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.

Fig. 1.

Cytoprotective effects of SNAP on the survival of PC12 cells in serum-free conditions. Cells were collected, washed extensively with serum-free RPMI 1640 medium, and replated onto collagen-coated 24-well plates at a density of 2.5 × 10⁵ cells per well in a volume of 1 ml of serum-free media. Cells were treated with the same concentration of the NO donor SNAP. At 24 hr, cell viability was determined by counting intact nuclei after lysing of the cell membrane (mean ± SD.n = 3). *p < 0.01 versus nonpretreatment.

Fig. 2.

Cytoprotective effects of SNAP and caspase inhibitors on survival of PC12 cells in serum-free media. Cells were treated with SNAP (100 μm), Ac-YVAD-cho (200 μm), or Ac-DEVD-cho (200 μm) in serum-free media. At 24 hr, cell viability was determined by counting intact nuclei (mean ± SD; n = 4). PC12 cells were collected, washed with ice-cold PBS, and lysed in 100 mmHEPES buffer, pH 7.4, containing protease inhibitors. The cytosolic fraction was obtained by centrifugation at 100,000 ×g for 30 min at 4°C. Caspase enzyme activity was measured with Ac-YVAD-pNA for caspase-1-like activity (or YVADase) and Ac-DEVD-pNA for caspase-3-like activity (or DEVDase) in a colorimetric assay (mean ± SD; n = 3). *p < 0.01 versus nonpretreatment.

Fig. 3.

A guanylyl cyclase inhibitor reversed the cytoprotective effect of SNAP on serum-deprived PC12 cells. Cells were treated with SNAP (100 μm) plus ODQ (40 μm) in the presence and absence of serum. Cell viability(A), DEVDase activity (B), DNA fragmentation (C), and PARP fragmentation(D) were determined as described in Materials and Methods. Data represent mean ± SD of three experiments. *p < 0.01; **p < 0.05.

Fig. 4.

A protein kinase G inhibitor reverses the cGMP-dependent protection of serum-deprived apoptosis of PC12 cells. Cells were cultured in serum-free media containing different concentrations of 8-Br-cGMP (A) or 100 μm8-Br-cGMP (B) with or without KT5823 (180 nm) or ODQ (40 μm). A, Cell viability was measured by counting intact nuclei (mean ± SD;n = 3). B, DEVDase activity was determined by a colorimetric assay using Ac-DEVD-pNA after incubation of lysate with or without 20 mm DTT for 30 min (mean ± SD; n = 3). Similar results were obtained by addition of dibutyl- cGMP into PC12 cell culture. *p < 0.01 versus nontreatment; **p < 0.05 versus nontreatment; ***p < 0.01.

Fig. 5.

SNAP and 8-Br-cGMP prevent mitochondrial cytochrome c release and caspase-3 activation in serum-deprived PC12 cells. Cells were treated with SNAP (100 μm), 8-Br-cGMP (1 mm), ODQ (40 μm), or KT5823 (180 nm) in serum-free media. After 6 hr for cytochrome c release and 12 hr for caspase-3 activation, cells were harvested and homogenized, and then the cytosolic fraction was prepared by centrifugation at 100,000 × g. Cytochrome c release (A) and caspase activation (B) were determined by Western blot analysis.

Fig. 6.

nNOS gene transfer inhibits apoptotic cell death, cytochrome c release, and DEVDase activity in PC12 cells. Plasmid (pCMV) containing the nNOS or LacZ cDNA was transferred into PC12 cells by the lipofection method. Transfectants were selected using 800 μg/ml G418. Isolated cells were treated with or without NMA (1.5 mm), ODQ (40 μm), and KT5823 (180 nm) in serum-free media. A, Cell viability was determined by counting intact nuclei (mean ± SD;n = 3). B, Cytochrome c release was measured by Western blot. C, DEVDase activity was measured by a colorimetric assay using Ac-DEVD-pNA (mean ± SD;n = 3). *p < 0.01 versus nontreatment.

Fig. 7.

NO donors and 8-Br-cGMP prevent differentiated PC12 cells from NGF withdrawal-induced apoptosis. PC12 cells were differentiated by treating with NFG (50 ng/ml) in RPMI 1640 medium containing 1% horse serum for 7 d. The culture media were switched to serum-free media with or without SNAP (100 μm), DETA/NO (100 μm), or 8-Br-cGMP (1000 μm), and cell viability (A) and DEVDase activity (B) were measured after 72 and 48 hr, respectively. *p < 0.01 versus nontreatment.