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

摘要

class="enumerated" style="list-style-type:decimal">In ovine ciliated tracheal epithelial cells, acetylcholine (ACh) activates signal transduction pathways that not only transiently increase cytoplasmic Ca²⁺ ([Ca²⁺]i) but also actively lower [Ca²⁺]i. The pathway for decreasing [Ca²⁺]i 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.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⁺]i decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it.The ACh-stimulated [Ca²⁺]i 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).The application of mitochondrial uncouplers (5 μM CCCP or 5 μM FCCP), eliminated the ACh-induced [Ca²⁺]i 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⁺]i.These data suggest that ACh-induced [Ca²⁺]i decreases in ciliated cells are caused by stimulated Ca²⁺ uptake into mitochondria.