Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling

Although nitric oxide (NO) induces neuronal cell death under some condition s, it also can prevent apoptosis resulting from growth factor withdrawal. W e investigated the molecular mechanism by which NO protects undifferentiate d and differentiated PC12 cells from trophic factor deprivation-induced apo ptosis. PC12 cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, poly(ADP-ribose) polymerase (PA RP) cleavage, and cytochrome c release after 24 hr of serum withdrawal. The apoptosis of PC12 cells was inhibited by the addition of NO-generating don or S-nitroso-N-acetylpenicillamine (SNAP) (5-100 mu M) and the specific cas pase-3-like protease inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho) bu t not the YVADase (or caspase-1-like protease) inhibitor N-acetyl-Tyr-Val-A la-Asp-aldehyde (Ac-YVAD-cho). SNAP and Ac-DEVD-cho prevented the increase in DEVDase (caspase-3-like protease) activity. The SNAP-mediated suppressio n of DEVDase activity was only minimally reversed by the incubation of cell lysate with dithiothreitol, indicating that NO did not S-nitrosylate caspa se-3-like proteases in PC12 cells. Western blot analysis showed that NO inh ibited the proteolytic activation of caspase-3. The cGMP analog 8-bromo-cGM P (8-Br-cGMP) blocked apoptotic cell death, caspase-3 activity and activati on, and cytochrome c release. The soluble guanylyl cyclase inhibitor 1-H-ox odiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (CODQ) significantly attenuated N O-mediated, but not 8-Br-cGMP-dependent, inhibition of apoptotic cell death , PARP cleavage, cytochrome c release, and DEVDase activity. Furthermore, t he protein kinase G inhibitor KT5823 reversed both SNAP- and 8-Br-cGMP-medi ated anti-apoptotic events. All these apoptotic phenomena were also suppres sed by NO production through neuronal NO synthase gene transfer into PC12 c ells. Furthermore, similar findings were observed in differentiated PC12 ce lls stimulated to undergo apoptosis by NO donors and NGF deprivation. These findings indicate that NO protects against PC12 cell death by inhibiting t he activation of caspase proteases through cGMP production and activation o f protein kinase G.