Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40 mM K+, or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the alleviation of pain and inflammation, but these drugs are also associated with a suite of negative side effects. Gastrointestinal (GI) toxicity is particularly concerning since it affects an estimated 70% of individuals taking NSAIDs routinely, and evidence suggests the majority of toxicity is occurring in the small intestine. Traditionally, NSAID-induced GI toxicity has been associated with indiscriminate inhibition of cyclooxygenase isoforms, but other mechanisms, including inhibition of cell migration, intestinal restitution, and wound healing, are likely to contribute to toxicity. Previous efforts demonstrated that treatment of cultured intestinal epithelial cells (IEC) with NSAIDs inhibits expression and activity of calpain proteases, but the effects of specific inhibition of calpain expression in vitro or the effects of NSAIDs on intestinal cell migration in vivo remain to be determined. Accordingly, we examined the effect of suppression of calpain protease expression with siRNA on cell migration in cultured IECs and evaluated the effects of NSAID treatment on epithelial cell migration and calpain protease expression in rat duodenum. Our results show that calpain siRNA inhibits protease expression and slows migration in cultured IECs. Additionally, NSAID treatment of rats slowed migration up the villus axis and suppressed calpain expression in duodenal epithelial cells. Our results are supportive of the hypothesis that suppression of calpain expression leading to slowing of cell migration is a potential mechanism through which NSAIDs cause GI toxicity.

Clinical use of non‐steroidal anti‐inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug‐affected signaling pathways that contribute to the toxic side‐effects of NSAIDs are poorly understood. Previous studies have shown that NSAIDs inhibit cell migration in vitro by depolarizing membrane potential and suppressing expression of voltage‐gated potassium channel subunits. Kv1.3, in particular, is known to play an important role in cell migration, and results from a microarray study suggested that its expression is affected by NSAID treatment. Accordingly, we investigated the effects of NSAIDs on Kv1.3 mRNA and protein expression in a rat duodenal cell line (IEC‐6) or the duodenum of whole rats treated with indomethacin or NS‐398 for 72 h. Treatment of IEC‐6 cells with indomethacin or NS‐398 significantly reduced total and plasma membrane protein expression of Kv1.3. In rat duodenum, our results showed that expression of Kv1.3 mRNA was significantly decreased by NS‐398, but not indomethacin, and that Kv1.3 protein expression was significantly decreased following treatment with either NSAID. These results suggest that inhibition of epithelial cell migration by NSAIDs is associated with a decreased expression of Kv1.3 channels. Grant Funding Source: Supported by National Institutes of Diabetes and Digestive and Kidney Diseases (1R15DK091791)