Pharmacological evidence that the activation of the Na(+)-Ca2+ exchanger protects C6 glioma cells during chemical hypoxia.

1. In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium-iodide positive dead cells. 2. Extracellular Na+ removal, which activates the Na(+)-Ca2+ exchanger as a Na+ efflux pathway and prevents Na+ entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells. 3. During chemical hypoxia, in the presence of extracellular Na+ ions, a progressive increase of [Ca2+]i occurred; in the absence of extracellular Na+ ions [Ca2+]i was enhanced to a greater extent. 4. The blockade of the Na(+)-Ca2+ exchanger by the amiloride derivative 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), lanthanum (La3+) and the Ca2+ chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ ions on C6 glioma cells exposed to chemical hypoxia. 5. The inhibition of the Na(+)-Ca2+ antiporter enhanced chemical hypoxia-induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ ions (145 mM), suggesting that the blockade of the Na(+)-Ca2+ antiporter during chemical hypoxia can lead to increased cell damage. 6. Collectively, these results suggest that activation of the Na(+)-Ca2+ exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

BACKGROUND: Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. AIM: To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. METHODS: Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). RESULTS: In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated TNFalpha release. Exposure to TNFalpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta release from endotoxin-stimulated macrophages. CONCLUSIONS: NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.