Prolonged Inactivation of Nicotinic Acid Adenine
Dinucleotide Phosphate-induced Ca2+ Release Mediates a
Spatiotemporal Ca2+ Memory*
Grant C.
Churchill
and
Antony
Galione
From the Department of Pharmacology, University of Oxford, Oxford
OX1 3QT, United Kingdom
Received for publication, October 12, 2000, and in revised form, December 11, 2000
 |
ABSTRACT |
Although numerous extracellular stimuli
are coupled to increases in intracellular Ca2+,
different stimuli are thought to achieve specificity by eliciting different spatiotemporal Ca2+ increases. We investigated
the effect of nicotinic acid adenine dinucleotide phosphate (NAADP)
inactivation on spatiotemporal Ca2+ signals in intact sea
urchin eggs. The photorelease of NAADP but not inositol
1,4,5-trisphosphate or cyclic ADP-ribose resulted in
self-inactivation. When NAADP was released first locally and subsequently globally, the spatial pattern of the first response shaped
that of the second. Specifically, the local release of NAADP created a
Ca2+ gradient that was reversed during the subsequent
global release of NAADP. Neither cyclic ADP-ribose nor inositol
1,4,5-trisphosphate showed a similar effect. In contrast to
homogenates, NAADP inactivation was reversible in intact eggs with
resensitization occurring in ~20 min. Because initial NAADP responses
affect later responses, NAADP can serve as a mechanism for a
Ca2+ memory that has both spatial and temporal components.
This NAADP-mediated Ca2+ memory provides a novel mechanism
for cells to control spatiotemporal Ca2+ increases.
 |
INTRODUCTION |
Numerous extracellular messengers control a diverse array of
intracellular functions through increases in intracellular
Ca2+ concentration (1). Nevertheless, intracellular targets
can be selectively activated by Ca2+ through spatial and
temporal control (1). One way of controlling spatiotemporal
Ca2+ signaling is by modulating the sensitivity of
intracellular Ca2+-release channels to
Ca2+-induced Ca2+ release
(CICR).1 Through positive
feedback, CICR results in regenerative Ca2+ release. Both
inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors
exhibit CICR, the sensitivity of which is controlled by IP3
(1) and cADPR (2), respectively. Thus, by altering the levels of these
messengers, Ca2+ signals can be confined to subcellular
regions or propagated throughout the cell (1).
The novel Ca2+-mobilizing messenger nicotinic acid adenine
dinucleotide phosphate (NAADP) was originally shown to mobilize
Ca2+ in the sea urchin egg (3) and subsequently has been
shown to be active in both mammalian (4-6) and plant tissues (7). Compared with IP3 and cADPR, NAADP has several unique
characteristics in the sea urchin egg (2, 8). First, the response to a
maximal NAADP concentration is eliminated by pretreatment with a
subthreshold concentration of NAADP (9, 10). Second, NAADP binds to its receptor irreversibly (9, 11, 12), which may relate to the
unique desensitization of NAADP (9) and increase the sensitivity of its receptor (12). Third, NAADP-mediated Ca2+ release is
not regulated by Ca2+ (6, 13, 14).
The unique properties of NAADP necessitate novel mechanisms for control
of spatiotemporal Ca2+ signaling. For example, we recently
reported that, in contrast to all previous Ca2+ waves that
propagate by CICR (1), NAADP-mediated Ca2+ increases
propagate by NAADP diffusion (15). We now show that NAADP
desensitization-resensitization can establish a subcellular Ca2+ memory that has both spatial and temporal components.
Thus, the unique properties of NAADP enables it to control
spatiotemporal Ca2+ signaling in a fundamentally distinct
manner from IP3 and cADPR.
 |
EXPERIMENTAL PROCEDURES |
Sea urchin eggs of Lytechinus pictus were obtained by
intracoelomic injection of 0.5 M KCl shed into artificial
sea water (in mM, NaCl 435, MgCl2 40, MgSO4 15, CaCl2 11, KCl 10, NaHCO3 2.5, EDTA 1), dejellied by passing through 90-µm nylon mesh, and then
washed twice by centrifugation. Eggs were transferred to polylysine-coated glass coverslips for microinjection and microscopy. Oregon Green 488 BAPTA
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid dextran; Molecular Probes) was pressure-microinjected
(Picospritzer; World Precision Instruments). The
Ca2+-sensitive dye was imaged by laser-scanning confocal
microscopy (Leica model TCS NT) using the 488-nm line of an
argon ion laser for excitation, and the emission was long pass-filtered
(515 nm) and detected with a photomultiplier tube. Caged NAADP
(2'P-(1-(2-nitrophenyl)ethyl) NAADP; Molecular Probes) was
purified further by high performance liquid chromatography to remove
small amounts of contaminating free NAADP (16). Caged cADPR
(P-(1-(2-nitrophenyl)ethyl) cADPR; Molecular Probes) and caged
IP3 (4,5P-(1-(2-nitrophenyl)ethyl) IP3;
Calbiochem) were not further purified. Caged compounds were photolyzed
with ultraviolet light (351- and 364-nm lines) from an argon ion laser
(Enterprise model 651; Coherent) that was directed into the scanning
head by a quartz fiber optic cable. The spatial location of photolysis
was controlled via a shutter that was placed in the light path of the
ultraviolet laser. This resulted in a band of UV across the image with
the position and width of the band being controllable. The confocal
images were processed with the software NIH Image to create a self
ratio by dividing the intensity (F) of each image on a pixel
by pixel basis by the intensity of an image acquired before stimulation
(Fo).
 |
RESULTS AND DISCUSSION |
In sea urchin egg homogenates, Ca2+ release mediated
by IP3, cADPR, and NAADP exhibit homologous
desensitization, but only the NAADP-sensitive Ca2+ release
system inactivates to nonreleasing concentrations of itself (2, 3, 9,
10, 17, 18). The time course of NAADP self-inactivation is both time-
and concentration-dependent (2, 9, 10). NAADP self-inactivation
also occurs in intact eggs (2, 9, 10). To investigate the time course
for messenger desensitization in intact eggs, each second messenger was
photoreleased repeatedly at different time intervals (Fig.
1). In response to repeated stimulation
with either IP3 or cADPR, the peak Ca2+
decreased, and the resting Ca2+ between pulses increased
when the pulses were separated by 20 s but were largely unaffected
when the pulses were separated by either 60- or 120-s intervals (Fig.
1). In contrast, in response to repeated stimulation with NAADP the
peak Ca2+ decreased regardless of the time between the
NAADP pulses (Fig. 1). The precise time course for NAADP inactivation
in intact eggs cannot be determined accurately because of egg to egg
variation and the fact that a given egg cannot be repeatedly stimulated with NAADP using different time courses.

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Fig. 1.
Time courses for desensitization to NAADP,
IP3, and cADPR in intact sea urchin eggs. Eggs
contained Oregon Green 488 BAPTA dextran (10 µM) and
caged IP3 (5 µM), caged cADPR (5 µM), or caged NAADP (0.5 µM) as labeled.
Compounds were photoreleased at various intervals as indicated by the
arrows. Responses are representative of those observed in
four to eleven similar experiments.
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A local release of NAADP resulted in a Ca2+ increase that
spread only part way across an egg, thus forming a Ca2+
gradient (Fig. 2). We have demonstrated
recently that NAADP-mediated Ca2+ increases spread via
NAADP diffusion (15), rather than via Ca2+-induced
Ca2+ release, which underlies all previously described
Ca2+ waves (1). When Ca2+ returned to near
resting levels, a global release of NAADP resulted in a
Ca2+ increase that was an inversion of the first
Ca2+ gradient (Fig. 2). This second Ca2+
gradient is a direct consequence of the first NAADP gradient that
formed subcellular zones where the NAADP response was completely inhibited, partially inhibited, and not inhibited.

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Fig. 2.
NAADP inactivation forms a spatial
Ca2+ memory. NAADP was photoreleased first
locally and then, after Ca2+ had returned to near resting
levels, globally. In the images the pseudocolor correspond to the
Oregon Green 488 BAPTA dextran self ratio
(F/Fo), and the images are taken from
the times indicated by the vertical tick marks above the
x axis of the graphs. The traces in
the graph are coded by color to the regions of
interest at the positions indicated in the diagram of the
egg. The shading in the diagram of the egg
indicates the area of photorelease, and the labeled horizontal
bars above the traces and images indicate
its duration. The response is representative of that observed in eight
similar experiments.
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In contrast to NAADP, a local release of either cADPR (Fig.
3a) or IP3 (Fig.
3b) did not affect the pattern of Ca2+ increase
elicited by a later global release of messenger. These experiments
illustrate a key difference between Ca2+ signals generated
by IP3 and cADPR and those generated by NAADP. Namely, that
NAADP responses exhibit a spatial memory of the previous response that
shapes the spatial pattern of subsequent Ca2+
increases.

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Fig. 3.
A spatial Ca2+ memory is not
formed by IP3 or cADPR. Compounds were
photoreleased first locally and then, after Ca2+ had
returned to near resting levels, globally. Eggs contained Oregon Green
488 BAPTA dextran (10 µM) and either caged cADPR (5 µM) or caged IP3 (5 µM). Other
details are as in Fig. 2. Responses are representative of those
observed in four similar experiments.
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In sea urchin egg homogenates, NAADP inactivation lasts at least 25 min
(9, 10) and correlates with irreversible binding of NAADP to its
receptor (9). Similarly, in intact sea urchin eggs, when either a
relatively large amount of NAADP was released (e.g. photoreleasing NAADP globally at a high UV
intensity for several frames) or free NAADP was injected to a final
intracellular concentration of 5 nM, the inactivation
persisted for at least 2 h (data not shown). To determine the
reversibility of spatially localized NAADP inactivation, eggs were
locally desensitized to NAADP and then given various periods of
recovery before a global release of NAADP. As shown in Fig. 2, when the
local and global NAADP increases were separated by about 1 min, there
was localized inactivation (Fig. 4). As
the recovery time between the two photoreleases of NAADP increased, the
egg progressively recovered from inactivation until after about 23 min
the NAADP-mediated Ca2+ increase was similar in both the
regions (Fig. 4). To verify that the local region was desensitizing to
NAADP, NAADP was released locally with a series of UV pulses. The
amplitude of the response decreased with each additional UV pulse
indicting desensitization. The subsequent response to global
photorelease of NAADP (Fig. 4; 25 min) demonstrates that the
recovery was genuine and not due to the lack of initial
desensitization. Following the NAADP-mediated Ca2+
increase, certain eggs exhibited either a Ca2+ pulse (Fig.
4; 23 min, blue trace) or a small and sustained
rise in Ca2+ (Fig. 4; 23 and 25 min).
These secondary Ca2+ rises likely correspond to the
Ca2+ oscillations induced by the photorelease of NAADP, as
described previously by Aarhus et al. (9).

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Fig. 4.
Localized NAADP inactivation in intact eggs
is reversible. NAADP was photoreleased first locally then
globally as shown in the schematic diagram. The
traces are labeled with the time between the local and
global pulses of UV. The traces are examples of the
responses observed in three to six similar experiments.
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Although the mechanism for recovery of the NAADP response is currently
unknown, the functional response (inhibition of NAADP-induced Ca2+ release) is relatively long-lasting in homogenates (9,
10) compared with intact eggs, suggesting that the sensitivity of the
egg to NAADP is highly regulated in vivo. Taken together, the experiments shown in Fig. 4 demonstrate that NAADP inactivation is
reversible when it is localized in intact eggs. Thus, the
NAADP-mediated spatiotemporal Ca2+ memory is short term
rather than permanent.
Our data demonstrate that in intact sea urchin eggs, NAADP inactivation
can be spatially restricted and is reversible. These unique properties
enable NAADP to form a spatiotemporal memory of past Ca2+
increases. Such a subcellular memory makes it ideal for controlling processes that require elevations in Ca2+ that are
spatially restricted and maintain an influence for 10-20 min.
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ACKNOWLEDGEMENTS |
We thank J. Thomas and S. Patel for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Wellcome Trust.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
Tel.: 01865-271-606; Fax: 01865-271-853; E-mail: grant. churchill@pharm.ox.ac.uk.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009335200
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ABBREVIATIONS |
The abbreviations used are:
CICR, Ca2+-induced Ca2+ release;
NAADP, nicotinic
acid adenine dinucleotide phosphate;
IP3, inositol
1,4,5-trisphosphate;
cADPR, cyclic ADP-ribose;
caged NAADP, 2'P-(1-(2-nitrophenyl)ethyl) NAADP;
caged IP3, 4,5P-(1-(2-nitrophenyl)ethyl) IP3;
caged cADPR, P-(1-(2-nitrophenyl)ethyl) cADPR;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
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