Our results show an increase in agonist-induced calcium mobilization associated with an increase in MLC phosphorylation and an increase in ASM cell shortening in favor of agonist-induced hyperresponsiveness in HASM cells derived from obese subjects. Our studies suggest that obesity induces a retained phenotype of hyperresponsiveness in cultured human airway smooth muscle cells.

The nongenomic mechanisms by which glucocorticoids modulate β2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on β2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and β2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

Airway remodeling in asthma manifests, in part, as enhanced airway smooth muscle (ASM) mass, due to myocyte proliferation. While the anti-proliferative effects of glucocorticoid (GC) were investigated in normal ASM cells (NASMC), little is known about such effects in ASM cells derived from asthma subjects (AASMC). We posit that GC differentially modulates mitogeninduced proliferation of AASMC and NASMC. Cells were cultured, starved, then treated with Epidermal growth factor (EGF) (10 ng/ml) and Platelet-derived growth factor (PDGF) (10 ng/ml) for 24 hr and/or fluticasone propionate (FP) (100 nM) added 2 hr before. Cell counts and flow cytometry analyses showed that FP failed to decrease the cell number of and DNA synthesis in AASMC irrespective of mitogens used. We also examine the ability of Insulin Growth Factor Binding Protein-1 (IGFBP-1), a steroid-inducible gene that deters cell growth in other cell types, to inhibit proliferation of AASMC where FP failed. We found that FP increased IGFBP1 mRNA and protein levels. Interestingly, the addition of IGFBP1 (1 μg/ml) to FP completely inhibited the proliferation of AASMC irrespective to the mitogens used. Further investigation of different signaling molecules involved in ASM growth and GC receptor functions (Protein kinase B (PKB/ AKT), Mitogen-activated protein kinases (MAPKs), Focal Adhesion Kinase (FAK)) showed that IGFBP-1 selectively decreased mitogen-induced p38 phosphorylation in AASMC. Collectively, our results show the insensitivity of AASMC to the anti-proliferative effects of GC, and

Glucocorticoids (GCs) and β2-adrenergic receptor (β2AR) agonists improve asthma outcomes in most patients. GCs also modulate gene expression in human airway smooth muscle (HASM), thereby attenuating airway inflammation and airway hyperresponsiveness that define asthma. Our previous studies showed that the pro-fibrotic cytokine, transforming growth factor- β1 (TGF-β1) increases phosphodiesterase 4D (PDE4D) expression that attenuates agonist-induced levels of intracellular cAMP. Decreased cAMP levels then diminishes β2 agonist-induced airway relaxation. In the current study, we investigated whether glucocorticoids reverse TGF-β1-effects on β2-agonist-induced bronchodilation and modulate pde4d gene expression in HASM. Dexamethasone (DEX) reversed TGF-β1 effects on cAMP levels induced by isoproterenol (ISO). TGF-β1 also attenuated G protein-dependent responses to cholera toxin (CTX), a Gαs stimulator downstream from the β2AR receptor. Previously, we demonstrated that TGF-β1 treatment increased β2AR phosphorylation to induce hyporesponsiveness to a β2 agonist. Our current data shows that expression of grk2/3, kinases associated with attenuation of β2AR function, are not altered with TGF-β1 stimulation. Interestingly, DEX also attenuated TGF-β1-induced pde4d gene expression. These data suggest that steroids may be an effective therapy for treatment of asthma patients whose disease is primarily driven by elevated TGF-β1 levels.

Epinephrine (EPI), an endogenous catecholamine involved in the body's fight-or-flight responses to stress, activates α1-adrenergic receptors (α1ARs) expressed on various organs to evoke a wide range of physiologic functions, including vasoconstriction. In the smooth muscle of human bronchi, however, the functional role of EPI on α1ARs remains controversial. Classically, evidence suggests that EPI promotes bronchodilation by stimulating β2-adrenergic receptors (β2ARs). Conventionally, the selective β2AR agonism of EPI was thought to be, in part, due to a predominance of β2ARs and/or a sparse, or lack of α1AR activity in human airway smooth muscle (HASM) cells. Surprisingly, we find that HASM cells express a high abundance of ADRA1B (the α1AR subtype B) and identify a spontaneous 'switch-like' activation of α1ARs that evokes intracellular calcium, myosin light chain phosphorylation, and HASM cell shortening. The switch-like responses, and related EPI-induced biochemical and mechanical signals, emerged upon pharmacological inhibition of β2ARs and/or under experimental conditions that induce β2AR tachyphylaxis. EPI-induced pro-contractile effects were abrogated by an α1AR antagonist, doxazosin mesylate (DM). These data collectively uncover a previously unrecognized feed-forward mechanism driving bronchospasm via two distinct classes of G protein-coupled receptors (GPCRs) and provide a basis for reexamining α1AR inhibition for the management of stress/exercise-induced asthma and/or β2-agonist insensitivity in patients with difficult-to-control, disease subtypes.