EAED inhibits TRs contraction
We first study the effect of EAED on TRs contraction. TRs were precontracted by 80mM KCl, then EAED was added when the contraction reaches 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 dissolved EAED was added in the same doses upon the stabilized contraction (Figure 1B) and no relaxation emerging subsequently. This suggests that EAED is indeed acting as a role to relax airway smooth muscle. The values of half maximal inhibitory concentration (IC50) of EAED was 0.063 ± 0.005 mg/mL (Figure 1C). It is also shown that contraction induced by 80 mM KCl was almost completely inhibited when the concentration of EAED reached 1 mg/mL. These results were from 7 TRs of 7 mice.
Figure 1. EAED inhibited high K+-induced tracheal ring contraction. (A) 80 mM K+ induced a sustained contraction in a mouse TR, which was blocked by EAED in a concentration dependent way. The dose-inhibition curve is presented. (B) Similar experiments were performed, except that vehicle (PSS containing 3% DMSO) was added 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 reached a peak and we could find a gradually obvious inhibition on the precontracted TRs (Figure 2A). At the same time, vehicle (PSS containing 3% DMSO) was also added in the same doses upon the steady contraction as a control (Figure 2B), which also exerted no relaxation. The dose-relaxation relationships were analyzed and the IC50 of EAED was 0.139 ± 0.04 mg/mL in this case (Figure 2C). We could also find the concentration of EAED inducing maximum relaxation was 3.16 mg/mL. These experiments above indicated that EAED could block high K+- and ACH-induced TRs precontraction. In addition, adding 3.16 mg/mL EAED without giving any agonist in advance exhibited a small contraction at once and returning to baseline later (Figure 2D), which indicated EAED had no effect on tracheal ring in resting state.
Figure 2. Contraction induced by ACH (100 𝜇M) was inhibited by cumulative addition of EAED. (A) Following addition of ACH, a TR reached a sustained contraction, which was inhibited following cumulative application of EAED. (B) Similar experiments were conducted, except that vehicle (PSS containing 3% DMSO) was added as control. (C) The summary results of EAED-induced relaxation in 7 TRs. The IC50 of EAED was 0.139 ± 0.04 mg/mL. (D) EAED had no effects 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, we observed the effect of EAED on lung slices. After adding 100 𝜇M ACH, the area of tracheal cavity decreased, but with the addition of EAED, the area of the lumen recovered (Figure 3A). The summary data are shown in Figure 3B from 6 lung slices of 5 mice. After adding 100 𝜇M ACH for 40 minutes, the area of the lumen reduced to approximately 48%, which extended to about 82% compared to the initial value with subsequent application of 3.16 mg/mL EAED for 120 minutes. These results suggested that EAED may also inhibit the contraction of the 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 after the addition of EAED. (B) The summary results are shown. Data were from 6 lung slices of 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 inhibiting ACH-induced contraction, 10 𝜇M nifedipine, a selective blocker of VDCCs, was added after contraction by ACH (Figure 4A). Then we could find the contractions were partial blocked. The relaxation value is about 18%. The remaining part were further blocked by EAED, relaxing to approximately 95% compared to baseline (Figure 4B). The above summary data were conducted from 7 TRs of 7 mice.
Then, we further investigate the nifedipine-resistant component of EAED-induced relaxation. Hence, we first incubated TRs with 10 𝜇M nifedipine for 15 minutes, adding ACH subsequently, and we then observed the effect of Pyr3. The summary results from 6 TRs of 6 mice showed that Pyr3 induced partial relaxation, about 25% (Figure 4C), and the remaining contractions were completely blocked by EAED, almost 100% (Figure 4D).
Figure 4. Nifedipine, Pyr3 partially inhibits ACH-induced contraction, respectively. (A) ACH (100 𝜇M) induced the contraction of mouse TRs, which was partially inhibited by 10 𝜇M Nifedipine, and the rest was inhibited by 3.16 mg/mL EAED. (B) The summary results are shown from 7 TRs. (C) Mouse TRs were preincubated with 10 𝜇M Nifedipine. ACH induced tracheal ring contraction, which was partially blocked by Pyr3, and the rest was completely inhibited by 3.16 mg/mL EAED. (D) The summary results are shown from 6 TRs.
EAED inhibit Ca2+ influx induced by high K+ and additional Ca2+ release induced by ACH
To further confirm the relationship between these channels and relaxation, the experiment of zero calcium and physiological calcium conversion was designed. The result in Figure 5A showed that when the tracheal ring was at 0 Ca2+, high K+ still activated the VDLCC channel but the intracellular Ca2+ concentration did not increase. So it could not cause tracheal ring contraction. When the extracellular [Ca2+]i returned to 2 mM, the extracellular Ca2+ flowing rapidly, the intracellular [Ca2+]i increased and the tracheal was constricted. This contraction could be 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 could be concluded that EAED relaxing precontracted tracheal smooth muscle induced by high K+ was mediated by inhibition of VDLCC mediating Ca2+ influx.
ACH can activate both VDLCC and NSCC channels, which leads to extracellular Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum into the cytoplasm, increased Ca2+ concentration and finally causes contraction of tracheal smooth muscle. Under the condition of zero calcium, ACH was added. Since there was no Ca2+ outside the cell, it caused a transient release of Ca2+ in the sarcoplasmic reticulum, leading a transient of the contraction. When the extracellular [Ca2+]i was restored to 2 mM, Ca2+ in cytoplasm was increased by the interaction of Ca2+ in sarcoplasmic reticulum and extracellular Ca2+ increasing (Figure 5C). So the trachea appeared a continuous and stable contraction and this contraction could be inhibited by 3.16 mg/mL EAED. Moreover, Under Ca2+-free conditions (0 Ca2+ and 0.5 mM EGTA) with conducted in the presence of EAED, ACH induced no transient contraction. Following the addition of 2 mM Ca2+, only a very weak contraction occurred, which was gradually back to baseline (Figure 5D). These results indicated that EAED-induced relaxation was through inhibiting 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 tracheal ring 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 1mg/ml EAED, and high K+-induced contraction did not appear after the restoration of 2 mM Ca2+. (C) After blocking the VDLCC channel with nifedipine, ACH was added in the bath solution without calcium, and the tracheal ring experienced an instantaneous internal calcium release process. After the calcium ions concentration recovered to 2 mM, the tracheal ring produced a stable contraction reaction, which could be completely inhibited by 3.16 mg/mL EAED added later to the baseline level. (D) After 3.16 mg/mL EAED pretreatment, the process of internal calcium release was significantly inhibited under the condition of 0 Ca2+ and 2 mM Ca2+ conversion experiment, and the contraction reaction caused by ACH was also significantly inhibited when the calcium ion concentration was restored to 2 mM.
EAED inhibits Ca2+ elevation in single ASMCs
Then, we attempted to observe the effect of EAED on intracellular Ca2+ in single ASMC conducted in the calcium imaging TILL system. The result showed that high K+ (Figure 6A) and ACH-induced (Figure 6C) increases of intracellular Ca2+ were inhibited by 1 mg/mL or 3.16 mg/mL EAED. The values of Ratio (340/380) at the sites indicated by a, b and c were obtained and summary results from 35/30 cells of 5 mice were shown (Figure 6B and D). After high K+ was added, the values of Ratio (340/380) increased from 0.51 ± 0.01 at point a to 0.75 ± 0.02 at point b, reducing to 0.35 ± 0.01 at point c with subsequent addition of 1 mg/mL EAED. Similar results were shown in the ACH-stimulated increasing [Ca2+]i, where the values of Ratio (340/380) increased from 0.44 ± 0.01 at point a to 0.55 ± 0.01 at point b, reducing to 0.33 ± 0.01 at point c with subsequent addition of 3.16 mg/mL EAED. It is suggested that the decreases of [Ca2+]i were owing to inhibition of 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) 80 mM K+ induced a transient and a sustained increase of intracellular Ca2+. The latter was inhibited following the addition of EAED. The values at the sites indicated by a , b and c were obtained. (B) Summary results from 35 cells of 5 mice. ***P < 0.001. (C) The increase of calcium level in tracheal smooth muscle cells induced by ACH was inhibited by 3.16 mg/mL of EAED. (D) Summary results from 30 cells of 5 mice. ***P < 0.001.
EAED effectively blocks VDLCCs and NSCCs currents
In order to further clarify the underlying mechanism, we meas-ured the currents regulated by VDLCCs and NSCCs. As shown in figure 7A, the VDLCCs current was completely blocked by 10 𝜇M nifedipine and 1 mg/mL EAED. The statistical data of 6 cases of cells in each of the two groups of experiments shows that at +10 mV, 1 mg/mL EAED and 10 𝜇M nifedipine completely blocked the current.
To test whether EAED affects the opening of NSCCs channels, nifedipine, niflumic acid and TEA were added to exclude the influence of VDLCCs, K+ and Cl- currents, respectively. The results showed that NSCCs current could be blocked by 3.16 mg/mL EAED under -70 mV voltage conditions (Figure 7B). Theseresults showedthat EAED could completely inhibit the opening of NSCCs channel induced by ACH.
Figure 7. EAED blocks VDLCCs and NSCCs currents. (A) Protocol for measuring VDLCCs current of a single tracheal smooth muscle. (B) VDLCCs current was blocked by EAED or Nifedipine under the condition of depolarization of cell membrane. (C) The I-V curve was drawn based on the experimental results of six different tracheal smooth muscle cells. (D) Protocol for recording NSCCs currents in a single tracheal smooth muscle. (E) At -70mv, point a is the NSCCs channel state when K+, Cl- and VDLCCs currents are excluded in the physiological state; Point b is the NSCCs channel opening after 100 μM ACH stimulation, reaching the plateau stage; Point c is the state of the NSCCs channel when 3.16 mg/mL EAED is added. (F) Broken line diagram obtained by experimental statistics of net slope current at three time points a, b and c based on figure B of 6. (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, we analyzed the toxicity of EAED on mouse tracheal rings. After the 3.16 mg/mL EAED completely blocked the contraction induced by ACH, the tracheal rings were eluted and balanced for a while, again with ACH stimulation, and the contraction apparently occurs again (Figure 8A). Statistics show that the second ACH-induced shrinkage was approximately 81% of the first (Figure 8B). The above results showed that EAED had little effect on the activity of trachea rings when relaxing them, and could be used in vivo experiments.
Figure 8. The tracheal rings could still be stimulated to shrink after relaxation by EAED. (A) After 3.16 mg/mL EAED was added to inhibit the contraction by ACH, the tracheal rings were stimulated to shrink again by ACH. (B) The contraction rate after the first ACH stimulation and the second were compared. The statistics were obtained from the six tracheal rings.*** P < 0.001.
EAED reduces the respiratory resistance induced by ACH in the control group and the asthma group
To investigate whether EAED could potentially improve airway hyperresponsiveness in mice, lung function of groups of healthy or asthmatic mice was assessed by FOT at baseline and following doubling concentrations of aerosolized ACH (3.125 - 50 mg/mL) dissolved with vehicle or EAED. The four experimental groups studied were indistinguishable under baseline conditions by FOT. When the ACH concentration was increased to 25 - 50mg/mL, the atomized EAED dissolved ACH significantly reduced the respiratory resistance of the control group and the asthma group compared with the vehicle group (Figure 9). As expected, the group of asthmatic mice demonstrated the ACH-sensitive hyperresponsiveness compared to control group, as illustrated especially after adding 25 and/or 50 mg/mL aerosol ACH.
Figure 9. EAED reduced the respiratory resistance induced by ACH in the control group and the asthma group. At the baseline level (B), there was no significant difference in respiratory resistance between the four groups. When the ACH concentration was added to 25 - 50 mg/mL, the atomized ACH dissolved with EAED significantly reduced the respiratory resistance of the control group and the asthma group 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).