Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.
For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.
Walker
B A
BA
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109.
Hagenlocker
B E
BE
Ward
P A
PA
D002118:Calcium / Q000502:physiology*; D003593:Cytoplasm / Q000502:physiology; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000502:physiology*; D010972:Platelet Activating Factor / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010972:Platelet Activating Factor; D013481:Superoxides; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium