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

Abstract

RATIONALE Cyclic AMP (cAMP) is one of the most important second messengers and is involved as a target for the therapy of chronic obstructive pulmonary disease (COPD), an airway 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. In the present study, we combine the precision cut lung slice (PCLS) technique in mice with fluorescence resonance energy transfer (FRET) to monitor cAMP in real time. METHODS To monitor the cAMP levels in lung tissues, transgenic mice (CAG-Epac1-camps) that express the FRET-based cAMP sensor Epac1-camps were used for the preparation of PCLS. The β2-adrenergic receptor agonist fenoterol was applied to elevate intracellular cAMP. To achieve PDE subtype specific inhibition, the PDE4 inhibitor rolipram, the PDE3 inhibitor cilostamide and the PDE2 inhibitor BAY60-7550 were used. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) served as a control. Moreover, lung slices were exposed to 2.5 % cigarette smoke extract (CSE) for 24 hours used as a COPD model in vitro. RESULTS and CONCLUSIONS We provide evidence that the FRET and PCLS technologies can be combined in CAG-Epac1-camps mice to measure global cAMP. 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 under fenoterol stimulated conditions. In contrast, we found that a significant increase could be observed 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. Therefore, as the major lung cyclic nucleotide hydrolyzing enzymes PDE3 and PDE4 are both involved in the local regulation of the cAMP levels in the β2-AR microdomain, our findings suggest that exposure to CSE induced alterations in the PDEs activity profile both under basal conditions and in the presence of the β2-agonist fenoterol.