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Br J Pharmacol. 2016 April; 173(8): 1339–1349.
Published online 2016 March 6. doi:  10.1111/bph.13431
PMCID: PMC4940814

Effects of new‐generation inhibitors of the calcium‐activated chloride channel anoctamin 1 on slow waves in the gastrointestinal tract

Abstract

Background and Purpose

High‐throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second‐generation. low MW inhibitors of the calcium‐activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5‐nitro‐2‐(3‐phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors.

Experimental Approach

Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose‐dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves.

Key Results

The efficacy of second‐generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh‐A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh‐A01 in both preparations.

Conclusions and Implications

We have identified several second‐generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.

Abbreviations

CaCCinh‐A01
6‐(1,1‐dimethylethyl)‐2‐[(2‐furanylcarbonyl)amino]‐4,5,6,7‐tetrahydrobenzo[b]thiophene‐3‐carboxylic acid
DIDS
4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid
GI
gastrointestinal
ICC
interstitial cells of Cajal
KRB
Krebs–Ringer buffer
NFA
niflumic acid
NPPB
5‐nitro‐2‐(3‐phenylpropylalanine) benzoate
RMP
resting membrane potential
T16Ainh‐A01
2‐[(5‐ethyl‐1,6‐dihydro‐4‐methyl‐6‐oxo‐2‐pyrimidinyl)thio]‐N‐[4‐(4‐methoxyphenyl)‐2‐thiazolyl]acetamide

Tables of Links

TARGETS
Other ion channels a
CaCC, Ano1 channels
Voltage‐gated ion channels b
L‐type Ca channel, Cav1.x

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (a bAlexander et al., 2015a, 2015b).

Introduction

Pacemaker activity in gastrointestinal (GI) muscles, originally thought to be myogenic in nature, has now been shown to be generated by a specialized population of cells termed interstitial cells of Cajal (ICC). Periodic pacemaker depolarizations that conduct into adjacent smooth muscle cells are termed slow waves and time the frequency and duration of phasic contractions in the GI tract, including segmentation and peristalsis (Sanders et al., 2014). The molecular entity carrying a Ca2 +‐activated Cl current responsible for generation and propagation of slow waves was identified recently. Microarray studies performed to identify prominent gene transcripts in ICC found the transmembrane protein 16A (Tmem16a, now termed Ano1) to be highly expressed in these cells (Chen et al., 2007). Ano1 was found to encode a Ca2 +‐activated Cl channel (CaCC; Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008), and immunohistochemical analyses showed that the protein product of Ano1, anoctamin 1 (Ano1) was highly and exclusively expressed in all populations of ICC throughout the GI tracts of mice, non‐human primates (Macaca fascicularis) and humans (Gomez‐Pinilla et al., 2009; Hwang et al., 2009; Rhee et al., 2011; Blair et al., 2012).

Utilization of a murine model that expresses copepod GFP (copGFP) in ICC (Ro et al., 2010) allowed identification of isolated ICC after enzymatic dispersion of GI muscles. When ICC of the small intestine were studied under voltage‐clamp conditions and stepped to depolarized potentials, they generated large‐amplitude inward currents (Zhu et al., 2009). The reversal potential of these currents shifted in a manner suggestive of a Cl current when the equilibrium potential for Cl (E Cl) was changed from 0 to −40 mV. Large tail currents, suggestive of a Ca2 +‐activated Cl conductance, were apparent when membrane potential was stepped from test potentials back to the holding potential, and analysis of tail currents showed reversal at approximately E Cl. These experiments demonstrated that the large‐amplitude inward currents elicited in freshly dispersed ICC were carried mainly by Cl ions.

To determine the functional role of Ano1 channels in ICC, pacemaker activity was investigated in the stomachs and intestines of mice with Tmem16a (Ano1) genetically deactivated (Tmem16a tm1Bdh/tm1Bdh) (Rock et al., 2008). Slow waves were recorded from gastric and intestinal muscles from all wildtype siblings but were absent in Ano1 / mice (Hwang et al., 2009; Singh et al., 2014). ICC networks appeared to develop normally in Ano1 / mice (Hwang et al., 2009). These findings suggested that Ano1 channels carry an important inward current that is responsible for slow waves.

Gastrointestinal slow waves are sensitive to Cl channel blocking drugs including niflumic acid (NFA) and 4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS) (Hwang et al., 2009). However, these pharmacological agents lack potency and specificity. High‐throughput screening of compound libraries has identified several new inhibitors of Ano1 channels, including, CaCCinh‐A01, T16Ainh‐A01, benzbromarone, hexachlorophene and dichlorophene. In the present study, we have examined the efficacy of these compounds in inhibiting mouse gastric and intestinal slow waves and compared the inhibition with that of a more classical CaCC blocker, 5‐nitro‐2‐(3‐phenylpropylalanine) benzoate (NPPB), in an attempt to find an effective means to block GI pacemaker activity.

Methods

Animals

All animal care and experimental procedures coomplied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Nevada. The animal studies follow the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015). No other methods to perform the described experiments (3Rs) were found.

Mice (C57BL/6; 30–60 days old; 20–30 g) were obtained from Jackson Laboratory, Bar Harbor, MN, USA). Mice were housed in a specific pathogen‐free environment at four adults to a cage in Tecniplast vent racks (Exton, PA, USA). Mice were maintained on a 12/12 h light/dark cycle at 21 ± 2°C with Corn Cob bedding and fed ad libitum on Harlan Teklad food with reverse osmosis filtered water. Mice in this age group were randomly selected for experiments by a laboratory technician, and gender was blinded. Tissues (gastric antrum and small intestine) were dissected after animals of both sexes had been exsanguinated following sedation with isoflurane and cervical dislocation. Tissues were placed in oxygenated Krebs–Ringer buffer (KRB; composition (in mM): NaCl 118.5; KCl 4.5; MgCl2 1.2; NaHCO3 23.8; KH2PO4 1.2; dextrose 11.0; CaCl2 2.4; when bubbled with 97% O2–3% CO2 at 37 ± 0.5°C, the pH of the KRB was 7.3–7.4.) for further preparation.

Electrophysiology

For electrophysiological measurements, antrums and intestines were prepared by first removing the mucosa by sharp dissection. Antrum and ileal muscles (10 × 5 mm) were cut and pinned to the Sylgard elastomer (Dow Corning Corp., Midland, MI, USA) floor of a recording chamber with the longitudinal (antrum) or circular (intestine) muscle facing upwards. GI muscles were restrained using fine diameter (80 μm) tungsten wire. Circular muscle cells were impaled with glass microelectrodes filled with 3 M KCl and having resistances between 80 and 100 MΩ. Transmembrane potentials were measured using a high input impedance amplifier (Axon Instruments/Molecular Devices Corp., Sunnyvale, CA, USA), and outputs displayed on a digital oscilloscope. Electrical signals were digitized using an analogue‐to‐digital converter (Digidata 1300 series; Axon Instruments/Molecular Devices Corp., Sunnyvale, CA, USA), recorded and stored on a computer running axoscope 10.0 software.

Five electrical parameters were analysed: (i) resting membrane potential (RMP); (ii) slow‐wave amplitude; (iii) slow‐wave duration; (iv) slow‐wave frequency; and (v) dV/dt of the upstroke component of slow waves. Dose‐dependent effects of CaCC blockers were determined cumulatively for each compound. IC50 values were calculated for different parameters from each experiment, and the average value was tabulated.

All experiments were performed in the presence of nifedipine. Nifedipine was dissolved in ethanol at a stock concentration of 10 mM before being added to the perfusion solution at a final concentration of 1 μM to inhibit contractile activity and facilitate long‐term cellular impalements.

Data and statistical analysis

The study design and analysis conform to the recent guidance on experimental design and analysis (Curtis et al., 2015). Data are expressed as means ± SEM. One‐way anova and Student's t‐test were used to evaluate any differences, and P values of less than 0.05 were considered a statistically significant difference. ‘n’ values refer to the number of animals used for each independent experimental protocol. Statistical analysis was performed using sigmastat 3.1 (Jandel Scientific Software, San Jose, CA, USA). IC50 values were calculated using a non‐linear regression fit. Figures were created from digitized data using Corel Draw X4 (Corel Corp., Ottawa, Ontario, Canada).

Materials

CaCCinh‐A01, T16Ainh‐A01 and NPPB were obtained from Tocris Bioscience (Bristol, UK). Benzbromarone, dichlorophene, hexachlorophene and (except nifedipine) were obtained from Sigma (St Louis, MO, USA). Drugs were dissolved in DMSO at a stock concentration of 10–100 mM before being added to the perfusion solution at the stated concentration.

Results

Effects of the inhibitors of Ano1 channels on gastric antral muscles

CaCCinh‐A01 has been shown to fully inhibit CaCC in bronchial and intestinal epithelial cells (Namkung et al., 2011). Therefore, we sought to determine whether this compound would also block gastric pacemaker activity. At concentrations between 1 and 3 μM, CaCCinh‐Ano1 caused a significant decrease in the amplitude, frequency, half‐maximal duration of slow waves and dV/dt of the slow‐wave upstroke (Figure 1). For example, CaCCinh‐Ano1 at 3 μM caused a reduction in slow‐wave frequency but did not significantly reduce upstroke dV/dt. CaCCinh‐Ano1 caused a slight depolarization in RMP (−67 ± 3 mV under control conditions and −64 ± 3 mV in 3 μM CaCCinh‐A01; P < 0.05). At 5 μM, CaCCinh‐Ano1 almost abolished gastric slow waves, with a significant reduction in all slow‐wave parameters and only a slight but significant depolarization to −63 ± 2 mV.

Figure 1
(A) Effects of CaCCinh‐A01 on gastric slow waves. CaCCinh‐A01 reduced slow‐wave amplitude, frequency and half‐maximal duration at concentrations between 1 and 3 μM and completely blocked slow waves at 5 μM. ...

Another low MW inhibitor of Ano1 channels, T16Ainh‐A01 (Figure 2), caused a dose‐dependent (1–30 μM) reduction in slow‐wave frequency and half‐maximal duration without a significant decrease in the amplitude of spontaneous antral slow waves. At 30 μM, T16Ainh‐A01 did not change RMP, the slow‐wave amplitude or dV/dt of the slow‐wave upstroke.

Figure 2
(A) T16Ainh‐A01 (1–30 μM) caused a dose‐dependent reduction in slow‐wave frequency and half‐maximal duration without a significant decrease in the amplitude of gastric slow waves. (B–E) Summary ...

The uricosuric compound benzbromarone (1–5 μM) caused a dose‐dependent reduction in gastric antrum slow‐wave parameters and inhibited slow waves at the highest concentration tested (Figure 3). For example, slow‐wave frequency and half‐maximal duration were more sensitive to benzbromarone at lower concentrations (1–3 μM), whereas amplitude was only affected at 3–5 μM. Benzbromarone (5 μM) did not change membrane potential but completely inhibited slow waves.

Figure 3
(A) Benzbromarone (1–5 μM) caused a dose‐dependent reduction in gastric antrum slow‐wave parameters and inhibited slow waves at the highest concentration tested. Both frequency and half‐maximal duration ...

Hexachlorophene (1–30 μM) produced a dose‐dependent inhibition in antral pacemaker activity, causing a partial inhibition in slow‐wave amplitude and frequency at 3 μM and displayed a dose‐dependent increase in slow‐wave blockade up to 30 μM (Figure 4). Slow‐wave amplitude; half‐maximal duration, frequency and dV/dt of the upstroke were reduced at a concentration of 10 μM. Hexachlorophene also caused a slight but significant depolarization in RMP from −62 ± 1 to −58 ± 1 mV. At a concentration of 30 μM, hexachlorophene caused a further and more significant reduction in all slow‐wave parameters.

Figure 4
(A) Effects of hexachlorophene on gastric pacemaker activity. Hexachlorophene showed a dose‐dependent inhibition of antral pacemaker activity, causing a partial inhibition in slow‐wave amplitude and frequency at 3 μM and ...

The final Ano1 channel inhibitor that was identified by high‐throughput screening was the antimicrobial agent, dichlorophene. Dichlorophene (1–30 μM) did not affect RMP but caused a significant reduction in amplitude, frequency and half‐maximal duration of slow waves. The inhibitory effects of dichlorophene were initially observed between 3 and 5 μM and were maximal at 30 μM (P < 0.05; Figure 5). Dichlorophene (30 μM) also decreased dV/dt of the slow‐wave upstroke.

Figure 5
(A) Effects of dichlorophene on antral slow waves. Dichlorophene inhibited slow waves at 30 μM but caused a reduction in amplitude, frequency and half‐maximal duration at 10 μM. (B–E) Summary of the effects ...

As a comparison, we also tested a more classical CaCC inhibitor, NPPB. Similar to the agents tested above, NPPB also caused a dose‐dependent reduction in slow‐wave amplitude, frequency, half‐maximal duration and dV/dt of the upstroke (Figure 6). There was a marked reduction in frequency at concentrations of 3 μM and greater and almost complete inhibition of slow waves at concentrations of 10 μM and greater. NPPB caused depolarization in membrane potential from −72 ± 1.3 to −66 ± 2.1 mV at 10 μM (n = 5; P < 0.05). The effects of Ano1 channel inhibitors on gastric slow‐wave parameters are summarized in Supporting Information Table S1. IC50 values are summarized in Supporting Information Table S2.

Figure 6
(A) Effects of NPPB on gastric slow waves. (A) NPPB caused a marked reduction in slow‐wave amplitude, frequency and half‐maximal duration at 10 μM, and slow waves were inhibited at 30 μM. (B–E) Summarized ...

Effects of Ano1 channel inhibitors on intestinal muscles

We have previously shown that intestinal muscles were less sensitive to the CaCC blockers, NFA and DIDS, suggesting the possibility that different Ano1 protein splice variants may be functional in these organs (Hwang et al., 2009). Therefore, we sought to determine whether the pacemaker activity in the stomach and the small intestine also displayed a difference in sensitivity to the identified second‐generation inhibitors.

Cumulative addition of CaCCinh‐A01 caused a dose‐dependent inhibition of intestinal slow waves (Figure 7). For example, slow waves were reduced in amplitude and half‐maximal duration at 10 μM CaCCinh‐A01. At 30 μM, CaCCinh‐A01, slow waves were further reduced in amplitude and half‐maximal duration. Slow‐wave frequency and dV/dt were also reduced. RMP remained unchanged at concentrations up to 10 μM but depolarized slightly, but not significantly, at 30 μM.

Figure 7
(A) Effects of CaCCinh‐A01 on intestinal pacemaker activity. CaCCinh‐A01 reduced intestinal slow‐wave amplitude, frequency and half‐maximal duration at concentrations above 5 μM and almost blocked slow waves ...

T16Ainh‐A01 caused only a small, dose‐dependent (1–30 μM) reduction in slow‐wave amplitude, frequency and half‐maximal duration (Figure (Figure8).8). At the highest concentration tested (30 μM), T16Ainh‐A01 did not significantly affect RMP, frequency or dV/dt of the upstroke but did slightly reduce half‐maximal duration.

Figure 8
(A) T16Ainh‐A01 (1–30 μM) was relatively insensitive at inhibiting intestinal slow waves. At 30 μM, there was little effect on slow‐wave amplitude, frequency and half‐maximal duration. (B–E) ...

The small intestine was also less sensitive to benzbromarone (Figure 9). For example, at 5 μM, when pacemaker activity was blocked in the antrum, slow waves still persisted in the intestine at a reduced level and eventually disappeared at 10 μM. Slow‐wave frequency and half‐maximal duration were also relatively insensitive to concentrations up to 5 μM. In 10 μM benzbromarone, intestinal slow waves were further reduced in frequency, half‐maximal duration and upstroke dV/dt. RMP was not significantly affected at concentration up to 10 μM.

Figure 9
(A) Benzbromarone (1–10 μM) caused a dose‐dependent reduction in intestinal slow waves. Slow‐wave amplitude was most sensitive to benzbromarone with minimal effects on frequency and half‐maximal duration ...

Hexachlorophene and dichlorophene also showed a similar tendency to resistance to inhibition of intestinal slow waves. Hexachlorophene had little effect on RMP until 10 μM, which caused a slight but non‐significant depolarization. Hexachlorophene caused a dose‐dependent (3‐10 μM) reduction in the amplitude of slow waves (Figure 10). Slow‐wave frequency was relatively insensitive and was not significantly changed at 3 μM or 10 μM. Small slow‐wave oscillations (approx. 1 mV) persisted in 30 μM hexachlorophene (P < 0.05). At 10 μM, the half‐maximal duration was also only slightly decreased, with greater effects on the upstroke dV/dt.

Figure 10
(A) Effects of hexachlorophene (1–30 μM) on intestinal pacemaker activity. Hexachlorophene showed a dose‐dependent inhibition of slow waves, causing a reduction in slow‐wave amplitude at 3 μM but ...

Dichlorophene caused an initial hyperpolarization in RMP from −65 ± 2 in control conditions to −71 ± 1 mV in 10 μM (P < 0.05) but subsequently slightly depolarized back to control levels in 30 μM dichlorophene (i.e. −66 ± 2 mV). Dichlorophene also showed a dose‐dependent reduction in slow‐wave amplitude at concentrations above 5 μM, i.e., at 10 μM and 30 μM (Figure 11). Slow‐wave frequency was relatively insensitive and was significantly reduced only at 30 μM; at this highest concentration, slow waves still persisted. The half‐maximal duration was significantly reduced at concentrations from 5 ‐30 μM. Dichlorophene also significantly decreased dV/dt of the slow‐wave upstroke at 10 μM and 30 μM (Figure 11).

Figure 11
(A) Effects of dichlorophene on intestinal slow waves. Dichlorophene inhibited slow‐wave amplitude at concentrations of 5 μM but had little effect on frequency until slow waves were almost abolished. (B–E) Summary of the ...

Similar to second‐generation inhibitors, NPPB was also relatively ineffective in blocking intestinal slow waves compared with those in the gastric antrum. In the small intestine, NPPB caused a slight and not significant membrane hyperpolarization at 3 μM and 10 μM. Slow‐wave amplitude was not significantly reduced at concentrations up to and including 30 μM NPPB (Figure 12). Likewise, intestinal slow‐wave frequency and half‐maximal duration were also insensitive to NPPB, being only slightly but significantly reduced at 30 μM. The dV/dt of the slow‐wave upstroke was significantly decreased only at 30 μM ( Figure 12). The effects of the Ano1 channel inhibitors on intestinal slow‐wave parameters are summarized in Supporting Information Table S1. IC50 values are summarized in Supporting Information Table S2.

Figure 12
(A) Insensitivity of intestinal slow waves to NPPB. NPPB caused a reduction in slow‐wave amplitude but only at concentrations greater than 10 μM. Even at 30 μM, there was little effect on slow‐wave frequency ...

Discussion and conclusion

This study shows that several second‐generation CaCC inhibitors are effective in blocking slow waves and that some of these compounds were more effective at inhibiting pacemaker activity than the more traditional CaCC blockers such as NPPB. Further, there were significant differences in the way that these compounds inhibited slow waves. Finally, there were significant differences in the sensitivity of slow waves in the stomach and small intestine, to these agents.

The CaCC are widely distributed in various organs and are involved in a wide range of physiological functions including epithelial secretion of mucin, electrolytes and water (Ousingsawat et al., 2009; Huang et al., 2012), neuronal and cardiac excitability (Lee et al., 2014, sensory transduction (Cho et al., 2012; Takayama et al., 2015), regulation of visceral muscle activity and vascular tone (Dixon et al., 2012; Forrest et al., 2012; Davis et al., 2013; Heinze et al., 2014) and GI pacemaker activity (Hwang et al., 2009; Zhu et al., 2009; Sanders et al., 2012; Singh et al., 2014). CaCC are also expressed in a variety of tumours, including GI stromal tumours where it was originally termed DOG1 (West et al., 2004; Espinosa et al., 2008; Miettinen et al., 2009), breast cancer (Britschgi et al., 2013) and head and neck cancer (Wanitchakool et al., 2014). These channels are thought to be the primary driver of the ‘grow or go’ model for cancer progression, in which expression of Ano1 channels acts to balance tumour proliferation and metastasis via methylation of its promoter (Shiwarski et al., 2014).

Identification of specific antagonists is valuable for determining the role of ion channels physiologically. Since the discovery that Tmem16a encoded a CaCC, now termed Ano1, there was a significant drive to develop selective inhibitors of this channel, especially because it has recently been reported that inhibition of Ano1 transcription and Ano1 inhibitors can suppress growth and invasion, thus inhibiting cancer progression (Liu et al., 2012; Britschgi et al., 2013). CaCC are known to be blocked by a variety of agents including DIDS, NFA, NPPB, flufenamic acid and tamoxifen (Eggermont, 2004). To date, however, few specific inhibitors are available for CaCC. Most require high concentrations to completely block CaCC channels and also have significant side‐effects on other ion channels (Hartzell et al., 2005).

In an attempt to identify novel inhibitors of CaCC, several high‐throughput screens of compound libraries have been performed (Namkung et al., 2011; Huang et al., 2012). The first of these screens included ~100 000 synthetic small molecules and ~7500 purified natural products and identified four novel chemical classes of Ano1 inhibitors including CaCCinh‐A01 and T16Ainh‐A01 (Namkung et al., 2011). CaCCinh‐A01 totally inhibited CaCC current in human bronchial and intestinal epithelial cells, whereas T16Ainh‐A01 inhibited total CaCC in these cells poorly but, in salivary gland epithelia cells, blocked an initial agonist‐stimulated, transient, chloride current and fully blocked a CaCC current (Namkung et al., 2011). In a second high‐throughput screen, three low MW inhibitors and three additional inhibitors of Ano1 channels were identified from an initial library screen of 2000 compounds. The assay in this screen is based on a genetically encoded YFP I ion flux assay. I ions are typically found in low intracellular concentrations and have few intrinsic exchangers or co‐transporters in HEK293 cells. Therefore, the ability of I ions to be transported through Cl channels into the cell allows the entry of I ions to serve as a surrogate for Cl ion flux. The effect of putative channel blockers can be quantified via a genetically encoded fluorescent I ion biosensor (YFP‐H148Q/I152L). HEK293 cells with stably expressed TMEM16A (mCherry tagged) and the YFP I ion biosensor show a bright basal YFP fluorescence. When exposed to ionomycin to increase intracellular calcium concentration, elevated Ano1 channel activity leads to I influx, resulting in quenching of the YFP fluorescence. Benzbromarone, hexachlorophene and dichlorophene had IC50 values of 10, 9.97 and 5.49 μM, compared with NPPB, which had an IC50 value of 150 μM in this assay (Huang et al., 2012).

We utilized these five second‐generation compounds to determine their efficacy in inhibiting gastric and intestinal pacemaker activity. We also compared these agents with the classical CaCC blocker, NPPB and previously published findings with NFA (Hwang et al., 2009). We found an order of potency on slow‐wave amplitudes of CaCCinh‐A01 ≥ benzbromarone ≥ hexachlorophene ≥ NPPB ≥ dichlorophene ≥ NFA ≥ T16Ainh‐A01 in gastric tissues. Similar order of potencies was also observed for slow‐wave frequency, 1/2 duration and dV/dt of the slow‐wave upstroke. Interestingly, in intestinal tissues, there was also a similar order of potency to that observed for gastric slow waves, that is, benzbromarone ≥ hexachlorophene ≥ dichlorophene ≥ CaCCinh‐A01 ≥ NFA ≥ NPPB ≥ T16Ainh‐A01, but the concentrations of all compounds that were needed to block slow waves were significantly greater. The IC50 value for NFA was much higher than that of several of the second‐generation blockers and, even at the highest concentration used, T16Ainh‐A01, and NPPB did not block intestinal slow waves completely (Supporting Information Tables [Link], [Link]).

We have previously reported a similar difference in the concentration–response relationships in gastric versus small intestine slow waves with the CaCC blockers NFA and DIDS in mouse, monkey and human GI muscles (Hwang et al., 2009). Further, there was also a relative lack of inhibition of slow‐wave amplitude and duration until slow waves were nearly blocked. Concentration–response data from Ano1 channels expressed in HEK cells suggest a greater than 90% inhibition of currents by 10 μM NFA and DIDS (Yang et al., 2008). Murine gastric slow waves were far more sensitive to NFA (Supporting Information Tables S2) and DIDS (IC50 = 150 μM) than intestinal slow waves (Supporting Information Tables S2 for NFA and 1368 μM for DIDS; Hwang et al., 2009) but never matched the sensitivity of Ano1 channels in HEK cells. All gastric slow‐wave parameters (amplitude, frequency, duration and upstroke dV/dt) were similarly sensitive to CaCC inhibitors, whereas intestinal slow‐wave amplitude and upstroke dV/dt were more sensitive to benzbromarone, hexachlorophene and dichlorophene than other parameters. These data suggest that CaCC activity may be greatest during the early phases of pacemaker activity. Three of the CaCC inhibitors (CaCCinh‐A01, hexachlorophene and NPPB) caused slight membrane depolarization in gastric tissues and two (CaCCinh‐A01 and hexachlorophene) in intestinal tissues. These data suggest that CaCC may contribute to RMP and that different CaCC blockers could be more effective at more negative membrane potentials. Alternatively, there may be non‐specific effects of these agents, especially at the higher concentrations tested.

Because of the sensitivity of gastric compared with intestinal slow waves to second‐generation CaCC inhibitors, it could be concluded that gastric slow waves are much more dependent on Ano1 channel activity than intestinal slow waves. However, slow waves were absent in both stomachs and intestines of Tmem16a or Ano1 / mice, demonstrating the importance of this channel protein in the generation of slow waves in both organs (Hwang et al., 2009). Further, we have also demonstrated that in isolated intestinal ICC, spontaneous inward currents and voltage oscillations were inhibited by low concentrations of NFA (<10 μM), revealing the potency of this inhibitor on Ano1 channels in isolated intestinal ICC.

CaCCinh‐A01, dichlorophene, hexachlorophene and NPPB all caused a significant reduction in slow‐wave amplitude and eventual frequency before slow waves were inhibited. Benzbromarone only had a slight decrease in frequency before complete block of slow waves in the stomach but inhibited the amplitude of slow waves in the small intestine before complete blockade. Likewise, there were differences in the degree of inhibition in the rate‐of‐rise of the upstroke component of slow waves. The differences in the way these agents inhibited slow waves suggest different sites of actions of these agents. Reduction in the rate‐of‐rise of the upstroke component of gastric and intestinal slow waves suggests that the Ano1 channel conductance contributes to the early phases of the slow wave. It is also possible that Ano1 channels may be important for the initiation of slow waves, but additional currents such as voltage‐dependent calcium currents may play a more significant role in intact muscle preparations for the propagation of slow waves in ICC networks of the small intestine versus stomach. A further complication is involvement of currents generated in electrically coupled smooth muscle cells that express different ionic conductances in the stomach versus small intestine that contribute to the overall excitability of GI muscles. Further studies are necessary to resolve the discrepancies in the pharmacology and sensitivity of pacemaker activity to Ano1 channels in different parts of the GI tract. Additional experiments will determine if the efficacy of these compounds on inhibiting Ano1 channels is additive.

It is possible that these recently identified Ano1 inhibitors also have side effects, apart from those on Ano1 channels. For instance, T16Ainh‐A01 and CaCCinh‐A01 caused relaxation of pre‐constricted resistance arteries and this relaxation was associated with membrane hyperpolarization. However, these responses occurred with or without extracellular chloride. T16Ainh‐A01 also inhibited nifedipine‐sensitive L‐type calcium currents (carried by barium) expressed in A7r5 cells (Boedtkjer et al., 2015). In the present study, both gastric and intestinal pacemaker activity was relatively insensitive to T16Ainh‐A01 (Figures 2, ,8)8) but were inhibited by CaCCinh‐A01. Mouse gastric and intestinal slow waves persist when L‐type calcium channels are inhibited by nifedipine (Ward et al., 1994; Hirst et al., 2002). To rule out side effects on L‐type calcium channels, we performed all our present experiments in the presence of nifedipine (1 μM), and, therefore, it is unlikely that inhibition of pacemaker activity by Ano1 channel inhibitors involved this side effect observed in the resistance arteries.

In conclusion, we have identified inhibitors of the CaCC Ano1 that block pacemaker activity in the gastric antrum and small intestine, further supporting the role of CaCC in the generation of slow waves in the GI tract. The efficacy of CaCCinh‐A01 and benzbromarone, inhibiting slow waves completely at concentrations less than 5 μM, makes these agents useful in analysing the importance of CaCC in pacemaker activity in the GI tract and other visceral organs from animals and from humans. These second‐generation CaCC inhibitors may also prove useful in the treatment of a variety of wide‐ranging visceral and vascular disorders including hypertension or as tocolytics in uterine contractility. The use of GI muscles to evaluate CaCC inhibitors could prove to be useful for screening potential therapeutic agents for the treatment of tumours or cancers.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1. Summary of the effects of second‐generation CaCC inhibitors on gastric and intestinal RMP and slow wave parameters.

Table S2. Summary of IC50 of second‐generation CaCC inhibitors on gastric antrum and small intestine slow wave parameters compared to NPPB and NFA.

Supporting info item

Supporting info item

Acknowledgements

This work was supported by NIH RO1 DK‐57236 to S.M.W. and P01 DK‐41315 to S.M.W. and K.M.S.

Notes

Hwang S. J., Basma N., Sanders K. M., and Ward S. M. (2016) Effects of new‐generation inhibitors of the calcium‐activated chloride channel anoctamin 1 on slow waves in the gastrointestinal tract. British Journal of Pharmacology, 173: 1339–1349. doi: 10.1111/bph.13431.

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