"Cytosolic" phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation.
The synthesis of arachidonic acid metabolites is initiated by activation of the sn-arachidonyl-dependent, 85-kd "cytosolic" phospholipase A2 (cPLA2) enzyme. We have investigated the subcellular localization of cPLA2 in resting and histamine-treated human and bovine endothelial cells (EC) using confocal immunofluorescence microscopy. In tightly confluent EC, cPLA2 was primarily localized in the cytoplasm. Surprisingly, in subconfluent EC, cPLA2 was also prominently located within the cell nucleus. By immunoblotting of cell lysates after SDS-PAGE, the cytoplasmic molecular species in subconfluent cells displayed the characteristic Mr 110,000, whereas nuclear extracts contained a predominant Mr 70,000. Nuclear localization of cPLA, in subconfluent EC is independent of cell cycle because it was observed in growth-arrested cells as well as in dividing cells. Nuclear localization was also observed in subconfluent MDCK and HeLa cells where, in contrast to EC, it persisted in tightly confluent monolayers. Treatment of subconfluent EC with histamine caused a rapid, dose-dependent redistribution of cPLA2, from the nucleus to the nuclear envelope. The same treatment of confluent EC produced translocation of cytoplasmic enzyme to the nuclear envelope and to the plasma membrane at the intercellular junctions. The cell density dependence of cPLA2, localization may contribute to altered arachidonic acid metabolism in injured vessels as compared with quiescent vessels.
Sierra-Honigmann
M R
MR
Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.
Bradley
J R
JR
Pober
J S
JS
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D001808:Blood Vessels / Q000293:injuries / Q000378:metabolism; D002417:Cattle; D002452:Cell Count; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007365:Intercellular Junctions / Q000201:enzymology; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2
D016718:Arachidonic Acid; D006632:Histamine; D010741:Phospholipases A; D054467:Phospholipases A2