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

Abstract

Key points: 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 A_2B increases 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.

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_2A and A_2B 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 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.

Keywords: CNG channel; Ca2+ influx; adenosine receptor; blood-brain barrier; cyclic adenosine monophosphate (cAMP); endothelial cell; gap junction.

Figure 1. Expression of adenosine receptor subtypes and Cx isoforms in hCMEC/D3 cells

The mRNA of the adenosine receptor subtypes A₁, A_2A and A_2B (A) and of the Cx isoforms Cx37, Cx40, Cx43 and Cx45 (B) was detected with RT‐PCR. Cx30, Cx32 and Cx36 were absent in hCMEC/D3 cells. ‐RT is the reverse transcription control with gapdh primers to confirm the absence of genomic DNA contamination. NTC is the negative control without template exemplarily shown for gapdh primers. C, immunofluorescence images of the different Cx isoforms in hCMEC/D3 cells (green). While Cx37 and Cx45 showed a diffuse intracellular staining (arrowheads), Cx40 and Cx43 staining showed gap junction plaques between neighbouring cells (arrows). Nuclei were counterstained with DAPI (blue). Scale bars represent 50 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Enhancement of gap junction coupling by 2‐PAA

A, representative micrographs of scrape loading/dye transfer experiments in hCMEC/D3 cells treated with the vehicle (cont., 0.3% ethanol) or 2‐PAA (20 μm) for 1 h. Scale bar represents 100 μm. B, the time‐dependent increase in the dye diffusion distance induced by 2‐PAA (20 μm) as found by scrape loading/dye transfer assays relative to the vehicle control (cont., 0.3% ethanol). C, the concentration dependency of 2‐PAA on the increased dye diffusion distance. The data points represent the relative dye diffusion distance achieved in cell monolayers after application of 2‐PAA for 1 h. All results were analysed using Student's t test. *Significant differences to the vehicle control: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3. 2‐PAA induced the formation of gap junction plaques between hCMEC/D3 cells

A, representative microscopic images of Cx43 (green) immunofluorescence in hCMEC/D3 cells after 20 μm 2‐PAA treatment for 1 h. Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm. B, the amount of hCMEC/D3 cells with Cx43 gap junction plaques was significantly increased after 2‐PAA treatment (20 μm) for 1 h compared to the vehicle control (cont., 0.3% ethanol). The results were analysed using Student's t test. ^*Significant differences to the vehicle control: ^* P < 0.05. C, exemplary confocal and STED images with increased resolution of individual Cx43 gap junction plaques. Scale bar represents 5 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. The pharmacology of the 2‐PAA‐related increase in gap junction coupling

A, 2‐PAA (20 μm) increased the intracellular cAMP concentration. This increase could be blocked by the adenylyl cyclase inhibitor SQ22536 (SQ, 400 μm). B, the increase in the dye diffusion distance in scrape loading/dye transfer assays was significantly attenuated by the adenylyl cyclase inhibitor SQ22536 (SQ, 400 μm). The adenylyl cyclase activator forskolin (for., 0.5 μm) and the cAMP analogue 8‐Br‐cAMP (1 mm) significantly increased the dye diffusion distance within 1 h similar to 2‐PAA. C, the increased dye diffusion distance by 2‐PAA (20 μm, 1 h) was not significantly affected by the A₁ adenosine receptor subtype antagonist DPCPX (DP, 25 nm) and only slightly affected by the A_2A adenosine receptor subtype antagonist SCH58261 (SCH, 0.5 μm). The A_2B adenosine receptor subtype antagonist MRS1754 (MRS, 0.5 μm) alone or together with DPCPX and SCH58261 nearly completely blocked the 2‐PAA‐induced increase in the dye diffusion distance. D, the A_2A adenosine receptor subtype‐specific agonist CGS21680 did not change the dye diffusion distance. E, transfection of the hCMEC/D3 cells with two different anti‐A_2B adenosine receptor siRNAs (A_2B siRNA) significantly decreased the mRNA amount of the A_2B adenosine receptor after 48 h compared to transfection with negative control (neg.) siRNA. Cells that were not treated with transfection reagent (‐transf.) served as control. F, the transfection with both anti‐A_2B adenosine receptor siRNAs (A_2B siRNA) attenuated the increase in the dye diffusion distance after 2‐PAA (20 μm) application compared to cells transfected with negative control (neg.) siRNA. The result of cells that were not treated with transfection reagent (‐transf.) is shown as control. The relative dye diffusion distances were normalized to vehicle‐treated cells transfected with negative control siRNA. All results were analysed using Student's t test. ^*Significant differences to the vehicle control: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001; ^#significant differences to 2‐PAA: ^# P < 0.05, ^## P < 0.01, ^### P < 0.001.

Figure 5. The signalling mechanism induced by 2‐PAA

A, the increase in the dye diffusion distance induced by 2‐PAA (20 μm, 1 h) was not blocked by the protein kinase A inhibitors Rp‐cAMPS (Rp, 200 μm) and KT5720 (KT, 1 μm). Additionally, the activator of the exchange protein directly activated by cAMP, 8‐pCPT‐O‐Me‐cAMP (100 μm) did not affect the dye diffusion distance. B, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem (L‐cis‐dil., 100 μm) could prevent the increase in the dye diffusion distance induced by 2‐PAA (20 μm, 1 h) relative to the vehicle control (cont., 0.3% ethanol) in scrape loading/dye transfer assays. C, RT‐PCR showed that the CNG channel subunits A1, A2 and B1 were expressed in hCMEC/D3 cells. D, furthermore, the cAMP‐sensitive subunit CNGA2 was confirmed to be expressed at protein level with β‐tubulin (β‐Tb) serving as loading control. E, in whole‐cell patch‐clamp experiments, 2‐PAA (20 μm) increased the current measured in hCMEC/D3 cells. This increased current was completely abolished by simultaneous application of the CNG channel blocker l‐cis‐diltiazem (L‐cis‐dil., 100 μm) with 2‐PAA. All results were analysed using Student's t test. ^*Significant differences to the vehicle control: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001; ^#significant differences to 2‐PAA: ^# P < 0.05.

Figure 6. The 2‐PAA‐induced increase in gap junction coupling was Ca²⁺ dependent

A, application of 2‐PAA (20 μm) increased the intracellular Ca²⁺ signal (fluorescence ratio F ₃₄₀/F ₃₈₀). The columns show the results obtained by counting the amount of cells with an increased [Ca²⁺]_i after treatment with the vehicle control (cont., 0.3% ethanol) or 2‐PAA (20 μm) in presence of 140 mm external Na⁺. B, a representative rapid transient Ca²⁺ signal induced by application of 2‐PAA (20 μm) when external Na⁺ was replaced by NMDG. C, the 2‐PAA‐induced increase in the dye diffusion distance was abolished when 2‐PAA (20 μm, 1 h) was applied on cells preloaded with the Ca²⁺ chelator BAPTA (10 μm) or in absence of external Ca²⁺ (‐ Ca²⁺ _extracell.). All results were analysed using Student's t test. ^*Significant differences to the vehicle control: ^* P < 0.05: ^*** P < 0.001; ^#significant differences to 2‐PAA: ^## P < 0.01.

Figure 7. Proposed mechanism of action of 2‐PAA to increase the gap junction coupling in hCMEC/D3 cells (continuous arrows)

The stimulation of A_2B adenosine receptors induces synthesis of cAMP which leads to a Ca²⁺ influx by opening CNG channels. We hypothesise that Ca²⁺ in turn induces the increase in gap junction plaques resulting in an enhancement of the gap junction coupling. The increase in the gap junction plaques is related by an as‐yet‐unidentified mechanism which is probably a fusion of connexon‐containing vesicles with the cell membrane. The green arrows indicate activation, the red bars indicate inhibition. The dotted arrows indicate other possible adenosine receptor‐dependent signalling mechanisms which were found not to be involved in the regulation of gap junction coupling in the cerebral microvascular endothelial cells in the present report. [Color figure can be viewed at wileyonlinelibrary.com]