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Differential Regulation of P2X₇ Receptor Activation by Extracellular Nicotinamide Adenine Dinucleotide and Ecto-ADP-Ribosyltransferases in Murine Macrophages and T Cells

Shiyuan Hong^*, Nicole Schwarz, Anette Brass, Michel Seman, Friedrich Haag, Friedrich Koch-Nolte, William P. Schilling^* and George R. Dubyak^1,*

^* Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44120; Institute of Immunology, University Hospital, Hamburg, Germany; and University of Rouen, Rouen, France

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X₇ purinergic receptor (P2X₇R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X₇R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X₇R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X₇R function in murine macrophages. Coexpression of the cloned murine P2X₇R with ART2.1 or ART2.2 in HEK293 cells verified that P2X₇R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X₇R. Rather, NAD potentiated ATP-dependent P2X₇R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X₇R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X₇R channel opening in T cells, this P2X₇R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X₇R signaling in murine macrophages and also suggest that the cellular context in which P2X₇R signaling occurs differs between myeloid and lymphoid leukocytes.

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¹ Address correspondence and reprint requests to Dr. George R. Dubyak, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland OH, 44120. E-mail address: george.dubyak{at}case.edu

² Abbreviations used in this paper: P2X₇R, P2X₇ receptor; ADP-R, ADP-ribose; ART, ADP-ribosyltransferase; BMDM, bone marrow-derived macrophage; BSS, basic salt solution; NAD, etheno-NAD; GSH, glutathione; iNOS, inducible NO synthase; mP2X₇, murine P2X₇; NZW, New Zealand White; PS, phosphatidylserine; WT, wild type.

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