Hypotonicity-Induced ATP Release Is Potentiated by Intracellular Ca²⁺ and Cyclic AMP in Cultured Human Bronchial Cells

Abstract

We have examined the cultured human bronchial epithelial cells (16HBE) to learn if changes in Cl⁻ concentration or osmolality stimulate the cells to release ATP and to determine whether its release is cyclic AMP (cAMP)- and/or Ca²⁺-dependent by using the luciferin-luciferase luminometric assay. In a control solution (290 mosmol kg H₂O⁻¹), the external ATP concentration and the rate of ATP release were 0.52 ± 0.20 nM and 0.036 ± 0.034 pmol min⁻¹, respectively. Upon hypotonicity (205 mosmol kg H₂O⁻¹), they increased to 7.0 ± 1.3 nM and 3.1 ± 0.6 pmol min⁻¹, respectively, at 6 min, then decreased. At the peak, the rate of ATP release is estimated to be 6.2×10⁴ ATP molecules s⁻¹ per cell. An accumulation of the released ATP for the initial 10 min increased significantly (p < 0.005) by 71.5% in the presence of forskolin (10 μM), adenylyl cyclase activator, however, it was abolished (p < 0.001) by pretreatment with BAPTA-AM (25 μM), a membrane permeable Ca²⁺ chelator. On the other hand, neither low Cl²⁻ (75 mM, isotonic) nor hypertonicity (+NaCl or +mannitol, 500 mosmol kg H₂O⁻¹) could significantly increase the ATP release. Further, forskolin or ionomycin (a Ca²⁺ ionophore) or, both, failed to stimulate ATP release under the isotonic condition. In conclusion, first, hypertonicity and changes in Cl⁻ concentrations are not effective signals for the ATP release; second, hypotonicity-induced ATP release is potentiated by the level of intracellular Ca²⁺ and cAMP; and third, 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, such as exocytosis, or through a volume-dependent, ATP-conductive anion channel.