Confocal fluorescent imaging of the interactions of cholinergic agonists with pulmonary neuroepithelial bodies (NEBs) using an in situ lung slice model

Abstract

Evidence strongly suggests that components of tobacco smoke induce changes in the pulmonary neuroendocrine system. In this study, potential receptor mediated cholinergic interactions with NEBs are postulated based on the expression of vesicular acetylcholine transporter (VAChT) in nerve endings between the NEB cells. Aim was to explore the possible direct effects of cholinergic agonists on NEBs via Ca2+ imaging in a mouse lung slice model. NEBs were visualized by 4-Di-2-ASP and Ca2+-dependent activation by Fluo-4. Application of high extracellular K+ (5s; 50mM) was used as a positive control for NEB activation and for the identification of Clara-like cells, which cover NEBs and show a characteristic slightly delayed Ca2+ rise. Surprisingly, no rise in Fluo-4 fluorescence could be detected in NEB cells after stimulation with 10–100μM nicotine, acetylcholine or carbachol. In Clara-like cells, however, the cholinergic stimulation resulted in a clear Ca2+ rise. In conclusion, NEBs in mouse lungs did not show a Ca2+-dependent activation by externally applied cholinergic agonists. Clara-like cells that intimately interact with NEBs did reveal a cholinergic activation and, considering their possible stem cell-like properties, may be more important players in the airways in health and disease than accounted for today. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)