Colocalization of Cytosolic Phospholipase A₂, 5‐Lipoxygenase, and 5‐Lipoxygenase‐Activating Protein at the Nuclear Membrane of A23187‐Stimulated Human Neutrophils

Abstract

The distribution of cytosolic phospholipase A, (cPLA,), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA, and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A231 87, both proteins accumulated primarily in nucleicontaining fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5-lipoxygenase and cPLA, were localized in a fraction that contained nuclear membranes. In comparison, 5-LAP was localized to the nuclearmembrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffinity labeling. In agreement with the immunoblot data, A231 87 stimulation markedly enhanced 5-lipoxygenase enzymatic activity in the nuclear-membrane fraction, which was accompanied by decreased cytosolic 5-lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA,, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state.

Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils

“…Presence of type IV cPLA 2 has been observed in distinct subcellular compartments, including the cytosol, plasma and nuclear membranes, as well as the endoplasmic reticulum and Golgi apparatus [35][36][37]; as such, sub-groups of type IV cPLA 2 (s) could be differentially sensitive to inhibition. Along these lines, we conducted experiments designed to compare sensitivity of type IV cPLA 2 (s) implicated in the generation of LTB 4 and of PGE 2 .…”

Involvement of Cytosolic Phospholipase A2 and Secretory Phospholipase A2 in Arachidonic Acid Release from Human Neutrophils

“…An 85-kDa cytosolic PLA 2 (cPLA 2 ) that selectively hydrolyzes glycerophospholipids with AA in the sn-2 position and translocates to the nucleus in a Ca 2ϩ -dependent manner in response to agonists (3,4) has been identified in PMN (5). Neutrophils also have Ca 2ϩ -independent PLA 2 (iPLA 2 ) activity (6).…”

“…Phospholipase A 2 in Arachidonic Acid Release from Human Neutrophils P hospholipase A 2 (PLA 2 ) 4 hydrolyzes arachidonic acid (AA) from the sn-2 position of glycerophospholipids (1). The hydrolysis of AA by PLA 2 is a critical regulatory step in the development of an inflammatory response.…”

Depletion of Human Monocyte 85-kDa Phospholipase A2 Does Not Alter Leukotriene Formation

“…This resulted in a concentration-dependent reduction in PGE 2 formation. Correlative evidence, such as coordinate enzyme expression (22)(23)(24)(25)(26) or subcellular localization (27,28), has been reported, implicating the 85-kDa PLA 2 as a possible participant in 5-LO product formation; however, there are no reports directly linking the two.…”