Bioluminescence detection of ATP release mechanisms in epithelia

Autocrine and paracrine release of and extracellular signalingby ATP is a ubiquitous cell biological and physiological process. Despite this knowledge, the mechanisms and physiological roles ofcellular ATP release are unknown. We tested the hypothesis thatepithelia release ATP under basal and stimulated conditions by using anewly designed and highly sensitive assay for bioluminescence detectionof ATP released from polarized epithelial monolayers. Thisbioluminescence assay measures ATP released from cystic fibrosis (CF)and non-CF human epithelial monolayers in a reduced serum mediumthrough catalysis of the luciferase-luciferin reaction, yielding aphoton of light collected by a luminometer. This novel assay measuresATP released into the apical or basolateral medium surroundingepithelia. Of relevance to CF, CF epithelia fail to release ATP acrossthe apical membrane under basal conditions. Moreover, hypotonicity isan extracellular signal that stimulates ATP release into bothcompartments of non-CF epithelia in a reversible manner; the responseto hypotonicity is also lost in CF epithelia. The bioluminescencedetection assay for ATP released from epithelia and other cells will beuseful in the study of extracellular nucleotide signaling inphysiological and pathophysiological paradigms. Taken together, theseresults suggest that extracellular ATP may be a constant regulator ofepithelial cell function under basal conditions and an autocrineregulator of cell volume under hypotonic conditions, two functions thatmay be lost in CF and contribute to CF pathophysiology.