Details
| Original language | English |
|---|---|
| Pages (from-to) | 606-622 |
| Number of pages | 17 |
| Journal | Cellular Physiology and Biochemistry |
| Volume | 53 |
| Issue number | 4 |
| Early online date | 25 Sept 2019 |
| Publication status | Published - 2019 |
Abstract
BACKGROUND/AIMS: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model.
METHODS: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity.
RESULTS: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate.
CONCLUSION: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway.
Keywords
- Adenosine A2 Receptor Agonists/pharmacology, Carbenoxolone/pharmacology, Cell Line, Connexin 26/antagonists & inhibitors, Cyclic AMP/metabolism, Cyclic AMP-Dependent Protein Kinases/metabolism, Dielectric Spectroscopy, Epithelial Cells/cytology, Gap Junctions/drug effects, Gene Expression Regulation/drug effects, Humans, Protein Isoforms/antagonists & inhibitors, RNA Interference, RNA, Small Interfering/metabolism, Receptor, Adenosine A2B/chemistry, Signal Transduction/drug effects, Adenosine receptors, Calu-3 cells, Metabolite uptake, Airway epithelium, Connexin hemichannels
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Physiology
Sustainable Development Goals
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In: Cellular Physiology and Biochemistry, Vol. 53, No. 4, 2019, p. 606-622.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Stimulation of the A2B Adenosine Receptor Subtype Enhances Connexin26 Hemichannel Activity in Small Airway Epithelial Cells
AU - Dierks, Anne
AU - Bader, Almke
AU - Lehrich, Tina
AU - Ngezahayo, Anaclet
N1 - Funding Information: The research was partly supported by the DFG project NG 瘂 猃爁猀 and the B?BF project TRANS-LARA. The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover.
PY - 2019
Y1 - 2019
N2 - BACKGROUND/AIMS: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model.METHODS: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity.RESULTS: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate.CONCLUSION: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway.
AB - BACKGROUND/AIMS: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model.METHODS: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity.RESULTS: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate.CONCLUSION: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway.
KW - Adenosine A2 Receptor Agonists/pharmacology
KW - Carbenoxolone/pharmacology
KW - Cell Line
KW - Connexin 26/antagonists & inhibitors
KW - Cyclic AMP/metabolism
KW - Cyclic AMP-Dependent Protein Kinases/metabolism
KW - Dielectric Spectroscopy
KW - Epithelial Cells/cytology
KW - Gap Junctions/drug effects
KW - Gene Expression Regulation/drug effects
KW - Humans
KW - Protein Isoforms/antagonists & inhibitors
KW - RNA Interference
KW - RNA, Small Interfering/metabolism
KW - Receptor, Adenosine A2B/chemistry
KW - Signal Transduction/drug effects
KW - Adenosine receptors
KW - Calu-3 cells
KW - Metabolite uptake
KW - Airway epithelium
KW - Connexin hemichannels
UR - http://www.scopus.com/inward/record.url?scp=85072560212&partnerID=8YFLogxK
U2 - 10.33594/000000160
DO - 10.33594/000000160
M3 - Article
C2 - 31550088
VL - 53
SP - 606
EP - 622
JO - Cellular Physiology and Biochemistry
JF - Cellular Physiology and Biochemistry
SN - 1015-8987
IS - 4
ER -