CYCLIC GMP-DEPENDENT PROTEIN-KINASE ACTIVITY IN RAT PULMONARY MICROVASCULAR ENDOTHELIAL-CELLS

Cyclic GMP-dependent protein kinase (cGPK) activity was determined in rat pulmonary microvascular endothelial cells(RPMVEC) using cGMP-stimu lated phosphorylation of BPDEtide and histone F2B substrates in the pr esence of PKI [peptide inhibitor of cAMP-dependent protein kinase (cAP K)]. RPMVEC cGPK activity was localized to the 100,000 x g cytosolic f raction. The EC(50) for cGMP activation in the presence of PKI was 0.1 6 mu M and H-89 inhibition under similar conditions showed an IC50 val ue of 0.16 mu M. Anion-exchange chromatography of RPMVEC and rat lung cytosolic fractions showed separation of the cGMP-dependent from the c GMP-independent protein kinase activity and similar elution conductivi ties. Further, Western blots of RPMVEC active DEAE-Trisacryl fractions showed immunoreactivity using bovine Type I cGPK antiserum. Prelimina ry studies reveal six potential substrates phosphorylated by cGPK in R PMVEC. These studies describe an endothelial cell (EC) cGMP-receptor, cGPK, in addition to cGMP-activated (Type II) phosphodiesterase (PDE). (C) 1994 Academic Press, Inc.