title: Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.
abstract: 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.
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000634:Aminoquinolines / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002630:Chemotactic Factors / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010762:Phosphorylase a / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism; D006005:Phosphorylases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000634:Aminoquinolines; D002630:Chemotactic Factors; D009842:Oligopeptides; D010566:Virulence Factors, Bordetella; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000001:Calcimycin; C024998:N-formylnorleucyl-leucyl-4-phenylalanine; D003994:Bucladesine; D010762:Phosphorylase a; D010763:Phosphorylase b; D006005:Phosphorylases; C035739:Quin2; D002118:Calcium