The Journal of Cell Biology, Vol 101, 1191-1197, Copyright © 1985 by The Rockefeller University Press
Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide
JL Slonczewski, MW Wilde and SH Zigmond
The activity of glycogen phosphorylase, an enzyme that is activated by both
cAMP and calcium, was used as an indicator of the state of the cytoplasm
after chemotactic stimulation of polymorphonuclear leukocytes
(neutrophils). The activity of the enzyme showed a clear dependence on
cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a
4-5-fold increase in activity of phosphorylase a. In the absence of
external Ca2+, A23187 caused only brief transient activation of
phosphorylase; probably reflecting release of sequestered intracellular
Ca2+. Addition of the chemotactic peptide N-
formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold
activation of the enzyme. The dose-dependence of activation by FNLLP showed
a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The
phosphorylase activity peaks by 90 s and then declines, returning to basal
levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with
10(-7) M peptide. This finding suggests that the cells do not need to
maintain elevated cytoplasmic calcium levels to exhibit stimulated
locomotion. Thus, if calcium continues to modulate the motility, there
either must be highly localized changes that are not detected in measures
of the total cytoplasm, or the sensitivity to calcium must be variable such
that basal levels are sufficient to maintain locomotion. Cells loaded with
the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of
external Ca2+ had elevated phosphorylase levels before addition of FNLLP.
Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it
clearly alters some aspects of the neutrophil physiology. Phosphorylase a
appears to be a sensitive, nonperturbing indicator of the cytoplasmic
calcium levels.
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