ATP release from human airway epithelial cells studied using a capillary cell culture system.
Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study. We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells. The Calu-3 cell line derived from human airway sub-mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a re-circulating medium pump. Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (approximately 250 microg (10(8) cells)(-1) day(-1)). Elevating apical flow rate 5-fold increased ATP secretion from approximately 200 to 6618 fmol min(-1). Reducing apical osmolarity by 25-43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained. Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis. Lowering apical [Ca(2+)] to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear-induced ATP release only in low-Ca(2+) solution, and therefore another secretory pathway may operate with physiological (i.e. mM) calcium. Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion.
Guyot
Annick
A
Department of Physiology, McGill University, 3655 Promemnade Sir William Osler, Montréal, Québec, Canada H3G 1Y6. annick.m.guyot@mcgill.ca
Hanrahan
John W
JW
D000255:Adenosine Triphosphate / Q000378:metabolism*; D019149:Bioreactors; D002462:Cell Membrane / Q000378:metabolism; D003584:Cytological Techniques; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009994:Osmolar Concentration; D013314:Stress, Mechanical; D014407:Tumor Cells, Cultured
D007038:Hypotonic Solutions; D000255:Adenosine Triphosphate