Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

Abstract

The basolateral Na ؉ -K ؉ -2Cl ؊ cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (␣, ␦, ⑀) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ϳ90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ-and shPKC␣-T84 cells, whereas the effect of PMA was not observed in shPKC␦-and shPKC⑀-cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMPstimulated chloride secretion in the uninfected, shLacZ-and shPKC␣-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKC␦-and shPKC⑀-T84 monolayers. In conclusion, the activated novel isoforms PKC␦ or PKC⑀, but not the conventional isoform PKC␣, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

Canonical Bcl-2 Motifs of the Na⁺/K⁺ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?

“…3) suggesting that NKCC1 inactivation is at variance with that of NKP. Unlike reports for TPA [45,68], NKCC1 remained in the membrane of CET-treated HLECs as tested by biotinylation experiments (data not shown). The cellular K + content data suggest that CET inhibition of NKP did not cause K + and probably Na + gradients to collapse and thus indirectly or secondarily inhibiting NKCC1, during the experimental time (20 min CET exposure and 10 min flux time).…”

Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1

“…1 suggested either that capsaicin blocked the FSK-I sc by acting on enteric nerves or directly on the epithelial cells responsible for chloride secretion, or both. To test the effect of capsaicin in a pure epithelial cell preparation, T84 cells, a well-established human colonic cryptlike cell line widely used to study chloride secretion (3,12,58), was used to test the effect of basolateral and apical capsaicin.…”

“…Basolateral biotinylation was performed as previously described (11,21,58). Briefly, T84 cells grown to confluence on Transwell were treated with vehicle (DMSO), or with 20 M capsaicin (applied either to the apical or basolateral side) for 1 h at 37°C in HPBSS.…”

“…The short-circuit current (I sc) in T84 cell monolayers was measured as previously described (58). Briefly, the I sc was defined as the external current needed to nullify the spontaneous transepithelial voltage (Vt 0), with positive values meaning anion flowing from the basolateral to the apical chamber.…”

“…Because excessive activation of chloride secretion underlies diarrheal disorders, substances that inhibit this process are of interest for targeted pharmacotherapy. Inhibition of chloride secretion can be accomplished at different levels such as blockade of apical chloride channels (43), blockade of basolateral potassium channels (38,39) and internalization of NKCC1 (11,58). Various natural compounds produced by luminal bacteria such as short-chain fatty acids and ammonium ions can inhibit chloride secretion (47,66).…”