Mechanical stress-induced ATP release from human bronchial cells

Abstract

Although adenosine triphosphate (ATP) released from epithelia is an important autocrine and paracrine signaling, it is difficult to study due to its sensitiveness to mechanical stress. We have examined the cultured human bronchial epithelial cells (16HBE) to determine whether changes in Cl⁻ concentration or osmolality or shaking stimulate the cell to release ATP by using the luciferin-luciferase luminometric assay. Upon hypotonicity (205 mosmol kgH₂O⁻¹), the rate of ATP release increased from 0.036 ± 0.034 to 3.1 ± 0.6 pmol min⁻¹ at 6 min and decreased. The peak value of ATP release is estimated to be 6.2 x 10⁴ ATP molecules s⁻¹ per cell. Forskolin (10 μM) increased the hypotonicity-induced ATP release (P < 0.005), which was abolished (P < 0.001) by pretreatment with BAPTA-AM (25 μM), a membrane permeable Ca²⁺ chelator. Interestingly, lowering Ca²⁺ in the extracellular solution stimulated ATP released during mechanical perturbation, such as hypotonicity and gentle shaking. On the other hand, neither low Cl⁻ (75 mM) nor hypertonicity (500 mosmol kgH₂O⁻¹) could significantly increase the ATP release. Further, either forskolin or ionomycin failed to stimulate ATP release under the isotonic condition. In conclusion, a biphasic increase in ATP release and its low rate at the peak support the hypothesis that ATP is released through a non-conducting pathway model including exocytosis. Intracellular Ca²⁺ may play a key role for hypotonicity-induced ATP release. [Jpn J Physiol 54 Suppl:S87 (2004)]