Functional effects of long‐term activation on human β2‐ and β3‐adrenoceptor signalling

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Abstract
The functional effects of long‐term activation of β‐adrenoceptors were investigated by measuring adenylyl cyclase activity, cyclic AMP accumulation and cyclic AMP‐dependent protein kinase activity in CHW and L cells expressing either human β2‐ or β3‐adrenoceptors. Pre‐incubation of CHW and L cells expressing β2‐adrenoceptors with 10 μm isoprenaline for 24 h produced a marked reduction in the total receptor number and dramatically reduced the capacity of the receptor to stimulate adenylyl cyclase maximally. In contrast, the ability of β3‐adrenoceptors to stimulate adenylyl cyclase maximally was not affected by pre‐incubation with the agonist in either cell type. However, a significant reduction of isoprenaline potency and a sustained down‐regulation of β3‐adrenoceptor number was observed in L but not in CHW cells. Maximal levels of intracellular cyclic AMP concentrations were reached during the first hour of receptor activation with isoprenaline in all four cell lines. In the absence of phosphodiesterase inhibitors, cyclic AMP decreased to basal levels within 24 h of continuous stimulation. This phenomenon occurred more rapidly in cells expressing the β2‐ than the β3‐adrenoceptors. These results confirm that, at the level of adenylyl cyclase stimulation and cyclic AMP accumulation, the β3‐adrenoceptor is more resistant than the β3‐adrenoceptor to long‐term desensitization. However, when cyclic AMP‐dependent protein kinase activity was considered, a 24 h stimulation of β2‐ and β3‐adrenoceptor expressing cells led to the desensitization of the kinase in L but not in CHW cells. In conclusion, long‐term desensitization may have distinct functional effects on cell signalling depending on the receptor subtype and the cell type considered. These findings might have practical implications for future strategies involving long‐term therapies with receptor agonists.