title: Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+influx through cyclic nucleotide-gated channels.
abstract: Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2Bincreases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier.
  
  
  
  The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2Aand A2Badenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+influx by opening CNG channels in a cAMP-dependent manner. Ca2+in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.
authors.forename: Almke
authors.initials: A
authors.lastname: Bader
authors.affiliation: Institute of Biochemistry, Charité Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Willem
authors.initials: W
authors.lastname: Bintig
authors.affiliation: Walter Brendel Centre of Experimental Medicine, Department of Cardiovascular Physiology and Pathophysiology, Biomedical Center, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Begandt
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Klett
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.
authors.forename: Ina G
authors.initials: IG
authors.lastname: Siller
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Carola
authors.initials: C
authors.lastname: Gregor
authors.affiliation: Institute of Biostatistics, Leibniz University Hannover, Hannover, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schaarschmidt
authors.affiliation: Weill Medical College of Cornell University, New York, NY, USA.
authors.forename: Babette
authors.initials: B
authors.lastname: Weksler
authors.affiliation: Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, UK.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Romero
authors.affiliation: INSERM, U1016, Institut Cochin, Paris, France.|CNRS, UMR8104, Paris, France.|Université Paris Descartes, Paris, France.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.affiliation: Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.|Center for Systems Neuroscience Hannover, University of Veterinary Medicine Hannover Foundation, Hannover, Germany.
authors.forename: Anaclet
authors.identifier: 0000-0002-3686-923X
authors.initials: A
authors.lastname: Ngezahayo
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism*; D043704:Receptor, Adenosine A2B / Q000502:physiology*
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D043704:Receptor, Adenosine A2B; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C026708:2-phenylaminoadenosine; D000241:Adenosine; D002118:Calcium
keywords: CNG channel; Ca2+ influx; adenosine receptor; blood-brain barrier; cyclic adenosine monophosphate (cAMP); endothelial cell; gap junction