Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A(2) occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A(2)-alpha.
Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.
Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Carine M
CM
Mounier
Farideh
F
Ghomashchi
Margaret R
MR
Lindsay
Scott
S
James
Alan G
AG
Singer
Robert G
RG
Parton
Michael H
MH
Gelb
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology*; D006025:Glycosaminoglycans / Q000502:physiology; D017261:Glycosylphosphatidylinositols / Q000502:physiology; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D054509:Group X Phospholipases A2; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018613:Microscopy, Confocal; D010741:Phospholipases A / Q000032:analysis / Q000502:physiology*; D014162:Transfection
D006025:Glycosaminoglycans; D017261:Glycosylphosphatidylinositols; D016718:Arachidonic Acid; D006493:Heparin; D006861:Hydrogen Peroxide; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D054509:Group X Phospholipases A2; C516365:PLA2G10 protein, human