Protein Kinase G May Exert Pro-Degradation Inhibition on Nitric Oxide Synthase

Nitric oxide synthase (NOS) is regulated by protein-protein interactions. We had earlier shown that PKG inhibits activated NOS in endothelial cells and speculated that PKG phosphorylation of NOS terminates its activity. The present work examines if PKG activation increases breakdown of NOS. Diamino-fluorescein fluorescence spectrometry of real time NO production was used to establish that isolated ovine lung microvascular endothelial cells responded to PKG modulation as previously reported. Fluorescence activated cell sorter (FACS) analysis was used to establish that 8-Br-cGMP, a PKG activator, caused carboxy terminal deletion on NOS, a sign of degradation. Western blot analysis was used to investigate NOS fragments in control and 5 min 8-Br-cGMP treated cells. PKG activator 8-Br-cGMP, at 20 nM, 200 nM, and 2 μM, decreased nitric oxide production in a dose dependent manner (p<0.05 in all cases). PKG inhibitors: 100 μM Rp-8-Br-PET-cGMPS, 50 nM Rp-8-pCPT-cGMPS, or 4 μM Rp-8-Br-cGMPS Na significantly increased NO production (p