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Cellular and Molecular Life SciencesVolume 73, Issue 24, 4 July 2016, Pages 4701-4716

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells(Article)

  • Coelho-Santos, V.,
  • Socodato, R.,
  • Portugal, C.,
  • Leitão, R.A.,
  • Rito, M.,
  • Barbosa, M.,
  • Couraud, P.-O.,
  • Romero, I.A.,
  • Weksler, B.,
  • Minshall, R.D.,
  • Fontes-Ribeiro, C.,
  • Summavielle, T.,
  • Relvas, J.B.,
  • Silva, A.P.
  • View Correspondence (jump link)
  • aInstitute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
  • bCNC.IBILI, University of Coimbra, Coimbra, Portugal
  • cInstituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal
  • dInstituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal
  • eNeurosurgery Service, Coimbra Hospital and University Centre (CHUC), Coimbra, Portugal
  • fFaculty of Medicine, University of Coimbra, Coimbra, Portugal
  • gInstitut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France
  • hDepartment of Life Sciences, Faculty of Science, The Open University, Milton Keynes, United Kingdom
  • iDepartment of Medicine, Weill Medical College of Cornell University, New York, NY, United States
  • jDepartment of Anesthesiology, University of Illinois at Chicago, Chicago, IL, United States
  • kDepartment of Pharmacology, University of Illinois at Chicago, Chicago, IL, United States

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 Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant 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. © 2016, Springer International Publishing.

Author keywords

Attention deficit hyperactivity disorderBlood–brain barrierNADPH oxidasePsychostimulantsVesicular transportVitamin C

Indexed keywords

EMTREE drug terms:ascorbic acidcaveolin 1methylphenidateprotein tyrosine kinaseRac1 proteinreactive oxygen metabolitereduced nicotinamide adenine dinucleotide phosphate oxidasecaveolin 1CSK tyrosine-protein kinasehorseradish peroxidasemethylphenidateoxidizing agentprotein Cdc42protein tyrosine kinaseRac1 proteinreactive oxygen metabolitereduced nicotinamide adenine dinucleotide phosphate oxidaseRhoA guanine nucleotide binding protein
EMTREE medical terms:Articleblood brain barrierbrain capillary endothelial cellcaveolacell membranecell membrane permeabilitycontrolled studyhumanhuman cellprotein phosphorylationsignal transductiontranscytosisbiological modelbraincapillary permeabilitycaveolacytologydrug effectsendothelium cellenzymologymetabolismphosphorylationtranscytosistransport at the cellular leveltransport vesicle
MeSH:Biological TransportBrainCapillary PermeabilityCaveolaeCaveolin 1cdc42 GTP-Binding ProteinEndothelial CellsHorseradish PeroxidaseHumansMethylphenidateModels, BiologicalNADPH OxidaseOxidantsPhosphorylationrac1 GTP-Binding ProteinReactive Oxygen SpeciesrhoA GTP-Binding ProteinSignal Transductionsrc-Family KinasesTranscytosisTransport Vesicles

Chemicals and CAS Registry Numbers:

ascorbic acid, 134-03-2, 15421-15-5, 50-81-7; methylphenidate, 113-45-1, 298-59-9; protein tyrosine kinase, 80449-02-1; reduced nicotinamide adenine dinucleotide phosphate oxidase, 9032-22-8;

Caveolin 1; cdc42 GTP-Binding Protein; CSK tyrosine-protein kinase; Horseradish Peroxidase; Methylphenidate; NADPH Oxidase; Oxidants; rac1 GTP-Binding Protein; Reactive Oxygen Species; rhoA GTP-Binding Protein; src-Family Kinases

Funding details

Funding sponsor Funding number Acronym
Programa Operacional Temático Factores de Competitividade
P01 HL60678
Kyoto University
University of Illinois
University of California, San Diego
Fundação para a Ciência e a Tecnologia
See opportunities
Federación Española de Enfermedades RarasSFRH/BD/85556/2012,SFRH/BPD/91962/2012,SFRH/BD/84408/2012,SFRH/BPD/91833/2012,UID/NEU/04539/2013,PEST-C/SAU/UI3282/2013FEDER

  • 1

    This work was supported by Project PTDC/NEU-OSD/0312/2012 from Foundation for Science and Technology (FCT Portugal) co-financed by COMPETE and FEDER funds, and strategic projects PEST-C/SAU/UI3282/2013 and UID/NEU/04539/2013. Also, Ph.D. fellowships SFRH/BD/85556/2012 and SFRH/BD/84408/2012 and, postdoctoral fellowship SFRH/BPD/91833/2012 and SFRH/BPD/91962/2012 from FCT Portugal co-financed by QREN. TSummavielle was supported by program Investigador FCT, POPH and Fundo Social Europeu, and RDMinshall by NIH P01 HL60678. We thank Dr. Shu Chien (University of California, San Diego) for providing KRas Src YPet and KRas Src (RV) YPet FRET probes. We thank Dr. Michiyuki Matsuda (Kyoto University) for kindly sharing with us the Raichu-RhoA, Raichu-Rac1 and Raichu-cdc42 FRET probes, and Dr. Andrei Karginov (University of Illinois, Chicago) for providing the RapR-Src construct.

  • ISSN: 1420682X
  • CODEN: CMLSF
  • Source Type: Journal
  • Original language: English
  • DOI: 10.1007/s00018-016-2301-3
  • PubMed ID: 27376435
  • Document Type: Article
  • Publisher: Birkhauser Verlag AG

  Silva, A.P.; Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
© Copyright 2017 Elsevier B.V., All rights reserved.

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