NAADP Mobilizes Calcium from the Endoplasmic Reticular Ca2+ Store in T-lymphocytes

Mareike Steen,Andreas H Guse,Tanja Kirchberger

doi:10.1074/jbc.m610925200

Abstract

The target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca(2+) signaling. However, the inhibitory effect was not specific since Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H(+)-ATPase, did not block or reduce NAADP-induced Ca(2+) signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca(2+) stores, did not antagonize subsequent NAADP-induced Ca(2+) signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca(2+) signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca(2+) signaling was obtained. The sensitivity of NAADP-induced Ca(2+) signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394-21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum.

Highlights

Bilizing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose that require micromolar concentrations for activity in mammalian cells [2, 3], nicotinic acid adenine dinucleotide phosphate (NAADP) acts at low nanomolar concentrations and displays a bell-shaped concentration-response curve in many different eukaryotic cell types (4 – 6)
glycyl-phenylalanine 2-naphthylamide (GPN) pretreatment resulted in loss of Ca2ϩmobilizing activity of all three second messengers, NAADP, cyclic ADP-ribose (cADPR), and InsP3, there was no inhibitory effect of bafilomycin A1 on any of the Ca2ϩ-mobilizing compounds, even when endogenous Ca2ϩ stores were previously emptied by TCR-CD3 stimulation in the presence of bafilomycin A1
In a compli- assumed to release proteases and other hydrolases into the mentary approach, the intracellular store sensitive to NAADP was partially depleted by microinjection of NAADP in the presence of GdCl3; the filling states of both the endoplasmic reticular (ER) and the lysosomes were analyzed by the addition of either thapsigargin cytosol, it is well imaginable that proteolytic attack of the InsP3 receptor or the ryanodine receptors (RyR) is the reason for the lack of effect of InsP3 or cADPR

Summary

Introduction

Bilizing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR) that require micromolar concentrations for activity in mammalian cells [2, 3], NAADP acts at low nanomolar concentrations and displays a bell-shaped concentration-response curve in many different eukaryotic cell types (4 – 6). Our data indicate a major role for the ER, but not for lysosomes, in NAADP-mediated Ca2ϩ signaling in T cells.

Results

Conclusion