- Email: [email protected]
P53. Low dose systemic nitrite infusion to healthy human subjects in a hypoxic environmental chamber
Poster abstracts / Nitric Oxide 19 (2008) S43–S72 2-methylbutane and rapidly converts to N2O4. From the semi-quantitative determination of mole fractions present in the gas phase by EPR spectroscopy, we estimated the rate constants of the steps which lead to ONOO and ONOONO from the known overall rate constant of the autoxidation reaction by assuming that a quasi-stationary mechanism applies. The constant for the rate-determining formation of ONOO is ca. 3.1 10 18 cm3 molecule 1 s 1(or 80 s 1 in mole fractions), the dissociation rate constant of ONOO is ca. 6.5 103 s 1 and ONOONO is formed with k = 7.7 10 14 cm3 molecule 1 s 1 (1.9 106 s 1 in mole fractions). From these constants we estimate that the equilibrium constant for the formation of ONOO from NO and O2, KONOO, is 4.8 10 22 cm3 molecule 1 (1.2 10 2) and therefore DrxnG° = +11.0 kJ mol 1. In water, the Gibbs energy change is close to 0. The presence of ONOO at steady-state concentrations under dioxygen excess may be important not only for reactions in the atmosphere, but especially for reactions in aerosols and biological environments, because the formation constant in solution is higher than in the gas phase and therefore the half-life of ONOO is longer. doi:10.1016/j.niox.2008.06.148
P52. Dietary nitrate and nitrite modulate the cellular response to ischemic stress Nicolaas J. Raat a, Audrey C. Noguchi a, Virginia Liu a, Nalini Raghavachari a, Delong Liu b, Xiuli Xu a, Sruti Shiva a, Peter M. Munson b, Mark T. Gladwin a,c a Pulmonary and Vascular Medicine Branch, National Heart, lung and Blood Institute, NIH b Mathematical and Statistical Computing Laboratory, Center for Information Technology, NIH c Critical Care Medicine Department, Clinical Center; NIH Nanomolar doses of injected sodium nitrite enhance cellular viability after ischemia reperfusion (IR) injury and increase nitrite and nitrate (NOx) levels in blood and tissue to a similar degree as after ingesting a nitrate rich diet. We wondered whether depletion or supplementation of dietary NOx would alter the susceptibility to IR injury and modulate basal and ischemic stress related gene expression. Therefore, mice were placed for one week on a standard rodent chow NIH-31, a low NOx diet and this diet supplemented with sodium nitrite in the drinking water. Plasma, liver and cardiac nitrite levels in mice on the low NOx diet were reduced compared with NIH-31 and could be restored to normal levels, or higher, with nitrite supplementation. To discriminate between an effect of the change in diet perse and nitrite levels, the effects of nitrite on basal cellular transcription were determined by comparing genes that were differentially regulated in both the NIH-31 diet and the water-nitrite repletion groups and the low NOx diet. Consistency testing provided evidence that consumption of either nitrate or nitrite, sufficient to normalize plasma and tissue levels, regulates basal transcriptional activity. We next characterized the effects of basal nitrite levels on gene expression profiles and cellular injury after liver IR. Increases in liver enzymes in the plasma of mice on the low NOx diet were 2- to 3-fold higher than on the NIH-31 diet. Supplementa-
S55
tion with 300 or 1500 mg/l nitrite completely prevented the liver injury comparable to mice fed with NIH-31. The early transcriptional response after cytoprotection was more robust than basal effects of nitrite, suggesting a hypoxic potentiation of nitritedependent transcriptional signaling. In conclusion, altering dietary NOx levels significantly modulates basal cellular signaling and the hypoxic stress response, supporting the hypothesis that dietary NOx are active components of the cardio-protective Mediterranean diet. doi:10.1016/j.niox.2008.06.149
P53. Low dose systemic nitrite infusion to healthy human subjects in a hypoxic environmental chamber Andrew G. Pinder a, Thomas E. Ingram a, Damian M. Bailey b, Alan G. Fraser a, Philip E. James a a Cardiology, Wales Heart Research Institute b Faculty of Health and Science, University of Glamorgan Local administration of increasing nitrite doses under normoxic conditions is relatively selective at dilating the human forearm venous capacitance bed over arteries. During application of a hypoxic mask pharmacological nitrite also increases local arterial forearm blood flow. We were keen to study the systemic effects of low dose nitrite infusion on arterial blood flow in subjects physiologically equilibrated in an environmental chamber maintained at 12% oxygen. Twenty-four healthy human subjects (mean age 23 ± 4) were investigated. Twelve undertook a 3 h equilibration period at 12% O2 prior to venous infusion of nitrite (1 lmol/ml/min) for 30 min. Six were given saline vehicle, a further six were given the same nitrite infusion under normoxic conditions. Contra-lateral forearm blood flow measures were taken using strain gauge plethysmography. Venous blood samples (contra-lateral) were taken for assessment of blood NO metabolites. Measurements were undertaken throughout the equilibration period, during infusion, and for 1 h following cessation of nitrite. For subjects in hypoxia, mean SaO2 reached 83% at 2hrs and remained unchanged during the study. Heart rate and blood pressure did not change during the infusion period (79 bpm at 2 hrs from mean baseline 66 bpm, 120/72 mmHg at 2 hrs from mean baseline 128/73). Forearm arterial blood flow increased to a maximum of 50% and plasma nitrite simultaneously increased 100% over pre-infusion values. Both FBF and plasma nitrite decreased with a decay profile of 44 min and 24min (50% decay) respectively. Of particular interest was the very transient increase in red blood cell Hb-bound NO corresponding to peak infusion and plasma nitrite, however, this returned to baseline hypoxic values within 15 min (50% @ 9 min). We present here the details of our findings and draw on results from isolated aortic vessel preparations that point to the mechanisms of nitrite arterial vasoactivity in hypoxia. doi:10.1016/j.niox.2008.06.150
Arginine availability and transport P54. Determination of arginase I in human gingival fibroblasts Farzaneh Daghigh, Benjason Nunez, Ellen Cho, Chalon Saunders Philadelphia College of osteopathic medicine In periodontitis, excessive nitric oxide (NO) is associated with tissue destruction and inflammation. The inducible form of nitric oxide synthase (iNOS) is responsible for the overproduction of NO. iNOS shares its substrate, arginine, with arginase which produces urea and ornithine, the precursor of polyamines. Polyamines and proline are required for cellular proliferation and collagen formation, respectively. Up-regulation of arginase results in limited arginine availability to iNOS and decreased production of NO in macrophage. Pro-inflammatory cytokines have been found to up-regulate iNOS in human gingival fibroblasts (HGF). However, regulation
of arginase in this tissue type has yet to be determined. Mammals express two isoforms of arginase, arginase I (ARGI) and arginase II (ARGII). Presence of ARGI or ARGII in HGF may provide a source of polyamines for collagen synthesis and cell proliferation in HGF. In this study, we have determined the expression of arginase I (ARGI) protein in HGF in presence of cytokines or other stimuli. Our major findings are as followings: (1) ARGI protein is expressed in HGF at basal levels. (2) cAMP significantly induces ARGI production while LPS, though not statistically significant, shows to increase ARGI. (3) IFN-c inhibits the ARGI expression and attenuated the enhanced expression of ARGI via cAMP. ARGI may modulate the inflammatory response in periodontitis, limiting NO production by iNOS and exerting an antiinflammatory effect. doi:10.1016/j.niox.2008.06.151
NO and physiology and pathophysiology P55. Nitrosyl complexes of hemoglobin: in vivo formation pathways and biological effects
a
Molec. Base Organ Failure, L. Boltzmann Inst. Exper. Clin. Traumatol. L. Boltzmann Inst. Exper. Clin. Traumatol., Vienna, Austria Department of Biophysics, Russian State Medical University, Moscow, Russia
b c
Andrey V. Kozlov a, Soheyl Bahrami b, Peter Dungel b, Rainer Mittermayr b, Anatoly Osipov c, Heinz Redl b
P52. Dietary nitrate and nitrite modulate the cellular response to ischemic stress Nicolaas J. Raat a, Audrey C. Noguchi a, Virginia Liu a, Nalini Raghavachari a, Delong Liu b, Xiuli Xu a, Sruti Shiva a, Peter M. Munson b, Mark T. Gladwin a,c a Pulmonary and Vascular Medicine Branch, National Heart, lung and Blood Institute, NIH b Mathematical and Statistical Computing Laboratory, Center for Information Technology, NIH c Critical Care Medicine Department, Clinical Center; NIH Nanomolar doses of injected sodium nitrite enhance cellular viability after ischemia reperfusion (IR) injury and increase nitrite and nitrate (NOx) levels in blood and tissue to a similar degree as after ingesting a nitrate rich diet. We wondered whether depletion or supplementation of dietary NOx would alter the susceptibility to IR injury and modulate basal and ischemic stress related gene expression. Therefore, mice were placed for one week on a standard rodent chow NIH-31, a low NOx diet and this diet supplemented with sodium nitrite in the drinking water. Plasma, liver and cardiac nitrite levels in mice on the low NOx diet were reduced compared with NIH-31 and could be restored to normal levels, or higher, with nitrite supplementation. To discriminate between an effect of the change in diet perse and nitrite levels, the effects of nitrite on basal cellular transcription were determined by comparing genes that were differentially regulated in both the NIH-31 diet and the water-nitrite repletion groups and the low NOx diet. Consistency testing provided evidence that consumption of either nitrate or nitrite, sufficient to normalize plasma and tissue levels, regulates basal transcriptional activity. We next characterized the effects of basal nitrite levels on gene expression profiles and cellular injury after liver IR. Increases in liver enzymes in the plasma of mice on the low NOx diet were 2- to 3-fold higher than on the NIH-31 diet. Supplementa-
S55
tion with 300 or 1500 mg/l nitrite completely prevented the liver injury comparable to mice fed with NIH-31. The early transcriptional response after cytoprotection was more robust than basal effects of nitrite, suggesting a hypoxic potentiation of nitritedependent transcriptional signaling. In conclusion, altering dietary NOx levels significantly modulates basal cellular signaling and the hypoxic stress response, supporting the hypothesis that dietary NOx are active components of the cardio-protective Mediterranean diet. doi:10.1016/j.niox.2008.06.149
P53. Low dose systemic nitrite infusion to healthy human subjects in a hypoxic environmental chamber Andrew G. Pinder a, Thomas E. Ingram a, Damian M. Bailey b, Alan G. Fraser a, Philip E. James a a Cardiology, Wales Heart Research Institute b Faculty of Health and Science, University of Glamorgan Local administration of increasing nitrite doses under normoxic conditions is relatively selective at dilating the human forearm venous capacitance bed over arteries. During application of a hypoxic mask pharmacological nitrite also increases local arterial forearm blood flow. We were keen to study the systemic effects of low dose nitrite infusion on arterial blood flow in subjects physiologically equilibrated in an environmental chamber maintained at 12% oxygen. Twenty-four healthy human subjects (mean age 23 ± 4) were investigated. Twelve undertook a 3 h equilibration period at 12% O2 prior to venous infusion of nitrite (1 lmol/ml/min) for 30 min. Six were given saline vehicle, a further six were given the same nitrite infusion under normoxic conditions. Contra-lateral forearm blood flow measures were taken using strain gauge plethysmography. Venous blood samples (contra-lateral) were taken for assessment of blood NO metabolites. Measurements were undertaken throughout the equilibration period, during infusion, and for 1 h following cessation of nitrite. For subjects in hypoxia, mean SaO2 reached 83% at 2hrs and remained unchanged during the study. Heart rate and blood pressure did not change during the infusion period (79 bpm at 2 hrs from mean baseline 66 bpm, 120/72 mmHg at 2 hrs from mean baseline 128/73). Forearm arterial blood flow increased to a maximum of 50% and plasma nitrite simultaneously increased 100% over pre-infusion values. Both FBF and plasma nitrite decreased with a decay profile of 44 min and 24min (50% decay) respectively. Of particular interest was the very transient increase in red blood cell Hb-bound NO corresponding to peak infusion and plasma nitrite, however, this returned to baseline hypoxic values within 15 min (50% @ 9 min). We present here the details of our findings and draw on results from isolated aortic vessel preparations that point to the mechanisms of nitrite arterial vasoactivity in hypoxia. doi:10.1016/j.niox.2008.06.150
Arginine availability and transport P54. Determination of arginase I in human gingival fibroblasts Farzaneh Daghigh, Benjason Nunez, Ellen Cho, Chalon Saunders Philadelphia College of osteopathic medicine In periodontitis, excessive nitric oxide (NO) is associated with tissue destruction and inflammation. The inducible form of nitric oxide synthase (iNOS) is responsible for the overproduction of NO. iNOS shares its substrate, arginine, with arginase which produces urea and ornithine, the precursor of polyamines. Polyamines and proline are required for cellular proliferation and collagen formation, respectively. Up-regulation of arginase results in limited arginine availability to iNOS and decreased production of NO in macrophage. Pro-inflammatory cytokines have been found to up-regulate iNOS in human gingival fibroblasts (HGF). However, regulation
of arginase in this tissue type has yet to be determined. Mammals express two isoforms of arginase, arginase I (ARGI) and arginase II (ARGII). Presence of ARGI or ARGII in HGF may provide a source of polyamines for collagen synthesis and cell proliferation in HGF. In this study, we have determined the expression of arginase I (ARGI) protein in HGF in presence of cytokines or other stimuli. Our major findings are as followings: (1) ARGI protein is expressed in HGF at basal levels. (2) cAMP significantly induces ARGI production while LPS, though not statistically significant, shows to increase ARGI. (3) IFN-c inhibits the ARGI expression and attenuated the enhanced expression of ARGI via cAMP. ARGI may modulate the inflammatory response in periodontitis, limiting NO production by iNOS and exerting an antiinflammatory effect. doi:10.1016/j.niox.2008.06.151
NO and physiology and pathophysiology P55. Nitrosyl complexes of hemoglobin: in vivo formation pathways and biological effects
a
Molec. Base Organ Failure, L. Boltzmann Inst. Exper. Clin. Traumatol. L. Boltzmann Inst. Exper. Clin. Traumatol., Vienna, Austria Department of Biophysics, Russian State Medical University, Moscow, Russia
b c
Andrey V. Kozlov a, Soheyl Bahrami b, Peter Dungel b, Rainer Mittermayr b, Anatoly Osipov c, Heinz Redl b