BIOLUMINESCENCE DETECTION OF ATP RELEASE MECHANISMS IN EPITHELIA

Autocrine and paracrine release of and extracellular signaling by ATP is a ubiquitous cell biological and physiological process. Despite thi s knowledge, the mechanisms and physiological roles of cellular ATP re lease are unknown. We tested the hypothesis that epithelia release ATP under basal and stimulated conditions by using a newly designed and h ighly sensitive assay for bioluminescence detection of ATP released fr om polarized epithelial monolayers. This bioluminescence assay measure s ATP released from cystic fibrosis (CF) and non-OF human epithelial m onolayers in a reduced serum medium through catalysis of the luciferas e luciferin reaction, yielding a photon of light collected by a lumino meter. This novel assay measures ATP released into the apical or basol ateral medium surrounding epithelia. Of relevance to CF, CF epithelia fail to release ATP across the apical membrane under basal conditions. Moreover, hypotonicity is an extracellular signal that stimulates ATP release into both compartments of non-OF epithelia in a reversible ma nner; the response to hypotonicity is also lost in CF epithelia. The b ioluminescence detection assay for ATP released from epithelia and oth er cells will be useful in the study of extracellular nucleotide signa ling in physiological and pathophysiological paradigms. Taken together , these results suggest that extracellular ATP may be a constant regul ator of epithelial cell function under basal conditions and an autocri ne regulator of cell volume under hypotonic conditions, two functions that may be lost in CF and contribute to CF pathophysiology.