Abstract
Dysregulation of blood-brain barrier (BBB) transport function is thought to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca2+, a condition mimicking acute ischemic stroke. In the absence of extracellular Ca2+, the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classic hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knockdown of Px1 and Cx43 with the small interfering RNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca2+ is at least partly attributable to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.
Footnotes
- Received September 29, 2014.
- Accepted February 9, 2015.
This study was supported in part by four Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) for Young Scientists (A) [KAKENHI: 23790170] and for Scientific Research (A) [KAKENHI: 24249011]; and by the JSPS and Centre National de la Recherche Scientifique (CNRS) under the Japan-France Basic Scientific Cooperation Program. T.T. and S.O. are full professors at Tohoku University and Kumamoto University, respectively, and are also directors of Proteomedix Frontiers Co. Ltd. This study was not supported by Proteomedix Frontiers Co. Ltd., and their positions at Proteomedix Frontiers Co. Ltd. did not affect the design of the study, the collection, analysis, and interpretation of the data, the writing of the manuscript, or the decision to publish, and does not present any financial conflicts. The remaining authors declare no competing financial interests.
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- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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