Roles for redox mechanisms controlling protein kinase G in pulmonary and coronary artery responses to hypoxia

Abstract

We previously reported that isolated endothelium-removed bovine pulmonary arteries (BPAs) contract to hypoxia associated with removal of peroxide- and cGMP-mediated relaxation. In contrast, bovine coronary arteries (BCAs) relax to hypoxia associated with cytosolic NADPH oxidation coordinating multiple relaxing mechanisms. Since we recently found that H2O2 relaxes BPAs through PKG activation by both soluble guanylate cyclase (sGC)/cGMP-dependent and cGMP-independent thiol oxidation/subunit dimerization mechanisms, we investigated if these mechanisms participate in BPA contraction and BCA relaxation to hypoxia. The contraction of BPA (precontracted with 20 mM KCl) to hypoxia was associated with decreased PKG dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. In contrast, exposure of 20 mM KCl-precontracted endothelium-removed BCAs to hypoxia caused relaxation and increased dimerization and VASP phosphorylation. Depletion of sGC by organoid culture of BPAs with an oxidant of the sGC heme (10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) increased aerobic force generation, decreased VASP phosphorylation, and inhibited further contraction to hypoxia and changes in VASP phosphorylation. Thiol reduction with dithiothreitol increased aerobic force in BPAs and decreased PKG dimerization, VASP phosphorylation, and the contraction to hypoxia. Furthermore, PKG-1α and sGC β1-subunit small interfering RNA-transfected BPAs demonstrated increased aerobic K+ force and inhibition of further contraction to hypoxia, associated with an attenuation of H2O2-elicited relaxation and VASP phosphorylation. Thus, decreases in both a sGC/cGMP-dependent and a dimerization-dependent activation of PKG by H2O2 appear to contribute to the contraction of BPAs elicited by hypoxia. In addition, stimulation of PKG activation by dimerization may be important in the relaxation of coronary arteries to hypoxia.