A pulmonary vascular redox O2 sensor
1
A PULMONARY VASCULAR REDOX 0, SENSOR Stephen Archer, Evangelos Michelakis, Div. of Cardiology, matching in the lun and is uni ue to PAS. The effector mechamsm of HP 4. mvolves m ltution of O,-sensitive, .R. . . volta e-gated f must e potassium channels (Kv) if PA smooth cells (PASMCs), results membrane depolarization, activation of voltage gated Ca” channels, increased intracellular Ca” and contraction. Whereas there is substantial agreement regarding the effector mechanism, the identity of the 0, sensor is unknown. Putative sensors include the Kv channels themselves and several 0, sensitive cytochromes. We suggest that PO, may be transduced by a redox sensor, separate from the channel. The sensitivity of certain R channels to redox modulation may relate to their redox-sensitive cysteine groups. Prior studies show that the normoxic lung is in an oxidized state (more radicals/peroxides) relative to the h I?+ poxic lung. Furthermore, exogenous oxidants increase channel activity and promote hyper-polarization and pulmonary vasodilatation; whilst reducm the opposite effect. We hypothesize that t k agents have ere IS a tonic production of a vasodilatory redox 2” messenger in the normoxic lung (e.g. H 0,) that is inhibited by hypoxia. The potential sources o# such PO,-sensitive redox stimuli include cytochromes in the mitochondrial electron transpoc chain (ETC) and NAD(P)H oxidase. In zFif.%~ b A utative 0, sensors we have shown that mice (P)H oxidase continue to sense 0, normally, despite impaired reduction of activated 0, species. Thts suggests that the I-phox containin 5 PA 0 sensor. The mitochondrial E 5 enzyme is not the C remains a likely candidate. Inhibitors of mitochondrial respiration, rotenone mimic hy oxia. They cause a reduced environment,. inhibit R+ current and elicit pulmonary vasoconstrictlon and systemic vasodilatation. Thus, mitochondria may be pulmonary vascular redox sensors. OXYGEN AND REDOX SENSITIVITY OF K+ CHANNELS IN THE DUCTUS ARTERIOSUS E. Kenneth Weir*, Simona Tolarova, Daniel P. Nelson, Stephen L. Archer, Helen L. Reeve. Dept of Medicine, VA Medical Center and University of Minnesota, Minneapolis, Minnesota USA We hypothesized that the closure of the ductus arteriosus (DA) after birth is initiated, in part, through the inhibition of oxygen-sensitive K+ channels by an increase in H202 or changes in related redox couples. Chemiluminescence measurements, with luminol (50 microM) or lucigenin (5 microh?), showed significantly higher levels of reactive 02 species in normoxic, compared to hypoxic DA. This increase was completely reversed by catalase (12OOU/ml). Prolonged normoxia caused a significant decrease in K+ current density (IK) and depolarization of membrane potential in single fetal DA smooth muscle cells. Removal of endogenous H202 with intracellular catalase (2OOU/ml), increased normoxic whole-cell K+ currents (IK) and hyperpolarized membrane potential, while intracellular H202 (1OOnM) and extracellular t-butyl H202 (100 microM) decreased IK and depolarized membrane potential. Superoxide dismutase (lOOU/ml) had no significant effect on normoxic K+ currents.The oxidizing agent 5,5’-dithiobis-(2-nitrobenzioc acid) constricted hypoxia-dilated rings, while N- mercaptopropionylglycine (NMPG), duroquinone and dithiothreitol all dilated normoxic-constricted DA rings. We conclude that increased H202 levels, associated with a cytosolic redox shift at birth, signal K+ channel inhibition and DA constriction. NADH OXIDASE AND REDOX CONTROL OF VASCULAR OXIDANT SIGNALING AND OXYGEN SENSING Michael S. Wolin. Dept. of Physiology, New York Medical College, Valhalla, NY 10595, USA. Our group initially recognized the importance of a NADH oxidase as an oxygen sensor in endothelium-removed bovine pulmonary (BPA) and coronary arteries (BCA) from studies on lactate increasing superoxide levels, causing a hydrogen peroxide- mediated relaxation and by it modulating responses to changes in oxygen tension. The actions of combinations of probes including lactate (10 mM) and pyruvate (10 mM) as modulators of cytosolic NAD(H) redox, inhibition of NADH oxidase with a flavoprotein probe (l-10 pM diphenyliodonium), inhibition of C&&-SOD (diethyldithiocarbamate), and modulation of peroxide metabolism by catalase (aminotriazole) and glutathione (GSH) peroxidase (0.1 mM ebselen) provide evidence that superoxide derived from NADH oxidase inhibits relaxation to NO when Zn,Cu-SOD is inhibited. Whereas, peroxidederived from this oxidase appears to mediate a catalase-dependent relaxation by the activation of cytosolic guanylate cyclase and a p42/p44 MAPK kinase- dependent enhancement of force generated by 30 mM KCI, a response activated by a 20 min exposure to increased passive force. Multiple components of responses of BPA and BCA to changes in oxygen tension or exposure to intermittent hypoxia appear to be mediated through oxidants derived from NADH oxidase. Thus, NADH oxidase appears to be a key source of oxidants involved in physiological oxidant signaling and oxygen sensing mechanisms in BPA and BCA. We hypothesize that the integrated function of &ox-controlled systems determine which signaling mechanisms are activated by oxidants derived from this ox&se. PPAR SIGNALING IN THE CONTROL OF CARDIAC ENERGY METABOLISM: LESSONS FROM GENETICALLY ALTERED MICE Daniel P. Kelly, John J. Lehman, Philip M. Barger, Janice M. Huss, Carla J. Weinheimer, Attiln Kovacs, Michael R. Courtois and Teresa C. Leone, Center for Cardiovascular Research, Washington University, St. Louis, MO 63110-1093, USA The energy demands of the mammalian heart are met by high capacity mitochondrial oxidative pathways specialized to catabolize fatty acid substrate to produce ATF’. The capacity for substrate flux through the cardiac mitochondrial fatty acid oxidation (FAO) pathway is regulated, in part, at the level of gene expression during development and in response to diverse physiologic conditions. This tight coordinate control of cardiac energy metabolic gene expression involves the lipid-activated nuclear receptor, PPARa. Recent studies in our laboratory have revealed that the inducible transcriptional coactivator PGC-1 is an important coregulator of PPARu function in heart. Gain-of-function studies in cardiac myocytes in culture and in transgenic mice have shown that PGC-1 overexpression not only activates the expression of PPARa genes involved in FAO, but also induces a dramatic mitochondrial biogenesis and increases coupled respiration consistent with augmented capacity for ATP production. The expression and activity of the PPARa transcriptional regulatory complex is also regulated in several cardiovascular disease states. Pathologic forms of cardiac hypertrophy and exposure to hypoxia deactivates the PPARtiGC-1 regulatory pathway at both transcriptional and post-transcriptional levels. Conversely, the activity of PPARa and PGC-1 are activated in the diabetic heart in which FAO rates are dramatically increased. The role of these transcriptional regulatory switches and corresponding metabolic alterations as adaptive versus maladaptive in the diseased heart will be discussed. A158
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Transcript of A pulmonary vascular redox O2 sensor
A PULMONARY VASCULAR REDOX 0, SENSOR Stephen Archer, Evangelos Michelakis, Div. of Cardiology,
matching in the lun and is uni ue to PAS. The effector mechamsm of HP 4. mvolves m ltution of O,-sensitive, .R. . . volta e-gated
f must e potassium channels (Kv) if PA smooth
cells (PASMCs), results membrane depolarization, activation of voltage gated Ca” channels, increased intracellular Ca” and contraction. Whereas there is substantial agreement regarding the effector mechanism, the identity of the 0, sensor is unknown. Putative sensors include the Kv channels themselves and several 0, sensitive cytochromes. We suggest that PO, may be transduced by a redox sensor, separate from the channel. The sensitivity of certain R channels to redox modulation may relate to their redox-sensitive cysteine groups. Prior studies show that the normoxic lung is in an oxidized state (more radicals/peroxides) relative to the h I?+
poxic lung. Furthermore, exogenous oxidants increase channel activity and promote hyper-polarization and
pulmonary vasodilatation; whilst reducm the opposite effect. We hypothesize that t k agents have ere IS a tonic production of a vasodilatory redox 2” messenger in the normoxic lung (e.g. H 0,) that is inhibited by hypoxia. The potential sources o# such PO,-sensitive redox stimuli include cytochromes in the mitochondrial electron transpoc chain (ETC) and NAD(P)H oxidase. In
zFif.%~ b A utative 0, sensors we have shown that mice (P)H oxidase continue to sense 0, normally,
despite impaired reduction of activated 0, species. Thts suggests that the I-phox containin 5 PA 0 sensor. The mitochondrial E 5
enzyme is not the C remains a likely
candidate. Inhibitors of mitochondrial respiration, rotenone mimic hy oxia. They cause a reduced environment,. inhibit R+ current and elicit pulmonary vasoconstrictlon and systemic vasodilatation. Thus, mitochondria may be pulmonary vascular redox sensors.
OXYGEN AND REDOX SENSITIVITY OF K+ CHANNELS IN THE DUCTUS ARTERIOSUS E. Kenneth Weir*, Simona Tolarova, Daniel P. Nelson, Stephen L. Archer, Helen L. Reeve. Dept of Medicine, VA Medical Center and University of Minnesota, Minneapolis, Minnesota USA
We hypothesized that the closure of the ductus arteriosus (DA) after birth is initiated, in part, through the inhibition of oxygen-sensitive K+ channels by an increase in H202 or changes in related redox couples. Chemiluminescence measurements, with luminol (50 microM) or lucigenin (5 microh?), showed significantly higher levels of reactive 02 species in normoxic, compared to hypoxic DA. This increase was completely reversed by catalase (12OOU/ml). Prolonged normoxia caused a significant decrease in K+ current density (IK) and depolarization of membrane potential in single fetal DA smooth muscle cells. Removal of endogenous H202 with intracellular catalase (2OOU/ml), increased normoxic whole-cell K+ currents (IK) and hyperpolarized membrane potential, while intracellular H202 (1OOnM) and extracellular t-butyl H202 (100 microM) decreased IK and depolarized membrane potential. Superoxide dismutase (lOOU/ml) had no significant effect on normoxic K+ currents.The oxidizing agent 5,5’-dithiobis-(2-nitrobenzioc acid) constricted hypoxia-dilated rings, while N- mercaptopropionylglycine (NMPG), duroquinone and dithiothreitol all dilated normoxic-constricted DA rings. We conclude that increased H202 levels, associated with a cytosolic redox shift at birth, signal K+ channel inhibition and DA constriction.
NADH OXIDASE AND REDOX CONTROL OF VASCULAR OXIDANT SIGNALING AND OXYGEN SENSING Michael S. Wolin. Dept. of Physiology, New York Medical College, Valhalla, NY 10595, USA.
Our group initially recognized the importance of a NADH oxidase as an oxygen sensor in endothelium-removed bovine pulmonary (BPA) and coronary arteries (BCA) from studies on lactate increasing superoxide levels, causing a hydrogen peroxide- mediated relaxation and by it modulating responses to changes in oxygen tension. The actions of combinations of probes including lactate (10 mM) and pyruvate (10 mM) as modulators of cytosolic NAD(H) redox, inhibition of NADH oxidase with a flavoprotein probe (l-10 pM diphenyliodonium), inhibition of C&&-SOD (diethyldithiocarbamate), and modulation of peroxide metabolism by catalase (aminotriazole) and glutathione (GSH) peroxidase (0.1 mM ebselen) provide evidence that superoxide derived from NADH oxidase inhibits relaxation to NO when Zn,Cu-SOD is inhibited. Whereas, peroxidederived from this oxidase appears to mediate a catalase-dependent relaxation by the activation of cytosolic guanylate cyclase and a p42/p44 MAPK kinase- dependent enhancement of force generated by 30 mM KCI, a response activated by a 20 min exposure to increased passive force. Multiple components of responses of BPA and BCA to changes in oxygen tension or exposure to intermittent hypoxia appear to be mediated through oxidants derived from NADH oxidase. Thus, NADH oxidase appears to be a key source of oxidants involved in physiological oxidant signaling and oxygen sensing mechanisms in BPA and BCA. We hypothesize that the integrated function of &ox-controlled systems determine which signaling mechanisms are activated by oxidants derived from this ox&se.
PPAR SIGNALING IN THE CONTROL OF CARDIAC ENERGY METABOLISM: LESSONS FROM GENETICALLY ALTERED MICE Daniel P. Kelly, John J. Lehman, Philip M. Barger, Janice M. Huss, Carla J. Weinheimer, Attiln Kovacs, Michael R. Courtois and Teresa C. Leone, Center for Cardiovascular Research, Washington University, St. Louis, MO 63110-1093, USA
The energy demands of the mammalian heart are met by high capacity mitochondrial oxidative pathways specialized to catabolize fatty acid substrate to produce ATF’. The capacity for substrate flux through the cardiac mitochondrial fatty acid oxidation (FAO) pathway is regulated, in part, at the level of gene expression during development and in response to diverse physiologic conditions. This tight coordinate control of cardiac energy metabolic gene expression involves the lipid-activated nuclear receptor, PPARa. Recent studies in our laboratory have revealed that the inducible transcriptional coactivator PGC-1 is an important coregulator of PPARu function in heart. Gain-of-function studies in cardiac myocytes in culture and in transgenic mice have shown that PGC-1 overexpression not only activates the expression of PPARa genes involved in FAO, but also induces a dramatic mitochondrial biogenesis and increases coupled respiration consistent with augmented capacity for ATP production. The expression and activity of the PPARa transcriptional regulatory complex is also regulated in several cardiovascular disease states. Pathologic forms of cardiac hypertrophy and exposure to hypoxia deactivates the PPARtiGC-1 regulatory pathway at both transcriptional and post-transcriptional levels. Conversely, the activity of PPARa and PGC-1 are activated in the diabetic heart in which FAO rates are dramatically increased. The role of these transcriptional regulatory switches and corresponding metabolic alterations as adaptive versus maladaptive in the diseased heart will be discussed.
A158
