Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIα and cGKIβ, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca²⁺ concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIα and CGKIβ. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca²⁺ transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIα and CGKIβ. The defective Ca²⁺ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIα and enhanced green fluorescent protein (EGFP) but not by a CGKIβ-EGFP construct. Fluorescence imaging indicated that the cGKIα-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the CGKIβ-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIα isoform, which decreases the noradrenaline-stimulated cytosolic Ca²⁺ level.