Interactions between intracellular Ca 2 + stores : Ca 2 + released from the NAADP pool potentiates cADPR-induced Ca 2 + release

! "! #$ %$ &$ ! ' ( ) " ( " ( * * " Correspondence E.N. Chini Department of Anesthesiology Mayo Clinic and Foundation

45 Ca uptake and release were measured by a filtration method using glass-fiber filters as described in Ref. 6.The remaining intravesicular 45 Ca was determined by filtration of 0.2 ml of a 1.25% (v/v) egg homogenate through a prewashed GF/C glass filter (Whatman) under vacuum, followed by rapid washing three times with 1 ml of an ice-cold intracellular medium containing 3 mM LaCl 3 .
The radioactivity retained on the filter was determined by standard scintillation counting.

Material
L. pictus and Aplysia californica were obtained from Marinus Inc., Long Beach, CA, USA.Fluo-3 was purchased from Molecular Probes, Eugene, OR, USA, and IP 3 , ryanodine, oligomycin and antimycin were from Calbiochem, San Diego, CA, USA.All other reagents, of the highest purity grade available, were supplied by Sigma Co., St. Louis, MO, USA.NAADP and cADPR were synthesized as described before (5).
The reported experiments were repeated at least three to six times.

NAADP and cADPR induce Ca 2+ release from different Ca 2+ pools
First we investigated the mechanisms of Ca 2+ uptake in sea urchin egg homogenates, which were found to have both thapsigarginsensitive and -insensitive Ca 2+ uptake systems.These data indicate that egg homogenates have both a sarcoplasmic-endoplasmic reticulum Ca 2+ ATPase (SERCA)-like pool and a second different mechanism of Ca 2+ uptake that is not mediated by a SERCA-like enzyme.As shown in Figure 1, the thapsigargin-insensitive system is slower.However, the maximum amount of Ca 2+ uptake was identical in the presence or absence of thapsigargin (Figure 1).Next we determined whether the intracellular Ca 2+ -releasing agents cADPR, IP 3 , and NAADP could activate Ca 2+ efflux in both thapsigarginsensitive and -insensitive pools (Figure 2).In agreement with data previously reported by Genazzani and Galione (15), the results indicated that cADPR and IP 3 promoted Ca 2+ release only through the thapsigargin-sensitive pools (Figure 2).In contrast, NAADP was able to induce Ca 2+ release from both thapsigargin-sensitive and -insensitive pools (Figure 2), indicating that the NAADP and cADPR Ca 2+ pools in sea urchin egg homogenates are at least partially independent.

Potentiation of the Ca 2+ -induced Ca 2+ release by Ca 2+ released from the NAADP pool
It has been previously reported that extravesicular Ca 2+ can not only potentiate but is also necessary for the Ca 2+ release induced by ryanodine receptor agonists such as cADPR and ryanodine (6,19).In contrast, the NAADP-induced Ca 2+ release does not behave like a Ca 2+ -induced Ca 2+ release (6,15).It has been proposed that the Ca 2+ released by NAADP could modulate the Ca 2+induced Ca 2+ release system activated by cADPR (18,20,21).However, no direct evidence for this action has been reported to date.Here we demonstrate that Ca 2+ release from the NAADP pool could potentiate the Ca 2+ release induced by ryanodine and cADPR.As shown in Figure 3, after the addition of 12 nM NAADP a small amount of Ca 2+ was released from the vesicles, and the addition of subthreshold concentrations of cADPR at the peak (steady state) of the Ca 2+ release led to a significant potentiation of the cADPR-induced Ca 2+ release (Figure 3).This effect was not mediated by NAADP itself but by the increase in extravesicular Ca 2+ , since when the Ca 2+ release induced by NAADP was abolished by previous desensitization of the NAADP receptor the cADPRinduced Ca 2+ release was not enhanced by NAADP (Figure 3C).The increase of extravesicular Ca 2+ induced by NAADP in-  pool can sensitize the ryanodine receptor to cADPR.In contrast, we found no effect of NAADP on the Ca 2+ release induced by IP 3 .Furthermore, Ca 2+ released from the IP 3 pool was not consistently able to sensitize the cADPR-induced Ca 2+ release (data not shown).This is probably due to the fact that cADPR and IP 3 induce Ca 2+ release from the same Ca 2+ pool in sea urchin egg homogenates (15).
A second mechanism for NAADP modulation of the cADPR-induced Ca 2+ release has been described by Churchill and Galione (12), who reported that in intact sea urchin eggs NAADP-induced Ca 2+ oscillations were mediated via a two-pool mechanism that primed the cADPR-and the IP 3 -sensitive Ca 2+ stores (12).In fact, priming the Ca 2+ pools with Ca 2+ (13) can increase the apparent affinity for cADPR and IP 3 .
The precise role of NAADP-modulated Ca 2+ release is not known.However, it has been proposed that in pancreatic acinar cells NAADP could be the trigger of Ca 2+ oscillations induced by cholecystokinin (20,21).The cited investigators proposed that Ca 2+ released by NAADP in response to cholecystokinin may activate the Ca 2+ -induced Ca 2+ release mediated by cADPR, leading to amplification of the Ca 2+ signaling and generation of the Ca 2+ oscillation (20,21).A similar role for NAADP has been proposed for the mobilization of Ca 2+ in starfish oocytes (18).The present study is the first to demonstrate a direct effect of the Ca 2+ released by NAADP on the apparent affinity of the ryanodine receptor for cADPR (Figure 4).This further indicates that NAADP may have an important role in the complex mechanism of intracellular Ca 2+ mobilization in several vertebrate and invertebrate cells (4,5,(16)(17)(18)20,21).In fact, the Ca 2+ released from the NAADP pool can modulate the intracellular Ca 2+ release by at least two different mechanisms: a) by priming the intracellular Ca 2+ pools ( 16) and b) by direct sensitization of the Ca 2+ -induced Ca 2+ release.creased the apparent affinity of the ryanodine receptor for cADPR and ryanodine (Figure 4).Increasing the extravesicular Ca 2+ could reproduce the effect of NAADP on the Ca 2+ release mediated by cADPR by the addition of Ca 2+ itself to the sea urchin egg homogenates (Figures 3E and 5).In fact, when normalized for the increase in extravesicular Ca 2+ upon the potentiation of the cADPR-induced Ca 2+ release, the effects of NAADP and of addition of Ca 2+ itself were near identical (Figure 5).These data indicate that Ca 2+ released from the NAADP A B Interactions between NAADP and cADPR-induced Ca 2+ release systems Multiple intracellular Ca 2+ stores are present in many cells (1,(4)(5)(6)20,21) and may play a role in several physiological processes including muscle contraction, exocrine and endocrine secretion, fertilization, neuronal activation and immune cell function (1,2,9,13,(16)(17)(18)20). Exactly how Ca 2+ exerts its intracellular effects is not completely understood.The answer may lie in the complex interaction between intracellular and extra-cellular Ca 2+ pools to generate specific spatial-temporal intracellular Ca 2+ signals.In this regard, the present results describing the direct interactions between NAADP (a non-Ca 2+ -induced Ca 2+ release) and cADPR (a Ca 2+ -induced Ca 2+ release) Ca 2+ stores may be of broad physiological importance.In fact, the determination of the specific role of different Ca 2+ stores in several cellular functions deserves further investigation.

Figure 1 .
Figure 1.Ca 2+ uptake in sea urchin egg homogenates.The determination of Ca 2+ uptake was performed using 45 Ca as described in Material and Methods.Sea urchin egg homogenates were incubated in the presence (open circles) or absence (filled circles) of 10 µM thapsigargin (a Ca 2+ ATPase inhibitor).

Figure 2 .Figure 3 .
Figure 2. Ca 2+ release induced by nicotinic acid adenine dinucleotide phosphate (NAADP) from the thapsigargin-insensitive pool.The sea urchin egg homogenates were loaded with 45 Ca as described in Figure 1.After 3 h of Ca 2+ uptake, Ca 2+ release was initiated by addition of 1 µM IP 3 , 100 nM cyclic ADP-ribose (cADPR) or 100 nM NAADP.The Ca 2+ release was performed in homogenates loaded in the absence (open circles) or the presence (filled circles) of 10 µM thapsigargin.

Figure 4 .
Figure 4. Effect of Ca 2+ released by NAADP on the apparent affinity of the ryanodine receptor for ryanodine and cyclic ADPribose (cADPR).Homogenates were treated with no addition (filled circles), or with the addition of 12 nM NAADP (open circles) as shown in Figure 3B.The dose-response dependence for ryanodine (A) and cADPR (B) was determined by the addition of different concentrations of the Ca 2+ -releasing compounds as shown in the figure.The addition of ryanodine and cADPR was performed after NAADP-induced Ca 2+ release was at its plateau level (see Figure 3B).The Ca 2+ released by NAADP potentiates the effect of both ryanodine and cADPR about 2.5 to 3 times.The data represent the mean ± SEM of four experiments.

Figure 5 .
Figure 5.Effect of extravesicular Ca 2+ on cADPR-induced Ca 2+ release.Ca 2+ release was monitored as described in Material and Methods.The figure indicates the Ca 2+ released by 16 nM cADPR under different levels of extravesicular Ca 2+ above baseline.The Ca 2+ released under ambient extravesicular Ca 2+ is indicated by a triangle.The extravesicular Ca 2+ was increased by the addition of different concentrations of NAADP (squares) or Ca 2+ (circles).The addition of cADPR was performed at the plateau level of Ca 2+ induced by NAADP or Ca 2+ itself, as shown in Figure 1.The data are the mean ± SEM of three independent experiments.