Nitric Oxide Protects PC12 Cells from Serum Deprivation-Induced Apoptosis by cGMP-Dependent Inhibition of Caspase Signaling

Abstract

Although nitric oxide (NO) induces neuronal cell death under some conditions, it also can prevent apoptosis resulting from growth factor withdrawal. We investigated the molecular mechanism by which NO protects undifferentiated and differentiated PC12 cells from trophic factor deprivation-induced apoptosis. PC12 cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and cytochrome c release after 24 hr of serum withdrawal. The apoptosis of PC12 cells was inhibited by the addition of NO-generating donor S-nitroso-N-acetylpenicillamine (SNAP) (5-100 microM) and the specific caspase-3-like protease inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho) but not the YVADase (or caspase-1-like protease) inhibitor N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-cho). SNAP and Ac-DEVD-cho prevented the increase in DEVDase (caspase-3-like protease) activity. The SNAP-mediated suppression of DEVDase activity was only minimally reversed by the incubation of cell lysate with dithiothreitol, indicating that NO did not S-nitrosylate caspase-3-like proteases in PC12 cells. Western blot analysis showed that NO inhibited the proteolytic activation of caspase-3. The cGMP analog 8-bromo-cGMP (8-Br-cGMP) blocked apoptotic cell death, caspase-3 activity and activation, and cytochrome c release. The soluble guanylyl cyclase inhibitor 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (CODQ) significantly attenuated NO-mediated, but not 8-Br-cGMP-dependent, inhibition of apoptotic cell death, PARP cleavage, cytochrome c release, and DEVDase activity. Furthermore, the protein kinase G inhibitor KT5823 reversed both SNAP- and 8-Br-cGMP-mediated anti-apoptotic events. All these apoptotic phenomena were also suppressed by NO production through neuronal NO synthase gene transfer into PC12 cells. Furthermore, similar findings were observed in differentiated PC12 cells stimulated to undergo apoptosis by NO donors and NGF deprivation. These findings indicate that NO protects against PC12 cell death by inhibiting the activation of caspase proteases through cGMP production and activation of protein kinase G.

Changes in nitric oxide synthase expression in young and adult rats after spinal cord injury

“…17 However, the neurotoxic and neuroprotective effects of NO depend on the time and site of release. 19,20 In the brain, NO has a neuroprotective effect and maintains blood vessel tone by increasing cerebral perfusion and decreasing platelet aggregation and interactions between leukocytes and endothelial cells. [21][22][23][24] How injury affects perfusion of the spinal cord has not yet been determined.…”

Regulation of p53 and suppression of apoptosis by the soluble guanylyl cyclase/cGMP pathway in human ovarian cancer cells

“…sGC also has basal activity and contributes to most of the basal cGMP levels found in mammalian cells (Waldman and Murad, 1987;Garthwaite et al, 1995;Fiscus, 2002). While activation of sGC and elevation of cGMP levels inhibits apoptosis in many cell types (Kim et al, 1999;Fiscus et al, 2001;Fiscus, 2002;Fiscus et al, 2002), inhibition of basal sGC activity with specific inhibitors (e.g. ODQ) lowers basal cGMP levels and induces apoptosis (Garthwaite et al, 1995;Flamigni et al, 2001;Fiscus, 2002;Chan and Fiscus, 2003).…”

Regulation and measurement of oxidative stress in apoptosis

“…NO dependent generation of intracellular cGMP has been shown to protect against apoptosis in lymphocytes (Genaro et al, 1995), eosinophils (Beauvais et al, 1995), embryonic motor neurons (Estevez et al, 1998), PC12 cells (Kim et al, 1999) and ovarian follicles (Chun et al, 1995). Exactly how cGMP protects against apoptosis is at present unclear.…”

“…Exactly how cGMP protects against apoptosis is at present unclear. In serum-deprived PC12 cells, it is thought to involve the activation of protein kinase G and the inhibition of caspase activation through the prevention of cytochrome c release (Kim et al, 1999). Recently, cGMP was found to suppress TNFα and ActD-induced apoptosis in hepatocytes (Li et al, 2000).…”