physical activity increases shear stress, which activates RBC nitric oxide synthase (NOS) leading to increased NO production and consequently increases RBC deformability. But the influence of chronic endurance training (>12 weeks) on RBC deformability and especially NO production has not been investigated so far. Therefore, the aim of the present study was to monitor RBC deformability, nitrite levels as marker for NO production and nitrosated/nitrosylated NO species (RxNO) in male and female athletes and non-athletes during a fivemonth investigation period. Thereby, the female groups were further divided depending on whether they used hormonal contraceptives (HC) or not. Twenty-five participants (12M, mean age 24.5 years /13F, mean age 26.5 years) were included in this study and persons with an endurance training content >4 h per week were classified as athletes. Deformability was measured weekly by ektacytometry while NO parameters were determined on a total of 7 investigation days by chemiluminescence detection. Data revealed significant higher deformability values in male athletes compared to male non-athletes as well higher values in female athletes than non-athletes. Also, deformability was higher in female athletes without HC than in athletes with HC. Additional investigations confirmed that estrogen positively influenced deformability. The results also showed that nitrite and RxNO levels in both, plasma and RBC, were higher in athletes than in nonathletes and that again, female athletes without HC showed higher values than athletes with HC. In conclusion, the data revealed a positive correlation between estrogen and RBC deformability in females. Further, data showed that chronic endurance training consequently increases RBC deformability due to an increase in NO production which possibly improves oxygen transport to the working muscle and thus sheds new light on physiological alterations in RBC deformability in female and male subjects due to exercise interventions. Keywords: RBC; Deformability; NO; NOS; Endurance exercise. P127. Influence of hypoxia on RBC–NOS activation, NO production and deformability of human red blood cells http://dx.doi.org/10.1016/j.niox.2014.09.074 Marijke Grau, Alexander Lauten, Steffen Hoeppener, Bjoern Goebel, Julian Brenig, Christian Jung, Wilhelm Bloch, Frank Suhr Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
The present study addressed the question whether hypoxia affects nitric oxide synthase activation (NOS) in red blood cells (RBC), NO generation and deformability of RBC. For this purpose,
123
oxygen saturation of the ambient air of fourteen healthy volunteers was reduced from 21% to 16.21%, 12.35% and 10% O2 every two hours before normal oxygen concentration was restored. Vital- and hypoxia associated parameters were determined at 21% and 10% O2. RBC–NOS phosphorylation sites and deformability were measured after each concentration. In a subsequent in vitro study, blood from ten healthy volunteers was adjusted to a hematocrit of 40% and gas-flushed with oxygen concentrations of 21%, 16.21%, 12.35% and 10% O2 for two hours each. At the end, samples were flushed for 45 min with 21% O2. RBC and plasma nitrite content as well as RBC deformability were measured after each concentration. Volunteers adapted to hypoxia with decreasing blood pressure and increases in heart rate and respiratory frequency. Blood oxygen saturation decreased while hypoxic parameters FCOHb and FMetHb increased from 21% O2 to 10% O2. Phosphorylation of RBC–NOS at its serine 1177 residue, indicating NOS activation, decreased with increasing hypoxia, while phosphorylation of serine 116 and threonine 495 residues, indicating decrease in NOS activation, increased. RBC deformability decreased until 12.35% O2, but significantly increased upon 10% O2 both in vivo and in vitro. In vitro experiments showed that plasma nitrite content was not affected by hypoxia while nitrite levels in RBC decrease with deceasing oxygen saturation suggesting a reduction of nitrite to NO in RBC under hypoxia. Both, in vivo and in vitro data first time provide clear evidence that hypoxia reduces RBC–NOS activation, and thus NO production, and RBC deformability to a certain extent. The results moreover suggest a compensatory mechanism by reducing RBC nitrite to NO, allowing the maintenance of RBC deformability and therefore sustaining microcirculation under high hypoxic conditions. The data gain new mechanistic understanding of hypoxic regulatory pathways and hypoxic vasodilatation, with a special emphasis on RBCs. Keywords: Hypoxia; RBC; Deformability; NO; NOS; Nitrite. P128. Production of nitric oxide by red blood cells in sickle cell anemia http://dx.doi.org/10.1016/j.niox.2014.09.075 Marijke Grau, Anais Mozar, Yann Lamarre, Frank Suhr, Keyn Charlot, Linda Weyel, Bianca Collins, Marc Romana, Marie-Dominique Hardy-Dessources, Nathalie Lemonne, Maryse Etienne-Julan, Wilhelm Bloch, Philippe Connes Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
Sickle cell anemia (SCA) is an inherited blood disorder caused by single nucleotide substitution in the b-globin gene leading to the synthesis of an abnormal hemoglobin (HbS) much less soluble than normal hemoglobin (HbA) when deoxygenated. The polymerization of HbS upon deoxygenation is the basic pathophysiologic event leading to red blood cell (RBC) sickling and stiffening, hemolysis, vasoocclusion and, ultimately, chronic organ damage. Sickle cell hemoglobin C disease (SC) is another sickle cell syndrome characterized by the presence of both HbS and HbC, with HbC being able to crystallize when oxygenated. Both SCA and SC disease are marked by anemia and the occurrence of vaso-occlusive events. Complications of these hemoglobinopathies, more particularly in SCA, have been associated with a reduction of nitric oxide (NO) bioavailability caused by the free hemoglobin and free arginase released into the plasma during red blood cell lysis. NO is enzymatically produced by NO synthases (NOS). RBC–NOS produced NO has been shown to regulate and maintain RBC deformability through direct S-nitrosylation of α- and β-spectrins in normal RBCs. SCA patients show reduced RBC deformability but so far RBC–NOS