Agonist-stimulated calcium decreases in ovine ciliated airway epithelial cells: Role of mitochondria

1. In ovine ciliated tracheal epithelial cells, acetylcholine (ACh) activates signal transduction pathways that not only transiently increase cytoplasmic Ca²⁺ ([Ca²⁺]₁) but also actively lower [Ca²⁺]₁. The pathway for decreasing [Ca²⁺]₁ is clearly revealed after depletion of intracellular Ca²⁺ stores by thapsigargin (Tg), 2,5-di-(tert-butyl)-1,4-benzohydroquinone or NiCl₂. Measurements with microinjected fura-2 excluded a [Ca²⁺] measurement artefact. 2. A four-compartment model to simulate calcium transients in non-excitable cells (consisting of a plasma membrane Ca²⁺ pump and channel; Ca²⁺ store with pump and channel; and cytosolic Ca²⁺ buffer) could not account for the observed [Ca²⁺]₁ decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it. 3. The ACh-stimulated [Ca²⁺]₁ decrease was not due to an inhibition of Ca²⁺ influx (Ca²⁺ channel blockers or absence of extracellular calcium had no effect), activation of a plasma membrane Ca²⁺-ATPase (two inhibitors, vanadate (30 mM) and lanthanum (10 mM), had no effect) or inhibition of the Na⁺-Ca²⁺ exchanger (replacing extracellular Na⁺ with N-methylglucamine had no effect). 4. The application of mitochondrial uncouplers (5 μM CCCP or 5 μM FCCP), eliminated the ACh-induced [Ca²⁺]₁ decrease. Addition of CCCP at the nadir of the decrease restored intracellular calcium levels of Tg-treated cells to baseline faster than controls not exposed to mitochondrial uncouplers. CCCP application to naïve cells did not block the ACh-induced transient increase in [Ca²⁺]₁. 5. These data suggest that ACh-induced [Ca²⁺]_i decreases in ciliated cells are caused by stimulated Ca²⁺ uptake into mitochondria.