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Soluble guanylate cyclase redox state under oxidative stress conditions in isolated monkey coronary arteries.

ABSTRACT: Coronary artery disease is associated with oxidative stress due to the excessive generation of free radicals in the vascular wall. This study investigated the impact of tert-butyl hydroperoxide (t-BuOOH), a peroxyl radical generator, on the redox state of soluble guanylate cyclase (sGC) in isolated monkey coronary arteries. Helically cut strips of endothelium-intact monkey coronary arteries treated with the nitric oxide synthase inhibitor N^G-nitro-L-arginine (10 μmol/L) were exposed for approximately 60 min to either no drug or t-BuOOH (100 μmol/L) in the presence and absence of α-tocopherol (300 μmol/L). Relaxation and cGMP levels in response to the sGC stimulator BAY 41-2272 and the sGC activator BAY 60-2770 were assessed by organ chamber technique and enzyme immunoassay, respectively. The relaxant response to BAY 41-2272 was significantly impaired by the exposure to t-BuOOH, whereas the response to BAY 60-2770 was significantly augmented. In addition, vascular cGMP accumulation caused by BAY 41-2272 was decreased by the exposure to t-BuOOH, whereas for BAY 60-2770, it was increased. These effects of t-BuOOH were abolished by coincubation with α-tocopherol. Furthermore, correlations were observed between BAY compound-induced relaxant magnitudes and cGMP levels. Therefore, it is concluded that increased oxidative stress leads to disruption of the sGC redox state in monkey coronary arteries. This finding is of great importance for understanding coronary physiology in primates.

SUBMITTER: Tawa M

PROVIDER: S-EPMC5045941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Heme oxygenase-1 knockout, H(mox)1(-/-), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in H(mox)1(-/-) mice. The contractile responses [superior mesenteric arteries (SMA), from female H(mox)1(-/-) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in H(mox)1(-/-) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in H(mox)1(-/-) than H(mox)1(+/+) mice (p < 0.001). Branches from H(mox)1(-/-) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. H(mox)1(-/-) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were H(mox)1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of H(mox)1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of H(mox)1(-/-) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both ?1 and ?1 subunit levels were reduced to <50% of H(mox)1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of H(mox)1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.

Project description:Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat β1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the β1 H-NOX construct (residues 1-194), the β1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1β1 sGC heterodimer (β1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.

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Soluble guanylate cyclase redox state under oxidative stress conditions in isolated monkey coronary arteries.

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