1. Cultured bovine bronchial epithelial cells express a large number of β-adrenoceptors of the β2 subtype which are coupled to activation of adenylate cyclase and synthesis of cyclic AMP. The mechanisms involved in agonist-induced desensitization of β-adrenoceptor function in these cells were investigated.
2. Preincubation of cells with the β-adrenoceptor agonist isoprenaline induced a rapid desensitization of adenylate cyclase activity stimulated by isoprenaline (40% at 30 min) with little or no decrease in forskolin stimulation or in the number of β-adrenoceptors detected by radioligand binding, indicating that uncoupling of β-adrenoceptors from adenylate cyclase had occurred.
3. Uncoupling of β-adrenoceptors from the stimulatory guanine-nucleotide-binding protein Gs was indicated by the loss of GTP effects on agonist competition for radioligand binding. Results from subcellular fractionation by sucrose density gradient centrifugation suggest that rapid internalization or sequestration of β-adrenoceptors occurred along with the desensitization.
4. Preincubation with isoprenaline for longer times led to a marked decrease in total receptor number (85% at 18 h), indicating that receptor down-regulation also occurs. Recovery of receptors after down-regulation was prevented by cyclobeximide and by actinomycin D, suggesting that degradation of both receptor protein and receptor mRNA may be involved in down-regulation.