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 de veloped that detects phosphorylated serine residue 239 of vasodilator-stimu lated phosphoprotein (VASP), a substrate of PKG-I. In this study, we used t his antibody to examine (i) possible functional differences between the alp ha and beta isoforms of PKG-I, (ii) ability of cAMP to activate PKG-I, as c ompared to cGMP, the principal PKG-I-activating cyclic nucleotide, and (iii ) time course and levels of PKG-I activation in vascular smooth muscle cell s (VSMC) of young and old rats. We created COS-7 cell clones that overexpre ssed PKG-I alpha or PKG-I beta, treated them with cAMP or cGMP, and analyze d their cell lysates for reactivity with VASP-16C2. The results showed that PKG-Ia phosphorylated VASP at a higher level than PKG-I beta, and cAMP was slightly weaker than cGMP in PKG-I activation. VSMC of young rats responde d to cAMP or cGMP stimulation in a dose-dependent manner with increasing le vels of PKG-I activation. The response was detected within 10 min and conti nued for at least 24 h. In contrast, VSMC of old rats showed no PKG-I activ ation 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 P KG-I activation may explain why vascular relaxation is decreased in older i ndividuals. (C) 2001 Academic Press.