Abstract 151: H2O2 Operates As An Endothelium-derived Hyperpolarizing Factor By Directly Activating Protein Kinase G (PKG) Iα Independently Of cGMP Via Disulfide Formation
H2O2 (5min, 100μM) perfusion of isolated rat hearts promoted interprotein disulfide formation (from 36% to 86%, P<0.01, observed on immunoblots) in PKG1α. Hearts showed a time and dose dependent correlation between H2O2-mediated disulfide formation and decreased perfusion pressure (vasodilation), implying direct activation of PKG1α by oxidation. To investigate this potentially novel form of activation we carried out aortic rings experiments using a soluble guanylate cyclase (ODQ, 5 μM) and PKG inhibitor (Rp-8-Bromo-cGMPS, 100 μM). Both inhibitors blocked NO mediated relaxation. However, H2O2 mediated relaxation was only attenuated by the PKG inhibitor indicating direct activation without cGMP production. In vitro Michaelis-Menten analysis with PKG1α, AT32P and Glasstide substrate showed cGMP increased Vmax by 45%, without changing the Km for substrate. In contrast disulfide oxidation left Vmax unaffected but profoundly decreased substrate Km from 247 to 37 μM. PKGIβ does not contain the redox cysteine, could not form a disulfide and was not oxidant activated. Similarly the dissociation constant (Kd) of WT PKG1α for its substrate RhoA was markedly enhanced by oxidation (4.6 to 0.36 μM); a response absent in the C42S mutant. Disulfide-enhanced substrate affinity explains H2O2-induced subcellular PKG1α translocation to myofilament, nucleus and sarcolemma observed by subcellular fractionation and immunoblotting in isolated hearts. Similarly FLAG-tagged WT (but not a “redox-dead” Cys42Ser mutant that cannot disulfide dimerize) PKG1α expressed in A10 cells was observed by confocal florescence to move to the nucleus and membrane in response to H2O2. Using 8-AET-cGMP-agarose to capture PKG (WT or C42S) expressed in A10 cells (± H2O2), we found that oxidation of WT caused association with BKCa channel and Myosin Phosphatase (MYPT1, which phosphoregulates Myosin Light Chain). Using MLC dephosphorylation as readout of PKG activity we found H2O2 activated WT, but not the C42S mutant, kinase. In summary we have shown a molecular basis for vasorelaxation by H2O2, an oxidant that operates as an EDHF distinct from NO. PKG Iα is activated by a novel cGMP-independent mechanism involving a disulfide between two aligned Cys42 on each chain of the kinase homodimer.