Ca2+-dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleoticles

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca2+-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca2+ for the elevation of intracellular Ca2+ concentration ([Ca2+](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca2+-dependent ATP release. We found that ATP release does not directly involve Ca2+ influx from extracellular spaces, but depends entirely on Ca2+ mobilization from intracellular stores. The [Ca2+](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca2+ release from TG-sensitive endoplasmic reticulum (ER) Ca2+ stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca2+-free extracellular solution; or blocking the Ca2+-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca2+], spike results from the autocrine stimulation Of IP3/Ca2+-coupled P2Y, predominantly P2Y(6), receptors, accounting for similar to 70% of total Ca2+-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells,is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nuclecitides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.