Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

Abstract

Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCα-dependent pathway.

Keywords: CFTR; KCNQ1 K+ channels; T84 cells; berberine; chloride secretion; colon ion transport.

Figure 1

Effect of berberine on forskolin-induced Cl⁻ secretion. (A) Representative short-circuit current recording of the effect of berberine (300 μM) on forskolin-stimulated I_SC in T84 cell monolayers. Drugs were added at arrow and remained in the bathing solutions throughout the experiment (B) Concentration-response curve for the antisecretory effect of berberine on forskolin-stimulated I_SC in T84 cell monolayers. Change in I_SC measured 15 min post berberine application at different concentrations. Values are mean ± SEM n = 8 for each point.

Figure 2

Effect of berberine on basolateral K⁺ current (I_K) across apically permeabilized T84 cell monolayers. (A) Effect of KCNQ1 channel inhibitors on I_K in T84 cell monolayers. Amphotericin B (10 μM) was added to apical bath after ouabain (100 μM) was added to basolateral bath. BaCl₂ (2 mM), chromanol 293B (10 μM), or HMR-1556 (500 nM) were added to the basolateral bath. Values were measured at the point of maximal inhibition of I_K by each inhibitor. (B) Short-circuit currents recording of the effect of berberine (300 μM, filled circles) on I_K across T84 cells monolayers compared to a vehicle control (DMSO 0.05%, open circles). Drugs were added at arrow and remained in the bathing solutions throughout the experiment. Values are mean ± SEM, **P < 0.01 compared to controls, n = 6 for each group. (C) The concentration dependence of the inhibitory effect of berberine on basolateral membrane K⁺ current showed a half-maximal inhibition at 150 μM.

Figure 3

(A) Current/voltage relationships illustrating the effect of berberine (100 μM) on the increase in whole-cell current stimulated by forskolin (10 μM) in T84 cells. Traces are control (○), forskolin (■), and forskolin + berberine (▲). Data are mean ± SEM (*P < 0.01 between the forskolin-stimulated current and berberine, n = 7). (B) Current/voltage relationships illustrating the effect of chromanol 293B (10 μM) on the increase in whole-cell current stimulated by forskolin (10 μM) in T84 cells. Traces are control (○), forskolin (■), and forskolin + chromanol 293B (▲). Data are mean ± SEM (*P < 0.01 between the forskolin-stimulated current and chromanol 293B, n = 3).

Figure 4

Berberine effect on PKC isoforms and PKA activity in T84 cells. (A) Representative blot of PKCα phosphorylation levels at Ser⁶⁵⁷ in cellular extracts from T84 cells after berberine treatment (300 μM) for 5 and 10 min compared to vehicle controls. (B) Representative blot of PKCδ phosphorylation levels at Ser⁶⁴³ in cellular extracts from T84 after berberine treatment (300 μM) cells for 5 and 10 min compared to vehicle controls. Beta-actin was used as an internal control for protein loading. The graphs represent densitometric analysis of PKC blots or PKA activity assays. Values are given as fold changes in PKC phosphorylation (activation) or PKA activity respect to an untreated control. Values are displayed as mean ± SEM (n = 3 for each group, **P < 0.01). (C) Representative blot of PKA phosphorylation levels in cellular extracts from T84 after berberine treatment (300 μM) cells for 10 min compared to vehicle controls and cells pretreated for 10 min with 100 μM HBDDE, a selective PKCα inhibitor.

Figure 5

Berberine effect on MAPK signaling in T84 cells. (A) Representative blot of p38 MAPK phosphorylation levels at Thr¹⁸⁰/Tyr¹⁸² in cellular extracts from T84 cells after berberine treatment (300 μM) for 5 and 10 min compared to vehicle controls. (B) Representative blot of p42/p44 MAPK phosphorylation levels at Thr²⁰²/Tyr²⁰⁴ in cellular extracts from T84 cells after berberine treatment (300 μM) for 5 and 10 min compared to vehicle controls. T84 cells were treated with EGF (100 ng·ml⁻¹) for 5 min to stimulate p42/p44 MAPK phosphorylation. PD98059 (20 μM) was added 15 min prior EGF-treatment. B-actin was used as an internal control for protein loading. The graphs represent densitometric analysis of blots. Values are given as fold changes in MAPK phosphorylation (activation) respect to an untreated control. Values are displayed as mean ± SEM (n = 3 for each group, *P < 0.05, ***P < 0.001).

Figure 6

Role of protein kinases on the antisecretory effect of berberine. (A) Short-circuit current recordings in T84 cell monolayers of the effect of protein kinases inhibitors on the antisecretory effect of berberine (300 μM). Cells were pre-incubated for 15 min with HBDDE 100 μM (▲), SB202190 10 μM (○), or vehicle DMSO 0.05% (•). (B) Mean ± SEM (n = 6) change in total I_SC caused by preincubation with several kinase inhibitors on the antisecretory effect of berberine on forskolin-stimulated I_SC. Values are mean ± SEM, n = 6, *P < 0.05, **P < 0.01 compared to controls.

Figure 7

Berberine effect on protein kinase association with KCNQ1 channel. (A) Representative blot of PKCα association with the KCNQ1 channel in response to berberine (300 μM) in T84 cells. (B). Representative blot of PKACI association with the KCNQ1 ion channel in response to berberine (300 μM) in T84 cells. Total KCNQ1 pools were immunoprecipitated from total cellular lysates using an antibody specific to KCNQ1. Associated PKCα and PKAcI were quantified by Western blot analysis. The graphs represent densitometric analysis at specific time points of berberine treatment compared to vehicle controls. Values are given as fold changes in kinase association with KCNQ1 respect to an untreated control. Values are displayed as mean ± SEM (n = 3 for each group, *P < 0.05, ***P < 0.001).

Figure 8

Hypothetical schema of the antisecretory action of berberine in intestinal epithelium. Berberine activates p38 MAPK, PKCα and PKA to phosphorylate the KCNQ1:KCNE3 K⁺ channel causing the channel to become inactive either through closure or removal from the plasma membrane. Inhibition of basolateral K⁺ channel reduces the electrical driving force for Cl⁻ exit across the apical membrane through CFTR and calcium-activated Cl⁻ channels. The receptor transducing the response to berberine may be the estrogen receptor or GPR30/GPCR as previously described for similar antisecretory and rapid protein kinase responses to estrogen in the intestine.