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Direct soluble guanylate cyclase activation improves vascular function in a mouse model of sickle cell disease
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Abstracts/Nitric Oxide 42 (2014) 99–153
Nitrite (NO2−) is an endogenous signaling molecule, a dietary constituent, and recent evidence suggests that nitrite mediates a number of biological effects including cytoprotection after ischemia/reperfusion injury. The conversion of nitrite to nitric oxide (NO), in a hypoxic condition, is required to achieve most, but not all of its cytoprotective effects. For example, nitrite confers cardioprotection after ischemia/reperfusion even when administered to a normoxic heart prior to the ischemic episode. We have previously shown that this effect of nitrite is dependent on the activation of protein kinase A (PKA) and subsequent phosphorylation of mitochondrial protein targets to enhance mitochondrial fusion. Here we investigate the mechanism by which nitrite activates PKA and the mitochondrial protein targets it phosphorylates. Preliminary data suggest that nitrite (25– 50 μM) inhibits phosphodiesterase activity, which potentially increases cAMP levels in the cell leading to PKA activation. Interestingly, the mitochondrially-localized phosphodiesterase appears to be a major target for nitrite. We show that PKA activation is responsible for the phosphorylation of electron transport complex IV, which results in an augmentation of mitochondrial respiration in cardiomyocytes and rat heart tissue. Ongoing studies are investigating the mechanism by which nitrite specifically activates the mitochondrial isoform of PKA. These data expand the role of nitrite as a signaling molecule. Further, these data contribute to understanding its physiological role and highlight the therapeutic potential in the prevention and treatment of cardiovascular diseases. Keywords: Nitrite; Mitochondria; Protein kinase A.
P172. Direct soluble guanylate cyclase activation improves vascular function in a mouse model of sickle cell disease http://dx.doi.org/10.1016/j.niox.2014.09.116 Karin Potoka, Christina Mucci a, Stephanie Mutchler a, Marta Bueno a, Eva Becker-Pelster b, Johannes-Peter Stasch c, Hubert Truebel b, Adam Straub a, Ana Mora a, Mark Gladwin a a University of Pittsburgh, VMI b Bayer Pharma AG, University of Witten/Herdecke c Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany
Rationale: Sickle cell disease (SCD) is caused by a point mutation in the hemoglobin (Hb) β gene, which generates hemoglobin S (HbS), a less soluble tetramer that undergoes polymerization. Endothelial dysfunction caused by chronic intravascular hemolysis is a hallmark of SCD affecting multiple organs, including the lung. A common complication of SCD is Pulmonary Arterial Hypertension (PAH) which is associated with early mortality. In the present study, we use an established animal model of SCD to investigate novel soluble guanylate cyclase (sGC) targeting in the pathobiology of global vascular dysfunction. Methods and results: We used 6 month old Berkley (BERK) transgenic mice that exclusively express human HbS and require knockout of the mouse α and β globins. Age matched wild type mice were used as controls. BERK mice were treated with sGC activator chow (17 mg/ kg/day; BAY 58–2667 × HCl (cinaciguat)) or placebo for 30 days. Following the treatment period, mice were sacrificed by terminal cardiac puncture. Superficial layers of the dorsal musculature were removed in order to access the thoracodorsal artery (TDA), lining the caudal side of the scapula. Segments 10– 15 mm long of the TDA was isolated and placed in cold KrebsHEPES to be used for pressure myography experiments. Isolated TDAs were mounted in a pressure arteriograph (Danish MyoTechnology) where they were maintained in a no-flow state and allowed to equilibrate for 30 min at 80 mmHg. Following
equilibration, arteries were stimulated with cumulative concentrations of the vasoconstrictor phenylephrine (PE, 10-8 to 10-4 M; Sigma). For each vessel, the luminal diameter was measured after stabilization of the response to the vasoconstrictor. The degree of contraction was calculated using the equation: DPE × 100/Dmax, where DPE is the diameter of the TDA after application of a given concentration of PE and Dmax is the maximal diameter of the TDA measured at the end of each experiment by applying Krebs-HEPES calcium free supplemented with acetylcholine. The basal tone was then calculated as: initial diameter/maximum diameter × 100. We found that BERK mice had an increased baseline tone and increased response to PE as compared with controls. SCD mice treated with sGC activator for 30 days showed improved baseline tone and improved constriction response to PE. Conclusions: The transgenic BERK sickle mouse develops significant vascular dysfunction with age. Direct activation of vascular smooth muscle cell sGC represents a novel therapeutic approach for global vascular dysfunction leading to PAH in SCD and other disease processes. Keywords: Sickle cell disease; Soluble guanylate cyclase; Vascular dysfunction.
P173. Protein S-nitrosylation facilitates the regulation of brain metabolism and neurotransmission by nitric oxide http://dx.doi.org/10.1016/j.niox.2014.09.117 Karthik Raju a, Paschalis-Thomas Doulis, Harry Ischiropoulos a University of Pennsylvania
Glutamate-based synaptic activity is an essential component of normal neuronal function. Changes in glutamatergic transmission are associated with corresponding adaptations in the metabolic support of neurons by astrocytes, although the mechanism(s) linking these two processes remain unclear. Nitric oxide (NO) may be a critical regulator of these pathways since loss of neuronal nitric oxide synthase (nNOS) leads to memory and behavioral deficits in mice, with profound alterations in glutamatergic transmission after deletion of both nNOS and endothelial NOS (eNOS). We performed a mass spectrometrybased proteomic interrogation of S-nitrosylated proteins in wildtype (WT), nNOS −/− and eNOS −/− brain that identified clusters of proteins linking synaptic transmission to astrocytic metabolism, particularly within the glutamate/glutamine cycle. 15Nbased profiling of this pathway in hippocampal slices revealed increased metabolic flux in the glutamine/glutamate cycle in nNOS −/− mice that may contribute to the phenotype observed in these animals. Furthermore, S-nitrosylation of sodiumdependent glutamate/aspartate transporter 2 (GLT1) profoundly decreases both its Vmax and Km. Taken together, the data indicate that nitric oxide, through reversible S-nitrosylation of key regulatory proteins, regulates the glutamate/glutamine cycle and influences glutamatergic transmission in vivo.
P174. Probing the mechanism of tetrahydrobiopterin radical reduction within NO synthases http://dx.doi.org/10.1016/j.niox.2014.09.118 Somasundaram Ramasamy a, Mohammad Mahfuzul Haque a, Jesus Tejero b, Mahinda Gangoda b, Dennis Stuehr a a Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA b Kent State University
Nitric-oxide synthase (NOS) enzymes are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from L-arginine. During the first reaction
Abstracts/Nitric Oxide 42 (2014) 99–153
Nitrite (NO2−) is an endogenous signaling molecule, a dietary constituent, and recent evidence suggests that nitrite mediates a number of biological effects including cytoprotection after ischemia/reperfusion injury. The conversion of nitrite to nitric oxide (NO), in a hypoxic condition, is required to achieve most, but not all of its cytoprotective effects. For example, nitrite confers cardioprotection after ischemia/reperfusion even when administered to a normoxic heart prior to the ischemic episode. We have previously shown that this effect of nitrite is dependent on the activation of protein kinase A (PKA) and subsequent phosphorylation of mitochondrial protein targets to enhance mitochondrial fusion. Here we investigate the mechanism by which nitrite activates PKA and the mitochondrial protein targets it phosphorylates. Preliminary data suggest that nitrite (25– 50 μM) inhibits phosphodiesterase activity, which potentially increases cAMP levels in the cell leading to PKA activation. Interestingly, the mitochondrially-localized phosphodiesterase appears to be a major target for nitrite. We show that PKA activation is responsible for the phosphorylation of electron transport complex IV, which results in an augmentation of mitochondrial respiration in cardiomyocytes and rat heart tissue. Ongoing studies are investigating the mechanism by which nitrite specifically activates the mitochondrial isoform of PKA. These data expand the role of nitrite as a signaling molecule. Further, these data contribute to understanding its physiological role and highlight the therapeutic potential in the prevention and treatment of cardiovascular diseases. Keywords: Nitrite; Mitochondria; Protein kinase A.
P172. Direct soluble guanylate cyclase activation improves vascular function in a mouse model of sickle cell disease http://dx.doi.org/10.1016/j.niox.2014.09.116 Karin Potoka, Christina Mucci a, Stephanie Mutchler a, Marta Bueno a, Eva Becker-Pelster b, Johannes-Peter Stasch c, Hubert Truebel b, Adam Straub a, Ana Mora a, Mark Gladwin a a University of Pittsburgh, VMI b Bayer Pharma AG, University of Witten/Herdecke c Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany
Rationale: Sickle cell disease (SCD) is caused by a point mutation in the hemoglobin (Hb) β gene, which generates hemoglobin S (HbS), a less soluble tetramer that undergoes polymerization. Endothelial dysfunction caused by chronic intravascular hemolysis is a hallmark of SCD affecting multiple organs, including the lung. A common complication of SCD is Pulmonary Arterial Hypertension (PAH) which is associated with early mortality. In the present study, we use an established animal model of SCD to investigate novel soluble guanylate cyclase (sGC) targeting in the pathobiology of global vascular dysfunction. Methods and results: We used 6 month old Berkley (BERK) transgenic mice that exclusively express human HbS and require knockout of the mouse α and β globins. Age matched wild type mice were used as controls. BERK mice were treated with sGC activator chow (17 mg/ kg/day; BAY 58–2667 × HCl (cinaciguat)) or placebo for 30 days. Following the treatment period, mice were sacrificed by terminal cardiac puncture. Superficial layers of the dorsal musculature were removed in order to access the thoracodorsal artery (TDA), lining the caudal side of the scapula. Segments 10– 15 mm long of the TDA was isolated and placed in cold KrebsHEPES to be used for pressure myography experiments. Isolated TDAs were mounted in a pressure arteriograph (Danish MyoTechnology) where they were maintained in a no-flow state and allowed to equilibrate for 30 min at 80 mmHg. Following
equilibration, arteries were stimulated with cumulative concentrations of the vasoconstrictor phenylephrine (PE, 10-8 to 10-4 M; Sigma). For each vessel, the luminal diameter was measured after stabilization of the response to the vasoconstrictor. The degree of contraction was calculated using the equation: DPE × 100/Dmax, where DPE is the diameter of the TDA after application of a given concentration of PE and Dmax is the maximal diameter of the TDA measured at the end of each experiment by applying Krebs-HEPES calcium free supplemented with acetylcholine. The basal tone was then calculated as: initial diameter/maximum diameter × 100. We found that BERK mice had an increased baseline tone and increased response to PE as compared with controls. SCD mice treated with sGC activator for 30 days showed improved baseline tone and improved constriction response to PE. Conclusions: The transgenic BERK sickle mouse develops significant vascular dysfunction with age. Direct activation of vascular smooth muscle cell sGC represents a novel therapeutic approach for global vascular dysfunction leading to PAH in SCD and other disease processes. Keywords: Sickle cell disease; Soluble guanylate cyclase; Vascular dysfunction.
P173. Protein S-nitrosylation facilitates the regulation of brain metabolism and neurotransmission by nitric oxide http://dx.doi.org/10.1016/j.niox.2014.09.117 Karthik Raju a, Paschalis-Thomas Doulis, Harry Ischiropoulos a University of Pennsylvania
Glutamate-based synaptic activity is an essential component of normal neuronal function. Changes in glutamatergic transmission are associated with corresponding adaptations in the metabolic support of neurons by astrocytes, although the mechanism(s) linking these two processes remain unclear. Nitric oxide (NO) may be a critical regulator of these pathways since loss of neuronal nitric oxide synthase (nNOS) leads to memory and behavioral deficits in mice, with profound alterations in glutamatergic transmission after deletion of both nNOS and endothelial NOS (eNOS). We performed a mass spectrometrybased proteomic interrogation of S-nitrosylated proteins in wildtype (WT), nNOS −/− and eNOS −/− brain that identified clusters of proteins linking synaptic transmission to astrocytic metabolism, particularly within the glutamate/glutamine cycle. 15Nbased profiling of this pathway in hippocampal slices revealed increased metabolic flux in the glutamine/glutamate cycle in nNOS −/− mice that may contribute to the phenotype observed in these animals. Furthermore, S-nitrosylation of sodiumdependent glutamate/aspartate transporter 2 (GLT1) profoundly decreases both its Vmax and Km. Taken together, the data indicate that nitric oxide, through reversible S-nitrosylation of key regulatory proteins, regulates the glutamate/glutamine cycle and influences glutamatergic transmission in vivo.
P174. Probing the mechanism of tetrahydrobiopterin radical reduction within NO synthases http://dx.doi.org/10.1016/j.niox.2014.09.118 Somasundaram Ramasamy a, Mohammad Mahfuzul Haque a, Jesus Tejero b, Mahinda Gangoda b, Dennis Stuehr a a Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA b Kent State University
Nitric-oxide synthase (NOS) enzymes are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from L-arginine. During the first reaction