Cancer Molecular Biology Section, Nevada Cancer Institute, Las Vegas, NV 89135, USA.
Cardiovasc Pathol. 2010 Nov-Dec;19(6):e221-31. doi: 10.1016/j.carpath.2009.11.001. Epub 2010 Jan 13.
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.
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).
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.
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.
蛋白激酶 G（PKG）是一氧化氮的公认下游介质，是心血管生理学和病理学的关键调节剂。使用高浓度的一氧化氮供体（模拟病理条件）高度刺激环鸟苷酸/ PKG 信号转导会诱导血管平滑肌细胞凋亡。相比之下，我们发现 PKG 在基础和适度升高的活性水平下可防止许多其他细胞中的自发性和毒素诱导的凋亡。我们假设 PKG 在血管平滑肌细胞中的凋亡调节作用取决于 PKG 的激活水平[低/基础水平的激活可防止凋亡，而高水平的激活（过度激活）则会导致凋亡]。此外，我们假设，尽管 PKG 过度激活会抑制血管平滑肌细胞增殖（可能产生抗动脉粥样硬化作用），但基础 PKG 活性可能会促进血管平滑肌细胞增殖/动脉粥样硬化形成。
在未经传代的来自小鼠主动脉的血管平滑肌细胞中确定 PKG 对凋亡和增殖的参与。使用 Western blot 分析来确定 PKG 的表达，并使用 PKG 活性的激活剂/抑制剂来确定其在凋亡（Hoechst 染色和 DNA 片段化 ELISA）和增殖（细胞计数、MTT 测定和 BrdU 掺入）中的参与。
表达了 PKG-Iα和 PKG-Iβ同工型。使用一氧化氮供体 S-亚硝基乙酰青霉胺（10、50 μM）以较低水平刺激 PKG 会显著（P<.05）降低自发性凋亡，而较高浓度（500、1000 μM）的 S-亚硝基乙酰青霉胺则会增加凋亡。24 小时预孵育心房利钠肽，一种 PKG 激活剂，可完全防止高浓度、一氧化氮诱导的凋亡。使用高度选择性的 PKG 抑制剂 DT-2 和 DT-3 抑制基础 PKG 活性，会显著（P<.001）增加凋亡并抑制 DNA 合成/增殖。
数据表明，基础/适度升高的 PKG 活性可防止高/病理性水平的一氧化氮诱导的凋亡，并促进血管平滑肌细胞中的 DNA 合成/增殖，这对动脉粥样硬化形成可能很重要。
