Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

Abstract

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.

Suppression of calpain expression by NSAIDs is associated with inhibition of cell migration in rat duodenum

“…Our results show that inhibition of calpain 1 or 2 protease expression slows cell migration in IEC-6 cells, and that treatment with NSAIDs suppresses migration of epithelial cells up the villus axis in rat duodenum, an effect that is associated with reductions in the expression of calpain 1 and 2 proteases. Our results provide a potential mechanism through which this suppression of migration may occur (through inhibition of calpain protease expression and/or activity) which is consistent with our other results in vitro and previously published research on the effects of NSAIDs in cultured intestinal epithelial cells (Raveendran et al 2008; Silver et al 2010; Silver et al 2015). …”

“…Most relevant to the current study, previous efforts show that NSAID treatment inhibits calpain protease activity by suppressing total and plasma membrane protein expression and/or depolarizing membrane potential in cultured rat intestinal epithelial cells (IEC-6; Raveendran et al 2008; Silver et al 2010; Silver et al 2015). Furthermore, inhibition of calpain activity by ALLM, or calpain inhibitor II (N-acetyl-L-leucyl-N-[(1S)-1-formyl-3-(methylthio)propyl]-L-leucinamide), results in a dose-dependent inhibition of cell migration in rat IEC-6 cells (Silver et al 2010).…”

“…A crypt-like intestinal cell line, IEC-6 (Quaroni et al 1979), derived from rat duodenal epithelia was grown as previously described (Freeman et al 2007; McCormack and Johnson 2001; Raveendran et al 2008; Silver et al 2012; Silver et al 2010; Silver et al 2015) on culture-treated plastic in DMEM (supplemented with 5% fetal bovine serum, 10 μg/mL insulin, and 50 μg/mL gentamicin) at 37 °C with 5% CO 2 . Only passages 15–20 were used in these experiments.…”

“…Further, mice genetically engineered to silence COX 1 expression are perfectly healthy under normal conditions, not showing any untoward gastric toxicity (Langenbach et al 1995). Though inhibition of COX remains important in evaluating the toxic effects of NSAIDs on GI epithelia, other mechanisms have also been proposed to contribute to GI toxicity that may be independent of inhibition of COX activity, including altering intestinal microflora, decreasing apoptosis, depolarizing membrane potential, uncoupling of oxidative phosphorylation (mitochondrial toxicity), interfering with neutrophil recruitment, and inhibiting cell migration/epithelial restitution (Ashton and Hanson 2002; Freeman et al 2007; Mahmud et al 1996; Pai et al 2001; Penney et al 1995; Raveendran et al 2008; Schmassmann et al 1995; Silver et al 2012; Silver et al 2010; Silver et al 2015; Somasundaram et al 2000). …”

“…In addition to wound healing, cell migration also permits movement of maturing cells from the intestinal crypts, where proliferation occurs, up the long axis of the villi to the apex to replace cells that have undergone apoptosis and sloughed into the lumen (Han et al 1993; Onishi et al 2007; Qi et al 2009; Shibahara et al 1995; Solanas and Batlle 2011; Takeuchi et al 1998, 1999). Cell migration in cultured intestinal epithelial cells (IEC) is inhibited by treatment with NSAIDs with ulcerogenic potential through a variety of affected targets or pathways, including depolarizing membrane potential, inhibiting voltage-gated potassium channel expression, and suppressing calpain protease expression and activity (Freeman et al 2007; Pai et al 2001; Penney et al 1995; Rahgozar et al 2001; Raveendran et al 2008; Silver et al 2012; Silver et al 2010; Silver et al 2015). Interestingly, in these experiments, an NSAID with low ulcerogenic potential, SC-560, had no inhibitory effect on IEC migration.…”