Male Wistar rat (200–250 g) and male A/J mice (18–20 g) were obtained from the Oswaldo Cruz Foundation breeding colony and used under the guidelines of the Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ, license L-030/2015). Animals were housed in groups of four under conditions of constant temperature and controlled illumination conditions, with food and water were available ad libitum.

Sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, magnesium sulfate heptahydrate, and calcium chloride dehydrate were purchased from Merck (Darmstadt, Germany). Glucose, EGTA, carbachol, methacholine, theophylline, mexiletine, propranolol, SQ22,536, lipopolysaccharide (LPS), sodium pentobarbital, pancuronium bromide, sodium orthovanadate, and tetraethylammonium (TEA) were purchased from Sigma-Aldrich (St. Louis, MO). Isoflurane was obtained from Cristália (São Paulo, Brazil). JME-173 was synthesized and provided by the Laboratory of Organic Chemistry (Farmanguinhos, FIOCRUZ, RJ, Brazil). GCMS spectrum revealed that JME-173 is a 100% pure compound as also attested by the ¹H NMR analysis and by its melting point 220–222°C. As predicted by the ChemAxon program (Version 19.22.0), the aqueous solubility at pH 6.4, 6.8, and 7.2 of JME-173 were 127.0, 50.6, and 20.2 mg/ml, respectively. There was no observed evidence of JME-173 degradation following long-lasting exposure to blood samples, storage at room temperature or −70°C, or after the repeated freeze and thaw cycles, pointing out the excellent physical stability of this compound (Pinto at al., under review in the Eur. J. Pharmacol.). All solutions were freshly prepared in distilled water and protected from light.

Rat clonal pituitary GH₃ cells were initially obtained from the Laboratory of Physiology and Molecular Biology, University of Buenos Aires, Buenos Aires, Argentina. GH₃ cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) and plated on a glass sheet at 37°C under a humidified atmosphere with 5% CO₂ from passages 3–6. Ion channel currents in GH₃ cells were recorded by the patch-clamp technique (Neher and Sakmann, 1992). The protocol used to promote the opening of Na⁺ channels was previously described (Da Costa et al., 2007).

Male Wistar rat tracheal rings were obtained and maintained in a 5-ml thermostatic organ bath, which was filled with the aerated physiological solution and connected to an isometric force transducer linked to a data acquisition system (PowerLab 16/30, AD Instruments, Australia) as previously reported (Vieira et al., 2013).

After stabilization for 60 min, tissues were subjected to consecutive cycles of carbachol (2.5 µM)-induced tracheal contraction and washouts until two consistent, reproducible contractions were elicited for each preparation. After carbachol washout and re-establishment of stable baseline tone, tissues were exposed to either carbachol (10⁻⁸–10⁻⁴ M) or ovalbumin (100 µg/ml) in the presence or absence of treatment. The preparations were pre-incubated with mexiletine or JME-173 for 15 min before the addition of the spasmogenic agent. All responses are presented as a percentage of the response to 2.5 μM carbachol. Rats used as tracheal tissue donors for the ovalbumin-induced contraction assay were sensitized and boosted with a subcutaneous injection of a saline suspension containing 50 μg of ovalbumin and 5 mg of Al(OH)3, in a final volume of 0.2 ml, on days 0 and 7, respectively. Then, the animals were killed in a CO₂ atmosphere 14 d after sensitization for tracheal removal as previously reported (Coelho et al., 2008).

To assess the effect of JME-173 on calcium extracellular influx, Ca⁺⁺ concentration-response curves were established. The responses of tracheal ring segments to 2.5 µM carbachol were recorded. Then, after the carbachol was washed out and a stable baseline tone was re-established, the tissues were subjected to successive cycles of 100 mM KCl stimulations/washouts in Ca⁺⁺-free Krebs solution containing 2 mM EGTA until complete desensitization to KCl (100 mM)-induced contraction was observed. Subsequently, tissues were placed in Ca⁺⁺-free Krebs solution containing 100 mM KCl, and the extracellular Ca⁺⁺ concentration was progressively increased by the cumulative addition of Ca⁺⁺ (10⁻⁵–10⁻¹ M) in the presence or absence of treatment (Serra et al., 2016). In some experiments, epithelial cells were mechanically removed, as previously reported (Coelho et al., 2008). The contractile response to the cumulative addition of carbachol was measured in intact or epithelium-denuded tracheas in the presence or absence of JME-173. To further investigate the mechanism of action, the tracheal rings were pretreated 10 min before JME-173 application with either a beta-blocker (1 µM propranolol), an inhibitor of nitric oxide synthase (100 µM L-NAME), an inhibitor of adenylyl cyclase (100 µM SQ 22536) or a nonselective K⁺ channel blocker (10 µM TEA). The preparations were pre-incubated with the tested compounds 30 min before addition of the spasmodic agents were added. All responses are expressed as a percentage of response to 2.5 µM carbachol.

Male C57BL6 mouse ventricular myocytes were enzymatically isolated as described previously (Shioya, 2007). Myocytes were freshly isolated and stored in Tyrode solution, containing 140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl₂, 0.33 mM NaH₂PO₄, 1.8 mM CaCl₂, 5 mM HEPES, and 11 mM glucose, with the pH adjusted to 7.4 with NaOH. The obtained cells were used for experiments within 6–8 h, and only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied. All current recordings were obtained using an EPC-10 patch-clamp amplifier (HEKA Electronics, Rheinland-Pfalz, Germany), at room temperature (20–25°C). After reaching the whole-cell configuration, the cells were maintained for 3 min at rest to allow for dialysis. Current recordings were filtered at 2.9 kHz and digitally sampled at 10 kHz. Patch pipettes had tip resistances of 0.8–1.8 MΩ, and myocytes presenting series resistance above 8.0 MΩ were not recorded. Pipettes were filled up with an internal solution containing 140 mM CsCl, 5 mM Na₂ATP, 5 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, and 5 mM EGTA, with pH adjusted to 7.2 with CsOH. Cardiomyocytes were maintained in a Tyrode solution, and during L-type calcium current recordings, cells were perfused with a modified Tyrode solution in which NaCl was fully replaced by tetramethylammonium chloride to abolish sodium and block potassium currents. Cells were sustained at a holding potential of −80 mV. After the steady-state was achieved, L-type calcium current was elicited by square voltage pulses to 0 mV with 300-ms square voltage pulses to 0 mV. To access the current-voltage relationship, cells were stepped from −80 to +60 mV from a holding potential of −80 V in steps of 10 mV, every 10 s. Data points were fitted using the following equation:

where G_max is the maximal conductance; V_m is the test membrane potential. E_i is the electrochemical equilibrium potential for the ion; V_0.5 is the membrane potential where 50% of the channels are activated, and S is the slope factor.

Airway obstruction and airway hyper-reactivity (AHR) measurements were performed by noninvasive and invasive whole-body plethysmography (DSI-Buxco, Wilmington, NC, USA) as previously reported (Serra et al., 2018). We measured the Penh responses in conscious, spontaneously breathing mice following PBS and methacholine provocations (150 mg/ml) at 1, 3, and 6 h after oral treatment with theophylline (30 and 100 mg/kg), JME-173 (10 and 30 mg/kg), or 0.9% sterile NaCl solution.

In another set of experiments, transpulmonary resistance (cm H₂O/ml/s) and elastance (cmH₂O/ml) were assessed 24 h after LPS challenge by using invasive whole-body plethysmography. Mice were anaesthetized with sodium pentobarbital (Nembutal) (60 mg/kg, i.p.) and then subjected to endotracheal intubation for AHR assessment in a FinePointe Buxco platform (Buxco Electronics, Sharon, CT). The assessments were performed in mechanically ventilated mice under neuromuscular blockade induced by pancuronium bromide (1 mg/kg, i.p.). Changes in lung resistance and elastance were measured at baseline after challenge with aerosol of PBS and then methacholine (3, 9, and 27 mg/ml). Data were recorded and processed using Buxco Biosystem XA software (DSI-Buxco Research System, USA). Area under the curve was calculated by Prism software based in the trapezoidal method. Prism calculates the area of each trapezoid by calculating the area of the equivalent rectangle. The area under the curve is the sum of areas of all the rectangles.

Data analyses were performed using a statistical software package (Prism version 5.0, Graph-Pad Software, San Diego, CA), and the results are expressed as the means ± SEM. Analyses were carried out with one-way ANOVA followed by the Newman-Keuls Student, or two-way ANOVA with post hoc Bonferroni correction. Differences in values were considered statistically significant if p< 0.05. Concentration-response curves were fitted via nonlinear regression using log (inhibitor) vs response-variable slope for patch-clamp data, and sigmoidal dose-response for tracheal ring contraction data.

We have compared the effectiveness of mexiletine and JME-173 (see chemical structures in Figure 1A ) on voltage-gated Na⁺ channel blockade in GH3 cells using a patch-clamp technique as previously reported (Da Costa et al., 2007). A concentration-dependent blockade of Na⁺ current was observed following exposure to increasing concentrations of mexiletine, with a 50% inhibitory concentration (IC₅₀) of 0.28 mM. In addition, we observed that JME-173 also inhibited voltage-gated Na⁺ current in GH3 cells, but with an IC₅₀ value of 183 mM, which is 653-fold higher than that of mexiletine ( Figure 1B ).

We examined the effect of JME-173 on the carbachol-induced contraction of isolated rat tracheal rings compared to that of mexiletine. As shown in Table 1 , both JME-173 (10–100 µM) and mexiletine (100–1,000 µM) concentration-dependently inhibited the maximal carbachol-mediated contraction. The concentration of mexiletine required to inhibit the maximal carbachol effect in 50% was about 15-fold higher than that of JME-173 (IC₅₀ value of 27 µM versus 418 µM, respectively). Despite, neither JME-173 (10–100 µM) nor mexiletine (30–600 µM) shifted the concentration-response curve of carbachol to the right as attested by the pEC₅₀ values of the agonist in the presence or absence of treatment. A slight shift to the right was observed only for 1,000 µM mexiletine ( Table 1 ).

Typical recordings to show the rat tracheal contractions induced by the anaphylactic challenge (ovalbumin, 100 μg/ml) without ( Figure 2A ), or in the presence of mexiletine (1,000 μM, Figure 2B ) or 30 μM JME-173 ( Figure 2C ) are illustrated in Figures 2A–C , respectively. Both mexiletine (300–1,000 μM) ( Figure 2D ) and JME-173 (3–30 μM) ( Figure 2E ) inhibited allergen-induced tracheal contraction in a concentration-dependent manner. The potency of blockade was significantly higher in the JME-173 treatment compared to that observed for mexiletine, with IC₅₀ values of 8 and 440 μM, respectively.

The anti-contraction effect of JME-173 was tested in tracheal rings pre-treated with either L-NAME (a nitric oxide synthesis inhibitor), propranolol (a β2 adrenergic receptor antagonist), SQ22,536 (an adenylate cyclase inhibitor), or TEA (a K⁺ channel blocker). The results showed that the protective effect of 30 μM JME-173 on carbachol-induced tracheal contraction remained unaltered when co-incubated with 100 µM L-NAME ( Figure 3A ), 100 µM SQ22,536 ( Figure 3B ), 1 µM propranolol ( Figure 3C ), or 10 µM TEA ( Figure 3D ). Furthermore, mechanic removal of epithelium did not modify the efficacy of JME-173 to inhibit the carbachol-induced contraction observed with intact epithelium. While 100 µM JME-173 reduced the maximal tension generated by increasing concentrations of carbachol (10⁻⁸–10⁻⁴ M) from 137.4 ± 1.9% to 0.9 ± 1.9% with intact epithelium, values reduced from 140.9 ± 4.6% to 1.2 ± 2.9%, (n=5, mean ± SEM) in de-epithelized tracheal rings.

Typical recordings of the rat tracheal contractions caused by changes in extracellular Ca⁺⁺ concentrations (10⁻⁵–10⁻¹M) during high K⁺-induced membrane depolarization in the absence or presence of 600 µM mexiletine or 60 µM JME-173 are shown in Figures 4A–C , respectively. Inhibitory effects of mexiletine (100–600 µM) or JME-173 (10–60 µM) on rat tracheal contraction caused by voltage-dependent Ca⁺⁺ channel activation are pointed out in Figures 4D, E , respectively. The concentrations of mexiletine and JME-173 required to inhibit the contractile response induced by voltage-dependent Ca⁺⁺ influx in 50% was 380 and 10 μM, respectively.

We further compared the effects of mexiletine and JME-173 on rat tracheal contraction caused by sodium orthovanadate, which acts downstream in the signaling cascade of smooth muscle contraction by shifting the kinase-phosphatase balance toward phosphorylation of tyrosine kinases (Yayama et al., 2014). Representative recordings to demonstrate the effectiveness of treatments on sodium orthovanadate-induced rat tracheal contractions are illustrated in Figures 4F–H . Exposure to mexiletine (300–1,000 μM) ( Figure 4I ) or JME-173 (30–100 μM) ( Figure 4J ) reduced the amplitude of sodium orthovanadate (10⁻⁵–10⁻² M)-induced contraction with IC₅₀ values of 415 and 73 µM, respectively.

To investigate the putative effect of mexiletine and JME-173 on L-type Ca⁺⁺ channels, we studied Ca_v 1.2 currents in mouse cardiomyocytes using the whole-cell patch-clamp technique. Figure 5A shows representative traces of Ca⁺⁺ currents before (control) and after exposure to 100 µM JME-173. Both mexiletine and JME-173 caused a concentration-dependent inhibition of L-type Ca⁺⁺ currents ( Figure 5B ), with estimated IC₅₀ values of 36 and 436 μM for JME-173 and mexiletine, respectively. Neither 30 μM JME-173 ( Figure 5C ) nor 300 μM mexiletine ( Figure 5D ) interfered with the V _0.5. lue for Ca⁺⁺ channel activation. However, JME-173 caused a slight shift in voltage-dependent Ca⁺⁺ channel inactivation ( Table 2 ).

We assessed the effect of JME-173 on methacholine-induced bronchoconstriction in A/J mice using noninvasive whole-body barometric plethysmography, which uses the increase in enhanced pause (Penh) as an indirect indicator of expiratory time (Mitzner and Tankersley, 2003). The provocations were performed at 1 h, and repeated at 3 and 6 h after JME-173 (10 and 30 mg/kg) or theophylline (30 and 100 mg/kg) oral treatments. Notably, the same group of animals was used for each treatment condition throughout the analyses (longitudinal evaluation). Untreated A/J mice reacted with a mild but significant increase in Penh response to the first aerosolized methacholine exposure (150 mg/ml) ( Figures 6A, B , closed circles) compared to that observed using PBS ( Figures 6A, B , open circles). We observed that JME-173 (30 mg/kg) ( Figure 6A , closed square) and theophylline (100 mg/kg) ( Figure 6B , closed square) significantly attenuated the methacholine-induced increase in Penh noted 1 h post-treatment. In line with previous studies (Ewart et al., 1996; Hadeiba et al., 2000), A/J mice reacted with an exaggerated Penh response following repeated exposure to methacholine within 3 and 6 h after treatment. Both, JME-173 (10 and 30 mg/kg) and theophylline (30 and 100 mg/kg) inhibited methacholine-induced Penh changes as provocation were repeated 3 h post-treatment ( Figures 6A, B , open and closed squares). In contrast, only JME-173 (30 mg/kg) ( Figure 6A , closed square) significantly inhibited the changes in Penh triggered by methacholine re-exposure 6 h post-treatment.

We assessed the effect of compound treatment on the methacholine-induced increase in lung resistance and elastance in A/J mice challenged with LPS by using invasive whole-body barometric plethysmography. Aerosolization with increasing methacholine concentrations (9–27 mg/ml) revealed a marked state of bronchial hyper-reactivity, indicated by increased lung airway resistance ( Figure 7A ) and elastance ( Figure 7B ), in animals challenged with LPS (25 µg/25 µl) compared to those challenged with saline. Figures 7C, D show that pre-treatment with JME-173 (50 mg/kg, oral) significantly inhibited both changes. Values of “area under the curve” to demonstrate the sensitivity of increased levels of airway resistance and lung elastance caused by intranasal instillation of LPS are shown in Figures 7C, D , respectively.