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(Circulation Research. 2002;90:1080.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Functional Reconstitution of Vascular Smooth Muscle Cells With cGMP-Dependent Protein Kinase I Isoforms

Robert Feil, Nicolai Gappa, Mark Rutz, Jens Schlossmann, Christine R. Rose, Arthur Konnerth, Sabine Brummer, Susanne Kühbandner, Franz Hofmann

From the Institut für Pharmakologie und Toxikologie der Technischen Universität München (R.F., N.G., M.R., J.S., S.B., S.K., F.H.) and Physiologisches Institut (C.R.R., A.K.), Universität München, München, Germany.

Correspondence to Robert Feil, Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Str. 29, 80802 München, Germany. E-mail feil{at}ipt.med.tu-muenchen.de

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKI and cGKIß, 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 cGKI and cGKIß. 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 cGKI and cGKIß. The defective Ca²⁺ regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKI and enhanced green fluorescent protein (EGFP) but not by a cGKIß-EGFP construct. Fluorescence imaging indicated that the cGKI-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIß-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 cGKI isoform, which decreases the noradrenaline-stimulated cytosolic Ca²⁺ level.

Key Words: smooth muscle • nitric oxide • calcium • gene targeting • cGMP kinase

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