Age-related decrease of protein kinase G activation in vascular smooth muscle cells.
Protein kinase G-I (PKG-I) activation is essential for vascular relaxation; however, its quantitative analysis in intact cells has been difficult. To overcome this difficulty, a monoclonal antibody, VASP-16C2, was recently developed that detects phosphorylated serine residue 239 of vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG-I. In this study, we used this antibody to examine (i) possible functional differences between the alpha and beta isoforms of PKG-I, (ii) ability of cAMP to activate PKG-I, as compared to cGMP, the principal PKG-I-activating cyclic nucleotide, and (iii) time course and levels of PKG-I activation in vascular smooth muscle cells (VSMC) of young and old rats. We created COS-7 cell clones that overexpressed PKG-Ialpha or PKG-Ibeta, treated them with cAMP or cGMP, and analyzed their cell lysates for reactivity with VASP-16C2. The results showed that PKG-Ialpha phosphorylated VASP at a higher level than PKG-Ibeta, and cAMP was slightly weaker than cGMP in PKG-I activation. VSMC of young rats responded to cAMP or cGMP stimulation in a dose-dependent manner with increasing levels of PKG-I activation. The response was detected within 10 min and continued for at least 24 h. In contrast, VSMC of old rats showed no PKG-I activation during the first hour of cAMP or cGMP stimulation and, at 24 h these cells showed only low-level PKG-I activation. We propose that the reduced PKG-I activation may explain why vascular relaxation is decreased in older individuals.
Lin
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Knuppe Molecular Urology Laboratory, University of California, San Francisco, CA 94143-1695, USA. clin@urol.ucsf.edu
Liu
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Chow
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D000375:Aging / Q000502:physiology*; D000818:Animals; D001011:Aorta; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D000882:Haplorhini; D006801:Humans; D008840:Microfilament Proteins; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D000242:Cyclic AMP; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP