EAED inhibits tracheal ring contraction
We first studied the effects of EAED on tracheal ring (TR) contraction. TRs were precontracted with 80 mM KCl and EAED was added when the contraction reached a plateau. The contraction was inhibited in a dose-dependent manner (Fig. 1a). As a comparison, vehicle (PSS containing 3% DMSO), which was used to dissolve the EAED, was added at the same doses when the contraction stabilized (Fig. 1b) and no relaxation was detected. This suggests that EAED indeed relaxes ASM. The half-maximal inhibitory concentration (IC50) of EAED was 0.063 ± 0.005 mg/mL (Fig. 1c). We also found that the contraction induced by 80 mM KCl was almost completely inhibited at an EAED concentration of 1 mg/mL. These results were obtained from 7 TRs from 7 mice.
Similarly, EAED was added after the contraction arising from 100 µM ACh) peaked, which induced a gradual but clear inhibition of the precontracted TRs (Fig. 2a). In addition, vehicle control (PSS containing 3% DMSO) was added at the same doses under steady contraction conditions (Fig. 2b), which again exerted no relaxant effects. Analysis of the dose-relaxation relationships determined an IC50 of EAED of 0.139 ± 0.04 mg/mL (Fig. 2c). The EAED concentration inducing maximum relaxation was 3.16 mg/mL. These experiments indicated that EAED could block high K+- and ACh-induced TR precontraction. In addition, the addition of 3.16 mg/mL EAED without pretreatment with any agonist resulted in a small immediate contraction and a subsequent return to baseline (Fig. 2d), which indicated that EAED had no effect on the TRs in the resting state.
EAED blocks bronchial smooth muscle contraction
To investigate whether EAED has a similar relaxant effect on mouse bronchial smooth muscle, the effects of EAED on lung slices were examined. Treatment with 100 µM ACh decreased the tracheal cavity area; the addition of EAED restored the lumen area (Fig. 3a). A summary of the data from 6 lung slices from 5 mice is shown in Fig. 3b. After the addition of 100 µM ACh for 40 min, the area of the lumen reduced to approximately 48%; subsequent application of 3.16 mg/mL EAED for 120 min further decreased the area by about 82% reduction compared with the initial value. These results suggested that EAED may also inhibit the contraction of bronchial smooth muscle.
EAED exerts diastolic effects by inhibiting L-type Ca2+, TRPC3, and/or STIM/Orai channels
To investigate the mechanism of the EAED inhibition of ACh-induced contraction, 10 µM nifedipine, a selective blocker of voltage-dependent calcium channels (VDCCs), was added after contraction was induced by ACh (Fig. 4a). The drug partially blocked the contractions, giving a relaxation value of about 18%. The remaining contractions were further blocked by EAED, with a relaxation of about 95% compared with baseline (Fig. 4b). These data were obtained from 7 TRs of 7 mice.
Next, we investigated the nifedipine-resistant components of EAED-induced relaxation. Hence, TRs were incubated with 10 µM nifedipine for 15 min and ACh was then added. The effect of Pyr3 was observed. The overall results from 6 TRs of 6 mice showed that Pyr3 induced partial relaxation (about 25%; Fig. 4c), with the remaining contractions completely blocked by EAED (almost 100%; Fig. 4d).
EAED inhibits Ca2+ influx induced by high K+ and additional Ca2+ release induced by ACh
To further confirm the relationship between these channels and relaxation, a calcium-free and physiological calcium conversion experiment was designed. As shown in Fig. 5a, when the TR was at 0 Ca2+, high K+ still activated the L-type voltage-dependent calcium channel (VDLCC) without increasing the intracellular Ca2+ concentration. Thus, it could not cause TR contraction. When the extracellular [Ca2+]i was returned to 2 mM, the extracellular Ca2+ flowed rapidly, the intracellular [Ca2+]i increased, and the TR constricted. This contraction was inhibited by 1 mg/mL EAED. Furthermore, incubation with EAED almost completely abolished the contraction induced by 2 mM Ca2+ (Fig. 5b). From these results, it can be concluded that EAED relaxation of precontracted tracheal smooth muscle induced by high K+ was mediated by inhibition of VDLCCs and Ca2+ influx.
ACh can activate both VDLCCs and non-selective cationic channels (NSCCs), which leads to extracellular Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum into the cytoplasm, increased Ca2+ concentration, and ultimately contraction of tracheal smooth muscle. ACh was added under calcium-free conditions. Because there was no Ca2+ outside the cell, it caused a transient release of Ca2+ from the sarcoplasmic reticulum, leading to a transient contraction. When the extracellular [Ca2+]i was restored to 2 mM, the Ca2+ in cytoplasm was increased by both the Ca2+ from the sarcoplasmic reticulum and the increase in extracellular Ca2+ (Fig. 5c). Thus, the trachea showed a continuous and stable contraction. This contraction was inhibited by 3.16 mg/mL EAED. Moreover, under Ca2+-free conditions (0 Ca2+ and 0.5 mM EGTA) in the presence of EAED, ACh did not induce a transient contraction. With the addition of 2 mM Ca2+, only a very weak contraction occurred, which gradually returned to baseline (Fig. 5d). These results indicated that EAED-induced relaxation was exerted through inhibition of the ACh-elicited Ca2+ influx and Ca2+ release.
EAED inhibits Ca2+ elevation in single ASMCs
Next, the effects of EAED on intracellular Ca2+ in single ASMCs were examined by use of the TILL calcium imaging system. High K+− (Fig. 6a) and ACh- (Fig. 6c) induced increases in intracellular Ca2+ were inhibited by 1 mg/mL or 3.16 mg/mL EAED. The 340/380 ratio at the sites indicated by a, b, and c were obtained and a summary of the results from 30–35 cells of 5 mice are shown (Fig. 6b and d). After the addition of high K+, the 340/380 ratio increased from 0.51 ± 0.01 at point a to 0.75 ± 0.02 at point b, before reducing to 0.35 ± 0.01 at point c with the subsequent addition of 1 mg/mL EAED. Similar results were found with the ACh-stimulated increase in [Ca2+]i, where the 340/380 ratio increased from 0.44 ± 0.01 at point a to 0.55 ± 0.01 at point b, before reducing to 0.33 ± 0.01 at point c with the subsequent addition of 3.16 mg/mL EAED. These results suggest that the [Ca2+]i decreases were due to inhibition of the above Ca2+-permeant ion channels by EAED.
EAED effectively blocks VDLCC and NSCC currents
To further clarify the underlying mechanism, the currents regulated by VDLCCs and NSCCs were measured. As shown in Fig. 7a, the VDLCC current was completely blocked by 10 µM nifedipine and 1 mg/mL EAED. The statistical data of 6 cells examined in each of the two experimental groups showed that + 10 mV, 1 mg/mL EAED, and 10 µM nifedipine completely blocked the current.
To test whether EAED affects the opening of NSCCs, nifedipine, niflumic acid, and TEA were added to exclude the influence of VDLCC, K+, and Cl− currents, respectively. The results showed that the NSCC current was blocked by 3.16 mg/mL EAED under − 70 mV voltage conditions (Fig. 7b). These results indicated that EAED can completely inhibit the opening of NSCCs induced by ACh.
The drug toxicity of EAED is very low at the tissue level
Next, the toxicity of EAED in mouse TRs was analyzed. After 3.16 mg/mL EAED completely blocked the contraction induced by ACh, the TRs were eluted and balanced for a period of time, again with ACh stimulation, and the contraction apparently occurred again (Fig. 8a). The second ACh-induced shrinkage was about 81% that of the first (Fig. 8b). The above results showed that EAED had little effect on the activity of TRs when relaxing them and could be used in in vivo experiments.
EAED reduces the respiratory resistance induced by ACh in control and asthma groups
To investigate whether EAED could potentially improve airway hyperresponsiveness in mice, the lung functions of groups of healthy or asthmatic mice were assessed by the forced oscillation technique at baseline and after exposure to doubling concentrations of aerosolized ACh (3.125–50 mg/mL) dissolved with vehicle or EAED. Under baseline conditions, the four experimental groups studied were indistinguishable with the forced oscillation technique. When the ACh concentration was increased to 25–50 mg/mL, the atomized EAED dissolved with ACh significantly reduced the respiratory resistance of the control and asthma groups compared with the vehicle group (Fig. 9). As expected, the asthmatic mouse group demonstrated ACh-sensitive hyperresponsiveness compared with the control group, particularly after the addition of 25 and/or 50 mg/mL aerosol ACh.