Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca2+ concentration ([Ca2+](i)) in a dose-dependent manner. An adequate hypo-osmotic stress (-40 mosM) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose-response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from -20 mosM to -90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca2+](i) and CBF, as well as potentiating ACh-stimulated rises in [Ca2+](i) and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X(4) purinergic receptor. A hypo-osmotic stress (-40 mosM) transiently increased [Ca2+](i) and potentiated the ACh-stimulated [Ca2+](i) increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca2+-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca2+](i) and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X(4).