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⁺). 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⁺ 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⁺ in VSMCs via activation of cGKIα might be a major mechanism of H₂O₂-induced vasodilation.