Protein kinase G activity prevents pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis/cell proliferation in vascular smooth muscle cells.
BACKGROUND
Protein kinase G (PKG), a recognized downstream mediator of nitric oxide, is a key regulator of cardiovascular physiology and pathology. High-level stimulation of cyclic guanosine monophosphate/PKG signaling using high concentrations of nitric oxide donors, mimicking pathological conditions, induces apoptosis in vascular smooth muscle cells. In contrast, we have found that PKG at basal and moderately elevated activity prevents both spontaneous and toxin-induced apoptosis in many other cells. We hypothesized that PKG's apoptosis-regulatory role in vascular smooth muscle cells depends on PKG activation levels [low/basal-level activation prevents apoptosis, whereas high-level activation (hyperactivation) causes apoptosis]. Furthermore, we hypothesized that, although PKG hyperactivation inhibits vascular smooth muscle cell proliferation (potentially causing anti-atherogenic effects), basal PKG activity may promote vascular smooth muscle cell proliferation/atherogenesis.


METHODS
Involvement of PKG in apoptosis and proliferation was determined in unpassaged vascular smooth muscle cells from mouse aorta. Western blot analysis was used to determine PKG expression, and activators/inhibitors of PKG activity were used to determine involvement in apoptosis (Hoechst staining and DNA-fragmentation ELISAs) and proliferation (cell count, MTT assay, and BrdU incorporation).


RESULTS
Both PKG-Iα and PKG-Iβ isoforms were expressed. Lower-level stimulation of PKG using the nitric oxide donor S-nitroso-acetylpenacillamine (10, 50 μM) significantly (P<.05) lowered spontaneous apoptosis, whereas S-nitroso-acetylpenacillamine at higher concentrations (500, 1000 μM) elevated apoptosis. Twenty-four-hour pretreatment with atrial natriuretic peptide, a PKG activator, completely prevented high-concentration, nitric oxide-induced apoptosis. Inhibition of basal PKG activity using highly selective PKG inhibitors, DT-2 and DT-3, significantly (P<.001) increased apoptosis and inhibited DNA synthesis/proliferation.


CONCLUSIONS
The data suggest that basal/moderately elevated PKG activity protects against high/pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis/proliferation in vascular smooth muscle cells, potentially important for atherogenesis.
Wong
Janica C
JC
Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, NV 89135, USA.
Fiscus
Ronald R
RR
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000633:toxicity; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013997:Time Factors
D020536:Enzyme Activators; D020030:Nitric Oxide Donors; D047428:Protein Kinase Inhibitors; D009569:Nitric Oxide; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569720:Prkg1 protein, mouse