Uncoupling of the beta-adrenergic receptor as a mechanism of in vitro neutrophil desensitization.
Human leukocytes have been useful in studying desensitization phenomena to beta-adrenergic agonists in a number of clinical conditions. For example, we have previously shown that oral terbutaline causes a time-dependent decrease in neutrophil (PMN) beta receptor number, using the beta antagonist ligand [3H]dihydroalprenolol (DHA), in conjunction with a significant loss of isoproterenol-induced adenylate cyclase activity. In the present in vitro study we have explored the mechanism for beta-adrenergic desensitization and have compared conditions for homologous and heterologous desensitization, using the intact PMN model. PMN preincubated with isoproterenol (10(-4)M), washed thoroughly, then restimulated, desensitize rapidly so that within 10 min 80% of control isoproterenol-induced cyclic AMP stimulation is lost. Cells washed free of isoproterenol recover full responsiveness in 1 to 2 hr. The estimated isoproterenol desensitization EC50 in cells washed and then restimulated is 1 X 10(-5)M, and the EC50 in unwashed cells that are restimulated is 9 X 10(-8)M. Rank-order potency studies of catecholamine desensitization show isoproterenol greater than epinephrine greater than norepinephrine, a beta-2 pattern. Isoproterenol-induced desensitization results in a small reduction in [3H]DHA binding sites, which becomes statistically significant (p less than 0.05) from control values at 1 hr (67% of control) and 3 hr (64%). Since the change in number of beta receptors did not explain the profound, rapid loss of beta agonist-induced cyclic AMP responsiveness, we explored the possibility of an uncoupling phenomenon. In the absence of GTP, isoproterenol binding is characterized by an EC50 of 6.6 +/- 2.6 X 10(-7)M, which is significantly different (p less than 0.05) from the EC50 of 38.1 +/- 9.1 X 10(-7)M found when cells are previously desensitized with isoproterenol for 10 min. GTP does not affect the EC50 of desensitized cells. These findings are consistent with the uncoupled receptor state fitting the model described by Su et al. Finally, prolonged (3 hr) isoproterenol preincubation results in a small but significant (p less than 0.05) loss of cyclic AMP responsiveness to histamine (67.7% +/- 11.7 of control) and PGE1 (59.3% +/- 7.4), suggesting heterologous desensitization. These studies suggest that the human PMN is a suitable model to study both homologous and heterologous desensitization in vitro.
D001798:Blood Proteins / Q000096:biosynthesis; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004082:Dihydroalprenolol / Q000378:metabolism; D004351:Drug Resistance; D004837:Epinephrine / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D011458:Prostaglandins E / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology*
D001798:Blood Proteins; D011458:Prostaglandins E; D011943:Receptors, Adrenergic, beta; D014475:Uncoupling Agents; D004082:Dihydroalprenolol; D006632:Histamine; D003513:Cycloheximide; D000242:Cyclic AMP; D007545:Isoproterenol; D009638:Norepinephrine; D004837:Epinephrine