Monitoring Local Pulmonary cAMP Levels

    Zuo, H. | Rijks Universiteit Groningen | 23 augustus 2016 | 7.1.16.104CO
American Thoracic Society Report 2016

As one of the most important international conferences, the American Thoracic Society (ATS) main meeting appeals the attendances of thousands of experts who work in pulmonary diseases, critical illnesses and sleep-related breathing disorders by providing a series of latest lectures regarding patients' perspectives, fundamental researches, workshop reports and training courses. As a second year PhD who is working in fundamental research of chronic obstructive pulmonary diseases, thanks to longfonds I have the opportunity to join ATS 2016 in San Francisco and present my research titled "Monitoring Local Pulmonary cAMP Levels: Combining Precision Cut Lung Slice (PCLS) and Fluorescence Resonance Energy Transfer (FRET) Technologies in Mice" via a poster.

Cyclic AMP (cAMP) is one of the most important second messengers and involved as a target for the therapy of chronic obstructive pulmonary disease (COPD), an airway disease primarily provoked by cigarette smoke (CS). 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. 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 2-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.

In our study, we do find PCLS and FRET could be combined and used as a novel method to monitor cAMP levels in airway. And moreover, it is also possible to measure the activities of relative receptors or enzymes which are involved in cAMP production or degradation. During ATS 2016, several researchers showed their interests about our unique method and also gave a lot of professional suggestions for the future study. Dr. Chen was curious about the reason why we would like to combine FRET with PCLS. Since FRET is widely used to perform real-time measurements of second messengers in living individual cell. But as a highly developed structure, airway is consisting of several different cell types, such as epithelial cells, airway smooth muscle cells and fibroblasts. These cells could communicate with each other through various ways, and also contribute to the morphology and function of airway together. Therefore, it will be more comprehensive to reveal cAMP compartmentalization in airway by our method. Also, Dr. Hanna suggested that cigarette smoke in vivo and in vitro exposures may be different. Actually, we do think about in vivo and in vitro smoke may have different effects on PDE distributions. And next in vivo cigarette smoke experiments will be planned.

I also joined a postgraduate course "Implementation of core components of a lung cancer screening program" in ATS 2016. Even though I am not a physician or nurse, basic information I learned from this course did help me to understand the critical components of a lung cancer screening program and the basic screening recommendations and requirements in the provision of lung cancer screening.

Another interesting study directly relating to my work was the poster by Dr. Abbott-Banner investigating the bronchodilator effect of a first in-class dual phosphodiesterase 3/4 inhibitor, RPL554. Their work demonstrated that inhalation RPL554 was more effective as a bronchodilator in asthmatics. Moreover, it showed fewer side effects in those asthma patients compared with traditional treatments. These outcomes fitted perfectly with our results that both PDE3 and PDE4 FRET response were significantly increased in the CSE treated groups. During our talk, we also agreed with each other that so far we don't have enough evidence to prove that PDE3 is specific on contractile modulation and PDE4 is responsible for inflammation, which is also important in my hypothesis.

Also, there are many researches regarding precision cut lung slices, which I also used in my studies. After discussion with some experts, such as Dr. Chen and Dr. Hanna, I did know that mouse strain, washing protocol and temperature control might also affect outcomes. I think it will help me to compare results, especially in literature study.

Thanks to longfonds and ATS, I had the opportunity to meet with numerous top scientists in this field during the conference and for further discussion. Also, I observed many latest progressions from others, which helps me to find my interested research topics in the future study. I had several constructive discussions about the technique PCLS, which helps me to reconsider more comprehensively about technique itself and also its contributions to my own study.

 

Keyword: ATS 2016 COPD Cyclic AMP

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