Radical Intermediates of Nitric Oxide Synthase &Myocardial Ischemia Reperfusion

Summary

Principal Investigator: Ah Lim Tsai
Abstract: DESCRIPTION (provided by applicant): Nitric oxide (NO) participates both in the normal cardiac physiology and various cardiac pathological events including myocardial ischemia and reperfusion injury. Dynamic expression and activation of specific NOS isozyme occurs at different stages of the disease processes. Whether NO is cardioprotective or cardiodestructive remains controversial due to the complexity of the chemical reactions catalyzed by NOS. Changing of the choreography of the substrate supply and cofactor binding could transform NO synthase to catalyst for the reactive oxygen species (ROS) or reactive nitrogen species (RNS) that are important intermediates for cardiac pathophysiology. Our recent studies disclosed very different radical intermediate profile and regulation mechanism in eNOS and nNOS catalysis. The central hypothesis of this proposal is that understanding the interplay of the various regulatory molecules and the dynamic changes of the ROS, RNS and other radical intermediates during coupled and uncoupled NOS catalysis are crucial to elucidation of the etiology of myocardial infarction and ischemia- reperfusion injury. Furthermore, previous studies using whole tissue, cells, or purified enzyme under steady-state condition with spin-trapping are insufficient to obtain direct structural and kinetic information and require other innovative approach. We plan to elucidate the mechanism of radical intermediates dynamics in three NOS isozymes: In Aim 1, we wish to test the hypothesis that different radical intermediates are formed in the nNOSox, eNOSox and iNOSox. Innovative rapid-freeze quench (RFQ) EPR kinetic measurements and other pulsed EPR methods will be used to characterize new radical intermediates as well as their kinetics. In Aim 2, we will test the hypothesis that thiol is required in preventing BH4 oxidation in all NOS isoforms but is also necessary for keeping structural integrity of the nNOS and iNOS. Similar RFQ EPR kinetic measurements will be conducted in the presence and absence of thiol. Site-specific mutants will be used to assess the role of the key cysteines. In Aim 3, we plan to test whether the reductase domain is the main source of radicals in iNOS but not eNOS or nNOS. Purified full length NOS and NOSred of three isoforms will be evaluated for oxygen-induced radical intermediates using CaM/Ca+2 or disruption of heme coordination to dissect the radical contribution from the NOSox and NOSred. Both cardiomyocytes and macrophage-like cells will be our models for ischemia/reperfusion to assess the regulatory roles of thiol, oxygen, substrate, cofactor and inhibitors on the radical intermediate profile in the last aim. These approaches will provide the most basic knowledge on the mechanism under coupled and uncoupled conditions of each NOS isoforms and can be useful in developing therapeutic regimens for treating reperfusion injury. PUBLIC HEALTH RELEVANCE: This project focuses on characterizing the structure and temporal dependence of the radical intermediates, including ROS and RNS, induced by oxygen in all three nitric oxide synthase isozymes. The regulation of these radical intermediates by substrate, cofactors and thiol also are studied, both in vitro and ex vivo, in order to elucidate the underlying disease mechanism of myocardial ischemia and reperfusion injury.
Funding Period: 2010-02-01 - 2015-01-31
more information: NIH RePORT

Top Publications

  1. pmc Opposite displacement of helix F in attractant and repellent signaling by sensory rhodopsin-Htr complexes
    Jun Sasaki
    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA
    J Biol Chem 286:18868-77. 2011
  2. pmc The selectivity of Vibrio cholerae H-NOX for gaseous ligands follows the "sliding scale rule" hypothesis. Ligand interactions with both ferrous and ferric Vc H-NOX
    Gang Wu
    Division of Hematology, Department of Internal Medicine, The University of Texas Medical School at Houston, 6431 Fannin Street, Houston, Texas 77030, United States
    Biochemistry 52:9432-46. 2013
  3. pmc Bloch equations for anisotropic paramagnetic centers with spin of 1/2
    Alexander G Maryasov
    The V V Voevodsky Institute of Chemical Kinetics and Combustion, The Siberian Branch of the Russian Academy of Science, 3 Institutskaya St, Novosibirsk 630090, Russia
    J Magn Reson 233:80-6. 2013
  4. pmc Skew projection of echo-detected EPR spectra for increased sensitivity and resolution
    Michael K Bowman
    Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 0336, USA
    J Magn Reson 231:117-25. 2013
  5. pmc Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation
    Yun Li
    Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Centre National de la Recherche Scientifique UMR8601, Universite Paris Descartes, 75270 Paris, France
    Proc Natl Acad Sci U S A 109:12526-31. 2012
  6. pmc Spin dynamics of paramagnetic centers with anisotropic g tensor and spin of 1/2
    Alexander G Maryasov
    The Institute of Chemical Kinetics and Combustion, The Siberian Branch of the Russian Academy of Science, 3 Institutskaya St, Novosibirsk 630090, Russia
    J Magn Reson 221:69-75. 2012
  7. pmc HAMP domain signal relay mechanism in a sensory rhodopsin-transducer complex
    Jihong Wang
    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, Houston, Texas 77030, USA
    J Biol Chem 287:21316-25. 2012
  8. pmc Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site
    Emil Martin
    Division of Cardiology, Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas 77030, United States
    Biochemistry 51:2737-46. 2012
  9. pmc How do heme-protein sensors exclude oxygen? Lessons learned from cytochrome c', Nostoc puntiforme heme nitric oxide/oxygen-binding domain, and soluble guanylyl cyclase
    Ah Lim Tsai
    Division of Hematology, University of Texas Health Science Center at Houston, Houston, Texas 77225, USA
    Antioxid Redox Signal 17:1246-63. 2012
  10. ncbi Enhancement of the electron spin resonance of single-walled carbon nanotubes by oxygen removal
    William D Rice
    Department of Electrical and Computer Engineering, Rice University, Houston, Texas 77005, USA
    ACS Nano 6:2165-73. 2012

Research Grants

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
  2. Genetic Mechanisms of Resistance against Cardiac Preconditioning
    Matthias L Riess; Fiscal Year: 2013
  3. Signaling Processes Underlying Cardiovascular Function
    Jeffrey Robbins; Fiscal Year: 2013
  4. Fast Kinetic Investigations of Nitric Oxide Synthase
    Raymond M Esquerra; Fiscal Year: 2013
  5. Neutralizing Antibody &AAV FIX Gene Therapy
    Richard J Samulski; Fiscal Year: 2013
  6. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013

Detail Information

Publications14

  1. pmc Opposite displacement of helix F in attractant and repellent signaling by sensory rhodopsin-Htr complexes
    Jun Sasaki
    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA
    J Biol Chem 286:18868-77. 2011
    ....
  2. pmc The selectivity of Vibrio cholerae H-NOX for gaseous ligands follows the "sliding scale rule" hypothesis. Ligand interactions with both ferrous and ferric Vc H-NOX
    Gang Wu
    Division of Hematology, Department of Internal Medicine, The University of Texas Medical School at Houston, 6431 Fannin Street, Houston, Texas 77030, United States
    Biochemistry 52:9432-46. 2013
    ..Characterization of both ferric and ferrous Vc H-NOX and their complexes with various ligands lays the foundation for understanding the possible dual roles in gas and redox sensing of Vc H-NOX...
  3. pmc Bloch equations for anisotropic paramagnetic centers with spin of 1/2
    Alexander G Maryasov
    The V V Voevodsky Institute of Chemical Kinetics and Combustion, The Siberian Branch of the Russian Academy of Science, 3 Institutskaya St, Novosibirsk 630090, Russia
    J Magn Reson 233:80-6. 2013
    ..The equations describe the dynamics of the magnetic moment including relaxation and only contain parameters that are experimentally measurable. ..
  4. pmc Skew projection of echo-detected EPR spectra for increased sensitivity and resolution
    Michael K Bowman
    Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 0336, USA
    J Magn Reson 231:117-25. 2013
    ..Examples of skew-projected spectra with single crystals, glasses and powders show resolution improvements as large as a factor of seven with sensitivity increases of as much as a factor of five...
  5. pmc Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation
    Yun Li
    Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Centre National de la Recherche Scientifique UMR8601, Universite Paris Descartes, 75270 Paris, France
    Proc Natl Acad Sci U S A 109:12526-31. 2012
    ..Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues...
  6. pmc Spin dynamics of paramagnetic centers with anisotropic g tensor and spin of 1/2
    Alexander G Maryasov
    The Institute of Chemical Kinetics and Combustion, The Siberian Branch of the Russian Academy of Science, 3 Institutskaya St, Novosibirsk 630090, Russia
    J Magn Reson 221:69-75. 2012
    ..It is generally impossible to set a uniform spin turning angle for simple pulses in an unoriented or 'powder' sample when g tensor anisotropy is significant...
  7. pmc HAMP domain signal relay mechanism in a sensory rhodopsin-transducer complex
    Jihong Wang
    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, Houston, Texas 77030, USA
    J Biol Chem 287:21316-25. 2012
    ....
  8. pmc Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site
    Emil Martin
    Division of Cardiology, Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas 77030, United States
    Biochemistry 51:2737-46. 2012
    ..Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO...
  9. pmc How do heme-protein sensors exclude oxygen? Lessons learned from cytochrome c', Nostoc puntiforme heme nitric oxide/oxygen-binding domain, and soluble guanylyl cyclase
    Ah Lim Tsai
    Division of Hematology, University of Texas Health Science Center at Houston, Houston, Texas 77225, USA
    Antioxid Redox Signal 17:1246-63. 2012
    ....
  10. ncbi Enhancement of the electron spin resonance of single-walled carbon nanotubes by oxygen removal
    William D Rice
    Department of Electrical and Computer Engineering, Rice University, Houston, Texas 77005, USA
    ACS Nano 6:2165-73. 2012
    ..We hypothesize that physisorbed molecular oxygen acts as an acceptor (p-type), compensating the donor-like (n-type) defects that are responsible for the ESR signal in bulk SWCNTs...
  11. pmc A "sliding scale rule" for selectivity among NO, CO, and O₂ by heme protein sensors
    Ah Lim Tsai
    Division of Hematology, Internal Medicine, University of Texas Medical School at Houston, Houston, Texas 77030, United States
    Biochemistry 51:172-86. 2012
    ..This multistep six-coordinate to five-coordinate mechanism appears to be common to all NO sensors that exclude O₂ binding to capture a lower level of cellular NO and prevent its consumption by dioxygenation...
  12. pmc Dynamic ligand exchange in soluble guanylyl cyclase (sGC): implications for sGC regulation and desensitization
    Ah Lim Tsai
    Divisions of Hematology, University of Texas Health Science Center in Houston, Medical School, Houston, Texas 77030, USA
    J Biol Chem 286:43182-92. 2011
    ..The implications of these observations for cellular NO/cGMP signaling and the process of rapid desensitization of sGC are discussed in the context of the proposed model of sGC/NO interactions and dynamic transformations...
  13. pmc Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes
    Gang Wu
    Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA
    J Inorg Biochem 105:1226-37. 2011
    ..Only iNOS(507-531) was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM...
  14. pmc Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin
    Todd L Mollan
    Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852, USA
    Free Radic Biol Med 69:265-77. 2014
    ....

Research Grants30

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
    ..Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program. ..
  2. Genetic Mechanisms of Resistance against Cardiac Preconditioning
    Matthias L Riess; Fiscal Year: 2013
    ..This CDA marks the indispensable basis for further investigations on the role of genetics in cardioprotection and will be a critical milestone to achieve investigative independence in cardiovascular research at the VA. ..
  3. Signaling Processes Underlying Cardiovascular Function
    Jeffrey Robbins; Fiscal Year: 2013
    ..These projects are supported by 3 Cores: Core A: The Administrative Core;Core B: The Physiology Core and Core C: The Imaging-Cell Culture Core. (End of Abstract) ..
  4. Fast Kinetic Investigations of Nitric Oxide Synthase
    Raymond M Esquerra; Fiscal Year: 2013
    ..Understanding how NOS is regulated and clarifying its catalytic mechanism are crucial both for designing therapies that control NO synthesis and for understanding how compromised NO physiology leads to deleterious health effects. ..
  5. Neutralizing Antibody &AAV FIX Gene Therapy
    Richard J Samulski; Fiscal Year: 2013
    ..The long-term objective of this PPG is to advance basic understanding of vector-cell-animal model interactions for safe gene delivery. ..
  6. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
    ..The ultimate goal of all the proposed studies is to define the molecular events that feature in the formation of human prions in order to develop therapeutics that cure the human prion diseases. ..