title: Induction of endothelial monolayer permeability by phosphatidate.
abstract: Released into the vasculature from disrupted cells or transported to the surface of adjacent effectors, phosphatidate and related lipids may potentiate endothelial cell activation. However, the effect of these lipids on endothelial monolayer barrier integrity has not been reported. The present study documents the induction of endothelial monolayer permeability by phosphatidate. Both long (di-C18:1) and medium (di-C10; di-C8) chain length phosphatidates increased permeability of bovine pulmonary artery endothelial cell monolayers assessed using a well characterized assay system in vitro. Barrier disruption effected by dioctanoyl (di-C8) phosphatidate was markedly potentiated by the addition of propranolol, an inhibitor of endothelial cell "ecto"-phosphatidate phosphohydrolase (PAP), a lipid phosphate phosphohydrolase (LPP) that efficiently hydrolyzes extracellular substrate. Disruption of barrier function by phosphatidate did not result from its non-specific detergent characteristics, since a non-hydrolyzable but biologically inactive phosphonate analog of dioctanoyl phosphatidate, which retains the detergent characteristics of phosphatidate, did not induce permeability changes. Furthermore, neither diacylglycerol nor lyso-PA effected significant increases in monolayer permeability, indicating the observed response was due to phosphatidate rather than one of its metabolites. Phosphatidate-induced permeability was attenuated by preincubation of endothelial cells with the tyrosine kinase inhibitor, herbimycin A (10 microg/ml), and enhanced by the tyrosine phosphatase inhibitor, vanadate (100 microM), implicating a role for activation of intracellular tyrosine kinases in the response. In addition, phosphatidate increased the levels of intracellular free Ca(2+) in endothelial cells and ligated specific binding sites on endothelial cell plasma membranes, consistent with the presence of a phosphatidate receptor. Since phosphatidate generated within the plasma membrane of adherent effectors potentially interacts with endothelial membranes, we evaluated the influence of phosphatidate-enriched neutrophil plasma membranes on endothelial monolayer integrity. The effects of ectopic phosphatidate on endothelial monolayer permeability were mimicked by phosphatidate confined to neutrophil plasma membranes. We conclude that phosphatidate may be a physiologic modulator of endothelial monolayer permeability that exerts its effects by activating a receptor-linked, tyrosine kinase-dependent process which results in mobilization of intracellular stored Ca(2+)and consequent metabolic activation.
authors.affiliation: Experimental Cell Research Laboratory, The Methodist Research Institute, Indianapolis, Indiana 46202, USA.dkenglish@msn.com
authors.forename: D
authors.initials: D
authors.lastname: English
authors.forename: Y
authors.initials: Y
authors.lastname: Cui
authors.forename: R
authors.initials: R
authors.lastname: Siddiqui
authors.forename: C
authors.initials: C
authors.lastname: Patterson
authors.forename: V
authors.initials: V
authors.lastname: Natarajan
authors.forename: D N
authors.initials: DN
authors.lastname: Brindley
authors.forename: J G
authors.initials: JG
authors.lastname: Garcia
mesh_terms: D000818:Animals; D016227:Benzoquinones; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects*; D004789:Enzyme Activation; D005070:Evans Blue / Q000378:metabolism; D047029:Lactams, Macrocyclic; D008055:Lipids / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism; D010712:Phosphatidic Acids / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011651:Pulmonary Artery / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D014638:Vanadates / Q000494:pharmacology
chemical_list: D016227:Benzoquinones; D004075:Diglycerides; D047029:Lactams, Macrocyclic; D008055:Lipids; D008246:Lysophospholipids; D010712:Phosphatidic Acids; D011809:Quinones; D017828:Rifabutin; D019000:Phosphotyrosine; D014638:Vanadates; D005070:Evans Blue; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D010739:Phospholipase D; D002118:Calcium