Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release

Braz J Med Biol Res, May 2002, Volume 35(5) 543-547

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

E.N. Chini

Departments of Anesthesia and Internal Medicine, Mayo Clinic and Foundation, Rochester, MN, USA

References

^{Correspondence and Footnotes}

Abstract

Cells possess multiple intracellular Ca²⁺-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca²⁺ release. In the present study the mechanism of interaction between three intracellular Ca²⁺ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP₃)-regulated Ca²⁺ stores, is explored. The data indicate that the NAADP Ca²⁺ pool could be used to sensitize the cADPR system. In contrast, the IP₃ pool was not affected by the Ca²⁺ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca²⁺ release, promoted by Ca²⁺ released from the NAADP pool, is mediated by the mechanism of Ca²⁺-induced Ca²⁺ release. These data raise the possibility that the NAADP Ca²⁺ store may have a role as a regulator of the cellular sensitivity to cADPR.

Key words: cADPR, NAADP, Calcium, Sea urchin eggs, Fertilization, IP₃

Introduction

The release of Ca²⁺ from intracellular stores is a widespread component of several signaling pathways (1-3). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently discovered nucleotide with intracellular Ca²⁺-releasing properties (4-11). NAADP-induced Ca²⁺ release was first described in sea urchin egg homogenates (5). The Ca²⁺ release mechanism elicited by NAADP differs in many ways from the Ca²⁺ release controlled by cyclic ADP-ribose (cADPR) and inositol 1',4',5'-trisphosphate (IP₃) (2,4-17). Properties of this Ca²⁺-releasing molecule include: i) absence of regulation by the intracellular divalent cations Mg²⁺ and Ca²⁺ (6-8,15); ii) NAADP-induced Ca²⁺ release is fully inactivated by exposure to low concentrations of NAADP (14), and iii) Ca²⁺ release induced by NAADP appears to be insensitive to changes of pH over a wide range (8,17). These characteristics make NAADP a unique trigger of intracellular Ca²⁺ (2,9,10). In addition to the NAADP-induced Ca²⁺ release system, cells also possess other intracellular Ca²⁺ messengers such as cADPR and IP₃ (5). The exact physiological role of three different intracellular Ca²⁺-releasing systems in cells is not known. However, it is possible that these different Ca²⁺ pools may interact in the complex mechanism of intracellular Ca²⁺ oscillation (9,12,13,16,18). In the present study I explored in vitro the mechanisms by which NAADP could modulate the Ca²⁺ release elicited by cADPR. It was found that NAADP could potentiate the cADPR-induced Ca²⁺ release by sensitization of the ryanodine receptor by a mechanism similar to the Ca²⁺-induced Ca²⁺ release. This result indicates that crosstalk between intracellular Ca²⁺ pools may modulate the complex mechanism of intracellular Ca²⁺ mobilization.

Material and Methods

Sea urchin egg homogenates

Homogenates from Lytechinus pictus egg were prepared as described previously (5). Frozen homogenates were thawed in a 17ºC water bath and diluted to 1.25% with an intracellular medium containing 250 mM N-methyl glutamine, 250 mM potassium gluconate, 20 mM HEPES buffer, pH 7.2, 1 mM MgCl₂, 2 U/ml creatine kinase, 4 mM phosphocreatine, 1 mM ATP, 3 µg/ml oligomycin, and 3 µg/ml antimycin. After incubation at 17ºC for 3 h, 3 µM fluo-3 was added. Fluo-3 fluorescence was monitored at 490 nm excitation and 535 nm emission in a 250-µl cuvette, held at 17ºC with a circulating water bath and continuously mixed with a magnetic stirring bar, in a Hitachi spectrofluorometer (F-2000).

⁴⁵Ca uptake and release were measured by a filtration method using glass-fiber filters as described in Ref. 6. The remaining intravesicular ⁴⁵Ca 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 LaCl₃. 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 IP₃, 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.

Results and Discussion

NAADP and cADPR induce Ca ²⁺ release from different Ca ²⁺ pools

First we investigated the mechanisms of Ca²⁺ uptake in sea urchin egg homogenates, which were found to have both thapsigargin-sensitive and -insensitive Ca²⁺ uptake systems. These data indicate that egg homogenates have both a sarcoplasmic-endoplasmic reticulum Ca²⁺ ATPase (SERCA)-like pool and a second different mechanism of Ca²⁺ 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 Ca²⁺ uptake was identical in the presence or absence of thapsigargin (Figure 1). Next we determined whether the intracellular Ca²⁺-releasing agents cADPR, IP₃, and NAADP could activate Ca²⁺ efflux in both thapsigargin-sensitive and -insensitive pools (Figure 2). In agreement with data previously reported by Genazzani and Galione (15), the results indicated that cADPR and IP₃ promoted Ca²⁺ release only through the thapsigargin-sensitive pools (Figure 2). In contrast, NAADP was able to induce Ca²⁺ release from both thapsigargin-sensitive and -insensitive pools (Figure 2), indicating that the NAADP and cADPR Ca²⁺ pools in sea urchin egg homogenates are at least partially independent.

Figure 1.
Ca

²⁺ uptake in sea urchin egg homogenates. The determination of Ca²⁺ uptake was performed using ⁴⁵Ca 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 Ca²⁺ ATPase inhibitor).

[View larger version of this image (6 K GIF file)]

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 ⁴⁵Ca as described in Figure 1. After 3 h of Ca²⁺ uptake, Ca²⁺ release was initiated by addition of 1 µM IP₃, 100 nM cyclic ADP-ribose (cADPR) or 100 nM NAADP. The Ca²⁺ release was performed in homogenates loaded in the absence (open circles) or the presence (filled circles) of 10 µM thapsigargin.

[View larger version of this image (15 K GIF file)]

Potentiation of the Ca ²⁺ -induced Ca ²⁺ release by Ca ²⁺ released from the NAADP pool

It has been previously reported that extravesicular Ca²⁺ can not only potentiate but is also necessary for the Ca²⁺ release induced by ryanodine receptor agonists such as cADPR and ryanodine (6,19). In contrast, the NAADP-induced Ca²⁺ release does not behave like a Ca²⁺-induced Ca²⁺ release (6,15). It has been proposed that the Ca²⁺ released by NAADP could modulate the Ca²⁺-induced Ca²⁺ release system activated by cADPR (18,20,21). However, no direct evidence for this action has been reported to date. Here we demonstrate that Ca²⁺ release from the NAADP pool could potentiate the Ca²⁺ release induced by ryanodine and cADPR. As shown in Figure 3, after the addition of 12 nM NAADP a small amount of Ca²⁺ was released from the vesicles, and the addition of subthreshold concentrations of cADPR at the peak (steady state) of the Ca²⁺ release led to a significant potentiation of the cADPR-induced Ca²⁺ release (Figure 3). This effect was not mediated by NAADP itself but by the increase in extravesicular Ca²⁺, since when the Ca²⁺ release induced by NAADP was abolished by previous desensitization of the NAADP receptor the cADPR-induced Ca²⁺ release was not enhanced by NAADP (Figure 3C). The increase of extravesicular Ca²⁺ induced by NAADP increased the apparent affinity of the ryanodine receptor for cADPR and ryanodine (Figure 4). Increasing the extravesicular Ca²⁺ could reproduce the effect of NAADP on the Ca²⁺ release mediated by cADPR by the addition of Ca²⁺ itself to the sea urchin egg homogenates (Figures 3E and ⁵). In fact, when normalized for the increase in extravesicular Ca²⁺ upon the potentiation of the cADPR-induced Ca²⁺ release, the effects of NAADP and of addition of Ca²⁺ itself were near identical (^{Figure 5}). These data indicate that Ca²⁺ released from the NAADP pool can sensitize the ryanodine receptor to cADPR. In contrast, we found no effect of NAADP on the Ca²⁺ release induced by IP₃. Furthermore, Ca²⁺ released from the IP₃ pool was not consistently able to sensitize the cADPR-induced Ca²⁺ release (data not shown). This is probably due to the fact that cADPR and IP₃ induce Ca²⁺ release from the same Ca²⁺ pool in sea urchin egg homogenates (15).

A second mechanism for NAADP modulation of the cADPR-induced Ca²⁺ release has been described by Churchill and Galione (12), who reported that in intact sea urchin eggs NAADP-induced Ca²⁺ oscillations were mediated via a two-pool mechanism that primed the cADPR- and the IP₃-sensitive Ca²⁺ stores (12). In fact, priming the Ca²⁺ pools with Ca²⁺ (13) can increase the apparent affinity for cADPR and IP₃.

The precise role of NAADP-modulated Ca²⁺ release is not known. However, it has been proposed that in pancreatic acinar cells NAADP could be the trigger of Ca²⁺ oscillations induced by cholecystokinin (20,21). The cited investigators proposed that Ca²⁺ released by NAADP in response to cholecystokinin may activate the Ca²⁺-induced Ca²⁺ release mediated by cADPR, leading to amplification of the Ca²⁺ signaling and generation of the Ca²⁺ oscillation (20,21). A similar role for NAADP has been proposed for the mobilization of Ca²⁺ in starfish oocytes (18). The present study is the first to demonstrate a direct effect of the Ca²⁺ 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 Ca²⁺ mobilization in several vertebrate and invertebrate cells (4,5,16-18,20,21). In fact, the Ca²⁺ released from the NAADP pool can modulate the intracellular Ca²⁺ release by at least two different mechanisms: a) by priming the intracellular Ca²⁺ pools (16) and b) by direct sensitization of the Ca²⁺-induced Ca²⁺ release.

Multiple intracellular Ca²⁺ stores are present in many cells (1,4-6,20,21) and may play a role in several physiological processes including muscle contraction, exocrine and endocrine secretion, fertilization, neuronal activation and immune cell function (1,2,9,13,16-18,20). Exactly how Ca²⁺ exerts its intracellular effects is not completely understood. The answer may lie in the complex interaction between intracellular and extracellular Ca²⁺ pools to generate specific spatial-temporal intracellular Ca²⁺ signals. In this regard, the present results describing the direct interactions between NAADP (a non-Ca²⁺-induced Ca²⁺ release) and cADPR (a Ca²⁺-induced Ca²⁺ release) Ca²⁺ stores may be of broad physiological importance. In fact, the determination of the specific role of different Ca²⁺ stores in several cellular functions deserves further investigation.

Figure 3.
Potentiation of the cyclic ADP-ribose (cADPR)-induced Ca

²⁺ release by Ca²⁺ released from the nicotinic acid adenine dinucleotide phosphate (NAADP) pool. Free Ca²⁺ concentrations were measured as described in Material and Methods using fluo-3. The arrow indicates the sequential addition of different Ca²⁺ channel agonists. In A the arrow indicates the addition of 16 nM cADPR that by itself does not promote Ca²⁺ release. In B the homogenate was first treated with 12 nM NAADP and 16 nM cADPR was added at the peak (steady state) of the Ca²⁺ release induced by NAADP. In C the homogenate was pretreated with 2 nM NAADP for 20 min (not shown) to promote self-desensitization of the NAADP receptor. After that the homogenate was treated with 12 nM NAADP and 16 nM cADPR. In D homogenates were treated with a saturating concentration of 60 nM NAADP and then, after the Ca²⁺ released by NAADP was taken up again, the homogenate was treated with 16 nM cADPR. In E the homogenate was treated with 4 nmol Ca²⁺ prior to the addition of 16 nM cADPR. The data are representative of 12 different experiments done with three different preparations of sea urchin egg homogenates.

[View larger version of this image (20 K GIF file)]

Figure 4.
Effect of Ca

²⁺ released by NAADP on the apparent affinity of the ryanodine receptor for ryanodine and cyclic ADP-ribose (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 Ca²⁺-releasing compounds as shown in the figure. The addition of ryanodine and cADPR was performed after NAADP-induced Ca²⁺ release was at its plateau level (see Figure 3B). The Ca²⁺ 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.

[View larger version of this image (11 K GIF file)]

[View larger version of this image (5 K GIF file)]

Address for correspondence: E.N. Chini, Department of Anesthesiology, Mayo Clinic and Foundation, 200 First Street, Rochester, MN 55905, USA. Fax: [phone]. E-mail: [e-mail]

Research supported by the Mayo Foundation. Received December 5, 2001. Accepted March 5, 2002.

• 1. Berridge MJ (1993). A tale of two messengers. Nature, 365: 388-389.
• 2. Dousa TP, Chini EN & Beers KW (1996). Adenine nucleotide diphosphate: emerging second messengers acting via intracellular Ca²⁺ release. American Journal of Physiology, 271: C1007-C1024.
• 3. Galione A & White A (1994). Ca²⁺ release induced by cyclic-ADP-ribose. Trends in Cell Biology, 4: 431-436.
• 4. Cheng J, Yusufi ANK, Thompson MA, Chini EN & Grande JP (2001). Nicotinic acid adenine dinucleotide phosphate (NAADP), a new Ca²⁺ releasing agent, in kidney. Journal of the American Society of Nephrology, 12: 54-60.
• 5. Chini EN, Beers KW & Dousa TP (1995). Nicotinate-adenine dinucleotide phosphate (NAADP) triggers a specific Ca²⁺ release in sea urchin eggs. Journal of Biological Chemistry, 270: 3216-3223.
• 6. Chini EN & Dousa TP (1996). Nicotinate-adenine dinucleotide phosphate-induced Ca²⁺ release does not behave as a Ca²⁺-induced Ca²⁺ release system. Biochemical Journal, 316: 709-711.
• 7. Chini EN & Dousa TP (1996). Palmitoyl-CoA potentiates the Ca²⁺ release elicited by cyclic ADP-ribose. American Journal of Physiology, 270: C530-C537.
• 8. Chini EN, Liang M & Dousa TP (1998). Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca²⁺ release systems. Biochemical Journal, 335: 499-504.
• 9. Galione A, Patel S & Churchill GC (2000). NAADP-induced calcium release in sea urchin eggs. Biology of the Cell, 92: 197-204.
• 10. Genazzani AA & Galione A (1996). A Ca²⁺ release mechanism gated by the novel pyridine nucleotide, NAADP. Trends in Pharmacological Sciences, 18: 108-110.
• 11. Lee HC & Aarhus R (1995). A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose. Journal of Biological Chemistry, 270: 2152-2157.
• 12. Churchill GC & Galione A (2001). NAADP-induces Ca²⁺-oscillations via a two-pool mechanism by priming IP₃- and cADPR-sensitive Ca²⁺ stores. EMBO Journal, 20: 2666-2671.
• 13. Galione A, McDougall A, Busa WB, Willmott N, Gillot J & Whitaker M (1993). Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea-urchin eggs. Science, 261: 348-352.
• 14. Genazzani AA, Empson RM & Galione A (1996). Unique inactivation properties of NAADP-sensitive Ca²⁺ release. Journal of Biological Chemistry, 271: 11599-11602.
• 15. Genazzani AA & Galione A (1996). Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca²⁺ from a thapsigargin-insensitive pool. Biochemical Journal, 315: 721-725.
• 16. Perez-Terzic CM, Chini EN, Shen SS, Dousa TP & Clapham DE (1995). Ca²⁺ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs. Biochemical Journal, 312: 955-959.
• 17. Yusufi ANK, Cheng J, Thompson MA, Chini EN & Grande JP (2001). NAADP elicits specific microsomal Ca²⁺ release from mammalian cells. Biochemical Journal, 353: 531-536.
• 18. Santella L, Kyozuka K, Genazzani AA, De Riso L & Carafoli E (2000). Nicotinic acid adenine dinucleotide phosphate-induced Ca²⁺ release: interaction among distinct Ca²⁺ mobilizing mechanisms in starfish oocytes. Journal of Biological Chemistry, 275: 8301-8306.
• 19. Lee HC (1993). Potentiation of calcium- and caffeine-induced calcium release by cyclic ADP-ribose. Journal of Biological Chemistry, 268: 293-299.
• 20. Cancela JM, Churchill GC & Galione A (1999). Coordination of agonist-induced Ca²⁺-signalling patterns by NAADP in pancreatic acinar cells. Nature, 398: 74-76.
• 21. Cancela JM, Gerasimenko OV, Gerasimenko JV, Tepikin AV & Petersen OH (2000). Two different but converging messenger pathways to intracellular Ca(²⁺) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate. EMBO Journal, 19: 25549-25572.

Figure 1. Ca²⁺ uptake in sea urchin egg homogenates. The determination of Ca²⁺ uptake was performed using ⁴⁵Ca 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 Ca²⁺ ATPase inhibitor).

Figure 2. Ca²⁺ release induced by nicotinic acid adenine dinucleotide phosphate (NAADP) from the thapsigargin-insensitive pool. The sea urchin egg homogenates were loaded with ⁴⁵Ca as described in Figure 1. After 3 h of Ca²⁺ uptake, Ca²⁺ release was initiated by addition of 1 µM IP₃, 100 nM cyclic ADP-ribose (cADPR) or 100 nM NAADP. The Ca²⁺ release was performed in homogenates loaded in the absence (open circles) or the presence (filled circles) of 10 µM thapsigargin.

Figure 3. Potentiation of the cyclic ADP-ribose (cADPR)-induced Ca²⁺ release by Ca²⁺ released from the nicotinic acid adenine dinucleotide phosphate (NAADP) pool. Free Ca²⁺ concentrations were measured as described in Material and Methods using fluo-3. The arrow indicates the sequential addition of different Ca²⁺ channel agonists. In A the arrow indicates the addition of 16 nM cADPR that by itself does not promote Ca²⁺ release. In B the homogenate was first treated with 12 nM NAADP and 16 nM cADPR was added at the peak (steady state) of the Ca²⁺ release induced by NAADP. In C the homogenate was pretreated with 2 nM NAADP for 20 min (not shown) to promote self-desensitization of the NAADP receptor. After that the homogenate was treated with 12 nM NAADP and 16 nM cADPR. In D homogenates were treated with a saturating concentration of 60 nM NAADP and then, after the Ca²⁺ released by NAADP was taken up again, the homogenate was treated with 16 nM cADPR. In E the homogenate was treated with 4 nmol Ca²⁺ prior to the addition of 16 nM cADPR. The data are representative of 12 different experiments done with three different preparations of sea urchin egg homogenates.

Figure 4. Effect of Ca²⁺ released by NAADP on the apparent affinity of the ryanodine receptor for ryanodine and cyclic ADP-ribose (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 Ca²⁺-releasing compounds as shown in the figure. The addition of ryanodine and cADPR was performed after NAADP-induced Ca²⁺ release was at its plateau level (see Figure 3B). The Ca²⁺ 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.

²⁺

Figure 5. Effect of extravesicular Ca 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.

Figure 5. Effect of extravesicular Ca²⁺ on cADPR-induced Ca²⁺ release. Ca²⁺ release was monitored as described in Material and Methods. The figure indicates the Ca²⁺ released by 16 nM cADPR under different levels of extravesicular Ca²⁺ above baseline. The Ca²⁺ released under ambient extravesicular Ca²⁺ is indicated by a triangle. The extravesicular Ca²⁺ was increased by the addition of different concentrations of NAADP (squares) or Ca²⁺ (circles). The addition of cADPR was performed at the plateau level of Ca²⁺ induced by NAADP or Ca²⁺ itself, as shown in Figure 1. The data are the mean ± SEM of three independent experiments.

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  06 May 2002
• Date of issue
  May 2002

History

• Accepted
  05 Mar 2002
• Received
  05 Dec 2001