TCA strongly inhibits the electromechanical activity of human colonic smooth muscle
The prescription of TCAs with high anticholinergic activity exhibits a number of anticholinergic signs and symptoms, such as dry mouth, blurred vision, urinary retention, constipation, and hallucinations (Remick 1988; King and Ashraf 2018). In addition, in TCA overdose, there are likely symptoms that raise suspicion for cardiovascular toxicity, such as arrhythmias and refractory hypotension (Thanacoody and Thomas 2005). Nevertheless, it has been shown to be extraordinarily beneficial to improve global IBS symptoms (Clouse 2003; Ford et al. 2014). However, in the action of TCA for treating IBS by inhibiting GI motility, molecular candidates and the exact mechanism have not been clearly established.
To experimentally prove the clinical benefit in patients with diarrhea by TCA treatment, we isolated normal tissues within human colon specimens from patients with colorectal cancer. As in our previous studies using mechanical tension recording (Ryoo et al. 2014), we first observed whether TCA inhibits the motility of isolated colon segments. The spontaneous generation or propagation of CMMCs is usually evaluated from the frequency and amplitude of contraction without removal of the muscularis mucosae and the inner portion of the submucosa from the colon. By the application of 10 µM TCAs in extracellular Krebs-Ringer solution, all three segments (proximal-to-distal) of the sigmoid colon showed potent inhibition of spontaneous motor activity. As shown in Figure 1A, AMI robustly reduced the amplitude of the proximal (21.20 ± 5.38%), middle (25.81 ± 10.22%) and distal (32.62 ± 8.14%) CMMCs by at least 70% compared to that before exposure to AMI. As mentioned, TCAs possessing a common polycyclic chemical structure [Fig. S1A] are known to exhibit similar biological effects (Moraczewski and Aedma 2021). DES and IMI also significantly reduced the amplitude of spontaneous CMMC (DES; 30.98 ± 5.09% in proximal, 21.45 ± 3.10% in middle, 26.45 ± 5.89% in distal, Fig. 1B, and IMI; 39.82 ± 13.03% in proximal, 36.79 ± 8.30% in middle, 30.07 ± 14.24% in distal, Fig. 1C).
Each ENS, smooth muscle (myocyte), and ICC cell type or all of these cell types might be responsible for the inhibition of spontaneous CMMC generation induced by TCA. To distinguish which cells TCA affects, we isolated the colon muscle strip from CMMCs with mucosa and submucosa removed. To mimic neurogenic contraction, electric field stimulation (EFS; 1-16 Hz, 100 V) induces ENS excitatory muscular motor neurons to release acetylcholine, resulting in cholinergic contraction. As shown in Figure 2A, EFS-induced contractions of the circular muscle myenteric plexus (CMMP) were completely suppressed by AMI. However, the contractile response by DES or IMI was gradually suppressed at the highest frequencies (Figs. S2A-D).
Although a colonic myocyte is the fundamental contractile unit of the colon and eventually myocytes should undergo excitation-contraction coupling, whether neurogenic or myogenic, the functional importance of myocytes on GI motility is often underestimated. To determine whether TCA directly inhibits the electromechanical properties of the myocyte itself, we focused on spontaneous contraction of the circular muscle strip. At a concentration of 10 µM, AMI caused a gradual decrease in the amplitude (34.64 ± 7.59%) of spontaneous contraction with no significant change in frequency compared to the basal level (Fig. 2B). In pretreatment with 1 µM TTX to block neural stimulation, spontaneous contractions before and after AMI treatment showed a similar pattern (28.30 ± 10.63%, Fig. 2D). As shown in the bar graphs of Figures 2C and E, DES and IMI also suppressed the amplitude (37.63 ± 2.83% of DES, 37.49 ± 8.40% of IMI, 26.11 ± 4.55% of DES in TTX pretreatment, 35.23 ± 5.75% of IMI in TTX pretreatment) (Figs. S2C-F).
The results of Figures 1 and 2 indicated that the spontaneous contraction of human CMMCs and CMMPs was strongly inhibited by TCAs, suggesting that the clinical effects of TCA-induced constipation and IBS treatment with TCA are supported through our experimental findings.
TRPC4 channels closely contribute to the regulation of human colonic smooth muscle contraction
We next attempted to identify the molecular candidate of TCA that inhibits colonic motility. Numerous studies have confirmed that TRPC4 channels in intestinal SMCs are gated by muscarinic receptors (Lee et al. 2005) and approximately 80% of mIcat are mediated by TRPC4 activity (Tsvilovskyy et al. 2009). To determine the functional role of TRPC4 in GI motility using its pharmacological agonist or antagonist, we investigated spontaneous CMMC activity and EFS-induced contractile activity as observed above. As shown in Figure 3A, 100 nM Englerin A (EA), a potent and selective activator of TRPC4, significantly increased the tonic contraction of proximal-to-distal CMMCs. These sustained (tonic) contractions could result from smooth muscle (Webb 2003), suggesting that TRPC4 has considerable potential for the depolarization of colonic myocytes. Tsvilovskyy et al. previously suggested that TRPC4 is indirectly activated by acetylcholine involved in neurogenic contraction (Tsvilovskyy et al. 2009). To rule out a contribution of TRPC4 to the neurogenic contraction of CMMPs, EFS-induced contraction was compared in the absence or presence of Pico145 (a remarkable inhibitor of TRPC4). Pico145 (100 nM) was slightly suppressed by approximately 30% at the highest (16 Hz) frequencies (Fig. 3B). Additionally, in the circular smooth muscle strip, Pico145 caused a substantial decrease in the frequency of spontaneous contraction rather than an amplitude of nearly half (Fig. S3A). Conversely, EA dramatically increased the amplitude only of spontaneous contraction (Fig. 3C). Even if TTX was pretreated, the increased contraction to EA was not altered (Fig. 3D).
These results indicated that blockade of the TRPC4 channel induces atrophy not only in ENS-mediated contractions but also in smooth muscle activation. Functional TRPC4 in neurogenic contraction should not be overlooked, but given that the dominant role of TRPC4 in the reports thus far is considered primarily to activate depolarization of intestinal myocytes, it is therefore considered to predominantly act to activate SMCs. These findings and suggestions indicated that TRPC4 is an essential determinant of colonic myocyte contraction causing intestinal motility. Thus, TRPC4 seems to be a reasonable candidate as a molecular target of TCA-induced constipation and IBS treatment with TCA.
Tca Evokes Direct Extracellular Inhibition Of Trpc4 Channel Activity
To investigate the electrical properties of the TRPC4 channel induced by TCA, we conducted patch clamp recordings in TRPC4-overexpressing HEK293 cells. As mentioned above about the relevance of TRPC4 to altered electromechanical activity in colonic contraction induced by TCA, we expected that TCA inhibits TRPC4 channel activity. Since the stimulation of the muscarinic acetylcholine receptor elicits mIcat for initiating cholinergic contraction, we measured the TRPC4 current by coexpression with muscarinic acetylcholine receptor type 2 (M2R) and type 3 (M3R), which are mainly expressed in smooth muscle (Dresviannikov et al. 2006; Tanahashi et al. 2021). As the Gαq-PLC pathway is a primary activation of the TRPC4 channel, carbamylcholine (CCh) stimulates M3R (Tang et al. 2000; Kim et al. 2012), apparently showing a typical doubly rectifying TRPC4 current by M3R stimulation (Fig. 4A). Pretreatment with 10 µM AMI completely inhibited the CCh-activated TRPC4 current (75.56 ± 12.92 to 1.05 ± 0.25 pA/pF). DES (115.23 ± 15.12 to 3.05 ± 1.00 pA/pF) and IMI (111.77 ± 15.30 to 2.74 ± 1.35 pA/pF) also showed a remarkable inhibition of inward current (Fig. 4B). Our group previously reported that the Gαi2 protein can directly activate the TRPC4 channel (Jeon et al. 2012). When coexpressed with M2R, all TCA compounds completely inhibited the CCh-induced TRPC4 current (AMI; 187.50 ± 24.87 to 1.05 ± 0.25 pA/pF, DES; 180.02 ± 42.91 to 2.74 ± 1.35 pA/pF, and IMI; 212.30 ± 55.77 to 6.41 ± 3.72 pA/pF), similar to M3R (Figs. 4C and D).
Since TCA produces anticholinergic effects, such as constipation, especially in the colon, we asked whether TCA directly inhibits TRPC4 channel activity in smooth muscle. As addressed in our previous experiments, TRPC4 activation with the Cs+ current could be clearly observed with a relatively high Cs+ permeability of TRPC channels (Jeon et al. 2013) when GTPγS in an internal solution is infused and Cs+-rich external solution is perfused. Similar to TRPC4 inactivation by TCAs, even in CCh-evoked activation, AMI significantly inhibited Cs+ current activation by 200 µM GTPγS (Fig. 4C). To assess the potency of TCAs against direct inhibition of TRPC4, we calculated the half maximal inhibitory concentration (IC50) values by applying various concentrations of TCAs. In GTPγS-evoked TRPC4 activity, the IC50 of AMI was approximately 1.51 µM (Fig. 4D), and those of DES and IMI were 5.37 µM and 6.12 µM, respectively (Figs. S4A and B). In contrast to the inhibition of the TRPC4 current by extracellular bath perfusion of TCAs (Figs. 4A, C, and E), intracellular infusions of AMI did not inhibit the current at all compared to vehicle controls (Figs. 4B, D, and F).
A previous report by Dennis et al. suggested that TCA compounds simultaneously block the hERG current and its surface expression by promoting ubiquitination and degradation (Dennis et al. 2011); thus, we needed to validate this possibility on the TRPC4 channel using a surface biotinylation method. Preincubation with TCAs for a short exposure (5 min) or even for over 16 hr overnight did not show any change in the expression level of TRPC4 protein on the plasma membrane or total expression (Figs. 4G and H).
These results indicated that TCA evokes direct extracellular inhibition of the TRPC4 current without changing TRPC4 expression. Therefore, TCA compounds absorbed into the gut have sufficiently negative potential to broadly block TRPC4 functions in intestinal smooth muscle.
TCA remarkably suppresses the m I cat formed by TRPC4 in isolated murine colonic myocytes
It is well defined that mIcat, observed in murine myocytes prominently elicited by a TRPC4-mediated cationic current (Tsvilovskyy et al. 2009; Melnyk et al. 2020). To further clarify whether TCA blocks the mIcat of the colonic myocyte response to CCh, we prepared myocytes from murine sigmoid colon tissue by enzymatic isolation following our previous procedure (Choi et al. 2006). Under the optimized conditions of TRPC4 recording similar to that of TRPC4-overexpressing HEK cells, the mIcat from a single myocyte was recorded. We ensured that the newly established data recorded in colonic myocytes met the following standards (Figs. 5A and S5A): (1) the current-voltage relationship (I-V curve) of the CCh-evoked inward current exhibited a typical doubly rectifying shape of TRPC4. (2) The selective and potent antagonist of TRPC4, Pico145, completely blocked the CCh-evoked current. (3) In colonic myocytes obtained from TRPC4-deficient mice, mIcat was not observed. To determine whether TCA suppresses the mIcat of colonic myocytes, we perfused TCA before or after the CCh-evoked current. As shown in Figure 5B, AMI substantially inhibited the mIcat, which responded to CCh, to the basal current level. The mIcat of TRPC4-deficient colonic myocytes was not evoked by CCh at all, and interestingly, the basal current was not further suppressed by AMI (Fig. 5C).
The following experiment was designed to evaluate whether TCA-induced myocyte inactivation could be improved by modulating TRPC4 activity as a therapeutic approach for constipation. As shown in Figure 6A, potent inhibition of spontaneous CMMC activity with reduced amplitude and frequency was rescued by TRPC4 activation with 10 nM EA. The higher concentration of 100 nM EA led to tonic contractions with a cumulative response in partial frequency recovery of the proximal (21.20 ± 5.38%), middle (25.81 ± 10.22%) and distal (32.62 ± 8.14%). Even under TTX pretreatment, the amplitude of spontaneous contraction, which was reduced by 15.28 ± 2.58% in AMI, was restored to 49.03 ± 8.84% by EA (Fig. 6B). In addition, EA significantly improved the spontaneous contractions suppressed by DES and IMI (37.57 ± 4.95 to 66.19 ± 7.98% of DES, 27.68 ± 5.82 to 78.71 ± 15.44% of IMI, Figs. 6C and D) compared to those without TTX (Figs. 6E-G).
These results of Figure 5 indicated that the mIcat suppressed by TCA is ultimately responsible for the inhibition of TRPC4 channels expressed in colonic myocytes. The results of Figure 6 mimicking the therapeutic evaluation of TCA-induced constipation and IBS-D indicated that colonic motility atrophied by TCA was improved by the restoration of TRPC4 activity.