Elsevier

Cell Calcium

Volume 46, Issue 4, October 2009, Pages 263-272
Cell Calcium

Extracellular NAD+ induces a rise in [Ca2+]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

https://doi.org/10.1016/j.ceca.2009.08.004Get rights and content

Abstract

Extracellular nicotinamide adenine dinucleotide (NAD+) is known to increase the intracellular calcium concentration [Ca2+]i in different cell types and by various mechanisms. Here we show that NAD+ triggers a transient rise in [Ca2+]i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca2+ from IP3-responsive intracellular stores and an influx of extracellular Ca2+. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD+-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD+. The identification of P2Y1 and P2Y11 as receptor subtypes responsible for the NAD+-triggered increase in [Ca2+]i was supported by several lines of evidence. First, specific P2Y1 and P2Y11 receptor antagonists inhibited the NAD+-induced increase in [Ca2+]i. Second, NAD+ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD+ caused an increase in [cAMP]i, prevented by the P2Y11 receptor-specific antagonist NF157.

Introduction

Besides playing key roles in almost all major aspects of energy metabolism [1], [2], there is mounting evidence that NAD+ also mediates various biological activities including calcium homeostasis [1], [3]. NAD+ can serve as a substrate for ADP-ribosylcyclases (e.g. CD38, BST-1) which generate cyclic ADP-ribose (cADPR), a potent activator of ryanodine receptor mediated calcium release [4]. The NAD+-derived metabolite nicotinic acid adenine dinucleotide phosphate (NAADP) also modulates the calcium metabolism by discharging intracellular Ca2+ stores [5]. Furthermore, mono-ADP-ribosylation of P2X7 receptors promoted by NAD+ results in an activation of the receptor and increased calcium influx [6].

Free ADP-ribose (ADPR), another degradation product of NAD+ activates TRPM2 receptors leading to calcium influx [7], [8], a property shared by 2′-O-acetyl-ADP-ribose (OAADPR) generated from NAD+ by Sir2 family proteins [9]. Additionally, when applied extracellularly ADPR has been shown to induce an increase in [Ca2+]i in freshly isolated resting human monocytes [10]. In most of these studies, the enzymes using NAD+ as a substrate are ectoenzymes.

For them to work, extracellular NAD+ concentrations should reach values exceeding those measured in plasma, which are in the submicromolar range [11], [12]. Considering that the intracellular NAD+ concentration is about 1 mM [13], [14], high local NAD+ concentrations may be reached in settings where NAD+ is released during mechanical tissue injury or infectious processes with severe cytolysis. Only recently it has been shown that high amounts of NAD+ are set free during inflammation [15].

The dinucleotide NAD+ shares structural characteristics with adenosine triphosphate (ATP). Similar to ATP, it contains an adenine based purine moiety. At present, the action of ATP is far better understood than that of NAD+ and it is already known to function as an extracellular messenger. Like NAD+, ATP is present in high concentrations (5–10 mM) in mammalian cells and will be released into the extracellular environment upon any kind of cell lysis.

Once released, ATP can be degraded into further metabolically active products like ADP, AMP, adenosine or it can exert its effects as an intact molecule through the specific activation of purinergic P2 receptors [16]. This class of receptors can be divided into two subfamilies: the ligand-gated ion channels (P2X) and the G-protein-coupled receptors (P2Y).

P2X receptors mediate fast permeability changes to mono- and divalent cations and P2Y receptors induce their functional effects via G-protein-coupling to phospholipase C and/or adenylate cyclase [17], [18].

Whereas ATP per se has been shown to function through the specific activation of P2 receptors, there is hardly any information about surface receptors triggering intracellular events induced by intact NAD+. Only recently Moreschi et al. showed, that NAD+ is an agonist of the human P2Y11 receptor in human granulocytes mediating an increase in [Ca2+]i, responsible for the functional activation of these cells [19].

In previous studies we demonstrated, that also in resting human monocytes extracellular NAD+ induces a rapid transient increase in [Ca2+]i, caused by calcium influx. Here, P2X receptors seem to interfere with the NAD+-induced calcium response [20].

Considering that in many cases activation of immune cells is a prerequisite for ATP to exert its immune modulatory functions [21], the focus of the present investigation was to study the effect of NAD+ on calcium signaling in human monocytes exposed to the inflammatory stimulus LPS, a component of the outer membrane of gram-negative bacteria. When challenging LPS-activated monocytes with NAD+ we observed a transient increase in [Ca2+]i, caused by the release of Ca2+ from intracellular stores combined with an uptake of extracellular Ca2+. These calcium signaling properties, which are in part similar to those of P2Y receptors, and the finding that their physiological agonist ATP applied prior to NAD+ completely abrogated the rise in [Ca2+]i made us consider P2Y receptors as putative interaction partners of NAD+. By the use of specific agonists and antagonists in Ca2+-imaging experiments and by cyclic AMP (cAMP) measurements we here present evidence, that P2Y1 and P2Y11 receptors mediate the NAD+-induced increase in [Ca2+]i in LPS-activated human monocytes.

Section snippets

Agonists, antagonists and antibodies

LPS (lipopolysaccharide; from Escherichia coli 055:B5), dimethyl sulfoxide (DMSO), ATP, adenosine, 2-MeS-ATP, thapsigargin, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-2,5-pyrrolidinedione (U-73343), pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), GdCl3 and

Extracellular NAD+ increases [Ca2+]i by internal calcium mobilization and calcium influx

Exposing human monocytes, cultured for 16 h in the presence and absence of LPS (100 ng/ml), to 200 μM NAD+, displayed differential effects. While in the absence of LPS the cells were unresponsive to NAD+, activated monocytes showed a rapid and transient increase in [Ca2+]i (Fig. 1A).

NAD+ can be cleaved by various ectoenzymes including CD38, which catalyzes the degradation of NAD+ to (c)ADPR and nicotinamide. By using capillary electrophoresis as described earlier [28], [29], we tested whether NAD+

Discussion

The function of extracellular ATP as a modulator of immunological responses has been well described [45], [46], [47], [48], [49]. For instance, ATP is a trigger of TNF-α secretion and a modulator of IL-1β release from activated murine and human macrophages [50], [51]. Thus, ATP released into the extracellular environment as a consequence of cell damage, could boost the proinflammatory response of LPS-activated macrophages [52]. ATP exerts its effects by activation of membrane bound P2 receptors

Conflict of interest

The authors state no conflict of interest.

Acknowledgements

We thank Dr. Jamshed Iqbal for performing the capillary electrophoresis experiments.

This study was supported by the Deutsche Forschungsgemeinschaft (HA 2484/3-1).

References (69)

  • H. Takemura et al.

    Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane

    J. Biol. Chem.

    (1989)
  • M. Oh-hora et al.

    Calcium signaling in lymphocytes

    Curr. Opin. Immunol.

    (2008)
  • G. Lambrecht et al.

    PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses

    Eur. J. Pharmacol.

    (1992)
  • I. von Kügelgen

    Pharmacological profiles of cloned mammalian P2Y-receptor subtypes

    Pharmacol. Ther.

    (2006)
  • F. Di Virgilio

    The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death

    Immunol. Today

    (1995)
  • B.B. Fredholm

    Purines and neutrophil leukocytes

    Gen. Pharmacol.

    (1997)
  • M. Tonetti et al.

    Extracellular ATP enhances mRNA levels of nitric oxide synthase and TNF-alpha in lipopolysaccharide-treated RAW 264.7 murine macrophages

    Biochem. Biophys. Res. Commun.

    (1995)
  • F. Di Virgilio et al.

    Nucleotide receptors: an emerging family of regulatory molecules in blood cells

    Blood

    (2001)
  • O.R. Baricordi et al.

    An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes

    Blood

    (1996)
  • C. el Moatassim et al.

    Extracellular ATP and cell signalling

    Biochim. Biophys. Acta

    (1992)
  • D. Communi et al.

    Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase

    J. Biol. Chem.

    (1997)
  • B. Torres et al.

    P2Y11 receptors activate adenylyl cyclase and contribute to nucleotide-promoted cAMP formation in MDCK-D(1) cells. A mechanism for nucleotide-mediated autocrine–paracrine regulation

    J. Biol. Chem.

    (2002)
  • C. Hague et al.

    Cell surface expression of alpha1D-adrenergic receptors is controlled by heterodimerization with alpha1B-adrenergic receptors

    J. Biol. Chem.

    (2004)
  • M. Margeta-Mitrovic et al.

    A trafficking checkpoint controls GABA(B) receptor heterodimerization

    Neuron

    (2000)
  • G. Milligan

    G-protein-coupled receptor heterodimers: pharmacology, function and relevance to drug discovery

    Drug Discov. Today

    (2006)
  • W. Ying

    NAD+ and NADH in cellular functions and cell death

    Front. Biosci.

    (2006)
  • M. Ziegler

    New functions of a long-known molecule. Emerging roles of NAD in cellular signaling

    Eur. J. Biochem.

    (2000)
  • A.H. Guse

    Second messenger function and the structure-activity relationship of cyclic adenosine diphosphoribose (cADPR)

    FEBS J.

    (2005)
  • A. Galione

    NAADP, a new intracellular messenger that mobilizes Ca2+ from acidic stores

    Biochem. Soc. Trans.

    (2006)
  • F.J. Kuhn et al.

    TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADP-ribose

    Pflugers Arch.

    (2005)
  • A. Gerth et al.

    Extracellular NAD+ regulates intracellular free calcium concentration in human monocytes

    Biochem. J.

    (2004)
  • C.A. Davies et al.

    Simultaneous analysis of nitrite, nitrate and the nicotinamide nucleotides by capillary electrophoresis: application to biochemical studies and human extracellular fluids

    Electrophoresis

    (1999)
  • S. Adriouch et al.

    NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo

    J. Immunol.

    (2007)
  • H. Zimmermann

    Extracellular metabolism of ATP and other nucleotides

    Naunyn Schmiedebergs Arch. Pharmacol.

    (2000)
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