Abstract：
High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17β-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17β-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.
摘要：
高血清雌激素浓度与哮喘的发展和严重程度有关。提示雌二醇与气道高反应性（AHR）之间存在联系。17β-雌二醇（E2）通过Ca2调节机制具有非基因组效应；然而，其对质膜Ca2ATPases（PMCA-1和-4）和肌浆网Ca2ATPase（SERCA）的影响尚不清楚。因此，在本研究中，我们的目的是通过涉及Ca2ATPases的机制，通过增加豚鼠气道平滑肌（ASM）中的细胞内Ca2浓度来证明E2是否有利于AHR。在豚鼠ASM中，Ca2+微量荧光法,肌肉收缩,和Westernblot进行评价。然后，我们在雌激素和Ca2+ATP酶之间进行了分子对接分析。在气管环中，E2对卡巴胆碱产生AHR。在豚鼠肌细胞中，急性暴露于生理水平的E2将咖啡因诱导的短暂Ca2峰值改变为Ca2平台。与PMCA抑制剂（镧和羧花素，CE）部分逆转了E2诱导的咖啡因反应持续平台。相比之下,环吡嗪酸（SERCA抑制剂），U-0126（ERK1/2抑制剂），氯化胆碱没有改变E2产生的Ca2平台。E2不影响线粒体单转运蛋白的活性和电容性Ca2进入。在豚鼠ASM中，Western印迹分析显示PMCA1和PMCA4表达。对接建模的结果表明E2与两种质膜ATP酶结合。在豚鼠气管平滑肌中，用CE抑制PMCA，诱导对卡巴胆碱的高反应性。17β-雌二醇通过抑制ASM中的PMCA产生高反应性，可能是导致女性哮喘危象增加的机制之一。
