Prolonged Inactivation of Nicotinic Acid Adenine Dinucleotide Phosphate-induced Ca2+ Release Mediates a Spatiotemporal Ca2+ Memory*

Grant C. ChurchillDagger 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

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.

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.

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.

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.


    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.

Dagger 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


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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