A threshold level of coupled G-proteins is required to transduce neutrophil responses.
Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.
Omann
G M
GM
Research Service (151), VA Medical Center, Ann Arbor, MI 48105.
Harter
J M
JM
Hassan
N
N
Mansfield
P J
PJ
Suchard
S J
SJ
Neubig
R R
RR
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D037342:Pertussis Toxin; D010587:Phagocytosis / Q000187:drug effects; D011108:Polymers; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D011108:Polymers; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D010566:Virulence Factors, Bordetella; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium