A Fluorescence-Activated Cell Sorter Analysis of the Relationship Between Protein Kinase G and Endothelial Nitric Oxide Synthase

The process of regulation of NOS after production of nitric oxide is not yet delineated. Protein kinase G may exert a feedback regulation of this enzyme. We used diaminofluorescein assays to detect changes in basal nitric oxide production caused by modulators of protein kinase G activity in freshly isolated ovine lung microvascular endothelial cells. We also used fluorescence activated cell sorter analysis (FACS) to determine molecular and phosphorylation changes caused by PKG activation with 8-Br-cGMP. The PKG activator, 8-Br-cGMP (100 mu M) produced a shift in the basal NO production curve downward. The inhibition began within 5 min and was sustained over 4.5 hr. The two protein kinase G inhibitors 100 mu M Rp-8-Br-PET-cGMPS and 50 nM guanosine 3'-5'-cyclic monophosphoro thionate-8-Br-Rp isomer Na salt and the cGMP inhibitor 4 mu M Rp-8-pCPT-cGMPS all enhanced NO production as seen by the upward shift in the basal NO curve. Conversely, the PKG activator drug, 100 mu M guanosine-3'-5'-cyclic monophosphate-beta-phenyl-1NF-ethano-8-bromo sodium salt decreased NO production causing a downward shift in the basal curve. FACS analysis revealed that 5 mu M 8-Br-cGMP in <5 min caused an increase in N-terminal labeling of NOS and a decrease in both C-terminal and serine 1177 labeling of NOS. 8-Br-cGMP appeared to increase PKG 1 alpha and to decrease PKG 1 beta labeling. Changes in other phosphorylation sites were less consistent but overall mean channel fluorescence increased from 19.92 to 217.36 for serine 116 and decreased from 329.27 to 254.03 for threonine 495 phosphorylation. Data indicated that PKG caused both molecular and phosphorylation changes in NOS. Anat Rec, 293:1755-1765, 2010. (C) 2010 Wiley-Liss, Inc.