ATP release from human airway epithelial cells studied using a capillary cell culture system

Abstract

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 (≈250 μg (108 cells)−1 day−1). Elevating apical flow rate 5‐fold increased ATP secretion from ≈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 [Ca2+] 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‐Ca2+ 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.

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…A major common mechanism involved in epithelial Ca 2+ signaling and propagation of intercellular Ca 2+ signaling in epithelial cell monolayers involves the regulated cellular release of ATP or related purines, which promote paracrine signaling by activating purinergic P2 receptors at the cell surface (136)(137)(138)(139). Various forms of chemical or mechanical injury, and cell stimulation by bacterial products or dsRNA, result in release of ATP at levels that are sufficient to activate P2 receptors and mediate Ca 2+ signaling (140)(141)(142)(143)(144).…”

Roles of gap junctions and hemichannels in bone cell functions and in signal transmission of mechanical stress

“…Extracellular domains of these undocked hemichannels are structurally different from connexons in docked gap junctional plaques. Hemichannels appear to provide a mechanism for ATP and NAD + release, which raises intracellular Ca 2+ levels and promotes Ca 2+ wave propagation in astrocytes, bone cells, epithelial cells, and retinal cells (103)(104)(105)(106)(107)(108)(109)(110)(111). Hemichannels in astrocytes are shown to be involved in the release of the neurotransmitter glutamate (112).…”

Normal and Cystic Fibrosis Airway Surface Liquid Homeostasis

“…Based on the observations that (i) tidal volume expansion and airflow impart shear to airway surfaces (13), (ii) shear releases nucleotides from many cell types into the extracellular environment (14,15), and (iii) extracellular nucleotides interact with P2Y 2 and P2X purinoceptors to regulate airway ion transport (16,17), we hypothesized that the failure to recapitulate in vitro the phasic motion associated with tidal breathing has hindered the identification of the pathways for PCL volume autoregulation. Accordingly, we developed a novel culture system that mimics the best characterized shear stress in the lung, i.e.…”