naadp-induced ca2+ release – a new signalling pathway

Review

NAADP-induced Ca2+ Release – A new signalling pathway

Sandip Patel *

Department of Physiology, University College London, London, United Kingdom

Received 7 November 2003; accepted 1 December 2003

Abstract

Changes in cystosolic Ca2+ concentration are critical for the regulation of numerous cellular events. Mobilisation of intracellular Ca2+

stores by Ca2+ mobilising messengers is a highly conserved mechanism whereby different agonists mediate their cellular effects. NAADP isthe newest of these messengers to be described. Accumulating evidence suggests that NAADP targets a novel intracellular Ca2+ releasechannel that under certain conditions can inactivate prior to opening. These channels are likely located on Ca2+ stores distinct from theendoplasmic reticulum, the activation of which evokes complex changes in Ca2+ involving cross-talk with other intracellular Ca2+ channels.Recent demonstrations of changes in cellular NAADP levels by physiologically relevant stimuli establish the importance of this molecule inthe control of calcium dynamics.

© 2003 Elsevier SAS. All rights reserved.

Many extracellular stimuli mediate their cellular effectsthrough manipulation of the cytosolic Ca2+ concentration(Berridge et al., 2000). Activation of eggs by sperm is oneexample; a large transient increase in cytosolic Ca2+ is a wellcharacterized event that occurs upon fertilization in all spe-cies (Stricker, 1999). Ca2+ signals play crucial roles in sub-sequent development, differentiation and finally cell death(Berridge et al., 2000). Understanding how a single ion cancontrol such a vast array of cellular processes, particularlywhen these processes can be opposing and occur within thesame cell, is, at present, a topic of intense study. The exactpresentation of the Ca2+ signal within the cytoplasm is morethan likely an important determinant. Temporally, Ca2+ sig-nals are often manifest as repetitive transient increases whichwhen analyzed at appropriate spatial resolution can be con-fined to a particular cell locus reflecting opening of clustersor even single channels (Berridge et al., 2000). These el-ementary Ca2+ signals culminate depending on stimulus in-tensity to more global signals that can travel throughout thecell and into neighbouring cells in a regenerative manner(Berridge et al., 2000). Complex Ca2+ signals are sustainedby entry of Ca2+ across the plasma membrane and initiatedby release of Ca2+ from intracellular Ca2+ stores, a processwhich results from activation of intracellular Ca2+ channelslocated on the endoplasmic reticulum (ER) (Berridge et al.,

2000). The best characterized of these Ca2+ channels are thereceptors for the second messenger, inositol trisphosphate(IP3) (Patel et al., 1999) and the related, ryanodine receptors(Fill and Copello, 2002). The latter are gated by the NADmetabolite, cyclic adenosine diphosphate ribose (cADPR)(Lee, 2001; Galione, 1994). A crucial feature shared by bothIP3 and ryanodine receptors is the regulation of channelopening by Ca2+ itself. Depending on its concentration, Ca2+

can exert both stimulatory and inhibitory effects and it is thisform of tight regulation that is thought to contribute to theclearly coordinated activation required in order to explainheterogeneity in the Ca2+ signal (Berridge et al., 2000).

In recent years, much attention has focused on the Ca2+

mobilizing properties of nicotinic acid adenine dinucleotidephosphate (NAADP) (Lee, 2001; Patel et al., 2001; Cancela,2001; Guse, 2002; Genazzani and Billington, 2002) (Fig-ure 1). This analogue of NADP stimulates Ca2+ mobilizationin a variety of organisms from plants (Navazio et al., 2000) tohumans (Berg et al., 2000). Several properties of NAADP-induced Ca2+ release challenge the current dogma regardingthe mechanism whereby intracellular Ca2+ stores are mobi-lized, pointing to the existence of an entirely new Ca2+

release pathway. In this article, exciting recent developmentsconcerning the actions of NAADP will be reviewed.

1. A new Intracellular Ca2+ Channel activated by NAADP

The Ca2+ mobilizing properties of NAADP were firstdescribed in sea urchin egg homogenates (Clapper et al.,

* Corresponding Author.E-mail address: [email protected] (S. Patel).

Biology of the Cell 96 (2004) 19–28

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© 2003 Elsevier SAS. All rights reserved.doi:10.1016/j.biolcel.2003.12.001

1987; Lee and Aarhus, 1995; Chini et al., 1995), a prepara-tion which remains important for the study of NAADP(Galione et al., 2000) nearly a decade after identification ofNAADP as a contaminant in commercial preparations ofNADP (Lee and Aarhus, 1995). Based on cross-desensitization studies (Lee and Aarhus, 1995; Chini et al.,1995), it is evident that NAADP activates a pathway that isdistinct to those activated by IP3 and cADPR. Indeed, antago-nists of IP3 and cADPR–induced Ca2+ release do not affectCa2+ release in response to NAADP (Lee and Aarhus, 1995;Chini et al., 1995). Unfortunately, no selective antagonists ofNAADP have been described to date although severalL-Type Ca2+ channel modulators and certain K+ channelantagonists do inhibit NAADP-induced Ca2+ release (Genaz-zani et al., 1996; Genazzani et al., 1997). A critical distin-guishing feature of this pathway is that unlike IP3 andcADPR-induced Ca2+ release, Ca2+ release in response toNAADP, is not modulated by divalent cations such as Ca2+

(Genazzani and Galione, 1996; Chini and Dousa, 1996). Thisfinding would suggest that NAADP receptors are unable tomediate Ca2+-induced Ca2+ release (see below). Radioligandbinding studies also support the idea that NAADP targets anew Ca2+ channel. Binding of radiolabelled NAADP to seaurchin egg homogenates is readily detectable (Aarhus et al.,1996; Patel et al., 2000a; Billington and Genazzani, 2000) -notably at maximal levels more than an order of magnitudehigher than that of cADPR (Thomas et al., 2001) - and isunaffected by IP3 and cADPR (Aarhus et al., 1996; Patel etal., 2000a) but see (Billington and Genazzani, 2000).

NAADP binding to membrane preparations from mousebrain (Patel et al., 2000b), rat heart (Bak et al., 2001) andMIN-6 cells (Masgrau et al., 2003) is also insensitive toestablished intracellular Ca2+ channel agonists. Bizarrely,NAADP appears to bind its receptor in the sea urchin egg inan irreversible manner (Aarhus et al., 1996; Patel et al.,2000a; Billington and Genazzani, 2000). Thus, whereas ra-dioligand binding is reduced to near background levels in thesimultaneous presence of even very low concentrations ofunlabelled NAADP (< 1 nM) indicative of an avid interac-tion, binding of the radioligand is unaffected by the same orhigher concentration of NAADP once association of radioli-gand is initiated (Aarhus et al., 1996; Patel et al., 2000a;Billington and Genazzani, 2000). No such behaviour is ob-served in the rodent heart and brain, where binding has beenshown to be fully reversible upon addition of NAADP afterequilibrium binding has been attained (Patel et al., 2000b;Bak et al., 2001). We have recently shown that the unusualnon-dissociating nature of NAADP binding to its receptor inthe sea urchin egg requires K+ (Dickinson and Patel, 2003).Thus, in media lacking K+, addition of NAADP resulted innear complete dissociation of bound NAADP (Dickinsonand Patel, 2003). NAADP dissociation could also be affectedby radioligand dilution under these conditions however, dis-sociation by this method was less effective indicating thatbinding is negatively cooperative (Dickinson and Patel,2003). Intriguingly the rates of dissociation by the two meth-ods were similar, only the final extent was increased byaddition of NAADP (Dickinson and Patel, 2003). This formof negative cooperativity is markedly different that describedin the heart (Bak et al., 2001) and clearly unusual, the physi-ological significance of which remains to be established.

Sadly, molecular details of the NAADP-sensitive Ca2+

channel are lacking. Although radioligand binding studieshave provided clues to the working of the receptor, whetherthe identified binding site resides on the putative Ca2+ chan-nel or an accessory protein is not known. The latter would bereminiscent of the interaction of cADPR with the ryanodinereceptor through a poorly-defined intermediate (Walseth etal., 1993). We have recently succeeded in solubilizing anNAADP binding protein from sea urchin egg homogenates(Berridge et al., 2002a). The binding characteristics of thedetergent solubilized and membrane-bound protein weresimilar with respect to affinity, analogue selectivity salt andpH dependence (Berridge et al., 2002a). Importantly, deter-gent extracts also bound NAADP irreversibly (Berridge etal., 2002a). We exploited the latter property to preparesoluble NAADP receptor-radioligand complexes by deter-gent treatment of membranes that had been first labelled withradioligand (Berridge et al., 2002a). “Tagged” receptors werereadily resolved by native gel electrophoresis, sucrose den-sity gradient centrifugation and gel filtration, the results ofwhich indicate that the target protein has properties distinctto IP3 and ryanodine receptors (Berridge et al., 2002a). Suchan approach should substantially facilitate purification of theNAADP receptor.

Fig. 1. Structure of NAADP. Constituent chemical groups are labelled.

20 S. Patel / Biology of the Cell 96 (2004) 19–28

2. Inactivation of NAADP-induced Ca2+ release

A remarkable property of the release mechanism activatedby NAADP is the manner in which it inactivates. Priorexposure of sea urchin egg homogenates to low concentra-tions of NAADP has been demonstrated to attenuate Ca2+

release in response to a normally maximal concentration ofNAADP (Aarhus et al., 1996; Genazzani et al., 1996; Dick-inson and Patel, 2003). This is not unusual - what is, is thatattenuation can occur in response to sub threshold concen-trations of NAADP (Aarhus et al., 1996; Genazzani et al.,1996; Dickinson and Patel, 2003). This effect is depictedschematically in Figure 2. Even more remarkable is theobserved time and concentration dependence of this effect.At a fixed sub threshold concentration of NAADP, the re-sponse to maximal challenge with NAADP progressivelydeclines as the time interval between the two NAADP chal-lenges is increased (Aarhus et al., 1996; Genazzani et al.,

1996). The rate of inactivation is slow (on the order of 1-2minutes to reach plateau) and appears independent ofNAADP concentration (Aarhus et al., 1996; Genazzani et al.,1996). Rather it is the final extent of attenuation that is afunction of NAADP concentration. A simple model to ex-plain this phenomenon, is outlined in Figure 2. It is proposedthat NAADP receptors possess two binding sites for NAADPof high and low affinity. The high affinity site mediateschannel inactivation whereas the low affinity site mediateschannel opening. At low concentrations of NAADP, the highaffinity site will be preferentially occupied and the channelwill therefore tend to inactivate. Thus, subsequent challengewith a higher concentration of NAADP will not mediatechannel opening. This inactivation of channel opening mustbe a slow process otherwise the channel would not to open inhomogenates treated with higher concentrations of NAADPconditions under which both sites would be occupied. Theobservation that the rate of inactivation of NAADP-induced

Fig. 2. Inactivation of NAADP receptors by their ligand. The top part of the figure is a schematic time-course of changes in cytosolic Ca2+ concentration inresponse to NAADP (black circles). A, In sea urchin eggs (SUE) a subthreshold concentration of NAADP does not mediate Ca2+ release but attenuatessubsequent response to a higher concentration of NAADP. In untreated homogenates (B), the higher concentration of NAADP effects a robust release of Ca2+.In mammalian cells (Mam.), Ca2+ mobilisation is stimulated by low (C) but not high (D) NAADP concentrations. A model to describe these experimentalobservations is shown in the bottom part of the figure. Unoccupied NAADP receptors are depicted as blue structures containing a high (H, circle) and low (L,square) affinity binding site for NAADP. In sea urchin eggs, the high affinity site mediates inactivation whereas the low affinity sites mediates channel openingsuch that prior treatment with low concentrations of NAADP (A) results in preferential labelling of the high affinity site (orange) which then initiates a slowinactivation process. The receptor is then unable to open in response to a high concentration of NAADP (red). When the receptor is exposed to highconcentrations of NAADP (B), both sites become occupied (orange) and because inactivation is slow, NAADP receptors are able to open through occupancy ofthe low affinity site (green). In mammalian cells, the situation is reversed; the high affinity site mediates channel opening whereas the low affinity site mediatesinactivation. Low concentrations of NAADP (C) therefore preferentially occupy the high affinity site (orange) to mediate opening (green). At higherconcentrations of NAADP (D), fast association to the low affinity site mediates fast inactivation such that binding to the high affinity site is prevented. See maintext for more details.

21S. Patel / Biology of the Cell 96 (2004) 19–28

Ca2+ release is independent of NAADP concentration (Aar-hus et al., 1996; Genazzani et al., 1996) is important as thismight suggest that the rate-limiting step governing channelfunction is the inactivation process (whatever this may be)and not ligand binding to the receptor. We therefore envisagea two-step process underlying inactivation. Step 1 is bindingof NAADP to the high affinity site and step 2 is conversion ofthe channel to an inactivated state. One would have to arguethat NAADP remains bound to site 1 in order to effectchannel closing. That binding of NAADP to its receptor isirreversible (Aarhus et al., 1996; Patel et al., 2000a; Billing-ton and Genazzani, 2000) would provide a mechanism forthis to occur. Non-reversible binding may also explain theclearly graded extent of inactivation. At submaximalNAADP concentrations, the concentration of NAADP mayapproach the concentration of NAADP receptors such thatonly a fraction of the receptors would be occupied (Patel etal., 2000a).

NAADP receptors in mammalian cells also appear toinactivate prior to activation but they do so in a fundamen-tally different manner to those expressed in sea urchin eggs.Concentration-effect relationships for NAADP-induced inCa2+ release in several cell types are biphasic. This observa-tion was first noted in pancreatic acinar cells (Cancela et al.,1999). Whereas NAADP mediated complex signals in thetens of nanomolar range, higher concentrations (micromolar)were ineffective (Cancela et al., 1999). These data suggestthat inactivation preceded activation but in contrast to the seaurchin that this occurred at concentrations well above thethreshold for Ca2+ release. In T lymphocytes (Berg et al.,2000), human pancreatic beta cells (Johnson and Misler,2002) and MIN-6 cells (Masgrau et al., 2003), where a rangeof NAADP concentrations were tested, clear “bell-shaped”activation curves have been described although due to thenature of the experiments (single cell microinjection), it isdifficult to accurately determine the absolute intracellularconcentration of NAADP. Lower concentrations of NAADP(10 nM) are also more effective than higher ones (100 µM) inevoking calcium rises in arterial smooth muscle cells (Boittinet al., 2003). The peculiar activation and inactivation proper-ties of NAADP receptors in these systems can also can alsobe described by a 2 site, high and low affinity model (Figure2C,D). However, in contrast to the sea urchin model, wepropose that it is the low affinity site which mediates channelclosing. Two requirements in order to explain closure of thechannel without activation are that i) the rate of association ofNAADP to the low affinity site must be faster than the rate ofassociation to the high affinity site and ii) that inactivationmust occur essentially immediately. Such a model allows forthe repeated opening of the channel concentrations ofNAADP above threshold since the low affinity site is notoccupied. In contrast, NAADP at concentrations abovethreshold in the sea urchin would result in occupancy of allinactivation sites (due to their higher affinity) such that fol-lowing the first NAADP-induced channel opening no furtherresponse would be expected. The scheme proposed for mam-

malian NAADP receptors does not require binding to pro-ceed in an irreversible manner. Indeed, as discussed abovebinding studies in mammalian systems, though limited, dodemonstrate reversible NAADP binding (Patel et al., 2000b;Bak et al., 2001).

3. Ca2+ Channel Chatter

As discussed above, there is ample evidence to suggestthat NAADP mediates its effects through the activation of adedicated, as of yet unidentified Ca2+ channel. Based on thepharmacology of NAADP-induced Ca2+ release in intactcells however, it is clear that NAADP receptors appear not toactivate in isolation. Instead, several studies reveal cross-talkbetween NAADP receptors and receptors for IP3 andcADPR/ryanodine. The most striking example of “channelchatter” (Patel et al., 2001) is that in the pancreatic acinar cell(Cancela, 2001). This cell is a particularly well studied one interms of Ca2+ signalling where it has been known for sometime that different agonists evoke quite distinct Ca2+ signal-ling patterns (Cancela, 2001). The Ca2+ signature for thebrain-gut peptide cholecystokinin for instance, is a mixtureof short and longer lasting Ca2+ oscillations corresponding toCa2+ increases localised to the secretory pole of the cell andmore global Ca2+ signals that traverse the cell entirety (Can-cela, 2001). Introduction of NAADP in to the cell through apatch-pipette mimicks these Ca2+ increases whereas intro-duction of IP3 or cADPR stimulates only the local Ca2+

changes (Cancela et al., 1999). Quite remarkably, the effectsof NAADP in pancreatic acinar cells were blocked by hep-arin or 8-NH2-cADPR, specific antagonists of IP3 andcADPR receptors, respectively (Cancela et al., 1999). Anattractive hypothesis put forward by the authors to explainthis finding is that NAADP provides a trigger release of Ca2+

that is subsequently propagated by IP3 and ryanodine recep-tors by Ca2+-induced Ca2+ release (Cancela et al., 1999)(Figure 3). That inactivation of NAADP receptors by highconcentrations of NAADP (see above) has little effect onresponses to IP3 and cADPR supports the idea that NAADPreceptors activate upstream of IP3 and ryanodine receptors(Cancela et al., 1999). NAADP receptors and receptors forIP3 and cADPR/ryanodine must be very tightly coupled sinceno Ca2+ release (be it measured indirectly through Ca2+

–dependent currents at the plasma membrane) is observed inresponse to NAADP following blockade of the latter chan-nels. Indeed, Ca2+ responses to cADPR are also blocked byheparin in these cells (Thorn et al., 1994) further highlightingthe interdependence of intracellular Ca2+ channel activation.

By examining the effects of different combinations of lowconcentrations of Ca2+ mobilizing messengers in the pancre-atic acinar cell, it is clear that NAADP potentiates both IP3

and cADPR-induced Ca2+ mobilization (Cancela et al.,2002). Similar effects of NAADP have also been docu-mented in the frog neuromuscular junction (Brailoiu et al.,2003). In this preparation, neurotransmission in response to


submaximal concentrations of IP3 or cADPR was substan-tially greater in the presence of NAADP than the sum of theresponse to NAADP and the appropriate individual messen-ger alone (Brailoiu et al., 2003). These data further highlightthe coordinating effects of NAADP on a downstream Ca2+-dependent process. Clear coupling between receptors forNAADP, IP3 and ryanodine is also evident in sea urchin eggs(Churchill and Galione, 2000) and starfish oocytes (Santellaet al., 2000). In both these cells, responses to NAADP can beinhibited by a combination of heparin and 8-NH2 cADPR,antagonists of IP3 and cADPR, respectively. Like the pancre-atic acinar cell, in the starfish oocyte inhibition of Ca2+-induced Ca2+ release eliminates the response to NAADP(Santella et al., 2000). In the sea urchin egg however, al-though attenuated, NAADP-induced Ca2+ release is neverthe less observable in the presence of IP3/ryanodine receptorantagonists (Churchill and Galione, 2000). These data mayreflect different densities of NAADP receptors in the differ-ent cells. In cells such as the pancreatic acinar cell andstarfish oocyte, NAADP receptors may be expressed in lownumbers together with Ca2+-induced Ca2+ release channelswhereas in the sea urchin egg, additional NAADP receptorsmay be located further from IP3 and ryanodine receptors suchthat the initial NAADP-dependent triggering event is, withthe current methods for measuring Ca2+, only discernable inthe latter case (Figure 3A,B). Presumably, coupling of intra-cellular Ca2+ release channels by Ca2+-induced Ca2+releaseis disrupted upon cell homogenisation thereby accounting forthe lack of effects of IP3 and ryanodine receptor antagonistson NAADP-induced Ca2+ release in many broken cell prepa-rations (see above).

The alternative interpretation of the sensitivity ofNAADP-induced Ca2+ release to inhibitors of establishedintracellular Ca2+ channels under certain conditions is thatNAADP directly activates IP3 and/or ryanodine receptors.Hohenegger et al have demonstrated NAADP-induced Ca2+

release from skeletal muscle microsomes, which based on itsregulation by Ca2+ and blockade by ryanodine is consistentwith direct activation of ryanodine receptors by NAADP(Hohenegger et al., 2002). Indeed, NAADP was shown tomodulate single channel opening of purified ryanodine re-ceptors incorporated into lipid bilayers (Hohenegger et al.,2002). NAADP has also been shown to regulate ryanodinereceptor opening from cardiac microsomes (Mojzisova et al.,2001) but Copello et al, using a similar electrophysiologicalapproach fail to find any modulation by NAADP of skeletalor cardiac ryanodine receptors (Copello et al., 2001). Insupport of the latter findings, NAADP mediates Ca2+ fluxfrom cardiac microsomes in a ryanodine-independent man-ner (Bak et al., 2001). However, recent studies demonstrateinhibitory effects of ryanodine and ruthenium red onNAADP-induced Ca2+ release from isolated nuclei of pan-creatic acinar cells (Gerasimenko et al., 2003). Modulationof ryanodine receptors by NAADP, possibly through an ac-cessory NAADP-binding protein as shown in Figure 3Ctherefore remains an alternative but clearly controversial

Fig. 3. Intracellular Ca2+ release channel cross talk. A, B Ca2+ (blackcircles) released via activation of NAADP receptors (yellow structures) bytheir ligand (green circles) may recruit neighbouring IP3 and/or ryanodinereceptors (red structures) by Ca2+-induced Ca2+ release. In cells such as thesea urchin egg, where NAADP receptors are likely expressed in high num-bers, blockade of Ca2+-induced Ca2+ release uncovers the NAADP-mediated trigger Ca2+ release event (A). In other cells such as pancreaticacinar cells and starfish oocytes, the trigger Ca2+ release may be too small toresolve due to the lower density of NAADP receptors (B). C, Anotherpossible mode of coupling between receptors for NAADP and IP3/ryanodinereceptors is that the different receptors are part of a protein complex. In thisscenario, NAADP binding to its target stimulates opening of the associatedchannel (possibly through a conformational change) without a trigger Ca2+

release. This could explain the activation by NAADP of ryanodine receptorspurified to apparent homogeneity and those expressed in broken cell prepa-rations, conditions unlikely to support Ca2+-induced Ca2+ release. D, NAA-DP–induced Ca2+ release may also be amplified by IP3 and ryanodinereceptors by uptake of Ca2+ in to the ER (grey structure) via Ca2+ ATPases(blue structures) following mobilisation of distinct Ca2+ stores sensitive toNAADP (green structures).


mechanism whereby NAADP mediates Ca2+ mobilisation.Activation of ryanodine receptors by NAADP either by Ca2+-induced Ca2+ release (Figure 3A,B) or through a more directinteraction (Figure 3C) is similar to the activation of ryanod-ine receptors by voltage-sensitive Ca2+ channels during theprocess of excitation-contraction coupling (Protasi, 2002). Inskeletal muscle, dihydropyridine receptors on the sarco-lemma activate juxtaposed ryanodine receptors primarilythrough direct interaction between the two proteins (Protasi,2002). This form of interaction is similar to the situationproposed for NAADP/ryanodine receptor interaction de-picted in Figure 3C. In cardiac muscle however, Ca2+ influxthrough similar voltage-sensitive channels is the primaryactivator of ryanodine receptors (Protasi, 2002), a processakin to the recruitment of ryanodine receptors by NAADPvia Ca2+-induced Ca2+ release (Figures 3A, B). The spatialarrangement of NAADP receptors and receptors for IP3 andryanodine may therefore dictate the mode of coupling be-tween the different intracellular Ca2+ release pathways.

4. A new NAADP-sensitive Ca2+ store

Removal of extracellular Ca2+ in several cell types includ-ing the sea urchin egg (Perez-Terzic et al., 1995), pancreaticacinar cells (Cancela et al., 1999) and arterial smooth musclecells (Boittin et al., 2003) does not abolish Ca2+ increases inresponse to NAADP. These data indicate that similar to IP3

and cADPR, NAADP mobilizes intracellular Ca2+ stores.The ER and its muscle counterpart the sarcoplasmic reticu-lum (SR), are established stores of Ca2+ expressing both IP3

and ryanodine receptors. Activation of these channels resultsin a decrease in luminal Ca2+ concentration as does inhibitionof Ca2+ ATPases which prevents store filling. Accumulatingevidence suggests that NAADP targets Ca2+ stores distinctfrom the ER/SR. For example, whereas fractionation of seaurchin egg homogenates on density gradients results in thecomigration of IP3 and cADPR-sensitive Ca2+ stores withmarkers for the ER, NAADP-sensitive Ca2+ stores show anunusual, much broader distribution (Lee and Aarhus, 1995).Ca2+ stores responsive to NAADP can also be separated fromthose sensitive to cADPR and IP3 in intact eggs (Lee andAarhus, 2000). Analysis of the spatial distribution of Ca2+

signals in response to Ca2+ mobilising messengers in eggsthat had been stratified by centrifugation revealed thatNAADP-sensitive Ca2+ stores were located in the oppositepole of the egg to where IP3/cADPR sensitive Ca2+ storeswere distributed (Lee and Aarhus, 2000). Ca2+ stores sensi-tive to NAADP are also distinguishable from those mobilisedby IP3 in permeabilsed pancreatic acinar cells (Krause et al.,2002). In this highly polarized cell type, NAADP mobilizedCa2+ from the basolateral pole of the egg whereas IP3 in-duced Ca2+ release from the apical pole (Krause et al., 2002).

Importantly, depletion of ER Ca2+ stores with thapsigar-gin in homogenates and intact eggs from the sea urchin, doesnot prevent release of Ca2+ by NAADP (Genazzani and

Galione, 1996; Churchill et al., 2002). Recently, Churchill etal have made substantial progress in the identification of theclearly distinct Ca2+ store activated by NAADP in sea urchineggs (Churchill et al., 2002). NAADP-induced Ca2+ releasewas found to be selectively abolished by glycyl-phenylalanyl-naphthylamide (GPN), a substrate of capthep-sin C which bursts compartments containing this enzyme byosmotic lysis (Churchill et al., 2002). Cathepsin C is a cys-teine proteinase and is often used as a marker for lysosomes.Further experiments characterised the mechanism of Ca2+

uptake in to this Ca2+ store by using vesicle preparationsdevoid of ER Ca2+ stores (Churchill et al., 2002). Ca2+ uptakein to these compartments was found to be inhibited by bafilo-mycin an inhibitor of V-Type ATPases and several otheragents (FCCP, NH3) that collapse proton gradients. Thesedata provide strong evidence that NAADP-sensitive Ca2+

stores are in fact acidic Ca2+ stores which in the sea urchinegg, based on the protocol used for their isolation maycorrespond to reserve granules, lysosomal-like organelles.

New studies in MIN-6 cells also provide evidence thatNAADP targets Ca2+ stores distinct form the ER. Again, as inthe sea urchin egg, the effects of NAADP are shown to bethapsigargin-independent (Mitchell et al., 2003). In support,using a Ca2+-sensitive probe targeted to the lumen of theendoplasmic reticulum, Mitchell et al show that NAADPreceptor activation is not associated with a decrease in Ca2+

concentration within this compartment (Mitchell et al.,2003). Instead, NAADP caused a decrease in concentrationof Ca2+ within the lumen of the secretory vesicles, measuredusing a second targeted Ca2+ probe (Mitchell et al., 2003). Itis more than noteworthy that secretory vesicles are alsocharacterised by their acidic interior. Thus, it is tempting tospeculate that NAADP may specifically mobilise acidic Ca2+

stores (Patel, 2003). This researcher at least eagerly awaitsthe identification of NAADP-sensitive Ca2+ stores in otherpreparations such as the frog neuromuscular junction(Brailoiu et al., 2001; Brailoiu et al., 2003) and smoothmuscle cells (Boittin et al., 2003) where the effects ofNAADP have also been reported to be thapsigargin-insensitive.

5. A two pool model for Ca2+ release by NAADP

In sea urchin eggs, fertilisation is associated with a seriesof Ca2+ oscillations (Poenie et al., 1985). Neither IP3 orcADPR mimick this temporal pattern of Ca2+ release al-though sperm-induced Ca2+ signals are sensitive to a combi-nation of heparin and ryanodine implicating a requirementfor IP3 and ryanodine receptors in this process (Galione et al.,1993; Lee et al., 1993). Intriguingly, long term Ca2+ oscilla-tions can be induced by NAADP (Aarhus et al., 1996). Giventheir propensity to inactivate and their insensitivity to Ca2+, itis perhaps, suprising that activation of NAADP receptorsresults in the generation of complex Ca2+ signals. NAADP-induced Ca2+ oscillations are however eliminated following


dual block of Ca2+-induced Ca2+ release channels (in furtheranalogy with fertilisation-induced Ca2+ transients) and bythapsigargin, which reduced but did not eliminate the initialNAADP-induced Ca2+ signal (Churchill and Galione,2001a). Based on these (and other) data Churchill andGalione propose that release of Ca2+ from NAADP-sensitiveCa2+ stores is followed by cycles of uptake into and releasefrom the ER (Churchill and Galione, 2001a). A “two pool”model for Ca2+ release may also underlie complex Ca2+

signals in arterial smooth muscle cells. Boittin et al reportlocalised Ca2+ release events in response to NAADP infusionthat precede larger global Ca2+ release (Boittin et al., 2003).As in the sea urchin egg, the initial Ca2+ release in responseto NAADP is demonstrable following depletion of Ca2+

stores with thapsigargin whereas the secondary Ca2+ signalsare not (Boittin et al., 2003). These effects are again consis-tent with sequential activation of NAADP sensitive and-insensitive Ca2+ stores. Thus, NAADP-induced Ca2+ signalsmay be amplified by both “channel” (Figure 3A-C) and“store” chatter (Figure 3D).

6. Role of Ca2+ entry in NAADP-mediated Ca2+

signalling

As mentioned, NAADP-induced Ca2+ release in severalcells is readily demonstrable in the absence of extracellularCa2+. Spatially resolved measurements of Ca2+ in sea urchineggs in the presence of extracellular Ca2+ however reveal thatNAADP-induced Ca2+ release in intact eggs is markedlybiphasic consisting of an initial Ca2+ increase confined to theperimeter of the egg followed by a global Ca2+ increasewhich extends through the egg cytoplasm (Churchill et al.,2003). While removal of extracellular Ca2+ (or addition ofthe Ca2+ channel blocker Cd2+) does not affect the secondmore robust Ca2+ increase (consistent with the mobilizationof intracellular Ca2+ stores, the localised Ca2+ increase at theplasma membrane is abolished by such maneouvers(Churchill et al., 2003). These data provide evidence that inaddition to mobilizing intracellular Ca2+ stores, NAADPmay also stimulate Ca2+ entry across the plasma membrane.Sperm, like NAADP, also stimulate a localised Ca2+ increaseat the plasma membrane termed the cortical flash, whichinvolves activation of voltage-sensitive Ca2+ channels (Shenand Buck, 1993). Intriguingly, inactivation of NAADP recep-tors abolishes the cortical flash at fertilization suggesting thatNAADP and sperm-induced Ca2+ entry are mediated bysimilar means (Churchill et al., 2003).

Ca2+ entry may also contribute to the generation ofNAADP-induced Ca2+ signals in starfish oocytes. Santella etal have reported that whereas in immature oocytes, removalof extracellular Ca2+ has a relatively modest effect on Ca2+

increases in response to photolysis of caged NAADP, theresponse to NAADP is effectively blocked in matured oo-cytes (Santella et al., 2000). Again, similar to the sea urchinegg, imaging of the Ca2+ increase in response to NAADP

reveals that NAADP-induced Ca2+ release initiates in thecortex of matured oocyte (Lim et al., 2001). An inwardlyrectifying Ca2+-selective membrane current activated byNAADP has recently been characterised (Moccia et al.,2003). This current is insensitive to a combination of heparinand 8-NH2-cADPR which (as discussed above) is sufficientto attenuate global Ca2+ increases in response to NAADP(Santella et al., 2000). Thus, NAADP may mediate its effectsin the starfish oocyte by triggering of Ca2+ entry across theplasma membrane followed by recruitment of IP3 and ryano-dine receptors by Ca2+-induced Ca2+ release. Interestingly,disruption of the actin cytoskeleton reduced the NAADP-sensitive current (Moccia et al., 2003). This finding is remi-niscent of the modulation by the cytoskeleton of store oper-ated (capacitative) Ca2+ entry – the mechanistically elusiveprocess whereby depletion of ER Ca2+ stores stimulatesentry of Ca2+ from the extracellular space (Venkatachalam etal., 2003).According to the “secretion” model, store operatedCa2+ entry is mediated via protein–protein interactions be-tween the ER and the plasma membrane (Venkatachalam etal., 2003). This likely involves the dynamic coupling of IP3

receptors with Ca2+ channels belonging to the transient re-ceptor potential (trp) family. In analogy, it is possible that inthe starfish oocytes, NAADP receptors located on corticalCa2+ stores couple to Ca2+ entry channels in the plasmamembrane, an interaction that may strengthen during matu-ration.

Further evidence for an interaction between NAADP re-ceptors and plasma membrane Ca2+ channels has been ob-tained in ascidian oocytes. In these cells, NAADP does notaffect global Ca2+ concentration yet inhibits depolarization-induced Ca2+ currents – an event mediated by increases incytosolic Ca2+ (Albrieux et al., 1998). NAADP-mediatedCa2+ signals in smooth muscle cells also appear to initiate inthe cell periphery consistent with a cortical location ofNAADP sensitive stores although in these cells, Ca2+ signalsin response to NAADP are not abolished by removal ofexternal Ca2+ (Boittin et al., 2003). Taken together these datasuggest that NAADP receptors can be localised close to orwithin the plasma membrane and that Ca2+ entry may con-tribute to the shaping of NAADP-induced Ca2+ signals.

7. A New Message

Although, NAADP receptors are expressed in many dif-ferent cells and tissues, including the brain (Bak et al., 1999;Patel et al., 2000b), information regarding the control ofcellular NAADP levels is scant. We have shown that metabo-lism of NAADP in brain membranes is activated by Ca2+

providing a possible Ca2+-dependent feedback mechanism tolimit NAADP-induced Ca2+ signals (Berridge et al., 2002b).Regarding its production, several studies have demonstratedthat Ca2+ signals in response to physiological Ca2+ mobiliz-ing extracellular stimuli are attenuated in cells exposed tohigh (inactivating) concentrations of NAADP. These data


provide evidence that the relevant stimuli engage NAADPreceptors upon cell stimulation presumably through elevat-ing NAADP levels. In pancreatic acinar cells for example,high concentrations of NAADP were shown to inhibit Ca2+

signals in response to cholecystokinin (Cancela et al., 1999)but not other secretagogues such as acetylcholine (Cancela etal., 2000) and bombesin (Burdakov and Galione, 2000). Asimilar strategy has been employed to show that NAADP isinvolved in mediating Ca2+ signals in neighbouring beta cellsof the endocrine pancreas stimulated with both glucose(Masgrau et al., 2003) and insulin (Johnson and Misler,2002) (see below) and also in antigen-mediated Ca2+ signal-ling in T lymphocytes (Berg et al., 2000). That high concen-trations of NAADP appears to inactivate its target proteinwithout elevating Ca2+ and that the effects of NAADP arespecific to a particular agonist (at least in pancreatic acinarcells) argue strongly against a non-specific action of NAADPon the response that it attenuates. However, as elegant asthese studies are they are clearly an indirect means to ascer-tain agonist-evoked NAADP generation. Two recent studieshave used a radioreceptor assay based on the selective bind-ing of NAADP to its target protein in sea urchin egg homo-genates (see above) (Patel et al., 2000a), to provide moredirect and quantitative evidence that NAADP levels are in-creased in response to physiological Ca2+ elevating stimuli.The first system in which NAADP levels have been measuredis sea urchin sperm. Contact of sperm with the extracellularmatrix of the egg in all animal species triggers an exocytoticevent within the sperm, termed the acrosome reaction, whichis vital for sperm penetration and eventual gamete union.Last year, exposure of sea urchin sperm to egg jelly wasshown to markedly increase (~4-fold) sperm NAADP levelsproviding much more direct evidence that NAADP levels canbe regulated by an extracellular stimulus (Churchill et al.,2003). That sperm contain unusually high concentrations ofNAADP (Billington et al., 2002; Churchill et al., 2003) andthat NAADP levels are further increased during contact withegg jelly (Churchill et al., 2003), raise the intriguing possi-bility that during fertilization, a bolus of NAADP is deliveredto the egg upon sperm-egg fusion. Since inactivation ofNAADP-induced Ca2+ release by low concentrations ofNAADP only very slowly reverses in living eggs (Churchilland Galione, 2001b) and persists indefinitely in brokenpreparations, delivery of NAADP may act as a simple mo-lecular memory marking the site of sperm entry. Indeed,localized liberation of NAADP within an egg shapes subse-quent Ca2+ release in response to global NAADP challenge(Churchill and Galione, 2001b). Moreover, NAADP-inducedCa2+ release is diminished in eggs (Perez-Terzic et al., 1995;Churchill et al., 2003) and homogenates (Churchill et al.,2003) after fertilization presumably as a consequence ofprior activation (and subsequent) inactivation of NAADPreceptors.

Measurement of NAADP levels in fertilised egg suspen-sions indicates that the observed increase in NAADP occursin two phases; an initial surge which may well be sperm-

derived and a second later increase peaking 4 - 5 minutes postfertilisation (Churchill et al., 2003). This latter increase inNAADP probably results from synthesis of NAADP withinthe egg and may contribute to the initiation of Ca2+ oscilla-tions that typically result at fertilisation (Poenie et al., 1985).

The second system in which NAADP levels have beenshown to increase upon extracellular stimulation is theMIN-6 cell, a clonal pancreatic beta cell line where like thesea urchin egg, NAADP-induced Ca2+ release proceedsthrough mobilisation of thapsigargin-insensitive stores (seeabove). Elevations in plasma glucose raise cytosolic Ca2+

levels in pancreatic beta cells, an event required for exocyto-sis of insulin-containing secretory vesicles (Rutter, 2003).How these Ca2+ signals are generated is subject to consider-able controversy although it is generally agreed upon thatCa2+ entry from the external medium (due to metabolicinhibition of K+ channels, depolarisation and the activationof voltage-sensitive Ca2+ channels) plays an important role(Rutter, 2003). Mobilization of intracellular Ca2+ stores isalso likely to contribute to glucose-induced Ca2+ signallingand may drive the so-called second phase of insulin release(Henquin, 2000). Masgrau et al report a 2-fold increase inNAADP levels upon glucose stimulation (Masgrau et al.,2003). Combined with their demonstration of i) NAADP-induced Ca2+ release, supported by independent studies inthe same cell type (Mitchell et al., 2003) and human betacells (Johnson and Misler, 2002), ii) a specific binding sitefor radiolabelled NAADP and iii), attenuation of glucose-induced Ca2+ signalling by inactivating concentrations ofNAADP, these data place NAADP central to the control ofCa2+ signals by glucose. Additionally, recent evidence sug-gests that released insulin may act in an autocrine manner toregulate its own release via changes in cytosolic Ca2+ (Rutter,2002). That inactivating concentrations of NAADP attenuateCa2+ signals in beta cells in response to insulin (Johnson andMisler, 2002), as well as glucose, extends the actions ofNAADP from its role in the generation of Ca2+ signals to animportant down stream event of major clinical relevance.

Molecular details of how NAADP is generated (and de-graded) are not clear. A strong candidate enzyme for itssynthesis is ADP ribosyl cyclase. This enzyme, which wasfirst purified from aplysia ovotestis (Hellmich and Strum-wasser, 1991) is a multifunctional enzyme that in addition tosynthesizing cADPR from NAD (by cyclisation) can alsocatalyse the synthesis of NAADP from NADP in the pres-ence of nicotinic acid (by base-exchange) (Lee, 2001). Base-exchange activity can be demonstrated in a variety of brokencellular/tissue preparations but not from several tissue prepa-rations from mice in which the gene for CD38, a mammalianADP-ribosyl cyclase homologue, is deleted (Chini et al.,2002). These data indicate that ADP ribosyl cyclase/CD38 islikely the enzyme responsible for NAADP synthesis in vitro.Whether this enzyme mediates NAADP synthesis in vivo isquestioned given the unphysiological conditions required tofavour base-exchange activity. The pH optimum of aplysiaADP-ribosyl cyclase and CD38 is acidic and far from that of


the cytosolic pH and the Km for nicotinic acid is high relativeto expected concentration within cells (Aarhus et al., 1995).It should be noted however that in the sea urchin egg, base-exchange activity under optimal conditions is far in excess ofcyclase activity such that even under sub-optimal pH andnicotinic acid concentrations, sufficient levels of NAADPmay be generated to activate high affinity NAADP receptors(Graeff et al., 1998).

8. Perspectives

Receptors for IP3 and ryanodine show many structural andfunctional similarities (Patel et al., 1999; Fill and Copello,2002). NAADP Receptors however appear quite distinct, asdo the Ca2+ stores upon which they reside. Clearly then,important goals include the molecular identification of theputative NAADP-sensitive Ca2+ channel and to establishwhether mobilisation of acidic stores of Ca2+ by NAADP isconserved in all NAADP-responsive cells. Although recentdemonstrations of changes in NAADP levels in response toCa2+ mobilizing stimuli provide strong evidence thatNAADP is indeed a new Ca2+ mobilizing messenger, theenzymatic route for its synthesis also remains to be defined.No doubt these outstanding issues will be addressed in whatis a rapidly advancing field.

Acknowledgements

I am grateful to Chi Li for useful discussion. Work in theauthors laboratory is supported by the Wellcome Trust (UK).

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naadp-induced ca2+ release – a new signalling pathway

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