title: Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.
abstract: Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14Fmutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.
authors.affiliation: Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.|CNC.IBILI, University of Coimbra, Coimbra, Portugal.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Coelho-Santos
authors.affiliation: Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.|Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
authors.forename: Renato
authors.initials: R
authors.lastname: Socodato
authors.affiliation: Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.|Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
authors.forename: Camila
authors.initials: C
authors.lastname: Portugal
authors.affiliation: Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.|CNC.IBILI, University of Coimbra, Coimbra, Portugal.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Leitão
authors.affiliation: Neurosurgery Service, Coimbra Hospital and University Centre (CHUC), Coimbra, Portugal.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rito
authors.affiliation: Neurosurgery Service, Coimbra Hospital and University Centre (CHUC), Coimbra, Portugal.|Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Marcos
authors.initials: M
authors.lastname: Barbosa
authors.affiliation: Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.affiliation: Department of Life Sciences, Faculty of Science, The Open University, Milton Keynes, UK.
authors.forename: Ignacio A
authors.initials: IA
authors.lastname: Romero
authors.affiliation: Department of Medicine, Weill Medical College of Cornell University, New York, NY, USA.
authors.forename: Babette
authors.initials: B
authors.lastname: Weksler
authors.affiliation: Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL, USA.|Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
authors.affiliation: Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.|CNC.IBILI, University of Coimbra, Coimbra, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Fontes-Ribeiro
authors.affiliation: Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.|Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Summavielle
authors.affiliation: Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.|Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
authors.forename: João B
authors.initials: JB
authors.lastname: Relvas
authors.affiliation: Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. apmartins@fmed.uc.pt.|CNC.IBILI, University of Coimbra, Coimbra, Portugal. apmartins@fmed.uc.pt.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Silva
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000166:cytology; D000081247:CSK Tyrosine-Protein Kinase; D002199:Capillary Permeability / Q000187:drug effects; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D008774:Methylphenidate / Q000494:pharmacology*; D008954:Models, Biological; D019255:NADPH Oxidases / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D057900:Transcytosis / Q000187:drug effects*; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D016877:Oxidants; D017382:Reactive Oxygen Species; D008774:Methylphenidate; D006735:Horseradish Peroxidase; D019255:NADPH Oxidases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein
keywords: Attention deficit hyperactivity disorder; Blood–brain barrier; NADPH oxidase; Psychostimulants; Vesicular transport; Vitamin C