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 |
| |
Authors: | Anderson Ronald Steel Helen C Tintinger Gregory R |
| |
Affiliation: | 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 |
| |
Abstract: | 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. |
| |
Keywords: | 2-APB, 2-aminoethoxydiphenyl borate DMSO, dimethylsulphoxide EGTA, ethylene glycol-bis (β-aminoethyl ether) N,N,N′N′-tetraacetic acid FMLP, N-formyl- smallcaps" >l-methionyl- smallcaps" >l-leucyl- smallcaps" >l-phenylalanine IP3, inositol 1,4,5-triphosphate U-73122, 1-[6[((17β)-3-methoxyestra-1,3,5(10)-trien-17-yl)amino]hexyl]-1-H-pyrrole-2,5-dione U-73343, 1-[6[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|