H2O2 lowers the cytosolic Ca2+ concentration via activation of cGMP-dependent protein kinase Iα

The cGMP-dependent protein kinase I (cGKI) is a key mediator of cGMP signaling, but the specific functions of its two isoforms, cGKIα and cGKIβ, are poorly understood. Recent studies indicated a novel cGMP-independent role for cGKIα in redox sensing. To dissect the effects of oxidative stress on the cGKI isoforms, we used mouse embryonic fibroblasts and vascular smooth muscle cells (VSMCs) expressing both, one, or none of them. In cGKIα-expressing cells, but not in cells expressing only cGKIβ, incubation with H2O2 induced the formation of a disulfide bond between the two identical subunits of the dimeric enzyme. Oxidation of cGKIα was associated with increased phosphorylation of its substrate, vasodilator-stimulated phosphoprotein. H2O2 did not stimulate cGMP production, indicating that it activates cGKIα directly via oxidation. Interestingly, there was a mutual influence of H2O2 and cGMP on cGKI activity and disulfide bond formation, respectively; preoxidation of the kinase with H2O2 slightly impaired its activation by cGMP, whereas preactivation of the enzyme with cGMP attenuated its oxidation by H2O2. To evaluate the functional relevance of the noncanonical H2O2–cGKIα pathway, we studied the regulation of the cytosolic Ca2+ concentration ([Ca2+]i). H2O2 suppressed norepinephrine-induced Ca2+ transients in cGKIα-expressing VSMCs and, to a lower extent, in VSMCs expressing only cGKIβ or none of the isoforms. Thus, H2O2 lowers [Ca2+]i mainly via a cGKIα-dependent pathway. These results indicate that oxidative stress selectively targets the cGKIα isoform, which then modulates cellular processes in a cGMP-independent manner. A decrease in [Ca2+]i in VSMCs via activation of cGKIα might be a major mechanism of H2O2-induced vasodilation.

► The cGMP-dependent protein kinase type I (cGKI) mediates many functions of cGMP.
► Oxidation by H2O2 activates the cGKIα but not the cGKIβ isoform in the absence of cGMP.
► H2O2 lowers [Ca2+]i in vascular smooth muscle cells mainly via cGKIα.
► The noncanonical H2O2–cGKIα–Ca2+ pathway might contribute to vasodilation.
► Oxidized cGKIα might be involved in many effects of oxidative stress.