Heme oxygenase-1 protects gastric mucosal cells against non-steroidal anti-inflammatory drugs.

Gastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAIDs) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions.

Low direct cytotoxicity of nabumetone on gastric mucosal cells.

Prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for clinical purposes because they are not harmful to the gastrointestinal mucosa. We recently showed that NSAIDs have direct cytotoxicity in NSAID-induced gastric lesions. We show here that under conditions where the NSAIDs indomethacin and celecoxib clearly induce cell death, an NSAID prodrug, nabumetone, and its active metabolite 6-methoxy-2-naphthylacetic acid (6MNA), did not have such effects. Moreover, nabumetone and 6MNA exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We recently reported that when an orally administered NSAID was used in combination with a low dose of intravenously administered indomethacin, the severity of gastric lesions produced in rats depended on the cytotoxicity of the orally administered NSAID. Using a similar protocol, we show here that gastric lesions were produced when the orally administered NSAID was celecoxib, but not when nabumetone was used. We thus propose that the low direct cytotoxicity of nabumetone observed in vitro is maintained in vivo, and that the use of nabumetone does not harm the gastric mucosa.

Involvement of intracellular Ca2+ levels in nonsteroidal anti-inflammatory drug-induced apoptosis.

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells.

Membrane permeabilization by non-steroidal anti-inflammatory drugs.

The cytotoxicity of non-steroidal anti-inflammatory drugs (NSAIDs) is involved in the formation of NSAID-induced gastric lesions. The mechanism(s) behind these cytotoxic effects, however, is not well understood. We found here that several NSAIDs tested caused hemolysis when employed at concentrations similar to those that result in cytotoxicity. Moreover, these same NSAIDs were found to directly permeabilize the membranes of calcein-loaded liposomes. Given the similarity in NSAID concentrations for cytotoxic and membrane permeabilization effects, the cytotoxic action of these NSAIDs may be mediated through the permeabilization of biological membranes. Increase in the intracellular Ca(2+) level can lead to cell death. We here found that all of NSAIDs tested increased the intracellular Ca(2+) level at concentrations similar to those that result in cytotoxicity. Based on these results, we consider a possibility that membrane permeabilization by NSAIDs induces cell death through increase in the intracellular Ca(2+) level.