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Biochim Biophys Acta. 2011 Aug;1813(8):1483-94. doi: 10.1016/j.bbamcr.2011.05.005. Epub 2011 May 13.

Acidic NAADP-releasable Ca(2+) compartments in the megakaryoblastic cell line MEG01.

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  • 1Department of Physiology (Cellular Physiology Research Group), University of Extremadura, Cáceres, Spain.

Abstract

BACKGROUND:

A novel family of intracellular Ca(2+)-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca(2+) mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca(2+) release from the acidic compartments.

OBJECTIVES:

The present study is aimed to investigate NAADP-mediated Ca(2+) release from intracellular stores in the megakaryoblastic cell line MEG01.

METHODS:

Changes in cytosolic and intraluminal free Ca(2+) concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR.

RESULTS:

Treatment of MEG01 cells with the H(+)/K(+) ionophore nigericin or the V-type H(+)-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca(2+) stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca(2+) from acidic lysosomal-like Ca(2+) stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca(2+) efflux from the acidic lysosomal-like Ca(2+) stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca(2+)-induced Ca(2+) release (CICR) from the complementary Ca(2+) compartment.

CONCLUSION:

Our results show for the first time NAADP-evoked Ca(2+) release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.

2011 Elsevier B.V. All rights reserved.

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