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Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2008-12-04)
Ian De Proost, Isabel Pintelon, William J Wilkinson, Sofie Goethals, Inge Brouns, Luc Van Nassauw, Daniela Riccardi, Jean-Pierre Timmermans, Paul J Kemp, Dirk Adriaensen
ABSTRACT

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atropine sulfate salt monohydrate, ≥97% (TLC), crystalline
Sigma-Aldrich
Anti-Calcitonin Gene Related Peptide antibody produced in rabbit, whole antiserum
Sigma-Aldrich
4-(4-Diethylaminostyryl)-1-methylpyridinium iodide, ≥97%