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, cGKIalpha and cGKIbeta, 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 Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-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 cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.
Feil
Robert
R
Institut für Pharmakologie und Toxikologie der Technischen Universität München, Universität München, München, Germany. feil@ipt.med.tu-muenchen.de
Gappa
Nicolai
N
Rutz
Mark
M
Schlossmann
Jens
J
Rose
Christine R
CR
Konnerth
Arthur
A
Brummer
Sabine
S
Kühbandner
Susanne
S
Hofmann
Franz
F
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006834:Hydrazines / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009589:Nitrogen Oxides; D009638:Norepinephrine / Q000037:antagonists & inhibitors; D020033:Protein Isoforms / Q000032:analysis / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D006834:Hydrazines; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C016276:8-bromocyclic GMP; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569720:Prkg1 protein, mouse; D006152:Cyclic GMP; D002118:Calcium; D009638:Norepinephrine