Depolarization and decreased surface expression of K+ channels contribute to NSAID-inhibition of intestinal restitution

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) contribute to gastrointestinal ulcer formation by inhibiting epithelial cell migration and mucosal restitution; however, the drug-affected signaling pathways are poorly defined. We investigated whether NSAID inhibition of intestinal epithelial migration is associated with depletion of intracellular polyamines, depolarization of membrane potential (E(m)) and altered surface expression of K(+) channels. Epithelial cell migration in response to the wounding of confluent IEC-6 and IEC-Cdx2 monolayers was reduced by indomethacin (100 microM), phenylbutazone (100 microM) and NS-398 (100 microM) but not by SC-560 (1 microM). NSAID-inhibition of intestinal cell migration was not associated with depletion of intracellular polyamines. Treatment of IEC-6 and IEC-Cdx2 cells with indomethacin, phenylbutazone and NS-398 induced significant depolarization of E(m), whereas treatment with SC-560 had no effect on E(m). The E(m) of IEC-Cdx2 cells was: -38.5+/-1.8 mV under control conditions; -35.9+/-1.6 mV after treatment with SC-560; -18.8+/-1.2 mV after treatment with indomethacin; and -23.7+/-1.4 mV after treatment with NS-398. Whereas SC-560 had no significant effects on the total cellular expression of K(v)1.4 channel protein, indomethacin and NS-398 decreased not only the total cellular expression of K(v)1.4, but also the cell surface expression of both K(v)1.4 and K(v)1.6 channel subunits in IEC-Cdx2. Both K(v)1.4 and K(v)1.6 channel proteins were immunoprecipitated by K(v)1.4 antibody from IEC-Cdx2 lysates, indicating that these subunits co-assemble to form heteromeric K(v) channels. These results suggest that NSAID inhibition of epithelial cell migration is independent of polyamine-depletion, and is associated with depolarization of E(m) and decreased surface expression of heteromeric K(v)1 channels.

Figure 1

IEC-6 migration in monolayers grown in the absence (Con) or presence of NSAIDs. Drug-treated cells were exposed to either indomethacin (Indo, 100 μM) or SC-560 (1 μM) for 72 h before a defect was created in the monolayer (wounding). Images were captured immediately (0 h) or 4 h after wounding.

Figure 2

Epithelial cell migration in IEC-6 (panel A) and IEC-Cdx2 (panel B) monolayers (n=6 per group) under control conditions (Con), after depletion of polyamines (DFMO, 5.0 mM), or after treatment with the NSAIDs indomethacin (Indo, 100 μM), phenylbutazone (PBZ, 100 μM), SC-560 (1 μM) and NS-398 (100 μM1). Monolayer cultures were exposed to drug for 72 h before wounding. Migration was measured 4 h post-wounding as the maximum linear distance from the created defect. Asterisks indicate significant difference from control (p < 0.05).

Figure 3

Cellular polyamine concentrations of IEC-6 exposed to NSAIDs. Panel A: Polyamine content of IEC-6 cells treated with DFMO (5 mM) in the absence and presence of exogenous spermadine (SPD, 5 μM). Panel B: IEC-6 treated with indomethacin (Indo, 100 μM), phenylbutazone (PBZ, 100 μM), SC-560 (1 μM) and NS-398 (100 μM). Cellular polyamine concentrations were measured by LC-MS at 78 h post-treatment (n= 3). Asterisks indicate significant difference from control (p < 0.05).

Figure 4

Relative E_m of IEC-6 (panel A) and IEC-Cdx2 (panel B) cells under control conditions (Con, n=86), after depletion of polyamines (DFMO, 5.0 mM, n=22 ), or after 72 h treatment with the NSAIDs indomethacin (Indo, 100 μM, n=23), phenylbutazone (PBZ, 100 μM, n=39), SC-560 (1 μM, n=44) and NS-398 (100 μM, n=22). Treatment-induced changes in E_m were detected by monitoring the fluorescence of cells loaded with DiBAC₄(3). Increased fluorescence indicates depolarization of E_m. Different superscripts indicate significant difference between groups (p < 0.05).

Figure 5

Effect of AVE0018 on epithelial cell migration in IEC-Cdx2 cells. Photomicrographs show control (left column) and drug-treated (right column) monolayers immediately (top row) and 4 h (lower row) after wounding. In the graph, epithelial migration is expressed as the percentage of a defined region adjacent to the defect that was occupied by migrating cells 4 h after monolayer wounding in the absence (Con, n=6) and presence of the K_v channel antagonist AVE0118 (1 μM, n=6). Asterisk indicates significant difference (p < 0.05).

Figure 6

K_v1 channel expression by IEC-Cdx2 cells. Immunblots of K_v1.5, K_v1.4 and K_v1.6 channel protein in total cell lysates (Lys), non-biotinylated (NB) and biotinylated (B) fractions obtained from IEC-Cdx2 cells subjected to cell surface biotinylation. After detection of K_v channel protein (upper panel of each set), each blot was stripped and reprobed with antibody against actin to assess loading (lower panel of each set). The apparent molecular masses of K_v1.5, K_v1.4 and K_v1.6 were ~75 kDa, ~96 kDa and ~56 kDa, respectively. The immunoblots shown are representative of n ≥ 3.

Figure 7

Co-association of K_v1.6 and K_v1.4 in IEC-Cdx2. Top: Immunoprecipitation (IP) was performed with antibody against K_v1.4, then the starting material (lane 1), IP (lane 2) and supernatant (lane 3) were immunoblotted (IB) directly with antibody against K_v1.6. Middle: IP was performed with antibody against K_v1.4, then the starting material (lane 1), IP (lane 2) and supernatant (lane 3) were IB with antibody against K_v1.6 pre-incubated with an excess of the fusion protein encoding the epitope used to generate the K_v1.6 antibody. Bottom: IP was performed with antibody against K_v10.1, then the starting material (lane 1), IP (lane 2) and supernatant (lane 3) were IB directly with antibody against K_v1.6. The immunoblots shown are representative of n ≥ 3.

Figure 8

NSAIDs influence the total (panel A), cell surface (panel B) and non-surface (panel C) expression of K_v1.4 by IEC-Cdx2. Monolayer cultures were exposed to drug for 72 h before surface biotinylation and lysis. Drug concentrations were identical to those used in Figures 1–4. Asterisks indicate significant differences (p < 0.05).

Figure 9

The NSAIDs that impair cell migration do not affect the total expression (panel A) of K_v1.6 by IEC-Cdx2; however, these drugs do diminish the cell surface expression (panel B) and increase the non-surface expression (panel C) of Kv1.6. Monolayer cultures were exposed to drug for 72 h before surface biotinylation and lysis. Drug concentrations were identical to those used in Figures 1–4. Asterisks indicate significant differences (p < 0.05).