Functional desensitization of the β 2 adrenoceptor is not dependent on agonist efficacy

Abstract

Chronic treatment with β 2 adrenoceptor agonists is recommended as a first-line maintenance therapy for chronic obstructive pulmonary disease (COPD). However, a potential consequence of long-term treatment may be the loss of functional response (tachyphylaxis) over time. In this study, we have investigated the tendency of such agonists, with a range of efficacies, to develop functional desensitization to cAMP responses in primary human bronchial smooth muscle cells following prolonged agonist exposure. The data show that upon repeat exposure, all agonists produced functional desensitization to the same degree and rate. In addition, β 2 adrenoceptor internalization and β-arrestin-2 recruitment were monitored using β 2·eGFP visualization and the PathHunter™ β-arrestin-2 assay, respectively. All agonists were capable of causing robust receptor internalization and β-arrestin-2 recruitment, the rate of which was influenced by agonist efficacy, as measured in those assays. In summary, although a relationship exists between agonist efficacy and the rate of both receptor internalization and β-arrestin-2 recruitment, there is no correlation between agonist efficacy and the rate or extent of functional desensitization.

Keywords: COPD; agonist efficacy; functional desensitization; β2 adrenoceptor.

Figure 1

β₂ adrenoceptor-mediated cAMP accumulation in hBSMc. Concentration effect curves for cAMP generation were determined in hBSMc following exposure to β₂ adrenoceptor agonists for 2 h. For each individual experiment, data have been normalized to the maximum amount of cAMP produced after addition of 10 μmol/L isoprenaline, and are expressed as means ± SEM for three independent experiments.

Figure 2

Loss of cAMP signaling in hBSMc following chronic β₂ adrenoceptor agonist exposure. hBSMc were pretreated with indicated concentrations of isoprenaline (A), salmeterol (B), indacaterol (C), or formoterol (D) for between 0 and 24 h, washed, and then exposed the same concentration of agonist for a further 2 h. For each individual experiment, data have been normalized to the maximum amount of cAMP produced by agonist in cells which have not been previously exposed to agonist. Data shown are expressed as means ± SEM for three independent experiments.

Figure 3

Rate of loss of cAMP signaling in hBSMc following chronic β₂ adrenoceptor agonist exposure. (A) Rate of loss of cAMP signaling in hBSMc following pretreatment with β₂ adrenoceptor agonists. Data shown are expressed as means ± SEM for three independent experiments. (B) Relative levels of cAMP (% response compared to isoprenaline) produced in human bronchial smooth muscle cells following treatment with different β₂ adrenoceptor agonists over a 24 h time period. Data shown are single determinations from a single experiment, which is representative of three independent experiments. (C) Rate of loss of cAMP signaling in human bronchial smooth muscle cells following pretreatment with a concentration of β₂ adrenoceptor agonist which produces an amount of cAMP equivalent to 20% of the isoprenaline response. Specifically, these concentrations were 4.5 nmol/L isoprenaline, 4 nmol/L indacaterol, 11.2 nmol/L salmeterol, and 1.2 nmol/L formoterol. These concentrations were taken from the data generated in Figure 2 and normalized to 20% isoprenaline. Data shown are expressed as means ± SEM for three independent experiments.

Figure 4

β₂ adrenoceptor agonist-mediated receptor internalization. Agonist-dependent formation of β₂·eGFP containing vesicles following stimulation of U2OS-β₂·eGFP cells with an EC₅₀ concentration of agonist (derived from Fig. 5) for 60 or 90 min in the case of salmeterol. Images were performed using ImageXpress Micro automated imaging system on the IX500 (Molecular Devices). Images were collected using a 40× objective and a Peltier cooled CCD camera. β₂·eGFP receptors were detected using fluorescein isothiocyanate filter and a 200 msec exposure time.

Figure 5

Concentration- and time dependency of receptor-mediated β₂-adrenoceptor internalization in U2OS-β₂·GFP cells. (A) Concentration-dependent increases in β₂ adrenoceptor internalization were assessed between 0 and 2 h stimulation with the indicated concentrations of β₂ adrenoceptor agonists. (B) Time-dependent increases in β₂ adrenoceptor internalization for between 0 and 2 h after stimulation of cells with approximately EC₈₀ concentrations of agonist (as determined from A). Slope factors (K) determined for initial rate of β₂ adrenoceptor internalization (between 0 and 45 min) from polynomial first order (straight) line fit. For each individual experiment, data have been normalized to the maximum amount of β₂ adrenoceptor internalization detected after addition of 10 μmol/L isoprenaline, and are expressed as mean ± SEM for 3–5 independent experiments.

Figure 6

Concentration- and time dependency of receptor-mediated β-arrestin-2 recruitment in PathHunter™ CHO-β₂:arrestin cells. (A) Concentration-dependent increases in β-arrestin recruitment were assessed between 0 and 4 h stimulation with the indicated concentrations of β₂ adrenoceptor agonists. (B) Time-dependent increases in β-arrestin-2 recruitment for between 0 and 4 h after stimulation of cells with approximately EC₈₀ concentrations of agonist (as determined from part A). Data were fit by nonlinear regression to a one phase exponential association to determine half-life of β-arrestin-2 recruitment. For each individual experiment, data have been normalized to the β-arrestin-2 recruitment detected after addition of 10 μmol/L isoprenaline for 4 h, and are expressed as mean ± SEM for three independent experiments.