Calcium signalling: NAADP comes out of the shadows.

Abstract

The molecular mechanisms through which extracellular stimuli mobilize intracellular Ca(2+) stores are still incompletely understood. Recent work suggests that nicotinic acid-adenine dinucleotide phosphate (NAADP) can finally join the ranks of genuine Ca(2+)-mobilizing second messengers.

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Biochem. J. (2003) 373 (10.1042/BJ20030472COM) (Printed in Great Britain)
COMMENTARY
Calcium signalling: NAADP comes out of the shadows
Guy A. RUTTER1
Henry Wellcome Signalling Laboratories and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, U.K.
The molecular mechanisms through which extracellular stimuli
mobilize intracellular Ca2+stores are still incompletely under-
stood.Recentworksuggeststhatnicotinicacid–adeninedinucleo-
tide phosphate (NAADP) can finally join the ranks of genuine
Ca2+-mobilizing second messengers.
Key words: Ca2+mobilization, cADP-ribose, CD38, inositol
phospholipids, insulin, nicotinic acid–adenine dinucleotide phos-
phate (NAADP).
Although inositol 1,4,5-trisphosphate (IP3) [1] and cADP-ribose
(cADPr) [2] are now well-accepted as mediators of Ca2+
release from intracellular stores, the role of a third putative
Ca2+-mobilizing molecule, nicotinic acid–adenine dinucleotide
phosphate (NAADP) [3], has until recently been more contro-
versial. A set of five new papers [4–8], and the very recent work
by Patel and co-workers [9] published in this issue of the
Biochemical Journal, now present evidence for a role of NAADP
as a bona fide and unique messenger molecule in a range of cell
types.
NAADP is synthesized by the same enzyme family as
cADPr, ADP-ribosyl cyclases, including CD38 [10]. Synthesis
of NAADP involves a modification of the enzymic reaction,
resulting in the exchange of nicotinamide for nicotinic acid
on the unphosphorylated ribose ring of NADP [10]. The latter
mechanism is favoured at low pHs such as those pertaining
in an endosomal compartment where CD38 may reside during
recycling from the cell surface. Replacement of an uncharged
amide in NADP for a negatively charge carboxyl function in
NAADP confers on the latter a potent (nanomolar affinity)
capacity to mobilize Ca2+from responsive stores. As first
described in sea urchin egg microsomes [11], NAADP mobilizes
Ca2+in a number of systems from higher organisms, including
mammalian T-lymphocytes [12], pancreatic acinar [13], kidney
[14] and heart [15] cells.
The new papers are important because (a) they provide
evidence, previously lacking, for an increase in NAADP
concentration during cell stimulation, and (b) they shed light on
the identity of the intracellular Ca2+stores targeted by NAADP
and the communication between these and other Ca2+stores.
Extending observations by Lee and Aarhus [16], Churchill et al.
[4] used live sea urchin eggs in which the organelles are
‘stratified’ in situ by centrifugation, as well as fractionated sea
urchin homogenates, to show that the NAADP-sensitive store co-
segregated with a lysosomal marker and was distinct from IP3-
and cADPr-sensitive stores (which fractionated with microsomal
markers). This finding explained the earlier observation [17]
that the NAADP-sensitive store was insensitive to an inhibitor
of the sarco(endo)plasmic reticulum Ca2+-ATPases, thapsigargin.
Confirming a lysosomal localization of the store targeted by
NAADP, incubation of intact eggs with an agent which disrupts
lysosomes significantly reduced Ca2+release in response to
photolysis (‘uncaging’) of an inactive precursor of NAADP [4].
1To whom correspondence should be addressed (e-mail g.a.rutter@bris.ac.uk).
In the context of higher organisms, microinjected NAADP
was found to mobilize intracellular Ca2+in single human
pancreatic β-cells [5], and high concentrations of NAADP,
sufficient to desensitize NAADP receptors, blocked the ability
of insulin to mobilize intracellular Ca2+in these cells. Corres-
pondingly, uncaging of NAADP increased intracellular free
[Ca2+] in clonal β-cells [6,7], an effect unaltered by the presence
of thapsigargin [7]. Importantly, increases in glucose con-
centration over the near-physiological range robustly increased
the intracellular concentration of NAADP in clonal MIN6 β-cells
and [32P]NAADP bound with high affinity to a crude membrane
fraction derived from these cells [6]. Strikingly, an independent
study in which intra-organellar free [Ca2+] was monitored with
recombinant targeted probes simultaneously reports that the
NAADP-sensitive store was likely to be localized principally to
dense-core secretory vesicles [7]. Thus addition of NAADP
to permeabilized MIN6 cells caused a significant decrease in
intravesiclular free [Ca2+], but had essentially no effect on
endoplasmic reticulum Ca2+concentrations [7]. In mice deleted
in both alleles of CD38, changes in β-cell NAADP generation,
and hence insulin secretion, therefore seem likely to contribute to
abnormal glucose homoeostasis [18].
It seems possible that, in the case of both insulin-containing
secretory vesicles and sea urchin lysosomes, the formation of
spatially constrained domains of high Ca2+concentration in the
immediate vicinity of an individual organelle may then serve
a role as an important local cue, either for the fusion of the
secretoryvesiclewiththeplasmamembrane[7]orforheterotypic
fusion between lysosomes and endosomes [19]. Given that
dense-core secretory vesicles share a similar route of biogenesis
withlysosomes[20],itisalsotemptingtospeculatethatthesetwo
organelle types may share common mechanisms for both Ca2+
accumulation and release. In fact, there appear to be important
differencesbywhichthesetwoacidicorganellesaccumulateCa2+
ions. Thus bafilomycin, an inhibitor of H+-ATPases, blocked
NAADP-induced release from the lysosome-associated store
[4], but had no effect on Ca2+accumulation by secretory vesi-
cles [22]. Only in the latter was Ca2+uptake blocked by
vanadate,aninhibitorP-typeATPases,wheresilencingofthegene
encoding the Ca2+-ATPase, PMR1, also inhibited Ca2+uptake
[21].
An interesting possibility is that other Ca2+channels, including
ryanodine receptors [22], are also involved in Ca2+-dependent
c ?2003 Biochemical Society

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Guy A. Rutter
amplification of the initial NAADP-mediated Ca2+release from
insulin-containing secretory vesicles. Such ‘intra-store chatter’
would complement ‘inter-store’ cross-talk, such as that which
occurs in T-lymphocytes, where NAADP receptors are necessary
for the release of Ca2+via IP3receptors [12]. The recent work
by Patel and co-workers [9] provides a direct demonstration of
the latter phenomenon in neurons by showing that NAADP-
sensitive stores also communicate with distinct Ca2+stores at
the frog neuromuscular junction. Thus NAADP applied via a
liposomedeliverysystemcausedincreasesinintracellular [Ca2+],
asmonitoredindirectlyviachangesinthereleaseofacetylcholine
andminiatureend-platepotentials.Importantly,NAADP-induced
[Ca2+] increases potentiated those evoked by the application of
IP3or cADPr. These experiments demonstrate for the first time
a Ca2+-induced Ca2+-release mechanism whereby Ca2+liberated
via NAADP receptors goes on to provoke further Ca2+release via
IP3and cADPr receptor channels.
Important questions remain regarding the role played by
NAADP in intracellular Ca2+signalling. Firstly, the molecular
identity of the NAADP receptor remains mysterious, although
the recent studies provide further convincing evidence that this is
almostcertainlydistinctfromIP3andcADPrreceptors.Secondly,
itremainsunclearjusthowextracellularsignalsprovokeincreases
in NAADP levels in target cells. In the case of glucose action on
β-cells, the sensitivity of the cyclase reaction of CD38 to ATP
(whichfavoursexchangeovercyclization)maybeinvolved,since
the free concentration of this nucleotide is significantly increased
in β-cells following exposure to elevated glucose concentrations
[23]. On the other hand, the mechanisms by which insulin might
provoke(asyetunmeasured)increasesinNAADPinthiscelltype
[5] remain obscure, since insulin has no effect on free [ATP] in
β-cells [24]. Likewise, the underlying mechanisms responsible
for the very large increase in NAADP concentration during the
acrosome reaction in sea urchin sperm [8] are unknown. Finally,
the role of NAADP in the propagation of complex Ca2+signals
(i.e. oscillations and waves) remains to be elucidated. Although
the search for answers to these questions is important, there can
be little doubt that NAADP has now come of age as an important
player in Ca2+signalling.
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Received 27 May 2003/28 May 2003; accepted 28 May 2003
Published on the Internet 8July 2003, DOI 10.1042/BJ20030472COM
c ?2003 Biochemical Society