Chronic effects of catecholamines on the beta(2)-adrenoreceptor system in cultured human airway epithelial cells

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 mu M) on beta AR density and the rate of beta AR degradation, steady-state beta(2)-adrenergic receptor (beta(2)AR) mRNA levels, and the content of G(s) alpha and G(i) alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). PAR density assessed by binding with [I-125]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 mu M), beta AR density decreased by similar to 82%. In contrast, forskolin (100 mu M) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha(i2) and G(s) alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 mu M) had no effect on steady-state levels of beta 2AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by similar to 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 mu M Iso). These results suggest that, in human airway epithelial cells, beta(2)AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta(2)AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.