Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins.
Previous work in our laboratory has shown that monocrotaline pyrrole (MCTP) interacts with actin and potentiates thrombin-mediated endothelial barrier permeability through increasing the overall surface area of intercellular gaps. To better characterize endothelial barrier leak in this model, we examined the effects of MCTP and thrombin on the localization and structure of three adhesion associated proteins that directly or indirectly interact with actin in regulating barrier function: cell-cell occludens junction molecule (ZO-1), the cell-cell adherens junction linker, ss-catenin, and the cell-matrix intermediary signaling protein, focal adhesion kinase (FAK). Immunohistochemistry demonstrated that thrombin treatment resulted in radial reorganization of focal adhesions and broader distribution of adherens and occludins junctions at the cell border suggestive of membrane stretching in contracture. MCTP pretreatment resulted in fewer and more disorganized focal adhesions and marked thinning of occludins and adherens junctions. MCTP pretreatment also interfered with thrombin stimulated junctional reorganization. Western blot analysis showed thrombin stimulated catalysis of ZO-1 and FAK while MCTP pretreatment resulted in FAK fragmentation similar to previous reports for apoptosis. We conclude that both MCTP and thrombin alter critical endothelial cell adhesion molecules and this may be an underlying mechanism for the potentiating effect MCTP has on thrombin induced vascular permeability in vitro.
Taylor
Debra W
DW
Department of Veterinary, University of California-Davis, 95616, USA.
Lamé
Michael W
MW
Nakayama
Lynn S
LS
Segall
H J
HJ
Wilson
Dennis W
DW
D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002469:Cell Separation; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000187:drug effects*; D008168:Lung / Q000166:cytology* / Q000187:drug effects; D016686:Monocrotaline / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D051176:beta Catenin
D000477:Alkylating Agents; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D051176:beta Catenin; C014114:monocrotaline pyrrole; D016686:Monocrotaline; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D013917:Thrombin