Protection of vascular nitric oxide by superoxide dismutase mimetics

MacKenzie, Andrew (1998) Protection of vascular nitric oxide by superoxide dismutase mimetics. PhD thesis, University of Glasgow.

Abstract

The reduced species of molecular oxygen, superoxide anion, rapidly destroys nitric oxide and thereby impairs the vital vasodilator role played by the endothelium. Therapeutic treatment with the endogenous scavenger of superoxide, superoxide dismutase (SOD) is of limited value since it cannot penetrate cell membranes to protect nitric oxide intracellularly. In this study, a number of structurally distinct membrane-permeant SOD mimetics were examined both biochemically for their ability to inhibit superoxide-catalysed reactions and functionally for their ability to protect nitric oxide from destruction by oxidant stress in rabbit aorta. The SOD mimetics investigated were the nitroxide spin traps PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide) and tempol (4-hydroxy 2,2,6,6- tetramethylpiperidine-1-oxyl), the superoxide scavenger tiron (4,5-dihydroxy-1,3-benzene-disulfonic acid), the metal-based compounds CuDIPS (Cu [II]- [diisopropylsalicylate]2) and MnTMPyP (Mn [III] tetrakis [1-methyl-4-pyridyl] porphyrin), and the metal salts CUSO4 and MnCl2. These mimetics were investigated for their ability to inhibit the superoxide anion- catalysed reduction of cytochrome c and nitro blue tetrazolium. The rank order of potency in inhibiting the reduction of cytochrome c was CUSO4 > MnCl2 > CuDIPS > MnTMPyP > tiron > tempol > PTIYO. The requirement of EDTA prevented the assessment of the activity of CUSO4, MnCl2 and CuDIPS in the assay involving inhibition of reduction of nitro blue tetrazolium. However, the rank order of potency for those agents which could be examined (MnTMPyP > tiron > tempol > PTIYO) was similar to that seen in the cytochrome c assay. Novel models of oxidant stress were generated in rabbit isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate DETCA (60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml-1) and hypoxanthine (HX; 0.1 mM). Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired both basal and acetylcholine-induced relaxation and exogenous SOD (250 u ml-1) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1 - 3 muM), CUSO4 (0.3 - 3 muM), MnCl2 (1 - 100 muM) and MnTMPyP (100 - 600 muM) also reversed the blockade powerfully. Tempol (30 muM - 1 mM) and tiron (0.3 - 10 mM) reversed the blockade weakly and PTIYO (10 - 300 muM) enhanced the blockade. ACh-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 muml-1), suggesting the oxidant stress was intracellular. MnTMPyP (600 muM and 1 mM) and MnCl2 (100 muM) were the only agents to reverse the blockade of ACh-induced relaxation. Thus, MnTMPyP was the only SOD mimetic to restore nitric oxide-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. It was found, however, that MnTMPyP can paradoxically destroy basal nitric oxide through the generation of superoxide. This undoubtedly compromises its therapeutic potential in the treatment of vascular pathologies associated with oxidant stress.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Billy Martin
• Subjects: Q Science > QP Physiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Date of Award: 1998
• Depositing User: Enlighten Team
• Unique ID: glathesis:1998-71832
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 18 Oct 2022 12:02
• Thesis DOI: 10.5525/gla.thesis.71832
• URI: https://theses.gla.ac.uk/id/eprint/71832