Signal processing times in neutrophil activation: dependence on ligand concentration and the relative phase of metabolic oscillations

Intracellular NAD(P)H oscillations exhibited by polarized neutrophils display congruent to 20 s periods, which are halved to 10 s upon stimulation with chemotactic peptides such as FNLPNTL (N-formyl-nle-leu-phe-nle-tyr-lys). By monitoring this frequency change, we have measured accurately the time interval between stimulus and metabolic frequency changes. A microscope flow chamber was designed to allow rapid delivery of FNLPNTL to adherent cells. Using fluorescein as a marker, we found delivery to be complete and stable throughout the chamber within similar to400 ms. Peptides were injected into the chamber at concentrations ranging from 10(-6) to 10(-9) M. Injections also varied with respect to the relative phase of a cell's NAD(P)H oscillations. The time interval between injection of 10(-6) M FNLPNTL and the acquisition of congruent to 10 s period metabolic oscillations was found to be 12.2 +/- 3.3 s when injections occurred at the NAD(P)H oscillation peak whereas the lag time was 22.5 +/- 4.8 s when coinciding with a trough. At 10(-8) M FNLPNTL, lag times were found to be 26.1 +/- 5.2 and 30.5 +/- 7.3 s for injections at NAD(P)H peaks and troughs, respectively. FNLPNTL at 10(-9) M had no effect on metabolic oscillations, consistent with previous studies. Our experiments show that the kinetics of transmembrane signal processing, in contrast to a simple transmembrane chemical reaction, can depend upon both ligand dose and its temporal relationship with intracellular metabolic oscillations. (C) 2003 Elsevier B.V. All rights reserved.