Experimental evaluation of the nonselective and selective TMEM16 family calcium-activated chloride channel blockers in the airways

TMEM16A (anoctamin-1) and TMEM16B (anoctamin-2) are the protein subtypes belonging to the group of transmembrane protein 16, a subclass of the calcium-activated chloride channel (CaCC) family. They differ in structure, affinity to intracellular calcium ([Ca²⁺]_i), and activation and deactivation kinetics. TMEM16A is widely expressed and involved in the functions of many cells, particularly epithelial secretory cells, found throughout the body. Among its implications is regulation of the processes involved in the progression and worsening of airway inflammation, contractions, secretion, mucus metaplasia, and consequential remodeling. Therefore, its dysfunction and possible manipulation may be significant factors in the development of airway inflammatory diseases (1).

The variety of signaling pathways influences the expression of TMEM16A, for example, the interleukins (ILs) released from Th₂ cells during inflammation. At the cellular level, these proinflammatory molecules activate the transcription factors JAK/STAT6 pathways. STAT6 binds to the TMEM16A promoter region and results in an increase of TMEM16A protein expression, which is particularly evident in asthmatics (2). Furthermore, this process is driven by [Ca²⁺]_i, which in turn changes the intracellular chloride concentration (efflux or influx depending on the current chloride gradient), which works as a second messenger system and promotes inflammation. This has been demonstrated by experiments in which inhibition of TMEM16A, using selective or nonselective agents of TMEM16A, resulted in a beneficial reduction of IL-4 and IL-13 secretion and IL-4 and IL-13–induced synthesis of airway epithelial mucin (MUC5AC) by asthmatic airway smooth muscle (ASM) when challenged with methacholine (3).

Furthermore, TMEM16A also modulates the contractility of asthmatic ASM, which contributes to excessive airway hyperresponsiveness (AHR) and plays a role in mucociliary clearance (MCC) (3). Manipulating TMEM16A can, therefore, be of interest as it affects the chloride levels, which promotes ASM depolarization and contraction succeeding stimulation of CaCCs and can be therapeutically exploited for inducing relaxation of ASM during bronchospasm seen in asthmatics, but potentially at the cost of affecting ciliary clearance and suppressing cough in the case of nonselective blocking, which also affects the neural TMEM16B (4, 5).

In the experimental study presented, nonselective pharmacological blocker (benzbromarone) and TMEM16A-selective blocker (CaCCinh-A01) have been tried to induce such favorable effects, but as we will discuss further, with additional questionable consequences during airway reactivity challenges. This article will focus on summarizing the most recent experimental findings of the study.

Animals: Adult male Dunkin-Hartley guinea pigs weighing 150–250 g were obtained from Velaz, Ltd. (Prague, Czech Republic; Veterinary Registration Number of the Breeding and Supply Facility in the Central Register-CZ 21760118). All protocols described in the study had been approved by the local ethics committee (IRB00005636, decision No. EK 40/2018) and followed the directive 2010/63/EU of the European Parliament, the council on the protection of animals used for experimental and other scientific purposes, and the Slovak law regulating animal experiments. Guinea pigs were housed in an approved animal holding facility for a 7-day adaptation period and for 2–3 days of adaptation to experimental conditions. A standard air conditioning system and a light/dark cycle with free access to food and water were guaranteed during the experiments.

A total of 72 animals were randomly divided into nine groups, each of which included eight guinea pigs. The groups were as follows: control groups (two negative, three positive) and four experimental groups. The nonselective blocker benzbromarone was administered intraperitoneally (dose 1 mg/kg) or by inhalation (10 μM/L, 5 min inhalation), and the selective blocker CaCCinh-A01 was administered intraperitoneally (dose 1 mg/kg) or by inhalation (10 μM/L, 5 min inhalation). The doses of substances were selected according to the manufacturer's instructions and literature data (6, 7) to achieve a selective effect on TMEM16. The negative groups received isotonic saline (0.9% NaCl, 5 min inhalation or 1 ml/kg intraperitoneally), and the positive control groups received salbutamol (4 mM/L, 5 min inhalation or 10 mg/kg bw intraperitoneally) or, in the experiments that focused on cough reflex sensitivity, codeine phosphate (10 mg/kg per os) was administered.

Responses to nonselective and selective TMEM16A blockers and control drugs were evaluated in unsensitized animals and after sensitization to an allergen.

Chemicals: Salbutamol, ovalbumin (OVA) from chicken egg white, and histamine were purchased from Sigma Aldrich (St. Louis, MO, USA). All other chemicals used were purchased as listed: codeine phosphate (Slovakofarma Hlohovec, Slovakia); sodium chloride solution and methacholine (ApliChem, Darmstadt, Germany); RPMI 1640 medium (Invitrogen/Gibco, USA); aluminum hydroxide (CentralChem, Bratislava, Slovakia); and citric acid (AC) (ACROS Organics, Bratislava, Slovakia). According to the manufacturer's instructions, the chemicals were dissolved in water for injection (salbutamol and codeine) and saline (OVA, methacholine, AC, aluminum hydroxide, and histamine).

An experimental model of chronic allergic airway inflammation was induced by 21 days of administration of an allergen, OVA, adsorbed on aluminum hydroxide (Al(OH)₃, which was applied in a repetitive parenteral manner (1^st day ip and sc, 4^th day ip, and 9^th day sc) at the same dose (5 mg OVA and 100 mg of Al(OH)₃). Then the allergen was administered by inhalation (on the 12^th, 15^th, 18^th, and 20^th days) using a double-chamber body plethysmograph box for small animals (HSE type 855, Hugo Sachs Elektronik, Germany). All tests with sensitized animals were performed 24 h after the last allergen administration (8).

The cough reflex was initiated by inhalation of 0.3 M citric acid aerosol generated by a nebulizer (PARI jet nebulizer, Paul Ritzau, Pari-Werk GmbH, Germany; output, 5 L/s; particle mass, 1.2 μm) and delivered to a nasal chamber during a 3-min interval in which the number of cough efforts was counted (9, 10). The cough response was measured 60 and 120 min after any drug inhalation or 60 and 240 min after intraperitoneal application.

In vivo airway reactivity was expressed as specific airway resistance values (sRaw) that were calculated according to Pennock et al. (11). The changes in the values of sRaw administration were recorded for 1 min immediately after histamine (1 μM/L) and methacholine (1 μM/L) inhalation 60 and 120 min after the single inhalation of the substances or 60 and 240 min when chloride channel blockers and control drugs were applied intraperitoneally.

Measurement of ciliary beating frequency (CBF): The ciliated epithelium specimen was obtained by brushing the trachea using the brushing method for in vitro investigation. The nutritive medium (RPMI 1640) and microscopic glass slides were maintained at a temperature of 37°C–38°C using a PeCon Temp Controller 2000-2 (PeCon GmbH, Erbach, Germany). Samples placed on glass slides were evaluated under an inverted phase-contrast microscope (Zeiss Axio Vert. A1; Carl Zeiss AG, Jena, Germany). Sequential 10-s video files were recorded at 1-min intervals for 15 min at a frame rate of 256–512 frames/s using a digital high-speed video camera (Basler A504kc; Basler AG, Ahrensburg, Germany). CBF was evaluated using the Ciliary Analysis software (LabVIEW™) to generate a ciliary region of interest (ROI) (12, 13).

Statistics: Data of cough reflex (the number of cough efforts) and sRaw were expressed as means ± standard error of mean (SEM). CBFs were expressed as the median value (Hz) for each ROI, followed by calculation of arithmetic means in each microscopic preparation. One-way analysis of variance (ANOVA) with post hoc Bonferroni test was selected as appropriate to test for statistically significant intergroup differences.

Statistical analysis of changes in the number of cough efforts and sRaw values revealed the large difference in responses of OVA-sensitized and unsensitized airways on both TMEM16 blockers. The healthy guinea pigs had a decrease in cough number only when treated with the positive control antitussive drug codeine, whereas OVA-sensitized guinea pigs had a significant reduction in cough efforts regardless of the treatment and route of administration used. However, from TMEM16 blockers, the antitussive effect's longer duration was monitored in inhaling the nonselective benzbromarone (Fig. 1).

Figure 1.

Treatment with inhalation or intraperitoneal administration of salbutamol in healthy animals led to a significant improvement in the airway response to the bronchoconstrictors histamine and methacholine. Except for the intraperitoneally administered benzbromarone's effect on histamine-induced bronchoconstriction, TMEM16 blockers did not significantly influence airway reactivity in unsensitized guinea pigs. However, notable inhibition of sRaw values was observed in OVA-sensitized animals treated with CaCCinh-A01 and benzbromarone, similar to salbutamol. Furthermore, CaCCinh-A01 inhalation was more effective in the suppression of histamine-induced bronchoconstriction than β-agonist and nonselective TMEM16 blocker (Fig. 2).

Figure 2.

In healthy animals, changes in ciliary movements occurred only among those treated with the nonselective drug benzbromarone. Sensitization with OVA altered ciliary kinematics, as evidenced by a decrease in CBF values of sensitized negative controls. Furthermore, as evident in Figure 3, CBF was consequentially altered in OVA-sensitized guinea pigs treated with the selective blocker of TMEM16A (Fig. 3).

Figure 3.

The pathogenesis of asthma involves chronic airway inflammation, increased mucus secretion, and hypertrophy and hyperresponsiveness of ASM. Inhaled corticosteroids are the mainstay in the treatment of asthma, whereas short-acting β-agonists rapidly reduce airway bronchoconstriction. However, the treatments have serious limitations, that is, steroid resistance and downregulation of β-adrenoreceptors. A better understanding of the pathogenetic mechanisms underlying asthma during the last 20 years has led to the development of biological therapies and has resulted in the proposal of various synthetic substances (9, 10, 12, 13) and natural products (14, 15, 16) with the potency to enlarge the group of antiasthmatics. However, an increasing number of patients with poorly controlled asthma supports the requirement of further research in this field.

CaCCs have been ascribed numerous cellular functions (17). Among these are epithelial fluid secretion and smooth muscle contraction, both of which contribute to the progression and severity of asthma. CaCCs are activated by an increase in [Ca²⁺]_i and membrane potential depolarization. Thus, they link Ca²⁺ signaling with the cell's electrical activity. CaCC currents have been documented in respiratory murine and human ASM and have been suggested to mediate depolarization, contributing to agonist-induced tracheobronchial constriction (18). Recently, Huang et al. demonstrated that pharmacological inhibition of CaCCs, especially TMEM16A, significantly reduces isolated mouse ASM contraction in response to cholinergic agonists (19). Indeed, TMEM16A was found to be highly expressed in murine ASM cells (20). The research using animal models of asthma is currently dominated by mouse models. Many of the identified therapeutic targets influencing airway hyperresponsiveness and inflammation in mouse models have, however, been disappointing when tested clinically in asthma (20). In contrast with mice, guinea pigs fulfill the primary and important assumption made when using animal models of human respiratory diseases for drug discovery, namely, that the physiological, immunological, and/or signal transduction mechanisms controlling the specific processes contributing to human pathology are recapitulated in the chosen animal system (21).

In our study, using healthy and OVA-sensitized guinea pigs, we demonstrated that antagonism of the CaCC TMEM16, resulting from acute inhalation or intraperitoneal administration of nonselective and TMEM16A-selective blockers, relaxes ASM and reduces bronchial hyperreactivity induced by histamine and methacholine in vivo. The doses of TMEM16 blockers used for the study should exactly modulate these ion channels’ activity (6, 7). These data suggest that the TMEM16A ion channel of ASM is specifically activated by the increase in [Ca²⁺]_i caused by stimulation of the G protein-coupled receptors (GPCRs) histaminic and muscarinic and is correlated to previously published findings (19).

Except for the bronchodilatory effect, both TMEM16 blockers exhibited the ability to suppress AC-induced cough in sensitized guinea pigs in our study. AC is generally accepted as the activator of bronchopulmonary C-fibers and myelinated cough receptors regulating coughing (22). Previous works (23, 24) suggest that the excitability of these afferents may involve an efflux of intracellular chloride (Cl⁻) ions through membrane chloride channels that were recently found to be TMEM16 (25). The majority of proinflammatory autacoids that directly activate C-fibers, including prostanoids, bradykinin, or histamine, are agonists for GPCRs, and thus increase Ca²⁺-activated Cl⁻ conductance. Consistent with our results, nonselective TMEM16 blocker niflumic acid was found to partially inhibit bradykinin-induced action potential discharge in jugular C-fiber terminals innervating the trachea and can lead to inhibition of cough (26). In sensory neurons, the intracellular Cl⁻ concentration is high and the opening of Cl⁻ channels leads to the efflux of Cl⁻, resulting in depolarization. In pathologic conditions, like chronic exposure to major inflammatory mediators, the intracellular Cl⁻ concentration is known to be elevated even further. Chloride channel opening may contribute more to significant firing than in noninflamed situations (27). This may explain the difference in cough responses of OVA-sensitized and unsensitized airways on TMEM16 blockers.

The physiological studies with knockout mice (28) and morphologic studies (29, 30) showed that airway epithelial cells expressed TMEM16A and are involved in the control of airway liquid surface formation. In noninflamed human lungs, TMEM16A was either only sparsely or not at all expressed in both surface epithelium and submucosal glands. In contrast, epithelial expression of TMEM16A was significantly elevated and TMEM16A was detected in the submucosal glands and ASM of asthma patients (31). CBF is an important component of MCC that responds to an elevation of [Ca²⁺]_i and thus also correlates with the activity of TMEM16A ion channels (32). Consistent with this, our in vitro analysis of cilia isolated from the airways of unsensitized guinea pigs showed no impact of selective TMEM16A blocker. Contrary to healthy animals, the same substance significantly inhibited the ciliary movement of the OVA-sensitized group. Unlike TMEM16A-selective blocker, benzbromarone significantly elevated the CBF value in unsensitized guinea pigs, but had only negligible effect on the ciliary kinematics of sensitized guinea pigs. These differences can be explained by the ability of benzbromarone to activate calcium-sensitive potassium channels (BK_Ca, K_Ca1.1) (33). The hyperpolarizing effects of BK_Ca-mediated K⁺ efflux increase the driving force for Cl⁻ secretion, enhancing the production of airway liquid surface and the frequency of ciliary beating (34).

In conclusion, we can summarize that the results demonstrate that treatment with TMEM16 blockers can be beneficial in reducing cough efforts and airway resistance; however, the effects are in favor of selective blocking of TMEM16A rather than a nonspecific blocker. In addition, selective blocking of TMEM16A also impairs the ciliary beat frequency in animals sensitized to OVA, which can be possibly considered as the limiting factor for its use in chronic bronchial asthma.