Differential amplification of antagonistic receptor pathways in neutrophils

In human neutrophils approximately 500 ligand-occupied beta-adrenergic receptors almost completely inhibit the superoxide production generated by at least 50,000 formyl peptide receptors, suggesting a massive amplification of the inhibitory receptor signals. We estimated two stages of amplification. In the first stage, we quantitated the ligand-dependent GTPase activities. For the formyl peptide receptor, the number of phosphates released from GTP in the presence of the saturating ligand is relatively modest, i.e. approximately 1/min/receptor, even though there are approximately 200 Gn (Gi type II) proteins/formyl peptide receptor in neutrophil membranes. In contrast, the number of GTPs cleaved in the presence of a beta-adrenergic agonist is approximately 100/min/beta-adrenergic receptor, and there are about 700 Gs/beta-adrenergic receptor in membranes. Thus the signal of the beta-adrenergic receptor is already massively amplified at the G protein, whereas the signal of the formyl peptide receptor is likely to be amplified at subsequent steps. New kinetic evidence from intact cells and biochemical evidence from permeabilized cells is provided that the second messenger of the inhibitory pathway is cAMP. To estimate the amplification of this step, we determined the cAMP concentration necessary to maximally inhibit superoxide anion production of formyl peptide-stimulated electropermeabilized cells, and we compare these concentrations to previously determined values of cAMP production in neutrophils. We conclude that each receptor may generate up to 10,000 molecules of cAMP.