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Monitoring local pulmonary cAMP levels: Combining precision cut lung slice (PCLS) and fluorescence resonance energy transfer (FRET) technologies in mice

Martina Schmidt, Haoxio Zuo, Wilfred Jelco Poppinga, Viacheslav Nikolaev
European Respiratory Journal 2016 48: PA5055; DOI: 10.1183/13993003.congress-2016.PA5055
Martina Schmidt
1Molecular Pharmacology, University of Groningen, Groningen, Netherlands
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Haoxio Zuo
1Molecular Pharmacology, University of Groningen, Groningen, Netherlands
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Wilfred Jelco Poppinga
1Molecular Pharmacology, University of Groningen, Groningen, Netherlands
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Viacheslav Nikolaev
24German Center for Cardiovascular Research (DZHK), Georg August University, Göttingen, Netherlands
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Abstract

Cyclic AMP (cAMP) is one of the most important second messengers and a target for the therapy of COPD, a disease primarily provoked by cigarette smoke. Cyclic nucleotide hydrolyzing phosphodiesterases (PDEs) are able to degrade cAMP or cGMP within subcellular compartments, thereby potentially altering pulmonary responses including airway contractility and inflammation. We combined the PCLS technique in mice with FRET to monitor cAMP in real time.

We provide evidence that the FRET and PCLS technologies can be combined in CAG-Epac1-camps mice to measure global cAMP levels. Moreover, we found in fenoterol-stimulated PCLS that PDE4 accounts for more than 80% of the total cAMP-PDE activity. Besides PDE4, PDE3 known as a cGMP-inhibited PDE also contributes to cAMP hydrolysis, indicating that cGMP may modulate the maintenance of local cAMP in PCLS. In contrast, the cGMP-activated PDE2 plays a limited role in cAMP hydrolysis.

Exposure to CSE did not alter the FRET signal in the presence of the PDE4 inhibitor in fenoterol-stimulated PCLS. In contrast, we found a significant increase in PDE3-dependent FRET responses (p<0.05). Under basal conditions, CSE treatment altered local cAMP levels by significantly increasing both PDE4 and PDE3 inhibitor effects. Both PDE4/3 are the most important cyclic nucleotide hydrolyzing enzymes responsible for the local regulation of cAMP in the β2-AR microdomain in the lung. In conclusion, our findings suggest that exposure to CSE causes alterations in the expression of both PDE4 and PDE3 and may thus alter functional responses in the airways.

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Monitoring local pulmonary cAMP levels: Combining precision cut lung slice (PCLS) and fluorescence resonance energy transfer (FRET) technologies in mice
Martina Schmidt, Haoxio Zuo, Wilfred Jelco Poppinga, Viacheslav Nikolaev
European Respiratory Journal Sep 2016, 48 (suppl 60) PA5055; DOI: 10.1183/13993003.congress-2016.PA5055

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Monitoring local pulmonary cAMP levels: Combining precision cut lung slice (PCLS) and fluorescence resonance energy transfer (FRET) technologies in mice
Martina Schmidt, Haoxio Zuo, Wilfred Jelco Poppinga, Viacheslav Nikolaev
European Respiratory Journal Sep 2016, 48 (suppl 60) PA5055; DOI: 10.1183/13993003.congress-2016.PA5055
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