Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca²⁺ influx through cyclic nucleotide‐gated channels

Abstract

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 A and A adenosine 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 Ca with BAPTA, or the absence of external Ca , 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 Ca influx by opening CNG channels in a cAMP-dependent manner. Ca 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.

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“…CMECs can directly or indirectly affect function of neurons and synapses and are involved in many neurological diseases, including cognitive impairment. Bader et al [22] used CMECs to characterize the physiological link between adenosine receptors and gap junction coupling in endothelial cells of the BBB. The report identifies CNG channels as a physiological link that integrates gap junction coupling and adenosine receptor-dependent signaling of endothelial cells of the BBB.…”

Unravelling a novel mechanism for the up‐regulation of connexin43 gap junctions between cells derived from the blood–brain barrier

“…With regard to the first possibility, cycloheximide (a protein synthesis inhibitor in eukaryotic cells) did not prevent the enlargement of Cx43 gap junctional plaques in cells treated with adenosine, which increases cAMP concentration, as observed through immunofluorescence in hCMEC/D3 (Bader et al . ). Curiously, though, cAMP does increase the transcriptional rate of Cx43 mRNA in a communication‐deficient Morris hepatoma cell line through a mechanism also resistant to cycloheximide (Mehta et al .…”

“…In an article in this issue of The Journal of Physiology , however, Bader et al . () detected only Cx43 at gap junction plaques from hCMEC/D3 cells, which is a cell line derived from the blood–brain barrier. The existence of one Cx type forming gap junctions should simplify the understanding of mechanisms that regulate this intercellular communication pathway.…”

“…The research group of Dr Anaclet Ngezahayo have now unravelled an unsuspected intracellular signalling step (Bader et al . ), on which I comment here, focusing exclusively on studies about gap junctions channels formed by Cx43 and their regulation by cAMP.…”

“…Thus, it remains to be studied whether the coordinated cross‐talk between a cAMP‐dependent pathway might activate Akt and/or CK1. A possible alternative was recently discovered in a systematic study by Bader et al () performed on hCMEC/D3 cells. These cells were treated with agents that increase cAMP intracellular concentration (e.g.…”