- Email: [email protected]
Reduced renal dimethylarginine dimethylaminohydrolase 1 (DDAH1) activity protects against progressive kidney fibrosis and eGFR decline
130
Abstracts/Nitric Oxide 42 (2014) 99–153
drome through Sirt3-AMPK-mediated signaling events, providing new therapeutic strategies in the treatment of PVH. Keywords: Nitrite signaling mechanism; Sirt3; AMPK; Metabolic syndrome; Pulmonary venous hypertension.
P149. Nitrosothiol formation and protection against Fenton chemistry by nitric oxide-induced dinitrosyliron complex formation from hypoxia-initiated cellular chelatable iron increase http://dx.doi.org/10.1016/j.niox.2014.09.093 Jack Lancaster Jr. a, Chuanyu Li b, Harry Mahtani b, Jian Du c, Aashka Patel d, Qian Li b a University of Pittsburgh School of Medicine b University of Alabama Birmingham c Anhui Medical University, China d Vestavia Hills High School, Vestavia Hills, AL
Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with •NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged •NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief •NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h hypoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron and brief •NO treatment prevents detection of this hypoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of •NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against hypoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of nitrosothiol under these high chelatable iron levels are comparable to DNIC levels and suggest that under these conditions both DNIC and RSNO are the most abundant cellular adducts of •NO. Keywords: Dinitrosyliron complexes; Chelatable iron; Hypoxia; Fenton reaction; Nitrosothiols.
P150. Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates nitric oxide production, haemodynamics and vascular responsiveness under basal conditions and outcome in polymicrobial sepsis http://dx.doi.org/10.1016/j.niox.2014.09.094 Peter Kelly a, Zhen Wang a, Blerina Ahmetaj a, Simon Lambden a, Manasi Nandi b, Belen Torondel a, Matthew Delahaye a, Laura Dowsett a, James Tomlinson a, Ben Caplin c, Olga Boruc a, Anna Slaviero a, Sanjay Khadayate a, Ben Lee a, James Leiper d a MRC b Kings College London c University College London d MRC Clinical Sciences Centre
Purpose: Nitric oxide (NO) is key to numerous physiological and pathophysiological processes. NO production is regulated endogenously by two methylarginines Asymmetric Dimethyl Arginine (ADMA) and L-N Monomethyl Arginine (L-NMMA). The enzyme that metabolises ADMA and L-NMMA is Dimethylarginine Dimethylaminohydrolase (DDAH). The first isoform, DDAH1, has previously been shown to be an important regulator of vascular reactivity in both health and disease. Our study explored for the first time the role of the DDAH2 enzyme in regulating cardiovascular physiology and also determined the functional impact of DDAH2 deletion on outcome and immune function in sepsis.
Methods: Mice, globally deficient in DDAH2 were compared to their wild type litter mates to determine the physiological role of DDAH2 using in vivo awake and anaesthetised telemetry and ex vivo assessment of vascular responsiveness. Using caecal ligation and puncture as a model of polymicrobial sepsis, global knockout animals were compared to their wild type litter mates and also to macrophage specific DDAH2 knockout animals to examine the role that DDAH2 plays on the response to sepsis. Methylarginine and nitrate/nitrite levels were measured as indices of nitric oxide regulation. Results: We show that global knockout of DDAH2 results in elevated blood pressure Mean (SEM) 118.5 (1.3) vs 112.7 mmHg (1.1) p < 0.001 and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue ADMA (SEM) 0.89 μM (0.06) vs 0.67 μM (0.05), p = 0.02 and systemic NO. In sepsis DDAH2 knockout determines outcome (120 hour survival 12% in DDAH2 knockouts vs 53% in wild type animals p < 0.001) and macrophage specific deletion of DDAH2 results in a similar pattern of increased severity to that seen in globally deficient animals. Immune dysfunction as a primary mechanism is further demonstrated by increased bacterial load in peritoneal lavage fluid (1519 CFU/ ml (IQR 580 to 4703) vs 484 CFU/ml (IQR 97.6 to 764), p = 0.037). Conclusions: DDAH2 has a regulatory role both in normal physiology and in determining outcome from severe polymicrobial sepsis. Elucidation of the roles of DDAH2 suggests a possible mechanism for the observed relationship between DDAH2 polymorphisms, cardiovascular disease and outcome in sepsis. Keywords: ADMA; DDAH; Hypertension; Sepsis. P151. Reduced renal dimethylarginine dimethylaminohydrolase 1 (DDAH1) activity protects against progressive kidney fibrosis and eGFR decline http://dx.doi.org/10.1016/j.niox.2014.09.095 James Tomlinson a, Ben Caplin b, Olga Boruc a, Pedro Cutillas a, Dirk Dorman a, Peter Faull a, Sanjay Khadayate a, V.R. Mas c, Zhen Wang a, Jill Norman b, David Wheeler b, James Leiper d a MRC b University College London c University of Virginia d MRC Clinical Sciences Centre
Introduction: Asymmetric dimethylarginine (ADMA) competitively inhibits nitric oxide (NO) synthesis whilst dimethylarginine dimethylaminohydrolase 1 (DDAH1) metabolises ADMA; thus representing an alternative regulatory pathway for NO production. Although an association between elevated circulating ADMA and poor cardiovascular and renal outcomes has been widely reported, a causal link is unresolved. We recently published evidence of a DDAH1 gene variant that leads to lower plasma ADMA but counter-intuitively, associates with a steeper rate of eGFR decline. Furthermore, we reported an association between renal allograft methylarginine metabolising enzyme gene expression and eGFR decline following transplantation. Hypothesis and methods: The principal site of renal DDAH1 expression is within the renal proximal tubule (PT). We tested the hypothesis that reduced kidney DDAH1 activity slows the progression of kidney function decline by: (A) generating a novel PT-specific DDAH1 gene knock-out (PTD1KO) mouse; (B) subjecting it to a folate model of CKD and (C) confirming associations between renal gene expression and functional decline in two independent human renal allograft cohorts. Results: (A) KO mice had elevated PT cell ADMA (60%, p < 0.05), with a reduction in NO synthesis (60%, p < 0.05) whilst no effect was observed in extra-renal tissues, urine, plasma or systemic BP. Urinary proteomic analysis revealed a baseline 8-fold
Abstracts/Nitric Oxide 42 (2014) 99–153
reduction in uromodulin (UMOD; p < 0.001) in PTD1KOs. (B) At 12 weeks following folate injury, PTD1KO mice were protected from kidney function decline (serum creatinine; 12.9 ± 0.7 μmol/L versus 24.4 ± 3.3 μmol/L in controls; p < 0.001); renal pro-fibrotic gene upregulation (Col12α and TGFβ; p < 0.05); and kidney collagen deposition (4.5% vs 7.2% in controls, p < 0.01). (C) A significant correlation between DDAH1 gene expression and eGFR decline was confirmed in human renal allograft protocol biopsies (p < 0.05). Furthermore, a positive association of renal tissue DDAH1 and UMOD gene expression was confirmed in the live-donor human allograft sub-group. Conclusions: Renal DDAH1 activity correlates with the progression of kidney function decline following injury in both an experimental in vivo model and human kidney allograft cohorts. Our work highlights the significance of NO-ADMA imbalances at a tissue level and suggests that circulating ADMA is an imprecise marker of renal disease. An association between UMOD and DDAH1 gene expression suggests a plausible mechanistic role of uromodulin in chronic kidney disease progression. Keywords: DDAH; ADMA; Renal failure.
P152. Characterization of human rhodanese and its role in sulfide oxidation pathway http://dx.doi.org/10.1016/j.niox.2014.09.096 Marouane Libiad, Ruma Banerjee Department of Biological Chemistry, University of Michigan Medical School
Hydrogen sulfide (H2S) is an important gaseous signaling molecule with effects on multiple physiological processes including neuromodulation, inflammation and cardiac function. Maintaining healthy levels of H2S in mammalian cells requires tight control between its biosynthesis and its catabolism. While the biogenesis of H2S has been extensively studied, the precise roles of the molecular players involved in the H2S degradation pathway are yet to be uncovered. The sulfide oxidation pathway requires the concerted action of the mitochondrial enzymes: sulfide quinone reductase (SQR), a persulfide dioxygenase (ETHE1), rhodanese and sulfite oxidase. With the exception of sulfite oxidase, the remaining enzymes are poorly characterized. Rhodanese belongs to the sulfurtransferase family of proteins that catalyze the transfer of a sulfur atom from sulfur donors such as thiosulfate or glutathione persulfide to a wide array of sulfur acceptors including cyanide. Pervious reports suggested that rhodanese is involved in cyanide detoxification converting cyanide to the less toxic thiocyanate. However, genomic and biochemical evidence points to a role for rhodanese in the sulfide oxidation pathway. In this study, we report the expression and first steadystate kinetic characterization of the recombinant human rhodanese. The kinetics of thiocyanate formation by human rhodanese yielded a KM of ~30 mM for cyanide indicating that the physiological role of the enzyme is unlikely to be in cyanide detoxification. Therefore, to identify its role in sulfide oxidation pathway we determined for the kinetic parameters for the sulfur transfer from glutathione persulfide or thiosulfate to a number of thiol acceptors. Our data support a role of the enzyme in the H2S degradation pathway by using glutathione persulfide as a persulfide donor and sulfite as a sulfur acceptor to generate GSH and thiosulfate.
P153. Hemoglobin is the primary erythrocytic nitrite reductase http://dx.doi.org/10.1016/j.niox.2014.09.097
131
Chen Liu a, Xiaohua Liu a, Nadeem Wajih a, John Janes a, Swati Basu a, Madison Marvel a, Christine Helms b, Debra Diz a, Paul Laurienti a, David Caudell a, Jun Wang c, Mark Gladwin c, Daniel Kim-Shapiro a a Wake Forest University b University of Richmond c University of Pittsburgh
Nitrite’s roles in therapeutics and physiology have been widely recognized and the extent and mechanisms of these roles continue to be explored. Nitrite signaling likely occurs through its reduction to nitric oxide (NO). However, the mechanisms of nitrite reduction in various tissues remain to be established. The importance of erythrocytes in nitrite reduction has been shown in platelet activation and aortic ring studies where NO production has been detected from erythrocytes under hypoxia. In this work we perform studies to determine whether the primary erythrocytic nitrite reductase is hemoglobin as opposed to other proteins in the red blood cell that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. We used specific inhibitors of other candidate erythrocytic nitrite reductases to study their effects on NO production including allopurinol to inhibit xanthine oxidoreductase, dorzolamide to inhibit carbonic anhydrase II, and L-NG-nitroarginine methyl ester to inhibit nitric oxide synthase. Our studies show no effect on NO production from nitrite in the presence of inhibitors of these other enzymes. Some experiments were performed with red blood cells from both normotensive and hypertensive individuals, in whom xanthine oxidoreductase has been reported to play a major role. In addition, we demonstrate that carbon monoxide blocks NO production from nitrite, supporting a primary role for hemoglobin. Thus, our results suggest that hemoglobin is the primary erythrocytic nitrite reductase. This work was supported by NIH Grants HL098032 and HL098032. Keywords: Nitrite; Nitric oxide; Hemoglobin. P154. Evaluation of methodologies to assess intracellular cGMP levels induced by soluble guanylate cyclase (sGC) stimulators in whole cells http://dx.doi.org/10.1016/j.niox.2014.09.098 Guang Liu a, Christina Butler a, Kim Long a, Renee Sarno b, Rob Solinga c, Kim Tang c, Yueh-tyng Chien a, Ken Carlson a, Jeff Segal a a Cellular and Molecular Pharmacology, Ironwood Pharmaceuticals, Inc., Cambridge, MA, USA b DMPK, Ironwood Pharmaceuticals, Inc., Cambridge, MA, USA c Discovery Pharmacology, Ironwood Pharmaceuticals, Inc., Cambridge, MA, USA
Objective/purpose: The objective of this study was to evaluate different methodologies to measure sGC stimulatorinduced cGMP responses in whole cells. Results: Two methodologies were evaluated to assess cGMP responses elicited by sGC stimulators (YC-1, BAY 41–2272 and several Ironwood compounds) in whole cells: an LC-MS/MS based bioanalytical assay to directly measure cGMP generated in Human Embryonic Kidney (HEK) cells or a cGMP Glosensor reporter-based method in HEK cells. Both assays had excellent signal to noise, low variability (%CV and Z prime values) and were highly reproducible. Each assay enabled efficient characterization of sGC stimulators with regard to potency, efficacy (Emax), and synergy with NO. The Glosensor assay had the benefits of rapid turnaround time and the ability to measure cGMP response in real time with endpoint and kinetic profiling. However, the Glosensor assay did not have the same resolution to differentiate compounds with different maximal levels of stimulation,
Abstracts/Nitric Oxide 42 (2014) 99–153
drome through Sirt3-AMPK-mediated signaling events, providing new therapeutic strategies in the treatment of PVH. Keywords: Nitrite signaling mechanism; Sirt3; AMPK; Metabolic syndrome; Pulmonary venous hypertension.
P149. Nitrosothiol formation and protection against Fenton chemistry by nitric oxide-induced dinitrosyliron complex formation from hypoxia-initiated cellular chelatable iron increase http://dx.doi.org/10.1016/j.niox.2014.09.093 Jack Lancaster Jr. a, Chuanyu Li b, Harry Mahtani b, Jian Du c, Aashka Patel d, Qian Li b a University of Pittsburgh School of Medicine b University of Alabama Birmingham c Anhui Medical University, China d Vestavia Hills High School, Vestavia Hills, AL
Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with •NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged •NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief •NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h hypoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron and brief •NO treatment prevents detection of this hypoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of •NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against hypoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of nitrosothiol under these high chelatable iron levels are comparable to DNIC levels and suggest that under these conditions both DNIC and RSNO are the most abundant cellular adducts of •NO. Keywords: Dinitrosyliron complexes; Chelatable iron; Hypoxia; Fenton reaction; Nitrosothiols.
P150. Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates nitric oxide production, haemodynamics and vascular responsiveness under basal conditions and outcome in polymicrobial sepsis http://dx.doi.org/10.1016/j.niox.2014.09.094 Peter Kelly a, Zhen Wang a, Blerina Ahmetaj a, Simon Lambden a, Manasi Nandi b, Belen Torondel a, Matthew Delahaye a, Laura Dowsett a, James Tomlinson a, Ben Caplin c, Olga Boruc a, Anna Slaviero a, Sanjay Khadayate a, Ben Lee a, James Leiper d a MRC b Kings College London c University College London d MRC Clinical Sciences Centre
Purpose: Nitric oxide (NO) is key to numerous physiological and pathophysiological processes. NO production is regulated endogenously by two methylarginines Asymmetric Dimethyl Arginine (ADMA) and L-N Monomethyl Arginine (L-NMMA). The enzyme that metabolises ADMA and L-NMMA is Dimethylarginine Dimethylaminohydrolase (DDAH). The first isoform, DDAH1, has previously been shown to be an important regulator of vascular reactivity in both health and disease. Our study explored for the first time the role of the DDAH2 enzyme in regulating cardiovascular physiology and also determined the functional impact of DDAH2 deletion on outcome and immune function in sepsis.
Methods: Mice, globally deficient in DDAH2 were compared to their wild type litter mates to determine the physiological role of DDAH2 using in vivo awake and anaesthetised telemetry and ex vivo assessment of vascular responsiveness. Using caecal ligation and puncture as a model of polymicrobial sepsis, global knockout animals were compared to their wild type litter mates and also to macrophage specific DDAH2 knockout animals to examine the role that DDAH2 plays on the response to sepsis. Methylarginine and nitrate/nitrite levels were measured as indices of nitric oxide regulation. Results: We show that global knockout of DDAH2 results in elevated blood pressure Mean (SEM) 118.5 (1.3) vs 112.7 mmHg (1.1) p < 0.001 and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue ADMA (SEM) 0.89 μM (0.06) vs 0.67 μM (0.05), p = 0.02 and systemic NO. In sepsis DDAH2 knockout determines outcome (120 hour survival 12% in DDAH2 knockouts vs 53% in wild type animals p < 0.001) and macrophage specific deletion of DDAH2 results in a similar pattern of increased severity to that seen in globally deficient animals. Immune dysfunction as a primary mechanism is further demonstrated by increased bacterial load in peritoneal lavage fluid (1519 CFU/ ml (IQR 580 to 4703) vs 484 CFU/ml (IQR 97.6 to 764), p = 0.037). Conclusions: DDAH2 has a regulatory role both in normal physiology and in determining outcome from severe polymicrobial sepsis. Elucidation of the roles of DDAH2 suggests a possible mechanism for the observed relationship between DDAH2 polymorphisms, cardiovascular disease and outcome in sepsis. Keywords: ADMA; DDAH; Hypertension; Sepsis. P151. Reduced renal dimethylarginine dimethylaminohydrolase 1 (DDAH1) activity protects against progressive kidney fibrosis and eGFR decline http://dx.doi.org/10.1016/j.niox.2014.09.095 James Tomlinson a, Ben Caplin b, Olga Boruc a, Pedro Cutillas a, Dirk Dorman a, Peter Faull a, Sanjay Khadayate a, V.R. Mas c, Zhen Wang a, Jill Norman b, David Wheeler b, James Leiper d a MRC b University College London c University of Virginia d MRC Clinical Sciences Centre
Introduction: Asymmetric dimethylarginine (ADMA) competitively inhibits nitric oxide (NO) synthesis whilst dimethylarginine dimethylaminohydrolase 1 (DDAH1) metabolises ADMA; thus representing an alternative regulatory pathway for NO production. Although an association between elevated circulating ADMA and poor cardiovascular and renal outcomes has been widely reported, a causal link is unresolved. We recently published evidence of a DDAH1 gene variant that leads to lower plasma ADMA but counter-intuitively, associates with a steeper rate of eGFR decline. Furthermore, we reported an association between renal allograft methylarginine metabolising enzyme gene expression and eGFR decline following transplantation. Hypothesis and methods: The principal site of renal DDAH1 expression is within the renal proximal tubule (PT). We tested the hypothesis that reduced kidney DDAH1 activity slows the progression of kidney function decline by: (A) generating a novel PT-specific DDAH1 gene knock-out (PTD1KO) mouse; (B) subjecting it to a folate model of CKD and (C) confirming associations between renal gene expression and functional decline in two independent human renal allograft cohorts. Results: (A) KO mice had elevated PT cell ADMA (60%, p < 0.05), with a reduction in NO synthesis (60%, p < 0.05) whilst no effect was observed in extra-renal tissues, urine, plasma or systemic BP. Urinary proteomic analysis revealed a baseline 8-fold
Abstracts/Nitric Oxide 42 (2014) 99–153
reduction in uromodulin (UMOD; p < 0.001) in PTD1KOs. (B) At 12 weeks following folate injury, PTD1KO mice were protected from kidney function decline (serum creatinine; 12.9 ± 0.7 μmol/L versus 24.4 ± 3.3 μmol/L in controls; p < 0.001); renal pro-fibrotic gene upregulation (Col12α and TGFβ; p < 0.05); and kidney collagen deposition (4.5% vs 7.2% in controls, p < 0.01). (C) A significant correlation between DDAH1 gene expression and eGFR decline was confirmed in human renal allograft protocol biopsies (p < 0.05). Furthermore, a positive association of renal tissue DDAH1 and UMOD gene expression was confirmed in the live-donor human allograft sub-group. Conclusions: Renal DDAH1 activity correlates with the progression of kidney function decline following injury in both an experimental in vivo model and human kidney allograft cohorts. Our work highlights the significance of NO-ADMA imbalances at a tissue level and suggests that circulating ADMA is an imprecise marker of renal disease. An association between UMOD and DDAH1 gene expression suggests a plausible mechanistic role of uromodulin in chronic kidney disease progression. Keywords: DDAH; ADMA; Renal failure.
P152. Characterization of human rhodanese and its role in sulfide oxidation pathway http://dx.doi.org/10.1016/j.niox.2014.09.096 Marouane Libiad, Ruma Banerjee Department of Biological Chemistry, University of Michigan Medical School
Hydrogen sulfide (H2S) is an important gaseous signaling molecule with effects on multiple physiological processes including neuromodulation, inflammation and cardiac function. Maintaining healthy levels of H2S in mammalian cells requires tight control between its biosynthesis and its catabolism. While the biogenesis of H2S has been extensively studied, the precise roles of the molecular players involved in the H2S degradation pathway are yet to be uncovered. The sulfide oxidation pathway requires the concerted action of the mitochondrial enzymes: sulfide quinone reductase (SQR), a persulfide dioxygenase (ETHE1), rhodanese and sulfite oxidase. With the exception of sulfite oxidase, the remaining enzymes are poorly characterized. Rhodanese belongs to the sulfurtransferase family of proteins that catalyze the transfer of a sulfur atom from sulfur donors such as thiosulfate or glutathione persulfide to a wide array of sulfur acceptors including cyanide. Pervious reports suggested that rhodanese is involved in cyanide detoxification converting cyanide to the less toxic thiocyanate. However, genomic and biochemical evidence points to a role for rhodanese in the sulfide oxidation pathway. In this study, we report the expression and first steadystate kinetic characterization of the recombinant human rhodanese. The kinetics of thiocyanate formation by human rhodanese yielded a KM of ~30 mM for cyanide indicating that the physiological role of the enzyme is unlikely to be in cyanide detoxification. Therefore, to identify its role in sulfide oxidation pathway we determined for the kinetic parameters for the sulfur transfer from glutathione persulfide or thiosulfate to a number of thiol acceptors. Our data support a role of the enzyme in the H2S degradation pathway by using glutathione persulfide as a persulfide donor and sulfite as a sulfur acceptor to generate GSH and thiosulfate.
P153. Hemoglobin is the primary erythrocytic nitrite reductase http://dx.doi.org/10.1016/j.niox.2014.09.097
131
Chen Liu a, Xiaohua Liu a, Nadeem Wajih a, John Janes a, Swati Basu a, Madison Marvel a, Christine Helms b, Debra Diz a, Paul Laurienti a, David Caudell a, Jun Wang c, Mark Gladwin c, Daniel Kim-Shapiro a a Wake Forest University b University of Richmond c University of Pittsburgh
Nitrite’s roles in therapeutics and physiology have been widely recognized and the extent and mechanisms of these roles continue to be explored. Nitrite signaling likely occurs through its reduction to nitric oxide (NO). However, the mechanisms of nitrite reduction in various tissues remain to be established. The importance of erythrocytes in nitrite reduction has been shown in platelet activation and aortic ring studies where NO production has been detected from erythrocytes under hypoxia. In this work we perform studies to determine whether the primary erythrocytic nitrite reductase is hemoglobin as opposed to other proteins in the red blood cell that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. We used specific inhibitors of other candidate erythrocytic nitrite reductases to study their effects on NO production including allopurinol to inhibit xanthine oxidoreductase, dorzolamide to inhibit carbonic anhydrase II, and L-NG-nitroarginine methyl ester to inhibit nitric oxide synthase. Our studies show no effect on NO production from nitrite in the presence of inhibitors of these other enzymes. Some experiments were performed with red blood cells from both normotensive and hypertensive individuals, in whom xanthine oxidoreductase has been reported to play a major role. In addition, we demonstrate that carbon monoxide blocks NO production from nitrite, supporting a primary role for hemoglobin. Thus, our results suggest that hemoglobin is the primary erythrocytic nitrite reductase. This work was supported by NIH Grants HL098032 and HL098032. Keywords: Nitrite; Nitric oxide; Hemoglobin. P154. Evaluation of methodologies to assess intracellular cGMP levels induced by soluble guanylate cyclase (sGC) stimulators in whole cells http://dx.doi.org/10.1016/j.niox.2014.09.098 Guang Liu a, Christina Butler a, Kim Long a, Renee Sarno b, Rob Solinga c, Kim Tang c, Yueh-tyng Chien a, Ken Carlson a, Jeff Segal a a Cellular and Molecular Pharmacology, Ironwood Pharmaceuticals, Inc., Cambridge, MA, USA b DMPK, Ironwood Pharmaceuticals, Inc., Cambridge, MA, USA c Discovery Pharmacology, Ironwood Pharmaceuticals, Inc., Cambridge, MA, USA
Objective/purpose: The objective of this study was to evaluate different methodologies to measure sGC stimulatorinduced cGMP responses in whole cells. Results: Two methodologies were evaluated to assess cGMP responses elicited by sGC stimulators (YC-1, BAY 41–2272 and several Ironwood compounds) in whole cells: an LC-MS/MS based bioanalytical assay to directly measure cGMP generated in Human Embryonic Kidney (HEK) cells or a cGMP Glosensor reporter-based method in HEK cells. Both assays had excellent signal to noise, low variability (%CV and Z prime values) and were highly reproducible. Each assay enabled efficient characterization of sGC stimulators with regard to potency, efficacy (Emax), and synergy with NO. The Glosensor assay had the benefits of rapid turnaround time and the ability to measure cGMP response in real time with endpoint and kinetic profiling. However, the Glosensor assay did not have the same resolution to differentiate compounds with different maximal levels of stimulation,