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

Rosethorne EM, Bradley ME, Kent TC, Charlton SJ - Pharmacol Res Perspect (2015)

Bottom Line: However, a potential consequence of long-term treatment may be the loss of functional response (tachyphylaxis) over time.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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research Wimblehurst Road, Horsham, West Sussex, RH12 5AB, United Kingdom ; School of Life Sciences, Queen's Medical Centre, University of Nottingham Nottingham, NG7 2UH, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig02: 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.

Mentions: When hBSMc were pretreated with a range of concentrations of each agonist for 3 h or more, washed to remove any exported cAMP, and then exposed to the same concentration of agonist for a further 2 h, each of the agonists tested showed a loss in maximal amount of cAMP production compared to control cells (Fig. 2). In addition, the degree of reduction in cAMP signaling correlated with the length of agonist pretreatment, with the greatest reduction observed after 24 h of agonist exposure. After this duration of agonist exposure the levels of cAMP produced by readdition of each agonist had been reduced by similar amounts, when compared to their own control responses (75.9% for isoprenaline; 76.2% for formoterol; 73.3% for indacaterol; 80.4% for salmeterol). Figure 3A shows that, additionally, the rate of loss of cAMP signaling, which plateaus between 6 and 24 h, is almost identical for each of the agonists tested. The data in Figure 2, 3A were calculated by comparing each agonist to itself in control conditions. The data in Figure 3B show the loss of cAMP signaling with time, but it is plotted as a percentage of the maximal control isoprenaline response. This highlights the differences in the initial levels of cAMP which are produced between the high–moderate and low-efficacy agonists and also shows that in the case of salmeterol the levels of cAMP after 24 h pretreatment are only approximately 5% of the maximal control isoprenaline response. In addition, it also shows that even after full desensitization (post 24 h), isoprenaline, formoterol, and indacaterol still generate approximately the same amount of cAMP in these cells as salmeterol does predesensitization (t = 0). Finally, the loss of cAMP signaling at equi-effective concentrations was studied. In order to look at this the concentration of agonist which produced 20% of the control isoprenaline response was determined from Figure 1 (4.5 nmol/L isoprenaline, 4 nmol/L indacaterol, 11.2 nmol/L salmeterol, and 1.2 nmol/L formoterol). This concentration of agonist was then used to calculate the % cAMP signal remaining at each time point (Fig. 3C). The data show that at equi-effective concentrations all of the agonists show a similar rate of loss of cAMP signaling. The % cAMP response remaining after 3 h treatment with each agonist was compared by one-way ANOVA, and shown to be significantly different between isoprenaline and salmeterol (P < 0.001), indacaterol and salmeterol (P < 0.05), and isoprenaline and formoterol (P < 0.05), suggesting that salmeterol is apparently faster to desensitize than the fuller agonists.


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

Rosethorne EM, Bradley ME, Kent TC, Charlton SJ - Pharmacol Res Perspect (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4317232&req=5

fig02: 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.
Mentions: When hBSMc were pretreated with a range of concentrations of each agonist for 3 h or more, washed to remove any exported cAMP, and then exposed to the same concentration of agonist for a further 2 h, each of the agonists tested showed a loss in maximal amount of cAMP production compared to control cells (Fig. 2). In addition, the degree of reduction in cAMP signaling correlated with the length of agonist pretreatment, with the greatest reduction observed after 24 h of agonist exposure. After this duration of agonist exposure the levels of cAMP produced by readdition of each agonist had been reduced by similar amounts, when compared to their own control responses (75.9% for isoprenaline; 76.2% for formoterol; 73.3% for indacaterol; 80.4% for salmeterol). Figure 3A shows that, additionally, the rate of loss of cAMP signaling, which plateaus between 6 and 24 h, is almost identical for each of the agonists tested. The data in Figure 2, 3A were calculated by comparing each agonist to itself in control conditions. The data in Figure 3B show the loss of cAMP signaling with time, but it is plotted as a percentage of the maximal control isoprenaline response. This highlights the differences in the initial levels of cAMP which are produced between the high–moderate and low-efficacy agonists and also shows that in the case of salmeterol the levels of cAMP after 24 h pretreatment are only approximately 5% of the maximal control isoprenaline response. In addition, it also shows that even after full desensitization (post 24 h), isoprenaline, formoterol, and indacaterol still generate approximately the same amount of cAMP in these cells as salmeterol does predesensitization (t = 0). Finally, the loss of cAMP signaling at equi-effective concentrations was studied. In order to look at this the concentration of agonist which produced 20% of the control isoprenaline response was determined from Figure 1 (4.5 nmol/L isoprenaline, 4 nmol/L indacaterol, 11.2 nmol/L salmeterol, and 1.2 nmol/L formoterol). This concentration of agonist was then used to calculate the % cAMP signal remaining at each time point (Fig. 3C). The data show that at equi-effective concentrations all of the agonists show a similar rate of loss of cAMP signaling. The % cAMP response remaining after 3 h treatment with each agonist was compared by one-way ANOVA, and shown to be significantly different between isoprenaline and salmeterol (P < 0.001), indacaterol and salmeterol (P < 0.05), and isoprenaline and formoterol (P < 0.05), suggesting that salmeterol is apparently faster to desensitize than the fuller agonists.

Bottom Line: However, a potential consequence of long-term treatment may be the loss of functional response (tachyphylaxis) over time.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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research Wimblehurst Road, Horsham, West Sussex, RH12 5AB, United Kingdom ; School of Life Sciences, Queen's Medical Centre, University of Nottingham Nottingham, NG7 2UH, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus