Translocation of the 85-kDa phospholipase A2 from cytosol to the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen.
The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.
Glover
S
S
Department of Chemistry, University of Washington, Seattle 98195, USA.
de Carvalho
M S
MS
Bayburt
T
T
Jonas
M
M
Chi
E
E
Leslie
C C
CC
Gelb
M H
MH
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology*; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D008407:Mast Cells; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000201:enzymology* / Q000648:ultrastructure; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012709:Serum Albumin / Q000494:pharmacology; D018411:Spodoptera; D013997:Time Factors; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012709:Serum Albumin; C049925:fura-2-am; D007073:Immunoglobulin E; D015759:Ionomycin; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D016257:Fura-2