The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes.
Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.
Fiekers
J F
JF
Department of Anatomy and Neurobiology, University of Vermont, College of Medicine, Burlington 05405, USA. jfiekers@salus.med.uvm.edu
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002999:Clone Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D015759:Ionomycin / Q000494:pharmacology; D008094:Lithium / Q000494:pharmacology; D008536:Meglumine / Q000494:pharmacology; D000077485:Meglumine Antimoniate; D051379:Mice; D009942:Organometallic Compounds / Q000494:pharmacology; D010755:Organophosphates / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D015398:Signal Transduction; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019789:Tetraethylammonium / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D006873:Hydroquinones; D009942:Organometallic Compounds; D010755:Organophosphates; D019831:Sodium-Calcium Exchanger; C012085:tris(2,3-dibromopropyl)phosphate; C019359:2,5-di-tert-butylhydroquinone; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D015759:Ionomycin; D019789:Tetraethylammonium; D011189:Potassium Chloride; D019284:Thapsigargin; D008536:Meglumine; D000077485:Meglumine Antimoniate; D008094:Lithium; D000252:Calcium-Transporting ATPases; D016257:Fura-2