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 (Figure 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 (Figure 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 (Figure 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.
Figure 1. EAED inhibited high K+-induced TR contraction. (A) K+ (80 mM) induced a sustained contraction in mouse TR, which was blocked by EAED in a concentration-dependent manner. The dose-inhibition curve is presented. (B) Similar experiments were performed with vehicle (PSS containing 3% DMSO) as control. (C) The dose-inhibition curve is presented. The IC50 of EAED was 0.063 ± 0.005 mg/mL. The data were obtained from 7 TRs.
Similarly, EAED was added after the contraction arising from 100 µM ACh) peaked, which induced a gradual but clear inhibition of the precontracted TRs (Figure 2A). In addition, vehicle control (PSS containing 3% DMSO) was added at the same doses under steady contraction conditions (Figure 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 (Figure 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 (Figure 2D), which indicated that EAED had no effect on the TRs in the resting state.
Figure 2. Contraction induced by ACh (100 µM) was inhibited by cumulative addition of EAED. (A) ACh induced a sustained contraction of TRs, which was inhibited by cumulative application of EAED. (B) Similar experiments were conducted but with vehicle (PSS containing 3% DMSO) as control. (C) Summary of the results of the EAED-induced relaxation in 7 TRs. The IC50 of EAED was 0.139 ± 0.04 mg/mL. (D) EAED had no effect on the basic tone of ASM.
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 (Figure 3A). A summary of the data from 6 lung slices from 5 mice is shown in Figure 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.
Figure 3. EAED inhibits contraction in lung slices. (A) The airway lumen area in a lung slice was decreased by ACh and was markedly increased by the addition of EAED. (B) Summary of the results obtained. Data were derived from 6 lung slices from 5 mice. *P < 0.05; **P < 0.01; ***P < 0.001.
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 (Figure 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 (Figure 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%; Figure 4C), with the remaining contractions completely blocked by EAED (almost 100%; Figure 4D).
Figure 4. Nifedipine and Pyr3 both partially inhibit ACh-induced contraction. (A) ACh (100 µM) induced the contraction of mouse TRs, which was partially inhibited by 10 µM nifedipine, with the remainder inhibited by 3.16 mg/mL EAED. (B) Summary of the results obtained from 7 TRs. (C) Mouse TRs were preincubated with 10 µM nifedipine. ACh induced TR contraction, which was partially blocked by Pyr3, with the remainder completely inhibited by 3.16 mg/mL EAED. (D) Summary of the results obtained from 6 TRs.
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 Figure 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+ (Figure 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+ (Figure 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 (Figure 5D). These results indicated that EAED-induced relaxation was exerted through inhibition of the ACh-elicited Ca2+ influx and Ca2+ release.
Figure 5. EAED blocks high K+-evoked Ca2+ influx and ACh-elicited Ca2+ influx and Ca2+ release. (A) A representative force tracing of 4 TRs. In the absence of calcium ions in the bath solution (0 Ca2+ and 0.5 mM EGTA), high K+ could not cause TR contraction. When the calcium ion concentration was restored to 2 mM, a sustained and stable contractile reaction was produced, which was inhibited by the subsequent addition of EAED. (B) Identical experiments were performed as described in the presence of 1 mg/mL EAED, and high K+-induced contraction did not appear after the restoration of 2 mM Ca2+. (C) After nifedipine blockade of the VDLCC, ACh was added in the bath solution without calcium, and the TR exhibited instantaneous internal calcium release. After the calcium ion concentration recovered to 2 mM, the TR showed a stable contraction reaction, which could be completely inhibited by 3.16 mg/mL EAED, returning the TR contraction to the baseline level. (D) After pretreatment with 3.16 mg/mL EAED, the process of internal calcium release was significantly inhibited in a 0 Ca2+ and 2 mM Ca2+ conversion experiment, with the contraction reaction caused by ACh significantly inhibited when the calcium ion concentration was restored to 2 mM.
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+- (Figure 6A) and ACh- (Figure 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 (Figure 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.
Figure 6. EAED inhibits high K+- and ACh-induced Ca2+ increases in single tracheal smooth muscle cells. (A) A transient and sustained increase in intracellular Ca2+ was induced by 80 mM K+. This increase was inhibited by the addition of EAED. The values were obtained at the sites indicated by a, b, and c. (B) Summary of the results from 35 cells of 5 mice. ***P < 0.001. (C) The increase in the calcium level in tracheal smooth muscle cells induced by ACh was inhibited by 3.16 mg/mL EAED. (D) Summary of the results from 30 cells of 5 mice. ***P < 0.001.
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 Figure 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 (Figure 7B). These results indicated that EAED can completely inhibit the opening of NSCCs induced by ACh.
Figure 7. EAED blocks VDLCC and NSCC currents. (A) Protocol for measuring the VDLCC current of a single tracheal smooth muscle. (B) The VDLCC current was blocked by EAED or nifedipine under depolarized cell membrane conditions. (C) The I-V curve was plotted based on the experimental results of six different tracheal smooth muscle cells. (D) Protocol for recording NSCC currents in a single tracheal smooth muscle. (E) At −70 mV, point a is the NSCC state when K+, Cl−, and VDLCC currents are excluded under physiological conditions; point b is the NSCC opening after 100 μM ACh stimulation, reaching the plateau stage; point c is the state of NSCCs when 3.16 mg/mL EAED is added. (F) The broken-line diagram obtained by the experimental statistics of the net slope current at time points a, b, and c is based on Figure 6B. (G) The average currents for time points b and c at −70 mV (n = 6). ***P < 0.001.
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 (Figure 8A). The second ACh-induced shrinkage was about 81% that of the first (Figure 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.
Figure 8. TRs can still be stimulated to shrink after relaxation by EAED. (A) After 3.16 mg/mL EAED was added to inhibit the ACh-induced contraction, the TRs were stimulated to shrink again by ACh. (B) A comparison of contraction rates after the first and second ACh stimuli. The results were obtained from 6 TRs. ***P < 0.001.
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 (Figure 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.
Figure 9. EAED reduces the respiratory resistance induced by ACh in control and asthma groups. At the baseline level (B), there was no significant difference in respiratory resistance between the four groups. When the ACh concentration was increased to 25–50 mg/mL, the atomized ACh dissolved with EAED significantly reduced the respiratory resistance of the control and asthma groups compared with the vehicle group. (*P < 0.05 Asthma+Vehicle vs Asthma+EAED, **P < 0.01 Asthma+Vehicle vs Control+Vehicle, #P < 0.05 Control+Vehicle vs Control+EAED; ANOVA).