Cyclic GMP-dependent protein phosphorylation in intact medial tissue and isolated cells from vascular smooth muscle.

cGMP-dependent protein kinase, CGMP-dependent phosphorylation of endogenous substrate proteins, and cGMP levels were studied in rabbit aortic medial tissue and in homogeneous primary cultures of isolated aortic medial smooth muscle cells. The photoaffinity ligand, ~-Ns-[~’P]cIMP, which labels the 74,000-dalton subunit of cGMP-dependent protein kinase, was used to study the distribution of this enzyme between soluble and total particulate fractions obtained from homogenates of aortic medial tissue. Approximately 25% of the total amount of 74,000-dalton protein present in homoge- nates of the tissue was recovered in the particulate fraction. Peptide mapping revealed that the 74,000-dd- ton protein present in the particulate fraction was identical with the 74,000-dalton subunit of the cGMP-de- pendent protein kinase present in the soluble fraction of rabbit aortic medial tissue. Using 8-N3-[32P]cIMP, cGMP-dependent protein kinase was also demon- strated in isolated, cultured aortic medial smooth muscle cells. total