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

Abstract

Background and purpose: Steroids prevent and reverse salbutamol-induced β(2)-adrenoceptor tolerance in human small airways. This study examines the effects of the long-acting β(2) agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization.

Experimental approach: Long-acting β(2) 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 β(2)-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 results: Following 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 β(2)-adrenoceptor fluorescence.

Conclusions and implications: Although both formoterol and salmeterol attenuate isoprenaline-induced bronchodilatation, they appear to induce β(2)-adrenoceptor tolerance via different mechanisms; formoterol, but not salmeterol, enhances receptor internalization. Budesonide protection against β(2)-adrenoceptor tolerance was correlated with the retention of receptor fluorescence on the plasma membrane, thereby suggesting a mechanism by which steroids alter β(2)-adrenoceptor function.

Figure 1

Concentration–response curves of human small airways to salbutamol, formoterol and salmeterol showing airway relaxation of a contractions induced by 30 µM carbachol. The dotted line indicates equi-effective concentrations to 0.1 µM salbutamol. Each data point is expressed as mean ± SEM. Each group contains three airways from each of the three donors (nine total airways).

Figure 2

Chronic exposure to salmeterol and formoterol attenuates β₂-adrenoceptor-mediated relaxation. Concentration–response curves to isoprenaline (10⁻⁹–10⁻⁴ M in the presence of 30 µM CCh) after a 12 h incubation of (A) formoterol or (B) salmeterol (0.3 nM) with lung slices containing human small airways. Budesonide (10 nM) was added to slices 1 h before the LABA and remained in contact with the airway for the entire LABA incubation period. In parallel studies, the LABA was vigorously washed out and fresh media was added to the slice for 6 h. Each data point is expressed as mean ± SEM. Each group contains two airways from each of four donors (eight airways in total).

Figure 3

Formoterol decreases the sensitivity of the airways to carbachol-induced bronchoconstriction to a greater extent than salmeterol. (A) Concentration–response curves to carbachol (10⁻⁸–10⁻⁴ M) in the absence and presence of formoterol or salmeterol (0.3 nM). (B) EC₅₀ values for carbachol were calculated from airways after a 2 h incubation with formoterol or salmeterol (0.3 nM). The LABAs remained in contact with the airway during the carbachol concentration–response curves. **P < 0.01; ***P < 0.001 versus control group, or ###P < 0.001 versus formoterol group. Each data point is expressed as mean ± SEM. Each group contained at least four airways from each of the three donors (at total of at least 12 airways).

Figure 4

Expression of Ad5-CMV-human β₂-adrenoceptor-YFP in human lung slices. (A) Low magnification view of receptor expression in the airway epithelial cells of a bisected ∼0.5 mm diameter bronchiole. The image is a projection of a 200 µm stack of confocal images collected at five micron intervals. See Figure S1 for an animation of this effect. (B) A higher magnification en face view of the of the airway epithelium demonstrating that expression in ∼30% of the cells is restricted to the plasma membrane. (C) Mean concentration–response curves to carbachol in mock- and β₂-adrenoceptor-YFP-infected airways. Each data point is expressed as mean ± SEM and each group contained four or five airways from a single donor.

Figure 5

β-Agonist-induced trafficking of human β₂-adrenoceptor-YFP in airway epithelial and smooth muscle cells. (A) Side view of airway epithelial cells demonstrating receptor internalization after treatment for 7 h with 1.0 µM isoprenaline. (B) En face view of airway epithelial cells after treatment for 8 h with 1.0 µM salbutamol. (A and B) Single confocal slices extracted from three-dimensional stacks over a 24 h time course. The complete time course can be viewed in Figures S2 and S3. (C) β₂-Adrenoceptor-YFP infected cells with smooth muscle morphology in a human cultured lung slice. (D) The same cell after treatment for 1 h with isoprenaline showing accumulation of particulate intracellular fluorescence. The scale bars are 40 µm.

Figure 6

Induction of trafficking of human β₂-adrenoceptor-YFP by formoterol and salmeterol. (A) En face view of living human airway epithelial cells in a lung slice with receptor expression largely restricted to the plasma membrane. (B) The same field after treatment for 12 h with 5 nM formoterol showing internalization of receptors coincident with a reduction in plasma membrane expression. (C) The same field after an 8 h treatment with 1.0 µM isoprenaline following formoterol exposure (3D time-lapse can be viewed in Figure S4). (D) En face view of living human airway epithelial cells immediately before salmeterol treatment. (E) The same field after treatment for 12 h with 50 nM salmeterol. (F) The same field after 8 h treatment with 1.0 µM isoprenaline following salmeterol exposure. Each image is a maximum intensity projection of a confocal stack. The scale bar is 20 µm (3D time-lapse can be viewed in Figure S5). (G, H) Single confocal slices from fixed human airways after treatment with 50 nM formoterol (G) or 250 nM salmeterol (H) for 12 h.

Figure 7

Budesonide suppresses the reductions in plasma membrane fluorescence induced by formoterol and salmeterol. (A, B) Confocal fluorescence images of β₂-adrenoceptor-YFP expression in airway epithelial cells in airways before (A) and after (B) they had been incubated with 10 nM budesonide and 5 nM formoterol for 18 h. (C, D) Images collected from airways before (C) and after (D) they had been incubated with 10 nM budesonide and 50 nM salmeterol.