title: Role of calcineurin in thrombin-mediated endothelial cell contraction.
abstract: Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.
authors.affiliation: Department of Medical Chemistry, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors.forename: Bernadett
authors.initials: B
authors.lastname: Kolozsvári
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Szíjgyártó
authors.forename: Péter
authors.initials: P
authors.lastname: Bai
authors.forename: Pál
authors.initials: P
authors.lastname: Gergely
authors.forename: Alexander
authors.initials: A
authors.lastname: Verin
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Eva
authors.initials: E
authors.lastname: Bakó
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D020134:Catalytic Domain / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D016572:Cyclosporine / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
chemical_list: D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D016572:Cyclosporine; D019703:Calcineurin; D013917:Thrombin; D016559:Tacrolimus