- Email: [email protected]
P47. Dietary nitrite levels influence hypoxia-induced pulmonary hypertension
S32
Nitrite 2011 meeting abstracts/Nitric Oxide 24 (2011) S16–S42
Background: Nitrite ðNO 2 Þ, a dietary constituent and intrinsic signaling molecule has been shown to regulate mitochondrial function, particularly respiration and reactive oxygen species generate in ischemic conditions. Here, we report for the first time that nitrite not only regulates mitochondrial function but also increases mitochondrial number during hypoxia by stimulating the mitochondrial biogenesis pathway. Methods: Primary rat aortic smooth muscle cells (RASMCs) were cultured and treated with different concentrations of nitrite (0–100 lM) in normoxia (21% O2) or hypoxia (1% O2) for 6 days continuously. Mitochondrial number was measured by MitoTracker green fluorescence in comparison to nuclear number. Results: We demonstrate here that six days of nitrite treatment in hypoxic conditions increases mitochondrial number by approximately three-fold, resulting in increased rates of coupled respiration and ATP production, with no change in reactive oxygen species generation. Nitrite-induced mitochondrial biogenesis occurs through the activation of sirtuin 1, the phosphorylation of AMP kinase, and an increase in the expression of mRNA for PGC-1a and the mitochondrial transcription factor Tfam. Interestingly, we find that nitrite-dependent stimulation of the classical mitochondrial biogenesis pathway deviates mechanistically from nitric oxide (NO) mediated biogenesis which is known to occur through the stimulation of cGMP. This is demonstrated by the fact that nitrite stimulates a greater extent of biogenesis at lower levels of cGMP production than NO. Additionally, ODQ, an inhibitor of sGC, does not abolish increases in PGC-1a mRNA expression and phosphorylation of AMP kinase induced by nitrite. We also provide evidence that nitrite-mediated mitochondrial biogenesis occurs in vivo. In a rat model of carotid injury, two weeks of nitrite treatment prevents a hyper-proliferative response in the smooth muscle after injury. This protection is accompanied by a nitrite-dependent upregulation of PGC-1a in the injured artery, an effect that is not observed in the corresponding uninjured carotid. Conclusion: These data demonstrate that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro partially independent on sGC/cGMP/NO pathway, and suggest that nitrite mediated mitochondrial biogenesis may play an important protective role in the setting of vascular injury. Disclosure: Sruti Shiva, corresponding author. Department of Pharmacology & Chemical Biology, Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA.
doi:10.1016/j.niox.2011.03.275
P45. The conception of nitric oxide and superoxide cycles: Biomedical aspects Valentin Reutov Institute of Higher Nervous Activity and Neurophysiology Russian Academy of Sciences, Laboratory of Functional Cytology, Moscow 115569, Russian Federation To construct a theory, one often needs to select the most general concept of the subject of study and its intuitively comprehensible conceptual content. In such cases, there is always a subconscious, intuitive feeling which attempts to suggest a hint that the phenomenon one is attacking is a key to something big and completely new. R. Feynman wrote at some point that we should extrapolate our knowledge into unknown areas; this is the only way for progress [1]. This is how the principle of cyclicity has emerged from analyses of metabolic cycles involving the four main atoms present in living organisms (C, N, O, and H) [2,3]. This principle made it possible to identity a general (cyclic) pattern in most various phenomena and processes that occur at practically every structural and functional level in living and nonliving matter [4,5]. Physiologists know that in order to learn how important one function or another is in an organism, one needs to switch it off and see what the consequences are. If we switched off all cyclic (or periodic) processes, then all biological rhythms would vanish, including periodic processes in the brain and in the heart. Hemoglobin would stop releasing and absorbing oxygen, blood circulation would come to a stop, as would all the pumps delivering certain ions into tissue cells and removing other ions from cells, and the heart would stop beating. All biochemical and energy transfer processes would stop, too, since enzymes operate in a cyclic mode. If all biological rhythms, all cyclic physiological and biochemical processes were switched off, life on planet Earth would come to an end. Therefore, both the topic of this discussion and the humans discussing this topic would disappear [3,4]. Consequently, cyclic organization can be identified at various structural and functional levels in living organisms, in the biosphere, and in stellar matter. This is the reason why we believe that the most important aspect in biology and the medical sciences is the question of cyclic organization. The report presents the conception of nitric oxide and superoxide radical anion cycles and discusses the biomedical aspects of these cycles. The developed concepts and their related ideas suggest, that the mechanism of cycle(s) shows naturally latent regularity and assigns a meaning to orderliness in the system of free radical compounds. By their chemical nature, the latter are well-known to tend to behave aggressively and commonly unpredictably, which frequently causes abnormalities of various genesis. In the author’s opinion, the mechanism of antiradical protection of cells and the body as a whole is built into very cyclic arrangement of metabolic processes which are attended by the formation of free radicals. Failure of this cyclic mechanism may be a cause of many diseases [2–5]. Disclosure: This presentation was supported in part by a grant from RFFI.
References [1] [2] [3] [4] [5]
R. Feynman, The Character of Physical Law, M.I.T. Press, Cambridge, 1965. V.P. Reutov, E.G. Sorokina, Biochemistry 63 (1998) 874–885. V.P. Reutov, Vest. Rus. Acad. Med. Sci. 4 (2000) 35–41. V.P. Reutov, Biochemistry (Moscow) 67 (3) (2002) 293–311. V.P. Reutov, A.N. Schechter, Physics-Uspekhi 53 (4) (2010) 377–396.
doi:10.1016/j.niox.2011.03.276
P46. Acute effects of dietary nitrate on glucose handling and insulin levels during an oral glucose tolerance test in healthy subjects Satnam Liddera,b, Joanne Hunta, Sami Omara, Andrew Webba King’s College London British Heart Foundation Centre, Clinical Pharmacology, London SE1 7EH, United Kingdom b Barts & The London NHS Trust, Clinical Pharmacology, London EC1M 6BQ, United Kingdom
a
Introduction: Recent evidence from observational studies suggests that diets rich in green leafy vegetables may reduce the risk of diabetes. This could relate to a greater intake of dietary nitrate. Indeed, M. Carlstrom et al. (2010) demonstrated that 10 weeks’ supplementation with sodium nitrate in eNOS-deficient mice improved several features of the metabolic syndrome, including impaired glucose tolerance. We and others have demonstrated that dietary nitrate has beneficial effects in humans on blood pressure, endothelial and platelet function, and exercise performance and we therefore hypothesised that dietary nitrate may improve glucose handling in healthy volunteers. Methods: Following ethical approval we conducted a randomised double blind cross-over study in 8 healthy subjects (4 male, weight 68.8 ± 3.0 kg, aged 26.5 ± 2.5 y) of the effects of potassium nitrate (KNO3, 24 mmol, in capsule form, nitrate content equivalent to 500 ml beetroot juice in a previous study) compared to potassium chloride (KCl, 24 mmol) on the plasma glucose excursion during a standard oral glucose tolerance test (75 g of glucose) performed 1 h later (the primary outcome). In addition to collecting blood for determination of plasma nitrite/nitrate concentrations by ozone chemiluminescence, exhaled nitric oxide (eNO) was measured at hourly intervals using a standard ATS exhaled NO breath protocol. Results: KNO3 increased plasma nitrate and nitrite concentrations and also the eNO curve (P < 0.0001): at 2 h post-ingestion the eNO value was 47.2 ± 7.0 ppb, compared to 27.4 ± 4.3 ppb with KCl (P < 0.01). KNO3 also resulted in a trend to a reduced systolic blood pressure of 7.6 ± 3.2 mmHg compared to KCl 2.5 h after ingestion (P = 0.057). KNO3 significantly reduced the excursion of the glucose curve compared to KCl (both 24 mmol) (P < 0.05, ANOVA), the mean AUC of the glucose curve being reduced by ~6%. This was associated with an increased excursion of the insulin curve with KNO3 compared to KCl (P = 0.01), and an increase in the mean AUC of the insulin curve of ~26%. However, no difference in insulin C-peptide was found. On linear regression analysis there was a trend for greater reductions in AUC glucose profiles with KNO3 to be associated with higher baseline AUC glucose profiles with KCl (r2 = 0.39, P = 0.05). Conclusions: A single acute ingestion of potassium nitrate results in an increase in bioavailable NO, as demonstrated by an almost doubling of eNO levels, and a modest improvement in the glucose curve during an oral glucose challenge in healthy subjects with normal glucose excursions. Subjects at the high normal end of the spectrum of glucose responses appeared to have the greatest reductions in glucose with nitrate. Increased insulin may account, at least in part, for this effect, but the mechanism may relate to reduced insulin clearance rather than increased insulin secretion. Increasing dietary nitrate, possibly in the form of green leafy salad starters, may be effective in limiting subsequent prandial hyperglycaemia with carbohydrate-rich main-courses and deserts, an effect that may be greater in people with impaired glucose tolerance; the long-term effects on improving glucose control remain to be established. Disclosure: This work was supported by King’s College London, British Heart Foundation Centre, and Barts & The London NHS Trust.
doi:10.1016/j.niox.2011.03.277
P47. Dietary nitrite levels influence hypoxia-induced pulmonary hypertension Roy Sutliffa,b, Alexander El-Alia,b, Erik Walpa,b, Benjamin Predmorec, David Leferc a Emory University, Medicine, Atlanta, GA 30033, United States b Atlanta VA Medical Center, Medicine, Decatur, GA 30033, United States c Emory University, Surgery, Atlanta, GA 30033, United States
Nitrite 2011 meeting abstracts/Nitric Oxide 24 (2011) S16–S42 Background: Pulmonary hypertension (PH), defined as elevation of mean pulmonary artery pressure above 25 mmHg at rest, produces significant morbidity and mortality. The pathobiology of PH involves endothelial dysfunction and vasoconstriction leading to abnormal proliferation of pulmonary vascular wall cells resulting in vascular remodeling and muscularization of small pulmonary vessels. Recent studies have suggested that inhaled nitrite protects against hypoxia-induced PH. The present study was performed to determine if dietary nitrite levels can influence hypoxia-induced PH. Methods: Male C57/Bl6 mice were fed one of several diets: low nitrite diet (Harlan 99366; 20.5 pmol/g); a standard diet (Purina 5001; 104.3 pmol/g); low nitrite diet supplemented with 50 mg/L sodium nitrite; a low nitrite diet supplemented with 100 mg/L sodium nitrite; or standard diet supplemented with 50 mg/L. After the mice were on the diet for 10 days they were exposed to room air or 10% oxygen for 3 weeks. At the end of the 3 weeks PH was determined by measurement of right ventricular (RV) systolic pressure (RVSP) and following euthanasia, hematocrits and right ventricular hypertrophy (measured as RV:LV + S weight ratios). Results: Hypoxia-induced changes in hematocrit persisted for all of the diets. The levels of nitrite in the diet had profound effects on hypoxia-induced PH. Mice on standard diet and low nitrite diet developed PH in response to hypoxia. Mice maintained on a low nitrite diet had an exaggerated PH response to hypoxia. Mice fed diets supplemented with nitrite did not develop an increase in RVSP or RV hypertrophy. Conclusion: Reductions in dietary nitrite levels results in an exaggerated PH response to hypoxia. Increasing dietary nitrite eliminates hypoxia-induced PH. Our studies suggest that dietary nitrite levels have a significant impact on hypoxiainduced pulmonary hypertension. Furthermore, dietary nitrite may be an effective therapy for the treatment of PH. Disclosure: The authors have no conflicts to disclose.
S33
identified so far [1]. The main biological purpose of NPs is to deliver NO into the tissue of a host species to promote vasodilatation and anticoagulation. The recent interests in the interaction of ferriheme proteins with plasma NO 2 lead to the present study. Methods: The interaction of nitrophorins with NO 2 was examined by various spectroscopic techniques, including absorbance, EPR, and resonance Raman spectroscopy. Crystallization of NP4 was performed as previously described and the crystals were soaked with KNO2 prior to the recording of the diffraction pattern.
doi:10.1016/j.niox.2011.03.278
P48. Nitrite disproportionation reaction: Investigations on the mechanism of the conversion of nitrite into nitric oxide at the ferriheme center of nitrophorins at blood plasma pH Markus Knipp, Chunmao He, Hideaki Ogata Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr, NRW 45470, Germany Background: Nitrophorins (NPs) comprise a class of small ferriheme proteins that appear in the saliva of bloodsucking insects. Five isoproteins (NP1-4 and 7) have been
Results: Unlike other ferrihemes [2], NPs exhibit a reaction with NO 2 , which re sults in the formation of NO. Quantification of NO, NO 2 , and NO3 revealed the produc tion of two NO per three NO2 consumed, showing that NO2 serves as both the electron acceptor and the electron donor [3]. The reaction can be written as þ 3NO 2 þ 2H ! 2NO þ H2 O þ NO3 :
This reaction is well-known as the nitrite disproportionation reaction that appears at low pH in aqueous solutions; remarkably, NPs are, to the best of our knowledge, the only examples among the ferriheme proteins and model compounds that promote the reaction at neutral pH. When the reaction is carried out in the presence of an NO scavenger, a continuous production of NO is observed; thus, the protein acts as a catalyst [3]. Unlike metHb and metMb, which form an g1-O nitrito complex [4,5], the spectroscopic and crystallographic characterization of the initial FeIII–NO 2 complex of NPs revealed the more common g1-N nitro conformation [6]. In metMb, deletion of the distal His64 residue lead to the change of the coordina1 tion state of NO 2 to the g -N nitro conformation where the concomitant insertion of an Arg into the distal heme pocket (Mb(H64V, V67R)) restored the unique g1-O nitrito complex [5]. Insertion of Arg into the distal pocket of NP4 (NP4(L130R)) lead to a significant decrease in the reaction rate. However, EPR spectroscopy revealed no significant change in the EPR parameters, suggesting that the ligand coordination is preserved. FeðIIIÞNO 2
NO 2 coordination
gA max
gB max
Ratio of spin systems A and B
Reference
metHb
g1-O
2.98
2.89
n.r.
3.11
2.97
4:6
[Nat. Chem. Biol. 5 (2009) 366] [Biochemistry 27 (1988) 2790]
metMb
1
g -O
Nitrite 2011 meeting abstracts/Nitric Oxide 24 (2011) S16–S42
Background: Nitrite ðNO 2 Þ, a dietary constituent and intrinsic signaling molecule has been shown to regulate mitochondrial function, particularly respiration and reactive oxygen species generate in ischemic conditions. Here, we report for the first time that nitrite not only regulates mitochondrial function but also increases mitochondrial number during hypoxia by stimulating the mitochondrial biogenesis pathway. Methods: Primary rat aortic smooth muscle cells (RASMCs) were cultured and treated with different concentrations of nitrite (0–100 lM) in normoxia (21% O2) or hypoxia (1% O2) for 6 days continuously. Mitochondrial number was measured by MitoTracker green fluorescence in comparison to nuclear number. Results: We demonstrate here that six days of nitrite treatment in hypoxic conditions increases mitochondrial number by approximately three-fold, resulting in increased rates of coupled respiration and ATP production, with no change in reactive oxygen species generation. Nitrite-induced mitochondrial biogenesis occurs through the activation of sirtuin 1, the phosphorylation of AMP kinase, and an increase in the expression of mRNA for PGC-1a and the mitochondrial transcription factor Tfam. Interestingly, we find that nitrite-dependent stimulation of the classical mitochondrial biogenesis pathway deviates mechanistically from nitric oxide (NO) mediated biogenesis which is known to occur through the stimulation of cGMP. This is demonstrated by the fact that nitrite stimulates a greater extent of biogenesis at lower levels of cGMP production than NO. Additionally, ODQ, an inhibitor of sGC, does not abolish increases in PGC-1a mRNA expression and phosphorylation of AMP kinase induced by nitrite. We also provide evidence that nitrite-mediated mitochondrial biogenesis occurs in vivo. In a rat model of carotid injury, two weeks of nitrite treatment prevents a hyper-proliferative response in the smooth muscle after injury. This protection is accompanied by a nitrite-dependent upregulation of PGC-1a in the injured artery, an effect that is not observed in the corresponding uninjured carotid. Conclusion: These data demonstrate that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro partially independent on sGC/cGMP/NO pathway, and suggest that nitrite mediated mitochondrial biogenesis may play an important protective role in the setting of vascular injury. Disclosure: Sruti Shiva, corresponding author. Department of Pharmacology & Chemical Biology, Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA.
doi:10.1016/j.niox.2011.03.275
P45. The conception of nitric oxide and superoxide cycles: Biomedical aspects Valentin Reutov Institute of Higher Nervous Activity and Neurophysiology Russian Academy of Sciences, Laboratory of Functional Cytology, Moscow 115569, Russian Federation To construct a theory, one often needs to select the most general concept of the subject of study and its intuitively comprehensible conceptual content. In such cases, there is always a subconscious, intuitive feeling which attempts to suggest a hint that the phenomenon one is attacking is a key to something big and completely new. R. Feynman wrote at some point that we should extrapolate our knowledge into unknown areas; this is the only way for progress [1]. This is how the principle of cyclicity has emerged from analyses of metabolic cycles involving the four main atoms present in living organisms (C, N, O, and H) [2,3]. This principle made it possible to identity a general (cyclic) pattern in most various phenomena and processes that occur at practically every structural and functional level in living and nonliving matter [4,5]. Physiologists know that in order to learn how important one function or another is in an organism, one needs to switch it off and see what the consequences are. If we switched off all cyclic (or periodic) processes, then all biological rhythms would vanish, including periodic processes in the brain and in the heart. Hemoglobin would stop releasing and absorbing oxygen, blood circulation would come to a stop, as would all the pumps delivering certain ions into tissue cells and removing other ions from cells, and the heart would stop beating. All biochemical and energy transfer processes would stop, too, since enzymes operate in a cyclic mode. If all biological rhythms, all cyclic physiological and biochemical processes were switched off, life on planet Earth would come to an end. Therefore, both the topic of this discussion and the humans discussing this topic would disappear [3,4]. Consequently, cyclic organization can be identified at various structural and functional levels in living organisms, in the biosphere, and in stellar matter. This is the reason why we believe that the most important aspect in biology and the medical sciences is the question of cyclic organization. The report presents the conception of nitric oxide and superoxide radical anion cycles and discusses the biomedical aspects of these cycles. The developed concepts and their related ideas suggest, that the mechanism of cycle(s) shows naturally latent regularity and assigns a meaning to orderliness in the system of free radical compounds. By their chemical nature, the latter are well-known to tend to behave aggressively and commonly unpredictably, which frequently causes abnormalities of various genesis. In the author’s opinion, the mechanism of antiradical protection of cells and the body as a whole is built into very cyclic arrangement of metabolic processes which are attended by the formation of free radicals. Failure of this cyclic mechanism may be a cause of many diseases [2–5]. Disclosure: This presentation was supported in part by a grant from RFFI.
References [1] [2] [3] [4] [5]
R. Feynman, The Character of Physical Law, M.I.T. Press, Cambridge, 1965. V.P. Reutov, E.G. Sorokina, Biochemistry 63 (1998) 874–885. V.P. Reutov, Vest. Rus. Acad. Med. Sci. 4 (2000) 35–41. V.P. Reutov, Biochemistry (Moscow) 67 (3) (2002) 293–311. V.P. Reutov, A.N. Schechter, Physics-Uspekhi 53 (4) (2010) 377–396.
doi:10.1016/j.niox.2011.03.276
P46. Acute effects of dietary nitrate on glucose handling and insulin levels during an oral glucose tolerance test in healthy subjects Satnam Liddera,b, Joanne Hunta, Sami Omara, Andrew Webba King’s College London British Heart Foundation Centre, Clinical Pharmacology, London SE1 7EH, United Kingdom b Barts & The London NHS Trust, Clinical Pharmacology, London EC1M 6BQ, United Kingdom
a
Introduction: Recent evidence from observational studies suggests that diets rich in green leafy vegetables may reduce the risk of diabetes. This could relate to a greater intake of dietary nitrate. Indeed, M. Carlstrom et al. (2010) demonstrated that 10 weeks’ supplementation with sodium nitrate in eNOS-deficient mice improved several features of the metabolic syndrome, including impaired glucose tolerance. We and others have demonstrated that dietary nitrate has beneficial effects in humans on blood pressure, endothelial and platelet function, and exercise performance and we therefore hypothesised that dietary nitrate may improve glucose handling in healthy volunteers. Methods: Following ethical approval we conducted a randomised double blind cross-over study in 8 healthy subjects (4 male, weight 68.8 ± 3.0 kg, aged 26.5 ± 2.5 y) of the effects of potassium nitrate (KNO3, 24 mmol, in capsule form, nitrate content equivalent to 500 ml beetroot juice in a previous study) compared to potassium chloride (KCl, 24 mmol) on the plasma glucose excursion during a standard oral glucose tolerance test (75 g of glucose) performed 1 h later (the primary outcome). In addition to collecting blood for determination of plasma nitrite/nitrate concentrations by ozone chemiluminescence, exhaled nitric oxide (eNO) was measured at hourly intervals using a standard ATS exhaled NO breath protocol. Results: KNO3 increased plasma nitrate and nitrite concentrations and also the eNO curve (P < 0.0001): at 2 h post-ingestion the eNO value was 47.2 ± 7.0 ppb, compared to 27.4 ± 4.3 ppb with KCl (P < 0.01). KNO3 also resulted in a trend to a reduced systolic blood pressure of 7.6 ± 3.2 mmHg compared to KCl 2.5 h after ingestion (P = 0.057). KNO3 significantly reduced the excursion of the glucose curve compared to KCl (both 24 mmol) (P < 0.05, ANOVA), the mean AUC of the glucose curve being reduced by ~6%. This was associated with an increased excursion of the insulin curve with KNO3 compared to KCl (P = 0.01), and an increase in the mean AUC of the insulin curve of ~26%. However, no difference in insulin C-peptide was found. On linear regression analysis there was a trend for greater reductions in AUC glucose profiles with KNO3 to be associated with higher baseline AUC glucose profiles with KCl (r2 = 0.39, P = 0.05). Conclusions: A single acute ingestion of potassium nitrate results in an increase in bioavailable NO, as demonstrated by an almost doubling of eNO levels, and a modest improvement in the glucose curve during an oral glucose challenge in healthy subjects with normal glucose excursions. Subjects at the high normal end of the spectrum of glucose responses appeared to have the greatest reductions in glucose with nitrate. Increased insulin may account, at least in part, for this effect, but the mechanism may relate to reduced insulin clearance rather than increased insulin secretion. Increasing dietary nitrate, possibly in the form of green leafy salad starters, may be effective in limiting subsequent prandial hyperglycaemia with carbohydrate-rich main-courses and deserts, an effect that may be greater in people with impaired glucose tolerance; the long-term effects on improving glucose control remain to be established. Disclosure: This work was supported by King’s College London, British Heart Foundation Centre, and Barts & The London NHS Trust.
doi:10.1016/j.niox.2011.03.277
P47. Dietary nitrite levels influence hypoxia-induced pulmonary hypertension Roy Sutliffa,b, Alexander El-Alia,b, Erik Walpa,b, Benjamin Predmorec, David Leferc a Emory University, Medicine, Atlanta, GA 30033, United States b Atlanta VA Medical Center, Medicine, Decatur, GA 30033, United States c Emory University, Surgery, Atlanta, GA 30033, United States
Nitrite 2011 meeting abstracts/Nitric Oxide 24 (2011) S16–S42 Background: Pulmonary hypertension (PH), defined as elevation of mean pulmonary artery pressure above 25 mmHg at rest, produces significant morbidity and mortality. The pathobiology of PH involves endothelial dysfunction and vasoconstriction leading to abnormal proliferation of pulmonary vascular wall cells resulting in vascular remodeling and muscularization of small pulmonary vessels. Recent studies have suggested that inhaled nitrite protects against hypoxia-induced PH. The present study was performed to determine if dietary nitrite levels can influence hypoxia-induced PH. Methods: Male C57/Bl6 mice were fed one of several diets: low nitrite diet (Harlan 99366; 20.5 pmol/g); a standard diet (Purina 5001; 104.3 pmol/g); low nitrite diet supplemented with 50 mg/L sodium nitrite; a low nitrite diet supplemented with 100 mg/L sodium nitrite; or standard diet supplemented with 50 mg/L. After the mice were on the diet for 10 days they were exposed to room air or 10% oxygen for 3 weeks. At the end of the 3 weeks PH was determined by measurement of right ventricular (RV) systolic pressure (RVSP) and following euthanasia, hematocrits and right ventricular hypertrophy (measured as RV:LV + S weight ratios). Results: Hypoxia-induced changes in hematocrit persisted for all of the diets. The levels of nitrite in the diet had profound effects on hypoxia-induced PH. Mice on standard diet and low nitrite diet developed PH in response to hypoxia. Mice maintained on a low nitrite diet had an exaggerated PH response to hypoxia. Mice fed diets supplemented with nitrite did not develop an increase in RVSP or RV hypertrophy. Conclusion: Reductions in dietary nitrite levels results in an exaggerated PH response to hypoxia. Increasing dietary nitrite eliminates hypoxia-induced PH. Our studies suggest that dietary nitrite levels have a significant impact on hypoxiainduced pulmonary hypertension. Furthermore, dietary nitrite may be an effective therapy for the treatment of PH. Disclosure: The authors have no conflicts to disclose.
S33
identified so far [1]. The main biological purpose of NPs is to deliver NO into the tissue of a host species to promote vasodilatation and anticoagulation. The recent interests in the interaction of ferriheme proteins with plasma NO 2 lead to the present study. Methods: The interaction of nitrophorins with NO 2 was examined by various spectroscopic techniques, including absorbance, EPR, and resonance Raman spectroscopy. Crystallization of NP4 was performed as previously described and the crystals were soaked with KNO2 prior to the recording of the diffraction pattern.
doi:10.1016/j.niox.2011.03.278
P48. Nitrite disproportionation reaction: Investigations on the mechanism of the conversion of nitrite into nitric oxide at the ferriheme center of nitrophorins at blood plasma pH Markus Knipp, Chunmao He, Hideaki Ogata Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr, NRW 45470, Germany Background: Nitrophorins (NPs) comprise a class of small ferriheme proteins that appear in the saliva of bloodsucking insects. Five isoproteins (NP1-4 and 7) have been
Results: Unlike other ferrihemes [2], NPs exhibit a reaction with NO 2 , which re sults in the formation of NO. Quantification of NO, NO 2 , and NO3 revealed the produc tion of two NO per three NO2 consumed, showing that NO2 serves as both the electron acceptor and the electron donor [3]. The reaction can be written as þ 3NO 2 þ 2H ! 2NO þ H2 O þ NO3 :
This reaction is well-known as the nitrite disproportionation reaction that appears at low pH in aqueous solutions; remarkably, NPs are, to the best of our knowledge, the only examples among the ferriheme proteins and model compounds that promote the reaction at neutral pH. When the reaction is carried out in the presence of an NO scavenger, a continuous production of NO is observed; thus, the protein acts as a catalyst [3]. Unlike metHb and metMb, which form an g1-O nitrito complex [4,5], the spectroscopic and crystallographic characterization of the initial FeIII–NO 2 complex of NPs revealed the more common g1-N nitro conformation [6]. In metMb, deletion of the distal His64 residue lead to the change of the coordina1 tion state of NO 2 to the g -N nitro conformation where the concomitant insertion of an Arg into the distal heme pocket (Mb(H64V, V67R)) restored the unique g1-O nitrito complex [5]. Insertion of Arg into the distal pocket of NP4 (NP4(L130R)) lead to a significant decrease in the reaction rate. However, EPR spectroscopy revealed no significant change in the EPR parameters, suggesting that the ligand coordination is preserved. FeðIIIÞNO 2
NO 2 coordination
gA max
gB max
Ratio of spin systems A and B
Reference
metHb
g1-O
2.98
2.89
n.r.
3.11
2.97
4:6
[Nat. Chem. Biol. 5 (2009) 366] [Biochemistry 27 (1988) 2790]
metMb
1
g -O