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+]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. 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+]i decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it. 3. 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). 4. The application of mitochondrial uncouplers (5 microM CCCP or 5 microM 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. 5. These data suggest that ACh-induced [Ca2+]i decreases in ciliated cells are caused by stimulated Ca2+ uptake into mitochondria.
Salathe
M
M
Division of Pulmonary and Critical Care Medicine and Department of Molecular and Cellular Pharmacology University of Miami School of Medicine, FL 33136, USA. msalathe@miami.edu
Ivonnet
P I
PI
Lieb
T
T
Bookman
R J
RJ
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D002923:Cilia / Q000187:drug effects / Q000502:physiology; D004396:Coloring Agents; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008845:Microinjections; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D012756:Sheep; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
D002120:Calcium Channel Agonists; D004396:Coloring Agents; D019831:Sodium-Calcium Exchanger; D014475:Uncoupling Agents; D000252:Calcium-Transporting ATPases; D000109:Acetylcholine; D002118:Calcium