Endocrine Abstracts (2008) 16 S14.1

Direct visualization of cyclic AMP levels in three dimensional cultures of thyroid follicles

Davide Calebiro1, Viacheslav Nikolaev1, Marco Bonomi2, Luca Persani2 & Martin Lohse1

1Department of Pharmacology and Toxicology, University of Wuerzburg, Wuerzburg, Germany; 2University of Milan and Istituto Auxologico Italiano IRCCS, Cusano Milanino, Italy.

Cyclic AMP (cAMP) is the principal intracellular mediator of TSH effects in the thyroid, inducing both thyroid hormone production and cell proliferation. This notion derives from a large series of evidences, mainly obtained by biochemical approaches on cell lines or primary thyrocytes. However, given the limited resolution in time and space of the techniques employed so far, little is know about the spatial localization and temporal dynamics of cAMP signaling in thyroid cells. Moreover, it is well known that the functional unit of the thyroid is a multicellular structure, i.e. the thyroid follicle. Therefore, a deeper understanding of TSH/cAMP cascade and of thyrocyte biology in general will probably require an in vivo approach, either on isolated thyroid follicles or on whole animals. The aim of this study was to develop a method for real-time monitoring of cAMP levels in living thyroid follicles. To this purpose we established a protocol for three dimensional culture of thyroid follicles, which results in good morphology and allows microscopic visualization. To monitor intracellular cAMP levels, we employed a Fluorescence Resonance Energy Transfer based sensor (Epac1-cAMPs), the fluorescence of which is inversely proportional to cAMP levels. Thyroid follicles obtained from a transgenic mouse expressing Epac1-cAMPs were then isolated and cultured as mentioned above. By this approach we were able to evaluate the kinetics of cAMP accumulation and clearance after TSH stimuli of different intensity and duration. The kinetics observed after a short application of TSH were unexpectedly fast, with t1/2 of ~1 and 3 min. for cAMP accumulation and degradation, respectively. These data are consistent with an unpredicted speed of TSH association and dissociation from its receptor. The newly developed method paves the way to a series of in vivo studies aimed at further elucidating the spatio-temporal organization of TSH/cAMP signaling cascade.

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