title: Caveolae as a target for Phoneutria nigriventer spider venom.
abstract: An important transcellular transport mechanism in the blood-brain barrier (BBB) involves caveolae, which are specialized delta-shaped domains of the endothelial plasma membrane that are rich in cholesterol, glycosphingolipids and the scaffolding protein Caveolina-1 (Cav-1). In this work, we investigated whether the increase in endocytosis and transendothelial vesicular trafficking in rat cerebellum after blood-brain barrier breakdown (BBBb) induced by Phoneutria nigriventer spider venom (PNV) was mediated by caveolae. The expression of Cav-1, phosphorylated Cav-1 (pCav-1), dynamin-2 (Dyn2), Src kinase family (SKF) and matrix-metalloproteinase-9 (MMP9), proteins involved in caveolar dynamics and BBB opening, was investigated. Immunofluorescence, western blotting (WB) and transmission electron microscopy were used to assess changes at 1, 2, 5, 24 and 72h post-venom. WB showed upregulation of Cav-1, Dyn2 and MMP9 at 1, 5 and 72h (corresponding, respectively, to intervals when intoxication was most evident, when signs of recovery were present, and when no intoxication was detectable). In contrast, pCav-1 and SKF, which are essential for internalization and transport, decreased when Cav-1 and Dyn2, proteins essential for caveolar formation, were increased. Overall, these changes indicated that vesicular trafficking across the endothelium (high pCav/SKF levels) coincided with lower numbers of caveolae (Cav-1/Dyn2 downregulation) and lower expression of MMP9. Thus, the internalization (disassembly) of caveolae alternates with caveolar neoformation (assembly), resulting in changes in caveolar density in the endothelium membrane. These caveolar dynamics imply tensional mechanical stress that is important in triggering key signaling mechanisms. We conclude that PNV-induced breakdown of transcellular transport in the BBB is caused by an increase in caveolae-mediated endocytosis; this effect was correlated with the progression of temporal signs of envenoming. Caveolar dynamics are probably involved in shear stress and BBBb regulatory mechanisms in this experimental model.
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), 13083-970 Campinas, SP, Brazil.
authors.forename: Edilene Siqueira
authors.initials: ES
authors.lastname: Soares
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), 13083-970 Campinas, SP, Brazil; Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), 13083-970 Campinas, SP, Brazil.
authors.forename: Monique Culturato Padilha
authors.initials: MCP
authors.lastname: Mendonça
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), 13083-970 Campinas, SP, Brazil; Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), 13083-970 Campinas, SP, Brazil. Electronic address: hofling@unicamp.br.
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: da Cruz-Höfling
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D021941:Caveolae / Q000187:drug effects* / Q000648:ultrastructure; D051242:Caveolin 1 / Q000378:metabolism; D002531:Cerebellum / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D034285:Dynamin II / Q000378:metabolism; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D051381:Rats; D017208:Rats, Wistar; D013111:Spider Venoms / Q000494:pharmacology*; D013997:Time Factors; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D051242:Caveolin 1; D013111:Spider Venoms; D019061:src-Family Kinases; D020780:Matrix Metalloproteinase 9; D034285:Dynamin II
keywords: Blood-brain barrier breakdown; Caveolae; Spider venom; Vesicle trafficking