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Compilation of References in Leaders in Pharmaceutical Intelligence about proteomics, metabolomics, signaling pathways, and cell regulation


Compilation of References in Leaders in Pharmaceutical Intelligence about
proteomics, metabolomics, signaling pathways, and cell regulation

Curator: Larry H. Bernstein, MD, FCAP

 

Proteomics

  1. The Human Proteome Map Completed
    Reporter and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/28/the-human-proteome-map-completed/
  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/06/24/proteomics-the-pathway-to-understanding-and-decision-making-in-medicine/
  1. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets
    Author and Curator, Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-of-therapeutic-targets/
  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
    Author and Curator, Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-metabolome/
  1. Synthesizing Synthetic Biology: PLOS Collections
    Reporter: Aviva Lev-Ari
    https://pharmaceuticalintelligence.com/2012/08/17/synthesizing-synthetic-biology-plos-collections/

 

Metabolomics

  1. Extracellular evaluation of intracellular flux in yeast cells
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
    https://pharmaceuticalintelligence.com/2014/08/25/extracellular-evaluation-of-intracellular-flux-in-yeast-cells/ 
  2. Metabolomic analysis of two leukemia cell lines. I.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
    http://pharmaceuticalintelligence.com/2014/08/23/metabolomic-analysis-of-two-leukemia-cell-lines-_i/ 
  3. Metabolomic analysis of two leukemia cell lines. II.
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
    https://pharmaceuticalintelligence.com/2014/08/24/metabolomic-analysis-of-two-leukemia-cell-lines-ii/ 
  4. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics
    Reviewer and Curator, Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/22/metabolomics-metabonomics-and-functional-nutrition-the-next-step-in-nutritional-metabolism-and-biotherapeutics/ 
  5. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator
    https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-metabolism-provides-homeomeostatic-regulation/

 

Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief
    Reviewer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/21/pentose-shunt-electron-transfer-galactose-more-lipids-in-brief/
  2. Mitochondria: More than just the “powerhouse of the cell”
    Reviewer and Curator: Ritu Saxena
    https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/
  3. Mitochondrial fission and fusion: potential therapeutic targets?
    Reviewer and Curator: Ritu saxena
    https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/ 
  4. Mitochondrial mutation analysis might be “1-step” away
    Reviewer and Curator: Ritu Saxena
    https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/
  5. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com
    Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-leaders-in-pharmaceutical-intelligence/
  6. Metabolic drivers in aggressive brain tumors
    Prabodh Kandal, PhD
    https://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/ 
  7. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes
    Author and Curator: Aviva Lev-Ari, PhD, RD
    https://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/
  8. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation
    Author and curator:Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/
  9. Therapeutic Targets for Diabetes and Related Metabolic Disorders
    Reporter, Aviva Lev-Ari, PhD, RD
    https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/
  10. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation
    Larry H. Bernstein, MD, FCAP, Reviewer and curator
    https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-metabolism-provides-homeomeostatic-regulation/
  11. The multi-step transfer of phosphate bond and hydrogen exchange energy
    Curator:Larry H. Bernstein, MD, FCAP,
    https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-bond-and-hydrogen-exchange-energy/
  12. Studies of Respiration Lead to Acetyl CoA
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/18/studies-of-respiration-lead-to-acetyl-coa/
  13. Lipid Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/15/lipid-metabolism/
  14. Carbohydrate Metabolism
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/13/carbohydrate-metabolism/
  15. Prologue to Cancer – e-book Volume One – Where are we in this journey?
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/04/13/prologue-to-cancer-ebook-4-where-are-we-in-this-journey/
  16. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-how-we-got-here/
  17. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/11/01/inhibition-of-the-cardiomyocyte-specific-kinase-tnni3k/
  18. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/05/15/the-binding-of-oligonucleotides-in-dna-and-3-d-lattice-structures/
  19. Mitochondrial Metabolism and Cardiac Function
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/
  20. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia
    Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/
  21. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo
    Author and Curator: SJ. Williams
    https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-tumor-growth-in-vivo/
  22. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/
  23. Overview of Posttranslational Modification (PTM)
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/
  24. Malnutrition in India, high newborn death rate and stunting of children age under five years
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-children-age-under-five-years/
  25. Update on mitochondrial function, respiration, and associated disorders
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-disorders/
  26. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
    Larry H. Bernstein, MD, FCAP, Curator
    https://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-in-renal-disease/ 
  27. Late Onset of Alzheimer’s Disease and One-carbon Metabolism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.
    https://pharmaceuticalintelligence.com/2013/05/06/alzheimers-disease-and-one-carbon-metabolism/
  28. Problems of vegetarianism
    Reporter and Curator: Dr. Sudipta Saha, Ph.D.
    https://pharmaceuticalintelligence.com/2013/04/22/problems-of-vegetarianism/

 

Signaling Pathways

  1. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
    Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN  https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-translational_medicine-part_2/
  2. Epilogue: Envisioning New Insights in Cancer Translational Biology
    Series C: e-Books on Cancer & Oncology
    Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant
    https://pharmaceuticalintelligence.com/2014/03/29/epilogue-envisioning-new-insights/
  3. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter  Writer and Curator: Larry H Bernstein, MD, FCAP and Curator and Content Editor: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy
  4. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
    Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
    Author and Curator: Larry H Bernstein, MD, FCAP and Article Curator: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/
  5. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
    Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/
  6. Identification of Biomarkers that are Related to the Actin Cytoskeleton
    Larry H Bernstein, MD, FCAP, Author and Curator
    https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/
  7. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage
    Author and Curator: Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-End-Stage/
  8. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology
    Demet Sag, PhD, Author and Curator
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
  9. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase
    Demet Sag, PhD, Author and Curator
    https://pharmaceuticalintelligence.com/2013/08/04/ido-for-commitment-of-a-life-time-the-origins-and-mechanisms-of-ido-indolamine-2-3-dioxygenase/
  10. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad
    Author and Curator: Demet Sag, PhD, CRA, GCP
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
  11. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute
    Reporter: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/06/26/signaling-pathway-that-makes-young-neurons-connect-was-discovered-scripps-research-institute/
  12. Naked Mole Rats Cancer-Free
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/06/20/naked-mole-rats-cancer-free/
  13. Amyloidosis with Cardiomyopathy
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/03/31/amyloidosis-with-cardiomyopathy/
  14. Liver endoplasmic reticulum stress and hepatosteatosis
    Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2013/03/10/liver-endoplasmic-reticulum-stress-and-hepatosteatosis/
  15. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/
  16. Nitric Oxide Function in Coagulation – Part II
    Curator and Author: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/
  17. Nitric Oxide, Platelets, Endothelium and Hemostasis
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/
  18. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/
  19. Nitric Oxide and Immune Responses: Part 1
    Curator and Author:  Aviral Vatsa PhD, MBBS
    https://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/
  20. Nitric Oxide and Immune Responses: Part 2
    Curator and Author:  Aviral Vatsa PhD, MBBS
    https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/
  21. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/
  22. New Insights on Nitric Oxide donors – Part IV
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/
  23. Crucial role of Nitric Oxide in Cancer
    Curator and Author: Ritu Saxena, Ph.D.
    https://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/
  24. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/
  25. Nitric Oxide and Immune Responses: Part 2
    Author and Curator: Aviral Vatsa, PhD, MBBS
    https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/
  26. Mitochondrial Damage and Repair under Oxidative Stress
    Author and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/
  27. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/
  28. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy
    Curator and Author: Ziv Raviv, PhD, RN 04/06/2013
    https://pharmaceuticalintelligence.com/2013/04/06/targeting-mitochondrial-bound-hexokinase-for-cancer-therapy/
  29. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/
  30. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/
  31. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I
    Curator and Author: Larry H Bernstein, MD, FACP
    https://pharmaceuticalintelligence.com/2012/11/26/biochemistry-of-the-coagulation-cascade-and-platelet-aggregation/

 

Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/06/what-is-the-meaning-of-so-many-rnas/
  2. RNA and the transcription the genetic code
    Larry H. Bernstein, MD, FCAP, Writer and Curator
    https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/
  3. A Primer on DNA and DNA Replication
    Writer and Curator: Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/07/29/a_primer_on_dna_and_dna_replication/
  4. Pathology Emergence in the 21st Century
    Author and Curator: Larry Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/03/pathology-emergence-in-the-21st-century/
  5. RNA and the transcription the genetic code
    Writer and Curator, Larry H. Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/02/rna-and-the-transcription-of-the-genetic-code/
  6. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H Bernstein, MD, FCAP
    Author: Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/07/16/commentary-on-biomarkers-for-genetics-and-genomics-of-cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/
  7. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies
    Author an Curator: Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2013/05/18/observations-on-finding-the-genetic-links/
  8. Silencing Cancers with Synthetic siRNAs
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
    https://pharmaceuticalintelligence.com/2013/12/09/silencing-cancers-with-synthetic-sirnas/
  9. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points
    Reporter: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/12/12/cardiometabolic-syndrome-and-the-genetics-of-hypertension-the-neuroendocrine-transcriptome-control-points/
  10. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets
    Larry H. Bernstein, MD, FCAP, Reviewer and Curator
    https://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-mellitus-and-treatment-targets/
  11. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis
    Reporter: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/11/15/ct-angiography-truevision-metabolomics-genomic-phenotyping-for-new-therapeutic-targets-to-atherosclerosis/
  12. CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics
    Genomics Curator, Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/08/30/cracking-the-code-of-human-life-the-birth-of-bioinformatics-computational-genomics/
  13. Big Data in Genomic Medicine
    Author and Curator, Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/
  14.  From Genomics of Microorganisms to Translational Medicine
    Author and Curator: Demet Sag, PhD
    https://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-microorganisms-to-translational-medicine/
  15.  Summary of Genomics and Medicine: Role in Cardiovascular Diseases
    Author and Curator, Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2014/01/06/summary-of-genomics-and-medicine-role-in-cardiovascular-diseases/

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Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP

 

This is an added selection of articles in Leaders in Pharmaceutical Intelligence after the third portion of the discussion in a series of articles that began with signaling and signaling pathways. There are fine features on the functioning of enzymes and proteins, on sequential changes in a chain reaction, and on conformational changes that we shall return to.  These are critical to developing a more complete understanding of life processes.  I have indicated that many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism3.1  Selected References to Signaling and Metabolic Pathways in Leaders in Pharmaceutical Intelligence
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Selected References to Signaling and Metabolic Pathwayspublished in this Open Access Online Scientific Journal, include the following:

Update on mitochondrial function, respiration, and associated disorders

Curator and writer: Larry H. Benstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-disorders/

A Synthesis of the Beauty and Complexity of How We View Cancer


Cancer Volume One – Summary

A Synthesis of the Beauty and Complexity of How We View Cancer

Author: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/03/26/a-synthesis-of-the-beauty-and-complexity-of-how-we-view-cancer/

Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-how-we-got-here/

 The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Author and Curator: Larry H Bernstein, MD, FCAP, 
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
And Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure

Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Author and Curator: Larry H. Bernstein, MD, FCAP
Curator:  Stephen J. Williams, PhD
and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Mitochondrial Metabolism and Cardiac Function

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Reversal of Cardiac mitochondrial dysfunction

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Advanced Topics in Sepsis and the Cardiovascular System  at its End Stage

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-End-Stage/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/

Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

 

Nitric Oxide, Platelets, Endothelium and Hemostasis (Coagulation Part II)

Curator: Larry H. Bernstein, MD, FCAP 

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/


Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Reporter and Curator: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

 

Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function

Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/


Mitochondria and Cancer: An overview of mechanisms

Author and Curator: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Mitochondria: More than just the “powerhouse of the cell”

Author and Curator: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Overview of Posttranslational Modification (PTM)

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/29/overview-of-posttranslational-modification-ptm/


Ubiquitin Pathway Involved in Neurodegenerative Diseases

Author and curator: Larry H Bernstein, MD,  FCAP

https://pharmaceuticalintelligence.com/2013/02/15/ubiquitin-pathway-involved-in-neurodegenerative-diseases/

Is the Warburg Effect the Cause or the Effect of Cancer: A 21st Century View?

Author: Larry H. Bernstein, MD, FCAP 

https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/

New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

Perspectives on Nitric Oxide in Disease Mechanisms [Kindle Edition]

Margaret Baker PhD (Author), Tilda Barliya PhD (Author), Anamika Sarkar PhD (Author), Ritu Saxena PhD (Author), Stephen J. Williams PhD (Author), Larry Bernstein MD FCAP (Editor), Aviva Lev-Ari PhD RN (Editor), Aviral Vatsa PhD (Editor)

https://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

 

Summary

Nitric oxide and its role in vascular biology

Signal transmission by a gas that is produced by one cell, penetrates through membranes and regulates the function of another cell represents an entirely new principle for signaling in biological systems.   All compounds that inhibit endothelium-derived relaxation-factor (EDRF) have one property in common, redox activity, which accounts for their inhibitory action on EDRF. One exception is hemoglobin, which inactivates EDRF by binding to it. Furchgott, Ignarro and Murad received the Nobel Prize in Physiology and Medicine for discovery of EDRF in 1998 and demonstrating that it might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta.  These investigators working independently demonstrated that NO is indeed produced by mammalian cells and that NO has specific biological roles in the human body. These studies highlighted the role of NO in cardiovascular, nervous and immune systems. In cardiovascular system NO was shown to cause relaxation of vascular smooth muscle cells causing vasodilatation, in nervous system NO acts as a signaling molecule and in immune system it is used against pathogens by the phagocytosis cells. These pioneering studies opened the path of investigation of role of NO in biology.

NO modulates vascular tone, fibrinolysis, blood pressure and proliferation of vascular smooth muscles. In cardiovascular system disruption of NO pathways or alterations in NO production can result in preponderance to hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis and thrombosis. The three enzyme isoforms of NO synthase family are responsible for generating NO in different tissues under various circumstances.

Reduction in NO production is implicated as one of the initial factors in initiating endothelial dysfunction. This reduction could be due to

  • reduction in eNOS production
  • reduction in eNOS enzymatic activity
  • reduced bioavailability of NO

Nitric oxide is one of the smallest molecules involved in physiological functions in the body. It is seeks formation of chemical bonds with its targets.  Nitric oxide can exert its effects principally by two ways:

  • Direct
  • Indirect

Direct actions, as the name suggests, result from direct chemical interaction of NO with its targets e.g. with metal complexes, radical species. These actions occur at relatively low NO concentrations (<200 nM)

Indirect actions result from the effects of reactive nitrogen species (RNS) such as NO2 and N2O3. These reactive species are formed by the interaction of NO with superoxide or molecular oxygen. RNS are generally formed at relatively high NO concentrations (>400 nM)

Although it can be tempting for scientists to believe that RNS will always have deleterious effects and NO will have anabolic effects, this is not entirely true as certain RNS mediated actions mediate important signalling steps e.g. thiol oxidation and nitrosation of proteins mediate cell proliferation and survival, and apoptosis respectively.

  • Cells subjected to NO concentration between 10-30 nM were associated with cGMP dependent phosphorylation of ERK
  • Cells subjected to NO concentration between 30-60 nM were associated with Akt phosphorylation
  • Concentration nearing 100 nM resulted in stabilisation of hypoxia inducible factor-1
  • At nearly 400 nM NO, p53 can be modulated
  • >1μM NO, it nhibits mitochondrial respiration

 

Nitric oxide signaling, oxidative stress,  mitochondria, cell damage

Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and ‘endocrine’ effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced.

NO itself in low concentrations have protective action on mitochondrial signaling of cell death.

The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes.

Oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, can cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload.  The normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease.

We reviewed the complex interactions and underlying regulatory balances/imbalances between the mechanism of vasorelaxation and vasoconstriction of vascular endothelium by way of nitric oxide (NO), prostacyclin, in response to oxidative stress and intimal injury.

Nitric oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function. The influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the resulting balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic.

ROS induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels.

NO and circulatory diseases

Blood vessels arise from endothelial precursors that are thin, flat cells lining the inside of blood vessels forming a monolayer throughout the circulatory system. ECs are defined by specific cell surface markers that characterize their phenotype.

Scientists at the University of Helsinki, Finland, wanted to find out if there exists a rare vascular endothelial stem cell (VESC) population that is capable of producing very high numbers of endothelial daughter cells, and can lead to neovascular growth in adults.

VESCs discovered that reside at the blood vessel wall endothelium are a small population of CD117+ ECs capable of self-renewal.  These cells are capable of undergoing clonal expansion unlike the surrounding ECs that bear limited proliferating potential. A single VESC cell isolated from the endothelial population was able to generate functional blood vessels.

Among many important roles of Nitric oxide (NO), one of the key actions is to act as a vasodilator and maintain cardiovascular health. Induction of NO is regulated by signals in tissue as well as endothelium.

Chen et. al. (Med. Biol. Eng. Comp., 2011) developed a 3-D model consisting of two branched arterioles and nine capillaries surrounding the vessels. Their model not only takes into account of the 3-D volume, but also branching effects on blood flow.

The model indicates that wall shear stress changes depending upon the distribution of RBC in the microcirculations of blood vessels, lead to differential production of NO along the vascular network.

Endothelial dysfunction, the hallmark of which is reduced activity of endothelial cell derived nitric oxide (NO), is a key factor in developing atherosclerosis and cardiovascular disease. Vascular endothelial cells play a pivotal role in modulation of leukocyte and platelet adherence, thrombogenicity, anticoagulation, and vessel wall contraction and relaxation, so that endothelial dysfunction has become almost a synonym for vascular disease. A single layer of endothelial cells is the only constituent of capillaries, which differ from other vessels, which contain smooth muscle cells and adventitia. Capillaries directly mediate nutritional supply as well as gas exchange within all organs. The failure of the microcirculation leads to tissue apoptosis/necrosis.

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G Protein–Coupled Receptor and S-Nitrosylation in Cardiac Ischemia

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

This recently published article delineates a role of G-protein-coupled receptor with S-nitrosylation in outcomes for acute coronary syndrome.

Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome to Cardiac Ischemic Injury

Z. Maggie Huang1, Erhe Gao1, Fabio Vasconcelos Fonseca2,3, Hiroki Hayashi2,3, Xiying Shang1, Nicholas E. Hoffman1, J. Kurt Chuprun1, Xufan Tian4, Doug G. Tilley1, Muniswamy Madesh1, David J. Lefer5, Jonathan S. Stamler2,3,6, and Walter J. Koch1*
1 Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA
2 Institute for Transformative Molecular Medicine, Case Western Reserve Univ SOM, Cleveland, OH
3 Department of Medicine, Case Western Reserve University, Cleveland, OH
4 Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA
5 Department  Surgery, Div of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA
6 University Hospitals Harrington Discovery Institute, Cleveland, OH

Sci. Signal., 29 Oct 2013; 6(299), p. ra95         http:dx.doi.org/10.1126/scisignal.2004225

Abstract

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving

  • G protein–coupled receptors (GPCRs) or nitric oxide (NO).

These regimens are thought to target distinct molecular pathways. We showed that

  • these pathways are interdependent and converge on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function.

Ischemic injury coupled to

  • GPCR activation, including GPCR desensitization and myocyte loss,
  • required GRK2 activation,

and we found that cardioprotection mediated by inhibition of GRK2 depended on

  • endothelial nitric oxide synthase (eNOS) and
  • was associated with S-nitrosylation of GRK2.

Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other,

the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into

  • the mechanism of action of classic drugs used to treat heart failure and
  • new therapeutic approaches to ischemic heart disease.

* Corresponding author. E-mail: walter.koch@temple.edu
Citation: Z. M. Huang, E. Gao, F. V. Fonseca, H. Hayashi, X. Shang, N. E. Hoffman, J. K. Chuprun, X. Tian, D. G. Tilley, M. Madesh, D. J. Lefer, J. S. Stamler, W. J. Koch, Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome t

 Editor’s Summary

Sci. Signal., 29 Oct 2013; 6(299), p. ra95 [DOI: 10.1126/scisignal.2004225]

NO More Heart Damage

Damage caused by the lack of oxygen and nutrients that occurs during myocardial ischemia can result in heart failure. A therapeutic strategy that helps to limit the effects of heart failure is to

  • increase signaling through G protein–coupled receptors (GPCRs)
  • by inhibiting GRK2 (GPCR kinase 2), a kinase that
    • desensitizes GPCRs.

Another therapeutic strategy provides S-nitrosothiols, such as nitric oxide, which can be

  • added to proteins in a posttranslational modification called S-nitrosylation.

Huang et al. found that the ability of S-nitrosothiols to enhance cardiomyocyte survival after ischemic injury required the S-nitrosylation of GRK2, a modification that inhibits this kinase. Mice bearing a form of GRK2 that could not be S-nitrosylated 

  • were more susceptible to cardiac damage after ischemia.

These results suggest that therapeutic strategies that promote the S-nitrosylation of GRK2 could be used to treat heart failure after myocardial ischemia.

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Nitric Oxide Synthase Inhibitors (NOS-I)

Author: Larry H Bernstein, MD, FCAP

Curator: Stephen J. Williams, PhD

and

Co-Curator: Aviva Lev-Ari, PhD, RN

 

This recent article sheds a new light on nitric oxide and the activity of NOS in reactive oxygen species generation and the effect of NOS inhibitors in bacteria.

Structural and Biological Studies on Bacterial Nitric Oxide Synthase Inhibitors

Jeffrey K. Holdena, Huiying Lia, Qing Jingb, Soosung Kangb, Jerry Richoa, Richard B. Silvermanb,1, and Thomas L. Poulosb,1
Agman@chem.northwestern.edu
Author contributions: J.K.H. designed research; J.K.H. and J.R. performed research; Q.J. and S.K. contributed new reagents/analytic tools; J.K.H., H.L., R.B.S., and T.L.P. analyzed data; and J.K.H., R.B.S., and T.L.P. wrote the paper.

PNAS Oct 21, 2013;       http://dx.doi.org/10.1073/pnas.1314080110
This article is a PNAS Direct Submission
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank
Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 23, 2013 (received for review July 29, 2013)
Keywords:  crystallography, antibiotics, nitric oxide, NOS inhibitors, Bacillus subtilis, gram positive bacteria

Significance

Nitric oxide (NO) produced by bacterial nitric oxide synthase has recently been shown to

Using Bacillus subtilis as a model system, we identified

  • two NOS inhibitors that work in conjunction with an antibiotic to kill B. subtilis.

Moreover, comparison of inhibitor-bound crystal structures between the bacterial NOS and mammalian NOS revealed an unprecedented

  • mode of binding to the bacterial NOS that can be further exploited for future structure-based drug design.

Overall, this work is an important advance in developing inhibitors against gram-positive pathogens.

Abstract

Nitric oxide (NO) produced by bacterial NOS functions as

  • a cytoprotective agent against oxidative stress in Staphylococcus aureusBacillus anthracis, and Bacillus subtilis.

The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds

  • impede the growth of B. subtilis under oxidative stress, and
  • crystal structures show that each compound exhibits a unique binding mode.

Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

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Curator/Reporter Aviral Vatsa PhD, MBBS

Based on: A review by (Wink et al., 2011)

This post is in continuation to Part 1 by the same title.

In part one I covered the basics of role of redox chemistry in immune reactions, the phagosome cauldron, and how bacteria bacteria, virus and parasites trigger the complex pathway of NO production and its downstream effects. While we move further in this post, the previous post can be accessed here.

REDOX REGULATION OF IMMUNE FUNCTION

Regulation of the redox immunomodulators—NO/RNS and ROS

In addition to eradicating pathogens, NO/RNS and ROS and their chemical interactions act as effective immunomodulators that regulate many cellular metabolic pathways and tissue repair and proinflammatory pathways. Figure 3 shows these pathways.

Figure 3. Schematic overview of interactive connections between NO and ROS-mediated metabolic pathways. Credit: (Wink et al., 2011)

Regulation of iNOS enzyme activity is critical to NO production. Factors such as the availability of arginine, BH4, NADPH, and superoxide affect iNOS activity and thus NO production. In the absence of arginine and BH4 iNOS becomes a O2_/H2O2 generator (Vásquez-Vivar et al., 1999). Hence metabolic pathways that control arginine and BH4 play a role in determining the NO/superoxide balance. Arginine levels in cells depend on various factors such as type of uptake mechanisms that determine its spatial presence in various compartments and enzymatic systems. As shown in Fig3 Arginine is the sole substrate for iNOS and arginase. Arginase is another key enzyme in immunemodulation. AG is also regulated by NOS and NOX activities. NOHA, a product of NOS, inhibits AG, and O2–increases AG activity. Importantly, high AG activity is associated with elevated ROS and low NO fluxes. NO antagonises NOX2 assembly that in turn leads to reduction in O2_ production. NO also inhibits COX2 activity thus reducing ROS production. Thus, as NO levels decline, oxidative mechanisms increase. Oxidative and nitrosative stress can also decrease intracellular GSH (reduced form) levels, resulting in a reduced antioxidant capability of the cell.

Immune-associated redox pathways regulate other important metabolic cell functions that have the potential for widespread impact on cells, organs, and organisms. These pathways, such as mediated via methionine and polyamines, are critical for DNA stabilization, cell proliferation, and membrane channel activity, all of which are also involved in immune-mediated repair processes.

NO levels dictate the immune signaling pathway

NO/RNS and ROS actively control innate and adaptive immune signaling by participating in induction, maintenance, and/or termination of proinflammatory and anti-inflammatory signaling. As in pathogen eradication, the temporal and spatial concentration profiles of NO are key factors in determining immune-mediated processes.

Brune and coworkers (Messmer et al., 1994) first demonstrated that p53 expression was associated with the concentrations of NO that led to apoptosis in macrophages. Subsequent studies linked NO concentration profiles with expression of other key signaling proteins such as HIF-1α and Akt-P (Ridnour et al., 2008; Thomas et al., 2008). Various levels of NO concentrations trigger different pathways and expectedly this concentration-dependent profile varies with distance from the NO source.NO is highly diffucible and this characteristic can result in 1000 fold reduction in concentration within one cell length distance travelled from the source of production. Time course studies have also shown alteration in effects of same levels of NO over time e.g. NO-mediated ERK-P levels initially increased rapidly on exposure to NO donors and then decreased with continued NO exposure (Thomas et al., 2004), however HIF-1α levels remained high as long as NO levels were elevated. Thus some of the important factors that play critical role in NO effects are: distance from source, NO concentrations, duration of exposure, bioavailability of NO, and production/absence of other redox molecules.

Figure and legend credits: (Wink et al., 2011)

Fig 4: The effect of steady-state flux of NO on signal transduction mechanisms.

This diagram represents the level of sustained NO that is required to activate specific pathways in tumor cells. Similar effects have been seen on endothelial cells. These data were generated by treating tumor or endothelial cells with the NO donor DETANO (NOC-18) for 24 h and then measuring the appropriate outcome measures (for example, p53 activation). Various concentrations of DETANO that correspond to cellular levels of NO are: 40–60 μM DETANO = 50 nM NO; 80–120 μM DETANO = 100 nM NO; 500 μM DETANO = 400 nM NO; and 1 mM DETANO = 1 μM NO. The diagram represents the effect of diffusion of NO with distance from the point source (an activated murine macrophage producing iNOS) in vitro (Petri dish) generating 1 μM NO or more. Thus, reactants or cells located at a specific distance from the point source (i.e., iNOS, represented by star) would be exposed to a level of NO that governs a specific subset of physiological or pathophysiological reactions. The x-axis represents the different zone of NO-mediated events that is experienced at a specific distance from a source iNOS producing >1 μM. Note: Akt activation is regulated by NO at two different sites and by two different concentration levels of NO.

Species-specific NO production

The relationship of NO and immunoregulation has been established on the basis of studies on tumor cell lines or rodent macrophages, which are readily available sources of NO. However in humans the levels of protein expression for NOS enzymes and the immune induction required for such levels of expression are quite different than in rodents (Weinberg, 1998). This difference is most likely due to the human iNOS promotor rather than the activity of iNOS itself. There is a significant mismatch between the promoters of humans and rodents and that is likely to account for the notable differences in the regulation of gene induction between them. The combined data on rodent versus human NO and O2– production strongly suggest that in general, ROS production is a predominant feature of activated human macrophages, neutrophils, and monocytes, and the equivalent murine immune cells generate a combination of O2– and NO and in some cases, favor NO production. These differences may be crucial to understanding how immune responses are regulated in a species-specific manner. This is particularly useful, as pathogen challenges change constantly.

The next post in this series will cover the following topics:

The impact of NO signaling on an innate immune response—classical activation

NO and proinflammatory genes

NO and regulation of anti-inflammatory pathways

NO impact on adaptive immunity—immunosuppression and tissue-restoration response

NO and revascularization

Acute versus chronic inflammatory disease

Bibliography

1. Wink, D. A. et al. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891 (2011).

2. Vásquez-Vivar, J. et al. Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. J. Biol. Chem. 274, 26736–26742 (1999).

3. Messmer, U. K., Ankarcrona, M., Nicotera, P. & Brüne, B. p53 expression in nitric oxide-induced apoptosis. FEBS Lett. 355, 23–26 (1994).

4. Ridnour, L. A. et al. Molecular mechanisms for discrete nitric oxide levels in cancer. Nitric Oxide 19, 73–76 (2008).

5. Thomas, D. D. et al. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic. Biol. Med. 45, 18–31 (2008).

6. Thomas, D. D. et al. Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 101, 8894–8899 (2004).

7. Weinberg, J. B. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol. Med. 4, 557–591 (1998).

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

https://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

https://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

https://pharmaceuticalintelligence.com/?s=Nebivolol

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

https://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

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Curator and Reporter: Aviral Vatsa PhD, MBBS

Based on: A review by Wink et al., 2011

This is the first part of a two part post

Nitric oxide (NO), reactive nitrogen species (RNS) and reactive oxygen species (ROS) perform dual roles as immunotoxins and immunomodulators. An incoming immune signal initiates NO and ROS production both for tackling the pathogens and modulating the downstream immune response via complex signaling pathways. The complexity of these interactions is a reflection of involvement of redox chemistry in biological setting (fig. 1)

Fig 1. Image credit: (Wink et al., 2011)

Previous studies have highlighted the role of NO in immunity. It was shown that macrophages released a substance that had antitumor and antipathogen activity and required arginine for its production (Hibbs et al., 1987, 1988). Hibbs and coworkers further strengthened the connection between immunity and NO by demonstrating that IL2 mediated immune activation increased NO levels in patients and promoted tumor eradication in mice (Hibbs et al., 1992; Yim et al., 1995).

In 1980s a number of authors showed the direct evidence that macrophages made nitrite, nitrates and nitrosamines. It was also shown that NO generated by macrophages could kill leukemia cells (Stuehr and Nathan, 1989). Collectively these studies along with others demonstrated the important role NO plays in immunity and lay the path for further research in understanding the role of redox molecules in immunity.

NO is produced by different forms of nitric oxide synthase (NOS) enzymes such as eNOS (endothelial), iNOS (inducible) and nNOS (neuronal). The constitutive forms of eNOS generally produce NO in short bursts and in calcium dependent manner. The inducible form produces NO for longer durations and is calcium independent. In immunity, iNOS plays a vital role. NO production by iNOS can occur over a wide range of concentrations from as little as nM to as much as µM. This wide range of NO concentrations provide iNOS with a unique flexibility to be functionally effective in various conditions and micro-environements and thus provide different temporal and concentration profiles of NO, that can be highly efficient in dealing with immune challenges.

Redox reactions in immune responses

NO/RNS and ROS are two categories of molecules that bring about immune regulation and ‘killing’ of pathogens. These molecules can perform independently or in combination with each other. NO reacts directly with transition metals in heme or cobalamine, with non-heme iron, or with reactive radicals (Wink and Mitchell, 1998). The last reactivity also imparts it a powerful antioxidant capability. NO can thus act directly as a powerful antioxidant and prevent injury initiated by ROS (Wink et al., 1999). On the other hand, NO does not react directly with thiols or other nucleophiles but requires activation with superoxide to generate RNS. The RNS species then cause nitrosative and oxidative stress (Wink and Mitchell, 1998).

The variety of functions achieved by NO can be understood if one looks at certain chemical concepts. NO and NO2 are lipophilic and thus can migrate through cells, thus widening potential target profiles. ONOO-, a RNS, reacts rapidly with CO2 that shortens its half life to <10 ms. The anionic form and short half life limits its mobility across membranes. When NO levels are higher than superoxide levels, the CO2-OONOintermediate is converted to NO2 and N2O3 and changes the redox profile from an oxidative to a nitrosative microenvironment. The interaction of NO and ROS determines the bioavailability of NO and proximity of RNS generation to superoxide source, thus defining a reaction profile. The ROS also consumes NO to generate NO2 and N2O3 as well as nitrite in certain locations. The combination of these reactions in different micro-environments provides a vast repertoire of reaction profiles for NO/RNS and ROS entities.

The Phagosome ‘cauldron’

The phagosome provides an ‘isolated’ environment for the cell to carry out foreign body ‘destruction’. ROS, NO and RNS interact to bring about redox reactions. The concentration of NO in a phagosome can depend on the kind of NOS in the vicinity and its activity and other localised cellular factors. NO and is metabolites such as nitrites and nitrates along with ROS combine forces to kill pathogens in the acidic environment of the phagosome as depicted in the figure 2 below.

Fig 2. The NO chemistry of the phagosome. (image credit: (Wink et al., 2011)

This diagram depicts the different nitrogen oxide and ROS chemistry that can occur within the phagosome to fight pathogens. The presence of NOX2 in the phagosomes serves two purposes: one is to focus the nitrite accumulation through scavenging mechanisms, and the second provides peroxide as a source of ROS or FA generation. The nitrite (NO2−) formed in the acidic environment provides nitrosative stress with NO/NO2/N2O3. The combined acidic nature and the ability to form multiple RNS and ROS within the acidic environment of the phagosome provide the immune response with multiple chemical options with which it can combat bacteria.

Bacteria

There are various ways in which NO combines forces with other molecules to bring about bacterial killing. Here are few examples

E.coli: It appears to be resistant to individual action of NO/RNS and H2O2 /ROS. However, when combined together, H2O2 plus NO mediate a dramatic, three-log increase in cytotoxicity, as opposed to 50% killing by NO alone or H2O2 alone. This indicates that these bacteria are highly susceptible to their synergistic action.

Staphylococcus: The combined presence of NO and peroxide in staphylococcal infections imparts protective effect. However, when these bacteria are first exposed to peroxide and then to NO there is increased toxicity. Hence a sequential exposure to superoxide/ROS and then NO is a potent tool in eradicating staphylococcal bacteria.

Mycobacterium tuberculosis: These bacterium are sensitive to NO and RNS, but in this case, NO2 is the toxic species. A phagosome microenvironment consisting of ROS combined with acidic nitrite generates NO2/N2O3/NO, which is essential for pathogen eradication by the alveolar macrophage. Overall, NO has a dual function; it participates directly in killing an organism, and/or it disarms a pathway used by that organism to elude other immune responses.

Parasites

Many human parasites have demonstrated the initiation of the immune response via the induction of iNOS, that then leads to expulsion of the parasite. The parasites include Plasmodia(malaria), Leishmania(leishmaniasis), and Toxoplasma(toxoplasmosis). Severe cases of malaria have been related with increased production of NO. High levels of NO production are however protective in these cases as NO was shown to kill the parasites (Rockett et al., 1991; Gyan et al., 1994). Leishmania is an intracellualr parasite that resides in the mamalian macrophages. NO upregulation via iNOS induction is the primary pathway involved in containing its infestation. A critical aspect of NO metabolism is that NOHA inhibits AG activity, thereby limiting the growth of parasites and bacteria including Leishmania, Trypanosoma, Schistosoma, HelicobacterMycobacterium, and Salmonella, and is distinct from the effects of RNS. Toxoplasma gondii is also an intracellular parasite that elicits NO mediated response. INOS knockout mice have shown more severe inflammatory lesions in the CNS that their wild type counterparts, in response to toxoplasma exposure. This indicates the CNS preventative role of iNOS in toxoplasmosis (Silva et al., 2009).

Virus

Viral replication can be checked by increased production of NO by induction of iNOS (HIV-1, coxsackievirus, influenza A and B, rhino virus, CMV, vaccinia virus, ectromelia virus, human herpesvirus-1, and human parainfluenza virus type 3) (Xu et al., 2006). NO can reduce viral load, reduce latency and reduce viral replication. One of the main mechanisms as to how NO participates in viral eradication involves the nitrosation of critical cysteines within key proteins required for viral infection, transcription, and maturation stages. For example, viral proteases or even the host caspases that contain cysteines in their active site are involved in the maturation of the virus. The nitrosative stress environment produced by iNOS may serve to protect against some viruses by inhibiting viral infectivity, replication, and maturation.

To be continued in part 2 …

Bibliography

Gyan, B., Troye-Blomberg, M., Perlmann, P., Björkman, A., 1994. Human monocytes cultured with and without interferon-gamma inhibit Plasmodium falciparum parasite growth in vitro via secretion of reactive nitrogen intermediates. Parasite Immunol. 16, 371–3

Hibbs, J.B., Jr, Taintor, R.R., Vavrin, Z., 1987. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235, 473–476.

Hibbs, J.B., Jr, Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1988. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157, 87–94.

Hibbs, J.B., Jr, Westenfelder, C., Taintor, R., Vavrin, Z., Kablitz, C., Baranowski, R.L., Ward, J.H., Menlove, R.L., McMurry, M.P., Kushner, J.P., 1992. Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleu

Rockett, K.A., Awburn, M.M., Cowden, W.B., Clark, I.A., 1991. Killing of Plasmodium falciparum in vitro by nitric oxide derivatives. Infect Immun 59, 3280–3283.

Stuehr, D.J., Nathan, C.F., 1989. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169, 1543–1555.

Wink, D.A., Hines, H.B., Cheng, R.Y.S., Switzer, C.H., Flores-Santana, W., Vitek, M.P., Ridnour, L.A., Colton, C.A., 2011. Nitric oxide and redox mechanisms in the immune response. J Leukoc Biol 89, 873–891.

Wink, D.A., Mitchell, J.B., 1998. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25, 434–456.

Wink, D.A., Vodovotz, Y., Grisham, M.B., DeGraff, W., Cook, J.C., Pacelli, R., Krishna, M., Mitchell, J.B., 1999. Antioxidant effects of nitric oxide. Meth. Enzymol. 301, 413–424.

Xu, W., Zheng, S., Dweik, R.A., Erzurum, S.C., 2006. Role of epithelial nitric oxide in airway viral infection. Free Radic. Biol. Med. 41, 19–28.

Yim, C.Y., McGregor, J.R., Kwon, O.D., Bastian, N.R., Rees, M., Mori, M., Hibbs, J.B., Jr, Samlowski, W.E., 1995. Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor. J. Immunol. 155, 4382–4390.

Further reading on NO:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

Author: Aviral Vatsa, PhD, September 23, 2012

https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

https://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

https://pharmaceuticalintelligence.com/?s=Nebivolol

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

https://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

 

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Author and Curator: Ritu Saxena, Ph.D.

 

Introduction

Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al

NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.

Regulation by Nitric oxide

NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.

The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.

Role of Nitric oxide in Cancer

One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.

NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.

S-nitrosylation and Cancer

The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.

Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.

NO mediated aberrant proteins in Cancer

Bcl2

Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.

p53

p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.

HIF-1a

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.

PTEN

Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.

C-Src

C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.

Reference:

Muntané J and la Mata MD. Nitric oxide and cancer. World J Hepatol. 2010 Sep 27;2(9):337-44. http://www.ncbi.nlm.nih.gov/pubmed/21161018

Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9. http://www.ncbi.nlm.nih.gov/pubmed/22425962

Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.http://www.ncbi.nlm.nih.gov/pubmed/20167555

Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34. http://www.ncbi.nlm.nih.gov/pubmed/11518674

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Author and Reporter: Meg Baker, Ph.D., Registered Patent Agent

The 1998 Noble Prize for medicine was for the discovery that nitric oxide (NO) was the chemical messenger responsible for relaxing vascular tissue and thereby increasing blood flow and reducing blood pressure. Alfred Noble himself had been prescribed nitro-glycerin for heart problems over 100 years before, a compound which is metabolized to NO.

NO, a gas at room temperature, has an exceedingly short half-life in the body. Normally, NO is produced from an amino acid, L-arginine (L-Arg), a normal component of the dietary protein, and molecular oxygen (O2) by the one of the several Nitric Oxide Synthases (EC 1.14.13.39): endothelial (eNOS, NOS III), inducible (iNOS, NOS II), and neural (nNOS, NOS I). In human studies, supplementation with l-arginine improved endothelium-dependent vasodilation.

The reaction of iNOS with L-Arg to produce NO leaves another amino acid, citrulline. Excess L-Arg can also be degraded by arginase (enzyme having two isoforms, I and II) which may be coinduced with iNOS in some cell types.

Citrulline formed as a by-product of the NOS reaction can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL).

Mori (2007)  http:// www.ncbi.nlm.nih.gov/ pubmed/ 17513437 found that AS and sometimes AL are coinduced with inducible NOS (iNOS) in various cells. In these cells, NO was synthesized from citrulline (via arginine) as well as from arginine, indicating operation of the citrulline-NO cycle.

Whereas, low concentrations of NO protect cells from apoptosis, excessive NO causes apoptosis. NO causes endoplasmic reticulum (ER) stress, induces a transcription factor, CAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), and leads to apoptosis.

The active site of NOS is formed by a heme-containing substrate-binding cavity, where L-arginine (Arg) and O2 are converted to L-citrulline and NO. The electrons required for reductive O2 activation are transferred from NADPH via the NOS-bound flavins (riboflavin, Vitamin B2) FMN and FAD. All NOS isoforms are only active as homodimers.

Generation of NO occurs in two discrete O2-requiring steps, with intermediate formation of N-hydroxy-L-arginine (NHA or NOHLA). NHA formation consumes one molecule of O2 and two electrons. Conversion of NHA to L-citrulline and NO requires another molecule of O2 and one more electron (http://en.wikipedia.org/wiki/Nitric_oxide_synthase).  The overall stoichiometry, reflecting the three electrons derived from NADPH, that pass through the flavin co-factors and are transferred one by one via the heme iron,  is then:

L-arginine + 3/2 NADPH + H+ + 2 O2 = citrulline + nitric oxide + 3/2 NADP+

Another factor affecting NOS activity is the availability of essential co-factors such as tetrahydrobiopterin (BH4) (Boeger et al. Cardiovasc Res (2003) 59 (4): 824-833 http://cardiovascres.oxfordjournals.org/content/59/4/824.full, Vasquez-Vivar J., et al . Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl. Acad. Sci. USA 1998;95:9220-9225 http://www.pnas.org/content/95/16/9220.full). H4-biopterin binds in the immediate vicinity of the heme at the dimer interface, interacting with residues from both subunits. When BH4 availability is limiting, electron transfer from NOS flavins becomes “uncoupled” from l-arginine oxidation and the ferrous-dioxygen complex formed as an intermediate in the reaction sequence, dissociates and superoxide(O2−·) is produced.

See Figure 1 in Werner et al.  2003 Exp Biol  Med 228: 1291-1302.

RADICALS

The conversion of Arg to NHA and of NHA to L-citrulline and NO both depend on the presence of H4-biopterin. In the absence of substrate or pterin, NADPH oxidation by NOS is accompanied by formation of O2 and peroxide (H2O2). Uncoupled eNOS is assumed to produce superoxide (O2−·) in addition to or instead of NO (·NO) which will react with itself, with NO, or with -hydroxyl, -sulfhydryl, or or side groups of proteins, lipids, or glycans. Reaction of ·NO produced by eNOS, with O2−· produced by eNOS or by other enzymes, such as NADPH and xanthine oxidases, decreases the amount of ·NO available to stimulate vascular relaxation. At the very low BH4 concentration of 100 nmol/L, recombinant human eNOS activity is fully developed. However, biopterin is formed from the pterin heterocycle also present in folic acid (Vitamin B9,
pteroyl-L-glutamate)
and which is synthesized from GTP. Human GTP cyclohydrolase I (GTPCH), is the rate-limiting enzyme in BH4 synthesis (Crabtree et al. JBC 2008, http://www.jbc.org/content/284/2/1136.full).

In addition to the NOS reaction, which generates a H3-biopterin radical cation, a neutral H3-biopterin radical is formed when H4-biopterin reacts with various radicals and which can be reduced back to H4-biopterin by ascorbate (Vitamin C). Folate species are also required to synthesize pyrimidines and purines (for DNA synthesis and repair and NADH and NADPH).

Enhancing NO Synthesis

The normal way to increase vascular nitric oxide is through vascular stress, such as exercise. As oxygen demand increases, cardiac output increases and the endothelial lining of the arteries releases nitric oxide into the blood, which, in turn, relaxes and widens the vessel wall, allowing for enhanced blood flow.

Enhancing the presence of L-Arg or the one or more of the NOS enzymes are obviously essential for NO production. However, NOS enzymes are co-valently bound to heme (heme, iron), and flavin co-factors (Vit B2), and require soluble co-factors NADPH (a dinucleotide phosphate, containing niacin, Vitamin B3), and BH4 (from Vit B9).

Foods high in Arginine and Citrulline include melons and cucumber, peanuts, salmon, and soy. Arginine is found in varying degrees (3-15% by weight) in all animal proteins. Blue-fin tuna has 1.8 g of arginine per 100 g so 2 oz. of tuna will provide about 1 g of arginine. Other sources of 1 g of L-Arg: 2.7 oz. of chicken thighs, about 4 oz. of chicken breast, 2 oz. of 75 percent lean hamburger or about 2.5 oz. of pork.

Foods rich in antioxidants and polyphenols will provide protection against free radical assault on proteins and, in particular, act to protect the NOS enzyme and cofactors. Almost all fruit and vegetables such as blueberries, cranberries, carrots, grapefruit, soybeans, apples, and spinach contain high levels of antioxidants. In addition, nuts, tea, seeds, dark chocolate, red wine, and seafood generally contain antioxidants such as resveratrol, ascorbate, and other phytochemicals. Other free radical scavengers, tocopherols (alpha-tocopherol, Vit E) work predominantly in the lipid environment such as in cell membranes, while the sulfur-containing soluble molecule, glutathione (GSH) protects the cytosolic milieu.

Supplements

Both L-Arg or L-citrulline can be purchased over the counter. Dietary L-arginine will be taken up by the intestine and transported directly to the liver by the hepatic artery as are most of the products of digestion. Much of this L-Arg will be used in metabolic steps related to the urea cycle which is co-ordinated with the kidney to rid the body of excess nitrogen and prevent ammonia concentration from building. A small amount will enter the blood stream and be used for NO synthesis.

Proargi-9 Plus® is one product being sold containing mutltigram doses of L-Arg plus L-Citrulline in combination with anti-oxidants and folate. Proargi-9 Plus® is a registered trademark and copyright of Nature’s Sunshine Products, Inc. L-arginine Plus™ is formulation with similar ingredients and stated amounts of L-Arg and L-Citrulline and is not affiliated with the makers of Proargi-9-Plus. Niteworks® is a registered trademark and copyright of Herbalife International, Inc. and is not affiliated with or a sponsor of L-arginine Plus™.

Dr. Joe Prendergast is an endocrinologist using L-Arg therapy who, over 19 years, never had to admit any of his 7200 diabetes patients to the hospital for peripheral artery disease, recommends supplemental L-Arg formulations to his patients. The combination of L-Arg with L-citrulline a longer acting NO forming product. http://www.livingwithoutdisease.com/?route=references/prendergast

Supplements of L-Arg and, in particular, in combination with L-citrulline other B-vitamins and antioxidents may be an effective way to boost vascular NO synthesis for anyone not exercising or eating a balanced diet, having a deficiency in any of the L-Arg recycling enzymes, NOS enzymes, co-factor recycling or synthetic enzymes, or other risk factor. Specific risk factors, such as inherently elevated levels of the natural NOS inhibitor ADMA (asymmetric-dimethyl-L-arginine) are beginning to be uncovered and will be the subject of another post.

 Additional References

Nitric Oxide: Biology and Pathobiology,  LJ Ignarro Editor, Sep 13, 2000 http://books.google.com/books?id=h5FugARr4bgC&dq=pterin+ring&source=gbs_navlinks_s

Mori, M. Regulation of nitric oxide synthesis and apoptosis by arginase and arginine recycling.  J Nutr. 2007 Jun;137(6 Suppl 2):1616S-1620S.   http://www.ncbi.nlm.nih.gov/pubmed/17513437

Werner, et al.  Tetrahydrobiopterin and Nitric Oxide: Mechanistic and Pharmacological Aspects Exp Biol Med December 2003 vol. 228 no. 11 1291-1302  Werner et al. Exp Biol Med 2003

Davel AP, Wenceslau CF, Akamine EH, Xavier FE, Couto GK, Oliveira HT, Rossoni LV. Endothelial dysfunction in cardiovascular and endocrine-metabolic diseases: an update.  Braz J Med Biol Res. 2011 Sep;44(9):920-32. Epub 2011 Aug 19. Davel et al. Braz J Med Biol Res 2011

Rainer H Boeger. Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism   Schwedhelm E, et al. Br J Clin Pharmacol. 2008_65_51-9

Louise Ignarro, UCLA, Nobel Prize Recipient, Author “NO More Heart Disease”

John Cook, Peripheral artery disease study, Author “Cardiovascular Cure”

Other aspects of Nitric Oxide involvement in biological systems in humans are covered in the following posts on this site:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

 

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

 

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

 

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

 

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

 

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

 

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

https://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

 

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

 

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

 

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

AuthorL Aviral Vatsa, PhD, September 23, 2012

https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

https://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

 

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

https://pharmaceuticalintelligence.com/?s=Nebivolol

 

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

 

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Curator: Aviva Lev-Ari, 10/4/2012.

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

 

 

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Curator: Aviral Vatsa, PhD, MBBS

Systemic sclerosis (SSc) is a type of autoimmune disease when the body’s immune system attacks and destroys body’s healthy tissue. It is characterised by lesions in the vessels and accumulation of collagen in the tissues. Although the pathogenesis of this disease is not clear, but one of the suggestions is that the endothelium fails to produce NO upon cold stimulation. Physiologically, NO acts as a vasodilator and its deficiency has been implicated in diseases such as hypertension and atherosclerosis.

In the body NO is generated when L-arginine is converted to L-citruline in the presence of NO synthase (NOS) enzyme, molecular oxygen, NADPH, and other cofactors. Principally, three isoenzymes of NOS are present in the body to catalyse the production of NO in various anatomic locations and under various physiological conditions. Three distinct genes encode for the three types of NOS i.e. endothelial (eNOS or NOS-3), neuronal (nNOS or NOS-1), and inducible (iNOS or NOS-2) NOS.

The inducible type 2 NOS (iNOS) may act as an immunoregulator. Several reports have provided evidence for the existence of a NO pathway in human mononuclear cells.

It is not well established if NO production increases or decreases in SSc patients. In one study by Allanore et al, NO production was shown to be reduced in plasma and PBMC supernatants, and iNOS synthesis in PBMCs.

The authors of this study investigated NO metabolites in plasma and PBMC supernatants, and iNOS synthesis in PBMC to see if the level of NO production by peripheral blood mononuclear cells (PBMC) was low in SSc, as this might contribute to the vasodilatory abnormalities observed in this disease.

Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica.The NO metabolites nitrite and nitrates were determined by flurometeric and spectrometeric assays respectively. iNOS expression was determined by using monoclonal anti‐NOS2 antibody and FACS analyses.

The data suggested a decrease in plasma NO concentration and iNOS production in PBMC in patients with systemic sclerosis as compared with patients with rheumatoid arthritis and sciatica. Subgroup analysis showed no difference between limited and diffuse SSc forms. Total plasma nitrite concentrations in five healthy volunteers were similar to those in patients with sciatica, which is consistent with this group being an appropriate control group.

Thus the authors suggest that low NO production in Ssc patients might be involved in the tendency towards vasospam.

However in other studies authors have shown an increase in NO production in SSc patients. Takagi et al set out to investigate this discrepancy in NO production in SSc patients. They sought to determine whether increased NO levels are associated with various clinical subsets of SSc patients, and to assess the contribution of fibroblasts in skin lesions to NO synthesis.

In this study Takagi et al measured the levels of serum NO metabolites in SSc patients and determined the contribution of the excessive production of NO synthase (NOS)-2 by skin fibroblasts to NO synthesis. Serum NO levels of 45 patients with SSc were significantly higher than those of 20 healthy volunteers. In addition, some clinical features of SSc (the extent of skin fibrosis, short disease duration, and the complication of active fibrosing alveolitis) were all correlated positively with the levels of NO metabolites in SSc patients. RT PCR was used to determine NOS-2 mRNA expression levels in cultured fibroblasts derived from SSc patients.

The authors showed that serum NO levels were significantly elevated in patients with SSc as compared to healthy normal controls. They also demonstrated that NOS-2 was produced spontaneously by cultured SSc fibroblasts, suggesting that increased serum NO levels might reflect in part the elevated expression of NOS-2 by fibroblasts derived from SSc patients.

The discrepancy in NO production could be explained by disease stage, severity of tissue fibrosis and various circumstances of endothelial damage. Takagi et al found increased NO production in early stages of SSc with tissue fibrosis and not in later stages of the disease. Hence they suggest that NO levels may be a sensitive marker of the early stages of the development of severe tissue fibrosis in SSc patients, although a longitudinal and prospective study is needed to confirm this.

NO is known to have dual functions in the body, both beneficial and cyototoxic. Generally it depends upon the concentration and the duration of NO production. Similarily in SSC, the dual functions of NO seem to be both beneficial (as a vasodilator) and harmful (as a cytotoxic effector) in regard to the clinical manifestations of SSc. One of the limitations of these studies is that they did not investigate the absolute concentrations of NO production but only its metaoblites were measured. This might be due the fact that NO is a short-lived molecule (half-life < 5 s) and it is challenging to quantify NO production at single cell level. Such techniques (e.g.DAR 4M AM flurophore) have been developed but are challenging to apply to various experimental set ups. DAR 4M AM has been used to quantify NO production online in single cells. In SSc determination of absolute NO concentrations at cellular or tissue level at various stages of the disease process will go a long way in solving the discrepancy of NO production in SSc patients.

Sources

Low levels of nitric oxide (NO) in systemic sclerosis: inducible NO synthase production is decreased in cultured peripheral blood monocyte/macrophage cells. Y. Allanore, D. Borderie, P. Hilliquin, A. Hernvann, M. Levacher, H. Lemaréchal, O. G. Ekindjian, and A. Kahan. (2001) 40(10): 1089-1096 doi:10.1093/rheumatology/40.10.1089.

Serum nitric oxide (NO) levels in systemic sclerosis patients: correlation between NO levels and clinical features. K. Takagi, Y. Kawaguchi, M. Hara, T. Sugiura, M. Harigai, N. Kamatani . Article first published online: 24 NOV 2003. DOI: 10.1111/j.1365-2249.2003.02320.x Clinical & Experimental Immunology: Volume 134, Issue 3, pages 538–544, December 2003

Extracellular NO signalling from a mechanically stimulated osteocyte. Aviral Vatsa, Theo H. Smit, Jenneke Klein-Nulend. Journal of biomechanics 1 January 2007 (volume 40 issue Pages S89-S95 DOI: 10.1016/j.jbiomech.2007.02.015)

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Bystolic’s generic Nebivolol – Positive Effect on circulating Endothelial  Progenitor Cells Endogenous Augmentation

Curator: Aviva Lev-Ari, PhD, RN

 

Bystolic’s generic Nebivolol – FDA approved for Treatment of Hypertension since 2008 – Pharmacological agent hypothesized to have positive effect on circulating Endothelial  Progenitor Cells (cEPCs) endogenous augmentation: Low number of cEPCs found to be associated with high Macrovascular Risk Events

Induction of NO Production and Stimulation of eNOS

Mechanism of Action (MOA) for Nitric Oxide (NO) and endothelial Nitric Oxide Syntase (eNOS) are described in George T. and P. Ramwell, (2004). Nitric Oxide, Donors, & Inhibitors. Chapter 19 in Katzung, BG., Basic & Clinical Pharmacology. McGraw-Hill, 9th Edition, pp. 313 – 318

http://books.google.com/books/about/Basic_and_Clinical_Pharmacology.html?id=4O7ghcthkt4C

Nitric oxide (NO) is a relative newcomer to pharmacology, as the paper which initiated the field was published only 25 years ago. In 2006, it is known that Arginine-vasopressin (AVP) is a hormone that is essential for both osmotic and cardiovascular homeostasis and exerts physiological regulation through three receptors, It causes a decrease in BP which occurs through mediated release of NO from the vascular endothelium (Koshimizu et al., 2006).

Dr. S. H. Snyder of Johns Hopkins University has established gases as a new class of neurotransmitters, beginning with his demonstrating the role of nitric oxide in mediating glutamate synaptic transmission and neurotoxicity. His isolation and molecular cloning of nitric oxide synthase led to major insights into the neurotransmitter functions of nitric oxide throughout the body. http://nrc88.nas.edu/pnas_search/memberDetails.aspx?ctID=50282

http://www.pnas.org/content/108/46/E1137.abstract

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3219156/

http://www.drproctor.com/O2NOpnas.htm

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability

http://www.pnas.org/content/98/5/2604.short

Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298

http://www.pnas.org/content/97/6/2832.short

Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase

http://www.pnas.org/content/95/15/8880.short

Superoxide generation by endothelial nitric oxide synthase: The influence of cofactors

NO impact is such that to date more than 31,000 papers have been published with NO in the title and more than 65,000 refer to it in some way. The identification of NO with endothelium-derived relaxing factor and the discovery of its synthesis from L-arginine led to the realization that the L-arginine: NO pathway is widespread and plays a variety of physiological roles. These include the maintenance of vascular tone, neurotransmitter function in both the central and peripheral nervous systems, and mediation of cellular defense. In addition, NO interacts with mitochondrial systems to regulate cell respiration and to augment the generation of reactive oxygen species, thus triggering mechanisms of cell survival or death.

Review of the role of NO in the cardiovascular system found, that in addition to maintaining a vasodilator tone, it inhibits platelet aggregation and adhesion and modulates smooth muscle cell proliferation. NO has been implicated in a number of cardiovascular diseases and virtually every risk factor for these appears to be associated with a reduction in endothelial generation of NO. Reduced basal NO synthesis or action leads to vasoconstriction, elevated blood pressure and thrombus formation. By contrast, overproduction of NO leads to vasodilatation, hypotension, vascular leakage, and disruption of cell metabolism. Appropriate pharmacological or molecular biological manipulation of the generation of NO will doubtless prove beneficial in such conditions (Moncada and Higgs, 2006).

http://onlinelibrary.wiley.com/doi/10.1038/sj.bjp.0706458/full

Evidence of HDL Modulation of eNOS in Humans

 Whereas the functional link between HDL and eNOS has been appreciated only recently, the relationship between HDL and endothelium-dependent vasodilation has been known for some time. In studies of coronary vasomotor responses to acetylcholine, it was noted in 1994 that patients with elevated HDL have greater vasodilator and attenuated vasoconstrictor responses (Zeiher et al., 1994).

Circulation, 89:2525–2532.

Studies of flow-mediated vasodilation of the brachial artery have also shown that HDL cholesterol is an independent predictor of endothelial function (Li et al., 2000).

Int. J. Cardiol., 73:231–236

The direct, short-term impact of HDL on endothelial function also has recently been investigated in humans. One particularly elegant study recently evaluated forearm blood flow responses in individuals who are heterozygous for a loss-of-function mutation in the ATP-binding cassette transporter 1 (ABCA1) gene. Compared with controls, ABCA1 heterozygotes (six men and three women) had HDL levels that were decreased by 60%, their blood flow responses to endothelium-dependent vasodilators were blunted, and endothelium-independent responses were unaltered. After a 4-hour infusion of apoAI/phosphatidylcholine disks, their HDL level increased threefold and endothelium-dependent vasomotor responses were fully restored (Bisoendial et al., 2003). It has also been observed that endothelial function is normalized in hypercholesterolemic men with normal HDL levels shortly following the administration of apoAI/phosphatidylcholine particles (Spieker et al., 2002).

Circulation, 105:1399–1402.

Thus, evidence is now accumulating that HDL is a robust positive modulator of endothelial NO production in humans (Shaul & Mineo, 2004).

J Clin Invest., 15; 113(4): 509–513.

HDL is more than an eNOS Agonist

 In addition to the modulation of NO production by signaling events that rapidly dictate the level of enzymatic activity, important control of eNOS involves changes in the abundance of the enzyme. In a clinical trial by the Karas laboratory of niacin therapy in patients with low HDL levels (nine males and two females), flow-mediated dilation of the brachial artery was improved in association with a rise in HDL of 33% over 3 months (Kuvin et al., 2002).

Am. Heart J., 144:165–172.

They also demonstrated that eNOS expression in cultured human endothelial cells is increased by HDL exposure for 24 hours. They further showed that the increase in eNOS is related to an increase in the half-life of the protein, and that this is mediated by PI3K–Akt kinase and MAPK (Ramet et al., 2003).

J. Am. Coll. Cardiol., 41:2288–2297.

Thus, the same mechanisms that underlie the acute activation of eNOS by HDL appear to be operative in upregulating the expression of the enzyme.

The current understanding of the mechanism by which HDL enhances endothelial NO production is summarized in Shaul & Mineo (2004), Figure 1.

J Clin Invest., 15; 113(4): 509–513.

It describes the mechanism of action for HDL enhancement of NO production by eNOS in vascular endothelium.

(a)   HDL causes membrane-initiated signaling, which stimulates eNOS activity. The eNOS protein is localized in cholesterol-enriched (orange circles) plasma membrane caveolae as a result of the myristoylation and palmitoylation of the protein. Binding of HDL to SR-BI via apoAI causes rapid activation of the nonreceptor tyrosine kinase src, leading to PI3K activation and downstream activation of Akt kinase and MAPK. Akt enhances eNOS activity by phosphorylation, and independent MAPK-mediated processes are additionally required (Duarte, et al., 1997). .Eur J Pharmacol, 338:25–33. HDL also causes an increase in intracellular Ca2+ concentration (intracellular Ca2+ store shown in blue; Ca2+ channel shown in pink), which enhances binding of calmodulin (CM) to eNOS. HDL-induced signaling is mediated at least partially by the HDL-associated lysophospholipids SPC, S1P, and LSF acting through the G protein–coupled lysophospholipid receptor S1P3. HDL-associated estradiol (E2) may also activate signaling by binding to plasma membrane–associated estrogen receptors (ERs), which are also G protein coupled. It remains to be determined if signaling events are also directly mediated by SR-BI (Yuhanna et al., 2001), (Nofer et al., 2004), (Gong et al., 2003), (Mineo et al., 2003).

Nat. Med., 7:853–857.

J. Clin. Invest.,113:569–581.

J. Clin. Invest., 111:1579–1587.

J. Biol. Chem., 278:9142–9149.

(b)   HDL regulates eNOS abundance and subcellular distribution. In addition to modulating the acute response, the activation of the PI3K–Akt kinase pathway and MAPK by HDL upregulates eNOS expression (open arrows). HDL also regulates the lipid environment in caveolae (dashed arrows). Oxidized LDL (OxLDL) can serve as a cholesterol acceptor (orange circles), thereby disrupting caveolae and eNOS function. However, in the presence of OxLDL, HDL maintains the total cholesterol content of caveolae by the provision of cholesterol ester (blue circles), resulting in preservation of the eNOS signaling module (Ramet et al., 2003), (Blair et al., 1999), (Uittenbogaard et al., 2000).

J. Am. Coll. Cardiol., 41:2288–2297.

J. Biol. Chem., 274:32512–32519.

J. Biol. Chem., 275:11278–11283.

Source for HDL-eNOS Figure: Shaul & Mineo (2004).

 

HDL enhances NO production by eNOS in vascular endothelium.

Nebivolol:  DRUG RESEARCH & CLINICAL TRIALS

Agent selection: Nebivolol

Rationale:            Patient’s pharmacological beneficial effects derived from usage of Nebivolol include the following but are not limited to this list

  •       Vasodilatory actions (Mukherjee et al., 2004).
  •      Inhibition of NADPH oxidase activity in inflammatory cells (Mollnau et al., 2003),
  •       Increase in arterial distensibility (McEniery et al., 2004)
  •       Reduction in nitroxidative stress and restores nitric oxide bioavailability in endothelium (Mason et al., 2005)
  •       Stimulation of nitric oxide release from endothelial cells through ATP efflux: a novel mechanism for antihypertensive   action (Kalinowski et al., 2003)
  •       {beta}-Adrenergic Receptor Stimulation and Nitric Oxide Release on Tissue Perfusion and Metabolism (Jordan et al., 2001)
  •       Correction of impaired adrenergic vasorelaxation in hypertension in use in conjunction to gene therapy implantation in the endothelium (Iaccarino, et al., 2002)
  •       Vasorelaxation of Coronary Microvessels (Dessy et al., 2005)
  •       Exploratory treatment for the Brugada syndrome, a disease caused by increased electrical heterogeneity between the right ventricular endo- and epicardium. The degree of electrical heterogeneity may be greater in the free wall in some patients, the outflow tract in others, or even in the inferior wall. The ST-segment elevation may then be recorded at the normal precordial position of V1–V3 in the first situation, at one or two intercostal spaces higher in the second, and in the inferior leads II, III and a VF in the third situation, representing a variant of the Brugada syndrome (Brugada et al., 2001).
  •       Endothelial ß2-Adrenergic Receptor–Mediated Nitric Oxide Production, two actions in one therapeutic agent for populations with prevalent polypharmacy due to multiple co-morbidities (Broeders et al., 2000).

The rationale for Agent selection supports the hypothesis that Nebovolol would have positive effect on cEPCs endogenous augmentation. It was a solution sought for the observations made be Werner in 2003 and in 2005 that Low number of cEPCs found in patient blood is statistically associated with high incidence of Macrovascular Risk Events.

 Nebivolol is a long-acting, cardioselective beta-blocker currently licensed for the treatment of hypertension. It has mild vasodilating properties attributed to its interaction with the L-arginine/nitric oxide pathway, a property not shared by other beta-blockers. To date this has been demonstrated in volunteers and small numbers of patients. If this mechanism is shown to result in improved clinical outcomes, nebivolol could be of value in managing hypertensive patients with endothelial dysfunction e.g., those with diabetes mellitus or hypercholesterolaemia and in patients with ischemic heart disease. It is an effective antihypertensive agent. Short-term (up to 12 weeks), published clinical studies in patients with mild-to-moderate essential hypertension have shown that it lowers sitting systolic and diastolic blood pressure to a similar extent as standard therapies – atenolol, metoprolol, enalapril, lisinopril, nifedipine and hydrochlorothiazide. One open non-comparative study showed that a significant reduction in BP is maintained over 1 year. It is well-tolerated; the frequency and severity of adverse events is similar to that reported for placebo, atenolol or enalapril in published studies. In the largest comparative study the numbers of patients complaining of fatigue was smaller for nebivolol compared with atenolol, although the numbers in both groups were too small for any meaningful comparisons to be made. In addition, in single comparative studies with nifedipine or metoprolol, the overall incidence of adverse events was smaller in the nebivolol groups. Although uncontrolled heart failure is listed as a contra-indication in the SPC, preliminary studies have shown that nebivolol has beneficial effects on left ventricular function in patients with hypertension and heart failure.

Nebivolol is considerably more expensive than atenolol, but costs less than carvedilol or celiprolol

How does it work?

Nebivolol belongs to a group of medicines called beta-blockers, which block beta receptors in the heart, lungs and other organs of the body. Blocking these receptors prevents the action of two chemicals called noradrenaline and adrenaline that occur naturally in the body. These are often referred to as the ‘fight or flight’ chemicals as they are responsible for the body’s reaction to stressful situations.

Blocking the beta receptors in the heart causes the heart to beat more slowly and with less force. This means that the pressure at which blood is pumped out of the heart to the rest of the body is reduced. This medicine also widens the blood vessels. These are two of the ways in which nebivolol helps to reduce blood pressure, however the whole mechanism is not fully understood.

What is it used for?

  •       High blood pressure (hypertension)

In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial ß2-adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca2+]i and endothelial NO synthase–dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans. Nebivolol is a ß1-selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties in humans. In this study, they explored whether nebivolol indeed induces NO production and, if so, by what mechanism. They hypothesized that not nebivolol itself but rather its metabolites augment NO production (Broeders et al., 2000).

Circulation, 102:677.

http://www.dailymedplus.com/monograph/view/setid/673f5ad2-c09b-4a89-9407-efdadd007917

Relation between Beta-adrenoceptor Stimulation and Nitric Oxide Synthesis in Vascular Control.

This commentary reviews recent evidence that implicates nitric oxide (NO) as a mediator of beta(2)-adrenoceptor (beta(2)-AR)-initiated vasodilatation. Emphasis is placed on the following: 1) in vivo studies that demonstrate potential physiological importance, 2) mechanistic studies performed in vitro in human umbilical vein endothelial cells (HUVEC), 3) effects of beta(2) agonists on arterial pulse wave reflection, and 4) therapeutic opportunities offered by the combination of beta(2) agonist action with selective beta(1) antagonism. Vascular beta(2)-AR-initiated mechanisms provide a physiologically important control mechanism during exercise. Activation of beta(2)-AR in HUVEC leads to vasodilatation that is partly NO-mediated via activation of protein kinase A (PKA) and of phosphatidylinositol-3 kinase (PI3K)/Akt pathways, leading to serine phosphorylation of the endothelial NO synthase (eNOS). In vivo, beta(2)-AR activation limits the rise in blood pressure during exercise and reduces arterial pulse wave reflection. Nebivolol is a selective beta(1)-AR antagonist with vasodilator actions operating through these pathways, offering novel therapeutic opportunities.

Ritter JM, Ferro A, Chowienczyk PJ., (2006). Relation between beta-adrenoceptor stimulation and nitric oxide synthesis in vascular control.

Eur J Clin Pharmacol., 62 (Supplement 13):109-113. 

eNOS is not Activated by Nebivolol in Human Failing Myocardium.

Nebivolol is a highly selective beta(1)-adrenoceptor blocker with additional vasodilatory properties, which may be due to an endothelial-dependent beta(3)-adrenergic activation of the endothelial nitric oxide synthase (eNOS). beta(3)-adrenergic eNOS activation has been described in human myocardium and is increased in human heart failure. Therefore, this study investigated whether nebivolol may induce an eNOS activation in cardiac tissue. Immunohistochemical stainings were performed using specific antibodies against eNOS translocation and eNOS serine(1177) phosphorylation in rat isolated cardiomyocytes, human right atrial tissue (coronary bypass-operation), left ventricular non-failing (donor hearts) and failing myocardium after application of the beta-adrenoceptor blockers nebivolol, metoprolol and carvedilol, as well as after application of BRL 37344, a specific beta(3)-adrenoceptor agonist. BRL 37344 (10 muM) significantly increased eNOS activity in all investigated tissues (either via translocation or phosphorylation or both). None of the beta-blockers (each 10 muM), including nebivolol, increased either translocation or phosphorylation in any of the investigated tissues. In human failing myocardium, nebivolol (10 muM) decreased eNOS activity. In conclusion, nebivolol shows a tissue-specific eNOS activation. Nebivolol does not activate the endothelial eNOS in end-stage human heart failure and may thus reduce inhibitory effects of NO on myocardial contractility and on oxidative stress formation. This mode of action may be of advantage when treating heart failure patients.

Brixius K, Song Q, Malick A, Boelck B, Addicks K, Bloch W, Mehlhorn U, Schwinger R, (2006). eNOS is not activated by nebivolol in human failing myocardium.

Life Sci. 2006 Apr 25

A Dose-response Trial of Nebivolol in Essential Hypertension.

Report by International Clinical R&D, Janssen Research Foundation, Beerse, Belgium.

A double-blind placebo-controlled dose-response trial of nebivolol, a cardioselective beta-blocking drug which also induces endothelium-dependent dilatation via nitric oxide, has been performed. Nebivolol reduced blood pressure (BP) in a dose dependent way, and was shown to be effective given once daily, without appreciable differences between peak and trough drug levels. There was no postural component to the BP fall. There was no clear inferiority of efficacy in black patients. A single daily dose of 5 mg was appropriate, with no evident advantage at 10 mg. The drug was well tolerated, even at 10 mg daily. BP control was achieved largely in the absence of typical side effects of beta-blockade. The combination of properties of nebivolol renders it an attractive addition to the antihypertensive repertoire.

Van Nueten L, Dupont AG, Vertommen C, Goyvaerts H, Robertson JI., (1997). A dose-response trial of nebivolol in essential hypertension.

J Hum Hypertens., 11(2):139-44.

Other eNOS Agonists – Exploration of Different Aspects related to eNOS Mechanism of Action

ACEI and NO stimulation

Carboxypeptidase cleavage of the C-terminal Arg of kinins generates specific agonists of the B1 receptor. Activation of B1 receptors produces nitric oxide via eNOS in bovine endothelial cells and iNOS in cytokine-stimulated human endothelial cells. Angiotensin-converting enzyme (ACE) inhibitors are direct agonists of B1 receptors in endothelial cells, although they release NO via a different signaling pathway than peptide ligands in bovine cells. This brief review discusses carboxypeptidase M as a required processing enzyme for generating B1 agonists, how ACE inhibitors and peptide ligands stimulate NO production and the evidence for, as well as some consequences of, the direct activation of B1 receptors by ACE inhibitors (Skidgel et al., 2006).

Biol Chem., 387(2):159-65.

Fenofibrate

 Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor alpha (Murakami et al., 2006). Biochem Biophys Res Commun., 341(4):973-8. Epub 2006 Jan 24.

Function of Ca2+ on NO response

Nitric oxide (NO) produced in the endothelium via the enzyme endothelial nitric-oxide synthase (eNOS) is an important vasoactive compound. Wild-type (WT) eNOS is localized to the plasma membrane and perinuclear/Golgi region by virtue of N-terminal myristoylation and palmitoylation. Acylation-deficient mutants (G2AeNOS) remain cytosolic and release less NO in response to Ca2+-elevating agonists; a disparity that we hypothesized was attributed to the greater distance between G2AeNOS and plasma membrane Ca2+ influx channels. The reduced activity of G2AeNOS versus WT was reversed upon disruption of cellular integrity with detergents or sonication. NO production from both constructs relied almost exclusively on the influx of extracellular Ca2+, and elevating intracellular Ca2+ to saturating levels with 10 microM ionomycin in the presence of 10 mM extracellular Ca2+ equalized NO production. To identify the contribution of calcium to the differences in activity between these enzymes, we created Ca2+/CaM-independent eNOS mutants by deleting the two putative autoinhibitory domains of eNOS. There was no difference in NO production between WT and G2A-targeted Ca2+-independent eNOS, suggesting that Ca2+ was the factor responsible. When eNOS constructs were fused in-frame to the bioluminescent probe aequorin, membrane-bound probes were exposed to higher [Ca2+] in unstimulated cells but upon ionomycin stimulation, the probes experienced equal amounts of Ca2+. The WT and G2A enzymes displayed significant differences in the phosphorylation state of Ser617, Ser635, and Ser1179, and mutating all three sites to alanine or restoring phosphorylation with the phosphatase inhibitor calyculin abolished the differences in activity. We therefore conclude that the disparity in NO production between WTeNOS and G2AeNOS is not caused by different localized [Ca2+] upon stimulation with ionomycin, but rather differences in phosphorylation state between the two constructs (Church & Fulton, 2006).

 J Biol Chem., 2006 Jan 20;281(3):1477-88. Epub 2005 Oct 28.

Muscarinic ACh and Purinergic (ADP) – mediated eNOS activation

Nitric oxide (NO) regulates flow and permeability. Acetylcholine (ACh) and platelet-activating factor (PAF) lead to eNOS phosphorylation and NO release. While ACh causes only vasodilation, PAF induces vasoconstriction and hyperpermeability. The key differential signaling mechanisms for discriminating between vasodilation and hyperpermeability are unknown. We tested the hypothesis that differential translocation may serve as a regulatory mechanism of eNOS to determine specific vascular responses. We used ECV-304 cells permanently transfected with eNOS-green fluorescent protein (ECVeNOS-GFP) and demonstrated that the agonists activate eNOS and reproduce their characteristic endothelial permeability effects in these cells. We evaluated eNOS localization by lipid raft analysis and immunofluorescence microscopy. After PAF and ACh, eNOS moves away from caveolae. eNOS distributes both in the plasma membrane and Golgi in control cells. ACh (10(-5) M, 10(-4) M) translocated eNOS preferentially to the Trans Golgi network (TGN) and PAF (10(-7) M) preferentially to the cytosol. We suggest that PAF-induced eNOS translocation preferentially to cytosol reflects a differential signaling mechanism related to changes in permeability, whereas ACh-induced eNOS translocation to the TGN is related to vasodilation (Sanchez et al., 2006).

Am J Physiol Heart Circ Physiol., May 5; [Epub ahead of print]

Nitric oxide (NO), derived from the endothelial isoform of NO synthase (eNOS), is a vital mediator of cerebral vasodilation. In the present study, we addressed the issue of whether the mechanisms responsible for agonist-induced eNOS activation differ according to the specific receptor being stimulated. Thus we examined whether heat shock protein 90 (HSP90), phosphatidylinositol-3-kinase (PI3K), and tyrosine kinase participate in ACh- versus ADP-induced eNOS activation in cerebral arterioles in vivo. Pial arteriolar diameter changes in anesthetized male rats were measured during sequential applications of ACh and ADP in the absence and presence of the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME), the neuronal NOS (nNOS)-selective inhibitor ARR-17477, the HSP90 blocker 17-(allylamino)-17-demethoxygeldanamycin (AAG), the PI3K inhibitor wortmannin (Wort), or the tyrosine kinase blocker tyrphostin 47 (T-47). Only NOS inhibition with L-NAME (not ARR-17477) reduced ACh and ADP responses (by 65-75%), which suggests that all of the NO dependence in the vasodilating actions of those agonists derived from eNOS. Suffusions of AAG, Wort, and T-47 were accompanied by substantial reductions in ACh-induced dilations but no changes in the responses to ADP. These findings suggest that muscarinic (ACh) and purinergic (ADP) receptor-mediated eNOS activation in cerebral arterioles involve distinctly different signal transduction pathways. (Xu et al., 2002).

Am J Physiol Heart Circ Physiol., 282:H237-H243

S-Nitrosylation of eNOS

Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005),

J. Biol. Chem. 280, 19888–19894).

In the present studies, we use mass spectrometry to confirm that the zinc-tetrathiolate cysteines of eNOS are S-nitrosylated. eNOS targeting to the plasma membrane is necessary for enzyme S-nitrosylation, and we report that translocation between cellular compartments is necessary for dynamic eNOS S-nitrosylation. We transfected cells with cDNA encoding wild-type eNOS, which is membrane-targeted, or with acylation-deficient mutant eNOS (Myr–), which is expressed solely in the cytosol. While wild-type eNOS is robustly S-nitrosylated, we found that S-nitrosylation of the Myr– eNOS mutant is nearly abolished. When we transfected cells with a fusion protein in which Myr– eNOS is ligated to the CD8-transmembrane domain (CD8-Myr–), we found that CD8-Myr– eNOS, which does not undergo dynamic subcellular translocation, is hypernitrosylated relative to wild-type eNOS. Furthermore, we found that when endothelial cells transfected with wild-type or CD8-Myr– eNOS are stimulated with eNOS agonist, only wild-type eNOS is denitrosylated; CD8-Myr– eNOS S-nitrosylation is unchanged. These findings indicate that subcellular targeting is a critical determinant of eNOS S-nitrosylation. Finally, we show that eNOS S-nitrosylation can be detected in intact arterial preparations from mouse and that eNOS S-nitrosylation is a dynamic agonist-modulated process in intact blood vessels. These studies suggest that receptor-regulated eNOS S-nitrosylation may represent an important determinant of NO-dependent signaling in the vascular wall (Erwin et al., 2006).

 J. Biol. Chem., 281:1, 151-157.

Phosphorylation of eNOS

 The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm) and/or myristoylation (myr) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine 1179 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr eNOS at this residue was nearly abrogated; the palm eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine 1179 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine 1179. In striking contrast to serine 1179, phosphorylation of eNOS at serine 116 was enhanced in the myr eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall (Gonzalez et al., 2002).

J. Biol. Chem., 277;42:39554-39560.

eNOS translocation and Ca2+

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser1177 (Ser1177 eNOS). We have recently shown that agonist-induced Ser1177 eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca2+ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser1177 eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca2+ chelator BAPTA before and after application of BRL 37344 (BRL), a 3-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser1177 eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca2+ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser1177 eNOS phosphorylation is dependent on intracellular Ca2+. 2) In atrial myocardium, eNOS-translocation and not Ser1177 eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca2+ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation (Pott et al., 2006).

Am J Physiol Cell Physiol 290: C1437-C1445.

Nebivolol  DRUG INFORMATION

http://www.intekom.com/pharm/adcock/nebilet.html – retrieved on 6/20/2006

 PHARMACOLOGICAL ACTION

 Pharmacodynamics

 Nebivolol is a racemate of two enantiomers, SRRR-nebivolol (or d-nebivolol) and RSSS-nebivolol (or l-nebivolol). It combines two pharmacological activities: –

• It is a competitive & selective B1-receptor antagonist which is attributable to the d-enantiomer

• It has mild vasodilating properties, possible due to an interaction with the L-arginine/nitric oxide pathway Nebivolol reduces heart rate & blood pressure at rest & during exercise. In healthy volunteers it has no significant effect on maximal exercise or endurance.

An in-vitro and in-vivo experiment in animals showed that nebivolol has no intrinsic sympathicomimetic activity and at pharmacological doses has no membrane stabilizing effect. It is also devoid of alpha-adrenergic antagonism at therapeutic doses.

Pharmacokinetics

Nebivolol can be given with or without meals with peak plasma concentrations occurring within 2 – 6 hours after dosing. It is extensively metabolized partly to active hydroxy metabolites. The bioavailability of nebivolol averages 12% in extensive metabolizers (EM’s) & is virtually complete in poor metabolizers (PM’s), but the mean bioavailability of the separate enantiomers and hydroxylated metabolites was fairly similar between EM’s & PM’s and no differences were found in the pharmacodynamic effects.

Steady-state plasma levels for nebivolol are reached within 24 hours in most subjects (EM’s). The elimination half-lives of the hydroxy-metabolites of both enantiomers average 24 hours in EM’s and are twice as long in PM’s. Plasma concentrations are dose proportional and the pharmacokinetics of nebivolol are unaffected by age. Nebivolol is highly protein bound; d-nebivolol being 98.1% and l-nebivolol 97,9% bound to albumin. About 52% of the dose is excreted in urine and about 15% in the faeces in PM’s one week after administration.

INDICATIONS: Treatment of mild to moderate essential hypertension.

 CONTRA-INDICATIONS

  • Hypersensitivity to Nebilet
  • Liver insufficiency or liver function impairment.
  • Pregnancy and lactation
  • Nebilet is contra-indicated in:

– Cardiogenic shock            – Untreated phaeochromocytoma

– Uncontrolled heart failure            – Metabolic acidosis

– Sick sinus syndrome, including            – Bradycardia (heart rate < 50 bpm)

– sino-atrial block            – Bronchial asthma

– 2nd & 3rd degree heart block            – Hypotension

– History of bronchospasm &             – Severe peripheral circulatory disorders

– bronchial asthma             – Verapamil therapy – Children, as safety and efficacy has not been demonstrated

 WARNINGS

Beta-adrenergic antagonists may increase the sensitivity to allergens and the severity of anaphylactic reactions

 SIDE-EFFECTS AND SPECIAL PRECAUTIONS:

 Side-Effects:

The most common side-effects (incidence between – 1-10%) are headache, dizziness, tiredness & paraesthesia. Other side-effects reported in 1% of patients are: diarrhea, constipation, nausea, dyspnea & edema. Typical beta-adrenergic antagonist side-effects reported in less than 1% of patients are: bradycardia, slowed AV conduction/AV-block, hypotension, heart failure, increase of intermittent claudication, impaired vision, impotence, depression, nightmare, dyspepsia, flatulence, vomiting, bronchospasm and rash.

The following side-effects have also been reported with some beta-adrenergic antagonists: hallucinations, psychoses, confusion, cold/cyanotic extremities, Raynaud phenomenon, dry eyes and mucocutaneous toxicity of the practolol-type, sleep disturbances and abdominal cramping.

Congestive heart failure or heart block may be precipitated in patients with underlying cardiac disorders. Pneumonitis, pleurisy, paraesthesia, peripheral neuropathy, overt psychosis, myopathies, skin rash, pruritis, and reversible alopecia have been reported. Ocular symptoms include decreased tear production, blurred vision and soreness.

Hematological reactions include nonthrombocytopenic purpura, thrombocytopenia, and less frequently agranulocytosis. Transient eosinophilia can occur.

Metabolic changes affect glucose control and cholesterol concentrations. Other side effects include a lupus like syndrome, male impotence, hypoglycemia, sclerosing peritonitis and retroperitoneal fibrosis. Severe peripheral vascular disease and even peripheral gangrene may be precipitated.

Special Precautions:

Cardiovascular:

Beta-adrenergic antagonists should not be used in patients with untreated congestive heart failure, unless their condition has been stabilized. One of the pharmacological actions of beta-blockers is to reduce the heart rate.

Abrupt discontinuation of therapy may cause exacerbation of angina pectoris in patients suffering from ischemic heart disease. Discontinuation of therapy should be gradual (over a period of 1-2 weeks) and patients should be advised to limit the extent of their physical activity during the period that their medicine may be discontinued. If the pulse rate drops below 50-55 bpm at rest and/or the patient experiences symptoms suggestive of bradychardia, the dosage should be reduced. Beta-adrenergic antagonists should be used with caution in:

• Peripheral circulatory disorders (Raynaud’s disease or syndrome, intermittent claudication) as the disorders may be aggravated

• 1st degree heart block because of the negative effect of beta-blockers on conduction time

• Prinzmetal’s angina due to unopposed alpha receptor mediated coronary artery vasoconstriction. Beta-blockers may increase the number and duration of anginal attacks

Metabolic/Endocrinological:

Symptoms of hypoglycemia (tachycardia, palpitations) may be masked in diabetic patients. Tachycardic symptoms may be masked in hyperthyroidism. Abrupt withdrawal may intensify symptoms.

Respiratory:

Bronchospasm may occur in patients suffering from asthma, bronchitis and other chronic pulmonary diseases.

Other:

Psoriasis may be aggravated. Patients with phaeochromocytoma should not receive beta-blockers without concomitant alpha-adrenoreceptor blocking therapy.

Beta-blockers may unmask myasthenia gravis.

Adverse reactions are more common in patients with renal decompensation, and in patients who receive beta-blockers intravenously.

INTERACTIONS

Calcium Antagonists:

Caution should be exercised when administering beta-blockers with calcium antagonists of the verapamil or diltiazem type because of their negative effect on contractility and atrio-ventricular conduction. Exaggeration of these effects can occur particularly in patients with impaired ventricular function and/or SA or AV conduction abnormalities. Neither medicine should therefore be administered intravenously within 48 hours of discontinuing the other.

Anti-arrhythmics:

Caution should be exercised when administering beta-blockers with Class I anti-arrhythmic drugs and amiodarone as their effect on atrial conduction time and their negative inotropic effect may be potentiated. Such interactions can have life threatening consequences.

Clonidine:

Beta-blockers increase the risk of rebound hypertension after sudden withdrawal of chronic clonidine treatment.

Digitalis:

Digitalis glycosides associated with beta-blockers may increase atrio-ventricular conduction times. Nebivolol does not influence the kinetics of digoxin & clinical trials have not shown any evidence of an interaction.

Special note: Digitalisation of patients receiving long term beta-blocker therapy may be necessary if congestive cardiac failure is likely to develop. The combination can be considered despite the potentiation of the negative chronotropic effect of the two medicines. Careful control of dosages and of individual patient’s response (notably pulse rate) is essential in this situation.

Insulin & Oral Antidiabetic drugs:

Glucose levels are unaffected, however symptoms of hypoglycemia may be masked.

Anaesthetics:

Concomitant use of beta-blockers & anaesthetics e.g. ether, cyclopropane & trichloroethylene may attenuate reflex tachycardia & increase the risk of hypotension

Other:

Provided Nebilet is taken with a meal & an antacid between meals, the two treatments can be co-prescribed.

Sympathicomimetic agents may counteract the effect of beta-blockers.

Concomitant administration of tricyclic antidepressants, barbiturates & phenothiazines may increase the blood pressure lowering effect.

Concomitant administration of serotonin re-uptake inhibitors or other compounds predominantly metabolized by the CYPZD6 pathway may delay oxidative metabolism of beta-blockers

 KNOWN SYMPTOMS OF OVERDOSAGE AND PARTICULARS OF ITS TREATMENT:

Symptoms:

Bradycardia, hypotension, bronchospasm and acute cardiac insufficiency

Treatment:

Blood glucose levels should be checked and symptomatic and supportive therapy given.

CONCLUSIONS

Nebvolol – one of the most interesting antihypertensive drugs on the market in 2012. Worldwide Sales of Nebivolol 2009-2011 in US $ (millions)

2009 – 179

2010 – 264  %increase 48

2011 – 348  %increase 32

http://www.evaluatepharma.com/Universal/View.aspx?type=Entity&entityType=Product&lType=modData&id=9552&componentID=1003

REFERENCES

Blair A, Shaul PW, Yuhanna IS, Conrad PA, Smart EJ., (1999). Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J. Biol. Chem., 274:32512–32519.

Broeders MAW, Doevendans PA, Bekkers BCAM, Bronsaer R, van Gorsel E, Heemskerk JWM. oude Egbrink MGA, van Breda E, Reneman RS, van der Zee R, (2000). Nebivolol: A Third-Generation ß-Blocker That Augments Vascular Nitric Oxide Release, Endothelial ß2-Adrenergic Receptor–Mediated Nitric Oxide Production.Circulation, 102:677.

Brugada P, Brugada J, Brugada R, (2001). Dealing with biological variation in the Brugada syndrome. Eur. Heart J., 22(24): 2231 – 2232.

Church JE, Fulton D., (2006). Differences in eNOS activity because of subcellular localization are dictated by phosphorylation state rather than the local calcium environment. J Biol Chem., 2006 Jan 20;281(3):1477-88. Epub 2005 Oct 28.

Dessy C, Saliez J, Ghisdal P, Daneau G, Lobysheva II, Frerart F, Belge C, Jnaoui K, Noirhomme P, Feron O, Balligand JL, (2005). Endothelial {beta}3-Adrenoreceptors Mediate Nitric Oxide-Dependent Vasorelaxation of Coronary Microvessels in Response to the Third-Generation {beta}-Blocker Nebivolol. Circulation, 112(8): 1198 – 1205.

Duarte J, Ocete MA, Perez-Vizcaino F, Zarzuelo A, Tamargo J, (1997). Effect of tyrosine kinase and tyrosine phosphatase inhibitors on aortic contraction and induction of nitric oxide synthase. Eur J Pharmacol, 338:25–33.

Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005), Receptor-regulated Dynamic S-Nitrosylation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells. J. Biol. Chem. 280, 19888–19894).

Erwin PA, Mitchell DA, Sartoretto J, Marletta MA, Michel T., (2006). Subcellular Targeting and Differential S-Nitrosylation of Endothelial Nitric-oxide Synthase. J. Biol. Chem., 281:1, 151-157.

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