Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells.
In human SH-SY5Y neuroblastoma cells, two distinct intracellular Ca2+ stores, a KCl-/caffeine-sensitive and a carbachol-/IP3-sensitive store, were demonstrated previously. In this study, responses of these two intracellular Ca2+ stores to thapsigargin were characterized. Ca2+-release from these stores was evoked either by high K+ (100 mM KCl) or by 1 mM carbachol, and changes in the intracellular Ca2+ level were monitored using Fura-2 fluorimetry. A sequential stimulation protocol (KCl-->carbachol or vice versa) allowed evaluation of the individual contribution of different Ca2+ stores to the evoked intracellular Ca2+ ([Ca2+]i)-transients and the dynamic interaction between them. Thapsigargin (0.05 nM - 20 microM) alone induced a [Ca2+]i-transient. Both the carbachol- and the KCl-evoked [Ca2+]i-transients were inhibited by thapsigargin, but with very different sensitivities. Thapsigargin inhibited the carbachol-evoked [Ca2+]i-transients with (IC50 = 0.353 nM) or without (IC50 = 0.448 nM) a KCl-prestimulation, but an additional small component, with a much lower sensitivity (IC50=4814 nM), was observed in the absence of a KCl-prestimulation. In contrast, the KCl-evoked [Ca2+]i-transients displayed only one component with a very low sensitivity to thapsigargin in both absence (IC50=3343 nM) and presence (IC50=6858 nM) of a carbachol-prestimulation. These findings suggest that the sarco-/endoplasmic reticular Ca2+ ATPases associated with the KCl-/caffeine- and carbachol-/IP3-sensitive intracellular Ca2+ stores differ from each other, either in types or in their post-translational modification. Such difference might play important role in the regulation of neuronal Ca2+ homeostasis.
Garavito-Aguilar
Zayra V
ZV
Department of Anesthesiology, New York University School of Medicine, 550 First Avenue, Tisch Building, 4th Floor, Room HE-438, New York, NY 10016, USA.
Recio-Pinto
Esperanza
E
Corrales
Alexandra V
AV
Zhang
Jin
J
Blanck
Thomas J J
TJ
Xu
Fang
F
D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D045744:Cell Line, Tumor; D018679:Cholinergic Agonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009447:Neuroblastoma; D011188:Potassium / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology*; D013997:Time Factors
D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D018727:Muscarinic Antagonists; D019284:Thapsigargin; D001285:Atropine; D002217:Carbachol; D011188:Potassium; D002118:Calcium; D016257:Fura-2