Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC113
Calcium mobilization by NAADP from acidic stores in the megakaryoblastic cell line MEG01
N. A. Dionisio1, L. Albarrán1, J. J. López2, R. Bobe2, G. M. Salido1, J. A. Rosado1
1. Department of Physiology, Cell Physiology Research Group, University of Extremadura, C
Changes in cytosolic free Ca2+ concentration ([Ca2+]c) are among the most universal signaling events regulating cell function. Activation of membrane receptors increases [Ca2+]c by Ca2+ release from agonist-sensitive Ca2+ compartments and Ca2+ entry through plasma membrane channels. Ca2+ release from the endoplasmic reticulum (ER) occurs through the occupation of two types of Ca2+-release channels, namely inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors (RyR), by IP3 and cyclic ADP-ribose, respectively (Cancela, 2001). A novel family of intracellular Ca2+-release channels termed two-pore channels (TPCs) have been presented as the receptors of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+ mobilizing intracellular messenger (Churchill et al., 2002). TPCs are exclusively localized to the endolysosomal system mediating NAADP-evoked Ca2+ release from the acidic compartments. We have investigated NAADP-mediated Ca2+ release from intracellular stores in the megakaryoblastic cell line MEG01. Changes in cytosolic and intraluminal free Ca2+ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively, and TPC expression was detected by reverse transcription polymerase chain reaction (RT-PCR). Treatment of MEG01 cells with the H+/K+ ionophore nigericin or the V-type H+-ATPase selective inhibitor bafilomycin A1 results in a rise in cytosolic free Ca2+ concentration (n=6), which revealed the presence of acidic Ca2+ stores in these cells. The acidic stores were also sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca2+ from TBHQ-sensitive acidic stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2, whose expression was detected by RT-PCR. Ca2+ efflux from the TBHQ-sensitive acidic Ca2+ stores or the TBHQ-insensitive ER results in ryanodine-sensitive activation of Ca2+-induced Ca2+ release (CICR) from the complementary Ca2+ compartment. In conclusion, here we report NAADP-evoked Ca2+ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.
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