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-stimulated phosphorylation of BPDEtide and histone F2B substrates in the presence of PKI [peptide inhibitor of cAMP-dependent protein kinase (cAPK)]. RPMVEC cGPK activity was localized to the 100,000 x g cytosolic fraction. The EC(50) for cGMP activation in the presence of PKI was 0.16 mu M and H-89 inhibition under similar conditions showed an IC50 value of 0.16 mu M. Anion-exchange chromatography of RPMVEC and rat lung cytosolic fractions showed separation of the cGMP-dependent from the cGMP-independent protein kinase activity and similar elution conductivities. Further, Western blots of RPMVEC active DEAE-Trisacryl fractions showed immunoreactivity using bovine Type I cGPK antiserum. Preliminary studies reveal six potential substrates phosphorylated by cGPK in RPMVEC. These studies describe an endothelial cell (EC) cGMP-receptor, cGPK, in addition to cGMP-activated (Type II) phosphodiesterase (PDE). (C) 1994 Academic Press, Inc.