Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells
Open accessAbstract
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 Tyr 14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1 Y14F 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.
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MPH modulates intracellular mediators and transcription factors involved in neuronal signalling, including AK1, MAPK3, FOS, CAMK4 and RAC1.
“Finally, MPH has been reported to modulate intracellular mediators and transcription factors involved in neuronal signalling, including adenylate kinase isoenzyme 1 (AK1), mitogen-activated protein kinase 3 (MAPK3), proto-oncogene c-Fos (FOS), calcium/calmodulin-dependent protein kinase type IV (CAMK4) and Ras-related C3 botulinum toxin substrate 1 (RAC1) [149].”
From Molecular Characterisation of the Mechanism of Action of Stimulant Drugs Lisdexamfetamine and Methylphenidate on ADHD Neurobiology: A Review (2022) · cites this paper as [149]
Coelho-Santos et al. demonstrated that MPH promotes ROS generation via activation of RAC1-dependent NADPH oxidase (NOX) and c-Src activation.
“In this sense, Coelho-Santos et al. demonstrated that MPH promotes ROS generation via activation of RAC1-dependent NADPH oxidase (NOX) and c-Src activation [149].”
From Molecular Characterisation of the Mechanism of Action of Stimulant Drugs Lisdexamfetamine and Methylphenidate on ADHD Neurobiology: A Review (2022) · cites this paper as [149]
MPH has been shown to promote ROS via RAC1 and CAMK4 activation, although the evidence regarding whether MPH use indeed induces or reduces oxidative stress is not clear.
“Moreover, MPH has been shown to promote ROS via RAC1and CAMK4 activation, although the evidence regarding whether MPH use indeed induces or reduces oxidative stress is not clear.”
From Molecular Characterisation of the Mechanism of Action of Stimulant Drugs Lisdexamfetamine and Methylphenidate on ADHD Neurobiology: A Review (2022) · cites this paper as [149]
Cites
Persidsky, Y., Heilman, D., Haorah, J., Zelivyanskaya, M., Persidsky, R., Weber, G. A., Shimokawa, H., Kaibuchi, K., & Ikezu, T. (2006) · Blood · doi:10.1182/blood-2005-11-4721 · Cited by 200 · see citing works
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Quintero, J., Gutiérrez-Casares, J. R., & Álamo, C. (2022) · Neurology and Therapy · doi:10.1007/s40120-022-00392-2 · Cited by 70 · see citing works
“Moreover, MPH has been shown to promote ROS via RAC1and CAMK4 activation, although the evidence regarding whether MPH use indeed induces or reduces oxidative stress is not clear.”
BackgroundInfluentialOxidopamine and oxidative stress: Recent advances in experimental physiology and pharmacology
Pantic, I., Cumic, J., Skodric, S. R., Dugalic, S., & Brodski, C. (2021) · Chemico-Biological Interactions · doi:10.1016/j.cbi.2021.109380 · Cited by 28 · see citing works
- Authors
- 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.
- Affiliations
- Universidade do Porto (PT)
- University of Coimbra (PT)
- Unidade Local de Saúde de Coimbra (PT)
- Centre National de la Recherche Scientifique (FR)
- The Open University (GB)
- Cornell University (US)
- University of Illinois Chicago (US)
- Corresponding author
- Silva, Ana P.
- ORCID
- Venue
- Cellular and Molecular Life Sciences
- Published
- 2016-07-04
- Volume
- 73
- Issue
- 24
- Pages
- 4701-4716
- ISSN
- 1420-682X (ISSN-L)
- 1420-9071
- PMC
- PMC11108272
- OpenAlex
- W2462985470
- Cited by
- 36
- References
- 1
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 4 · 2023: 5 · 2022: 4 · 2021: 2 · 2020: 3 · 2019: 5 · 2018: 4 · 2017: 4
- FWCI
- 2.98
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Cellular and Molecular Life Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Heart, Lung, and Blood Institute (P01HL060678)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Caveolin-1 and cellular processes
- Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Keywords
- methylphenidate, reactive oxygen species, caveolae, transcytosis, Rac1 signaling, NADPH oxidase, Src kinase, caveolin-1 phosphorylation, brain endothelial cells, transendothelial permeability, vesicle trafficking, attention deficit hyperactivity disorder, stimulants
- MeSH terms
- CSK Tyrosine-Protein Kinase, Biological Transport, Brain, Capillary Permeability, Horseradish Peroxidase, Humans, Methylphenidate, Models, Biological, Phosphorylation, Signal Transduction, Oxidants, Reactive Oxygen Species, src-Family Kinases, NADPH Oxidases, rhoA GTP-Binding Protein, cdc42 GTP-Binding Protein, rac1 GTP-Binding Protein, Caveolae, Transport Vesicles, Endothelial Cells, Caveolin 1, Transcytosis
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