Inhibition of TRPC4 channel activity in colonic myocytes by tricyclic antidepressants disrupts colonic motility causing constipation

Abstract

Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca²⁺ -permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and which mechanism in colonic myocytes consequently causes constipation. To prove the clinical benefit in patients with diarrhoea caused by TCA treatment, we performed mechanical tension recording of repetitive motor pattern (RMP) in segment, electric field stimulation (EFS)-induced and spontaneous contractions in isolated muscle strips. From these recordings, we observed that all TCA compounds significantly inhibited contractions of colonic motility in human. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes. In TRPC4-overexpressed HEK cells, we observed TCA-evoked direct inhibition of TRPC4. Compared with TRPC4-knockout mice, we identified that muscarinic cationic current (mI_cat ) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Collectively, we suggest that TCA action is responsible for the inhibition of TRPC4 channels in colonic myocytes, ultimately causing constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy.

Keywords: TRPC4; irritable bowel syndrome; mIcat; smooth muscle; tricyclic antidepressant.

FIGURE 1

TCA‐induced inhibitory effect on MMC in the human sigmoid colon. In all panels, representative mechanical traces (left) showed that three types of TCAs induced MMCs in the human sigmoid colon at the proximal, middle and distal sites. Compounds were applied at times indicated by bars, and the baseline tension was indicated by dotted lines. The summarized bar graph (right) before (grey) and after (black) TCA treatment. (A) 10 μM AMI (n = 5). (B) 10 μM DES (n = 5). (C) 10 μM IMI (n = 4)

FIGURE 2

TCA‐induced inhibitory effect on EFS‐induced contraction and spontaneous contraction in human colonic muscle strips. (A) Representative traces (left) of EFS‐induced contraction suppressed by 10 μM AMI. Inset traces showing contractile responses to EFS at 16 Hz before (i) and after (ii) application of AMI. Summarized amplitude data (right) on the inhibition rate at 16 Hz (n = 10). (B–E) The representative traces (left) and summarized bar graph (right) of the human sigmoid colonic circular muscle strips before (grey) and after (black) TCA treatment. (B and D) 10 μM AMI (n = 5 of B, n = 5 of D). (C and E) 10 μM DES (n = 5 of C, n = 8 of E) or 10 μM IMI (n = 7 of C, n = 6 of E). (D and E) Pretreatment with 1 μM TTX for 10 min

FIGURE 3

Effect of TRPC4 activation on human colonic muscle contraction. (A) Representative mechanical traces (left) of RMPs evoked by 1 μM EA. Summarized bar graph (right) before (grey) and after (black) EA application (n = 4). (B) Representative traces (left) of EFS‐induced contraction suppressed by 100 nM Pico145. Inset traces showing EFS‐induced amplitude before (i) and after (ii) application of Pico145. Summarized data (right) show the inhibition rate of amplitude (n = 4). (C and D) The representative traces (left) and summarized bar graph (right) before (grey) and after (black) EA application (n = 8 of vehicle, n = 6 of 0.1 μM and n = 8 of 1 μM in C). (D) Pretreatment with 1 μM TTX (n = 8)

FIGURE 4

Inhibition of TRPC4 activity induced by extracellular TCA treatment. (A and C) Representative current trace (left) and current (I)–voltage (V) curve (right) showed TRPC4β current induced by M₃R (A) or M₂R (C) stimulation. Pretreatment with 10 μM AMI for 3 min before 100 μM CCh application. (B and D) Summarized bar graph of current density (pA/pF) by extracellular (left) or intracellular (right) treatment of 10 μM TCAs. (B) External solution (left) (n = 4 of AMI, n = 3 of DES and n = 3 of IMI) and internal solution (right) (n = 4 of vehicle and n = 4 of AMI). (D) External solution (left) (n = 3 of AMI, n = 4 of DES and n = 4 of IMI) and internal solution (right) (n = 5 of vehicle and n = 5 of AMI). (E) Representative current trace (left) and IV curve (middle) of 200 μM GTPγS‐activated current suppressed by 10 μM AMI. The dose‐dependent curve (right) of the inhibition rate depending on the AMI concentration. (F) Summarized bar graph of current density (pA/pF) by intracellular treatment of 10 μM AMI (n = 4 of vehicle, n = 5 of AMI). (G and H) Representative western blot (left) and quantified data of the ratio (right, H) of the PM expression level of TRPC4β‐GFP (n = 4 of 5 min and n = 3 of 16 h)

FIGURE 5

Inhibition of the CCh‐induced TRPC4 current by AMI in murine myocytes. In all panels, representative current trace (left) and IV curves (middle) showing the whole‐cell current in murine sigmoid colonic myocytes evoked by 100 μM CCh. Summarized data (right) showing the current density (pA/pF) at −60 mV. (A) 100 nM Pico145 (n = 5). (B and C) 10 μM AMI (n = 5 of B and n = 5 of C)

FIGURE 6

Restoration of TCA‐induced colonic motility inhibition against TRPC4 activation. Representative trace (left) and summarized bar graph (right) showing AUC induced by 1 μM EA (grey) after TCA treatment (black). (A) RMPs (n = 5). (B–D) Spontaneous contraction pretreated with 1 μM TTX for 10 min. (B) 10 μM AMI (n = 5). (C) 10 μM DES (n = 4). (D) 10 μM IMI (n = 5)