Formoterol and salmeterol induce a similar degree of β₂‐adrenoceptor tolerance in human small airways but via different mechanisms

Abstract

BACKGROUND AND PURPOSESteroids prevent and reverse salbutamol-induced b2-adrenoceptor tolerance in human small airways. This study examines the effects of the long-acting b2 agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization. EXPERIMENTAL APPROACHLong-acting b2 agonists in the presence and absence of budesonide were incubated with human precision-cut lung slices containing small airways. Tolerance was deduced from measurements of reduced bronchodilator responses to isoprenaline and correlated with b2-adrenoceptor trafficking using a virally transduced, fluorescent-tagged receptor. The ability of the LABAs to protect airways against muscarinic-induced contraction was also assessed. KEY RESULTSFollowing a 12 h incubation, both formoterol and salmeterol attenuated isoprenaline-induced bronchodilatation to a similar degree and these effects were not reversible by washing. Pre-incubation with budesonide prevented the desensitization induced by formoterol, but not that induced by salmeterol. Formoterol also protected the airways from carbachol-induced bronchoconstriction to a greater extent than salmeterol. In the epithelial cells of small airways, incubation with formoterol promoted receptor internalization but this did not appear to occur following incubation with salmeterol. Budesonide inhibited the formoterol-induced reduction in plasma membrane b2-adrenoceptor fluorescence. CONCLUSIONS AND IMPLICATIONSAlthough both formoterol and salmeterol attenuate isoprenaline-induced bronchodilatation, they appear to induce b2-adrenoceptor tolerance via different mechanisms; formoterol, but not salmeterol, enhances receptor internalization. Budesonide protection against b2-adrenoceptor tolerance was correlated with the retention of receptor fluorescence on the plasma membrane, thereby suggesting a mechanism by which steroids alter b2-adrenoceptor function. AbbreviationsCCh, carbachol; COPD, chronic obstructive pulmonary disease; LABA, long-acting b2-agonist; PCLS, precision-cut lung slice; SABA, short-acting b2-agonist; YFP, yellow fluorescent protein BJP British Journal of Pharmacology

Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of β2-Adrenergic Receptors

“…In summary, our BRET findings strongly support the conclusion that short-term salmeterol desensitization via the GRK pathway is diminished by its reduced efficacy for arrestin binding. In support of this conclusion, the observation of a deficit in salmeterol-induced internalization via GRK/arrestin was also demonstrated in HAMC cells by immunolocalization of yellow fluorescent protein-tagged b2AR after salmeterol stimulation (Cooper et al, 2011), in mouse myoblast C2C12 cells using a galactosidase complementation assay (Carter and Hill, 2005), and in HEK 293 cells by arrestin translocation (van der Westhuizen et al, 2014) and fluorescence resonance energy transfer (Drake et al, 2008). Furthermore, it was also shown that knockdown of arrestin reduces functional desensitization (Deshpande et al, 2008).…”

“…Our studies demonstrated that short-term treatment of the b2AR with salmeterol caused much reduced desensitization compared with strong agonists in membrane assays (Clark et al, 1996;January et al, 1998). On the basis of studies comparing the effects of equieffective concentrations of salmeterol and strong agonists on functional desensitization in human airway smooth muscle (HASM) cells by measure of either cAMP accumulation or bronchodilation and bronchoprotection, it was concluded that salmeterol was a strong agonist (Düringer et al, 2009;Cooper et al, 2011).…”

“…Furthermore, salmeterol's rapid uptake and sequestration into plasma membranes has presented a major obstacle to quantitative assessment of desensitization by the classic washout and restimulation protocols. Whether in measurements of either cell-free membrane preparations of AC (Clark et al, 1996;January et al, 1998) or functional desensitization in intact cells (Düringer et al, 2009;Nino et al, 2009;Cooper et al, 2011), membrane-sequestered salmeterol has a profoundly confounding effect. Our studies demonstrated that short-term treatment of the b2AR with salmeterol caused much reduced desensitization compared with strong agonists in membrane assays (Clark et al, 1996;January et al, 1998).…”

Airway smooth muscle in airway reactivity and remodeling: what have we learned?

“…For example, inhibiting GRK2/3 or blocking ␤-arrestin-2 can selectively enhance ␤ 2 AR signaling in human ASM (60). Here, corticosteroids, a mainstay of asthma therapy, can also help restore ␤-agonist sensitivity and prevent desensitization although such effects may be specific to particular ␤-agonist agents (47). With TAS2R, recent but limited data based on prevention of desensitization by dynamin but lack of effect of PKA or PKC antagonists (256) also suggest a role for GRK/␤-arrestins.…”

Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii

“…To determine if hAMs are the true C. burnetii target cell, we extended our studies to intact ex vivo human lung tissue to study initial interactions between C. burnetii and its human host. Lung tissue was sectioned into 750-m-thick slices termed precision-cut lung slices (PCLS) (17) and incubated in tissue culture plates. We visualized PCLS by bright-field microscopy ( Fig.…”