Inositol 1,4,5-triphosphate-mediated shuttling between intracellular stores and the cytosol contributes to the sustained elevation in cytosolic calcium in FMLP-activated human neutrophils.
The current study was designed to probe Ca2+ shuttling between intracellular stores and the cytosol as a potential mechanism contributing to the prolongation of elevated Ca2+ transients in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation were measured using spectrofluorimetric and radiometric procedures, respectively, while inositol 1,4,5-triphosphate (IP3) was measured using a radioreceptor assay. The Ca2+-chelating agent, ethylene glycol-bis (beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA; 10mM), was used to exclude store-operated influx of Ca2+ into neutrophils, while the IP3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB, 100 microM), added to the cells 10s after FMLP (0.01 and 1 microM), at which time the increases in IP3 and cytosolic Ca2+ were maximal, was used to eliminate both sustained release from stores and influx of Ca2+. Addition of FMLP at 0.01 or 1 microM resulted in equivalent peak increases in cytosolic Ca2+, while the increase in IP3 was greater and the rate of clearance of Ca2+ from the cytosol slower, in cells activated with 1 microM FMLP. Treatment of the cells with either EGTA or 2-APB following addition of 1 microM FMLP, completely (EGTA) or almost completely (2-APB) abolished the influx of Ca2+ and accelerated the rate of clearance of the cation from the cytosol. Post-peak cytosolic Ca2+ concentrations were lower, and the Ca2+ content of the stores higher, in cells treated with 2-APB. The involvement of IP3 was confirmed by similar findings in cells treated with U-73122 (1 microM), a selective inhibitor of phospholipase C. Taken together, these observations are compatible with IP3-mediated Ca2+ shuttling in neutrophils activated with FMLP.
Anderson
Ronald
R
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, PO Box 2034, Pretoria 0001, South Africa. randerso@medic.up.ac.za
Steel
Helen C
HC
Tintinger
Gregory R
GR
D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000097:blood / Q000378:metabolism*; D002469:Cell Separation; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000097:blood / Q000502:physiology*; D007424:Intracellular Fluid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D018375:Neutrophil Activation / Q000187:drug effects / Q000502:physiology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
D001896:Boron Compounds; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium