Mueller H, Motulsky H J, Sklar L A
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Mol Pharmacol. 1988 Sep;34(3):347-53.
The binding to catecholamines to the beta-adrenergic receptors on human polymorphonuclear leukocytes rapidly inhibits cell responses stimulated by chemoattractant ligands. As a first step in understanding the mechanism of the inhibition, we investigated the number of beta-receptors required to optimally block superoxide anion production, a response that is measured kinetically by a convenient spectrophotometric assay for the reduction of cytochrome c. We found that after blockade of 50-60% of the beta-adrenergic receptors with an irreversible antagonist, maximal inhibition of the response was still elicited by isoproterenol (ISO). Next we investigated the kinetics with which superoxide generation is inhibited. We found that half-maximal inhibition was observed at 3 x 10(-8) M ISO, which approximates the Kd because many of the receptors are involved. Cell responsiveness recovered when propranolol was added between the time of ISO and chemoattractant addition. From the recovery we estimated that the half-time for ISO dissociation is less than 10 sec. Finally, we examined the rate at which cell responses decay following ISO administration after chemoattractant. Optimal rates of inhibition, turning off oxidant production in seconds, occur at ISO concentrations greater than or equal to 3 x 10(-7) M. Taken together, these observations are consistent with an association rate constant for ISO estimated to be greater than or equal to 10(8) M-1min-1 and a dissociation rate constant greater than or equal to 4 min-1. These results are discussed in terms of the available data concerning the binding of agonists to beta-adrenergic receptors.
