Citations

Gender disparity in incidence and severity of asthma is clinically recognized. While estrogen‐induced vasodilation is well‐known, effects on airway smooth muscle (ASM) are controversial. In human ASM, we found that both estrogen receptors (ERs), ERα‐66 and ERβ‐47 are expressed, as are two truncated ERα isoforms (ERα‐36 and ERα‐47). In fura‐2 loaded human ASM cells, acute exposure to 17β‐estradiol (17βE 2 ; 1nM) blunts intracellular calcium ([Ca 2+ ] i ) response to ACh and histamine, effects prevented by ER antagonist. We tested the hypothesis that ERα is the main receptor involved in nongenomic Ca 2+ regulation. Real‐time calcium imaging showed greater decrease in [Ca 2+ ] i with ERα specific agonist R,R THC (10nM) compared to 17βE 2 . TNFα (50ng/ml; 24h) upregulated ERα‐66 but downregulated ERα‐36 expression. TNFα‐induced increase in its receptor TNFRI was blunted by 17βE 2 . Finally, TNFα‐induced enhancement of [Ca 2+ ] i response to histamine was attenuated by ERα activation. These data suggest that in human ASM: 1) nongenomic [Ca 2+ ] i regulation by E 2 involves ERα; b) ERα activation may be beneficial in attenuating inflammatory response to TNFα. Overall, ERs may be a novel therapeutic target in airway diseases such as asthma. Supported by Mayo Graduate School and NIH grant UL1RR024150.

TSLP is a newly‐identified cytokine thought to be involved in airway inflammation. TSLP levels have been shown to significantly increase in airway smooth muscle (ASM) with chronic obstructive pulmonary disease and other inflammatory diseases. This study evaluated the expression of TSLP in human ASM cells and tissues exposed to cigarette smoke extract (CSE) or to pro‐inflammatory cytokines TNF‐α and IL‐13. In parallel studies, TSLP expression was evaluated in house dust mite sensitized mice with or without cigarette smoke exposure. The presence of TSLP and TSLP‐receptors were verified using Western blot analysis. Exposure to TNF‐α and IL‐13 (50 ng/mL each) for 24 hours resulted in increased TSLP and TSLP‐R expression. Simultaneous exposure to functional TSLP antibody blunted cytokine induced TSLP‐R expression. Interestingly, acute CSE (30 min) down‐regulated TSLP expression, while chornic exposure increased expression. Similarly, [Ca 2+ ] i responses of human ASM to histamine were blunted with chronic CSE exposure, effects reversed by TSLP and TSLP‐R antibodies. Sensitized mice demonstrated an increase in TSLP expression in whole lung samples. This effect was even further enhanced in dust mite sensitized mice exposed to chronic cigarette smoke. These results indicate a complex role for TSLP in ASM inflammation with or without cigarette smoke exposure. Supported by NIH grants HL088029, HL090595, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

Asthma is a disease characterized by airway hyperresponsiveness and inflammation. One‐third of pregnant women with pre‐existing disease experience a decrease in symptoms during the third trimester, when estrogen levels are highest. Previous studies have shown that estrogens relax vascular smooth muscle, however, little is known about estrogen effects on airway smooth muscle (ASM). We tested the hypothesis that estrogen modulates human ASM contractility. Western blot analysis and immunohistochemical studies showed the presence of both full‐length and truncated isoforms of ERα and β. Furthermore, ER isoforms were localized to caveolae. Caveolin‐1 siRNA downregulated ERα isoform in human ASM. Exposure to the pro‐inflammatory cytokine TNFα substantially increased ERα expression in ASM. In fura‐2 loaded human ASM cells, acute exposure to biologically‐active 17β‐estradiol decreased the intracellular Ca 2+ response to contractile agonists such as histamine and bradykinin; this effect was blunted by the ER antagonist ICI 182, 780. Overall, these data suggest that estrogens may have a bronchodilatory effect. Accordingly, increased ER expression with airway inflammation may actually be protective during pregnancy in that bronchodilation resulting from higher estrogen levels (acting via ERs) may alleviate the increased ASM contractility of asthma. Supported by the Mayo Graduate School and NIH grant UL1RR024150‐01(Mayo Clinic CTSA awards to YSP and CMP)

Acute administration of estrogen induces vasodilation in several vascular beds including coronary artery smooth muscle (CSM). We tested the hypothesis that the response of CSM to estrogen is mediated via the binding of estrogen to estrogen receptors (ER) and that the response will be influenced by hormonal status. Coronary smooth muscle was prepared from gonadally intact (INT) and. ovariectomized (OVX) female pigs. Immunocytochemistry and receptor binding assays were performed to confirm presence vs. absence of ER. In INT CSM cells, but not OVX CSM, acute administration of 17β‐estradiol resulted in a concentration‐dependent decrease in [Ca 2+ ] i responses to endothelin‐1. [Ca 2+ ] i responses to 17β‐estradiol were comparable between groups. The effect of 17β‐estradiol was inhibited by the ER antagonist ICI 182,780. 17β‐estradiol decreased [Ca 2+ ] i in INT (but not OVX) CSM cells when sarcoplasmic reticulum (SR) Ca 2+ reuptake was inhibited, but to a lesser extent than when extracellular Ca 2+ was present. This effect was abolished by extracellular lanthanum. These data suggest that 17β‐estradiol modulates [Ca 2+ ] i in CSM via a likely non‐genomic and necessary ER signaling cascade that mediates both plasma membrane Ca 2+ influx and efflux, but does not involve SR Ca 2+ release. Supported by NIH grants UL1RR024150‐01(Mayo Clinic CTSA awards to YSP & CMP) and HL74309 (GCS)

Women have a higher incidence and severity of asthma exacerbations compared to men. Asthma symptoms fluctuate with hormonal status suggesting a role for sex steroids in modulating bronchoconstriction. We have previously shown that acute estrogen decreases intracellular calcium in human airway smooth muscle thereby facilitating bronchodilation, similar to estrogen‐induced vasodilation. Akin to endothelium, airway epithelium is a source of nitric oxide (NO), a bronchodilator. We hypothesized that estrogens facilitate bronchodilation by generating NO in bronchial epithelium. In acutely dissociated human bronchial epithelial cells, 5–10 min exposure to 1 μM ACh or 17β‐estradiol (E2) (1 and 10 nM) resulted in increased DAF‐2 fluorescence (NO indicator). Both ER specific agonists THC (ERα) and DPN (ERβ) (10 nM,) induced NO production comparably. E2 effects were abrogated by the estrogen receptor (ER) antagonist ICI 182,780 (Faslodex®). eNOS phosphorylation was enhanced by E2 while E2‐induced NO production was attenuated by Ca2+ chelation (BAPTA; 20 μM, 30 min) and eNOS inhibition (L‐NAME). Overall, these data indicate a role for airway epithelium‐derived NO in the bronchodilatory effects of estrogens in human ASM. Supported by NIH grants HL090595 (CMP), HL088029 & HL56470 (YSP), and Mayo Graduate School (EAT).

Clinically observed differences in airway reactivity and asthma exacerbations in women at different life stages suggest a role for sex steroids in modulating airway function although their targets and mechanisms of action are still being explored. We have previously shown that clinically relevant concentrations of exogenous estrogen acutely decrease intracellular calcium ([Ca2+]i) in human airway smooth muscle (ASM), thereby facilitating bronchodilation. In this study, we hypothesized that estrogens modulate cyclic nucleotide regulation, resulting in decreased [Ca2+]iin human ASM. In Fura-2-loaded human ASM cells, 1 nM 17β-estradiol (E2) potentiated the inhibitory effect of the β-adrenoceptor (β-AR) agonist isoproterenol (ISO; 100 nM) on histamine-mediated Ca2+entry. Inhibition of protein kinase A (PKA) activity (KT5720; 100 nM) attenuated E2effects on [Ca2+]i. Acute treatment with E2increased cAMP levels in ASM cells comparable to that of ISO (100 pM). In acetylcholine-contracted airways from female guinea pigs or female humans, E2potentiated ISO-induced relaxation. These novel data suggest that, in human ASM, physiologically relevant concentrations of estrogens act via estrogen receptors (ERs) and the cAMP pathway to nongenomically reduce [Ca2+]i, thus promoting bronchodilation. Activation of ERs may be a novel adjunct therapeutic avenue in reactive airway diseases in combination with established cAMP-activating therapies such as β2-agonists.

The severity of asthma, a disease characterized by airway hyperresponsiveness and inflammation, is enhanced in some women during the menstrual cycle and during pregnancy but relieved in others. These clinical findings suggest that sex steroids modulate airway tone. Based on well-known relaxant effects of estrogens on vascular smooth muscle, we hypothesized that estrogens relax airway smooth muscle (ASM), thus facilitating bronchodilation. In ASM tissues from female patients, Western and immunocytochemical analyses confirmed the presence of both estrogen receptor (ER) isoforms, ERα and ERβ. In fura 2-loaded, dissociated ASM cells maintained in culture, acute exposure to physiological concentrations of 17β-estradiol (E2; 100 pM to 10 nM) decreased the intracellular Ca2+ ([Ca2+]i) response to 1 μM histamine, an effect reversed by the ER antagonist ICI-182,780. The ERα-selective agonist (R,R)-THC had a greater reducing effect on [Ca2+]i responses to histamine and 1 μM ACh compared with the ERβ-selective agonist (DPN). The effects of E2 on [Ca2+]i were mediated, at least in part, via decreased Ca2+ influx through l-type channels and store-operated Ca2+ entry but not via Ca2+-activated K+ channels, receptor-operated entry, or sarcoplasmic reticulum reuptake. Overall, these data support our hypothesis that estrogens relax ASM and suggest a potentially novel therapeutic target in airway hyperresponsiveness.