Graded G-protein uncoupling by pertussis toxin treatment of human polymorphonuclear leukocytes.
Pertussis toxin (PT) inhibits polymorphonuclear leukocyte (PMN) function by ADP-ribosylating and inactivating guanine nucleotide binding proteins (G-proteins) that transduce activation by chemoattractants such as N-formyl peptides (FP). Studies of PMN activation during the time course of PT treatment yielded these results. 1) Responses were differentiated based on their sensitivity to PT treatment. Suboptimal PT treatment that resulted in 50% inhibition of the FP-induced actin-associated right angle light scatter response resulted in greater than 90% inhibition of oxidant production. Exhaustive PT treatment was required to completely inhibit the right angle light scatter response. This is consistent with previous observations that, relative to oxidant production, actin polymerization requires 100-fold fewer active N-formylpeptide receptors to elicit the response. This differential sensitivity to PT treatment has important implications for studies that use pertussis toxin to determine if the neutrophil G-protein is involved in the signaling of responses. If inhibition of oxidant production is used as the only indicator of the effectiveness of PT treatment, significant cytoskeletal changes may still be activated in these cells. Inhibition of actin polymerization is a much more rigorous indicator of complete G-protein inhibition by PT. 2) Analysis of FP-induced actin polymerization and cytosolic calcium elevation using flow cytometry, which measures individual cell responses, revealed that PT treatment resulted in the conversion of PMN from a responding to a non-responding population. In contrast, in control PMN, submaximal doses of FP caused submaximal stimulation of all the cells. The all-or-none effect of PT may result from heterogeneous insertion of the A-promoter of PT into the cell or it may result from a sharp threshold of coupled G-proteins required to transduce the responses.
Omann
G M
GM
Department of Surgery, University of Michigan Medical School, Ann Arbor.
Porasik-Lowes
M M
MM
D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D037342:Pertussis Toxin; D015398:Signal Transduction / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000199:Actins; D010566:Virulence Factors, Bordetella; D013481:Superoxides; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium