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Rationale: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca(2+) homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.Objectives: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken.Methods: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots.Measurements and Main Results: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR(inh)172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-alpha.Conclusions: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF. [ABSTRACT FROM AUTHOR]

Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.Objectives: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor-deficient mice.Methods: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.Measurements and Main Results: Estrogen receptor-alpha knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-alpha also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.Conclusions: These data suggest that estrogen receptor-alpha is a critical regulator of airway hyperresponsiveness in mice. [ABSTRACT FROM AUTHOR]

Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling. Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo. Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen. Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge. Conclusion: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma. [ABSTRACT FROM AUTHOR]