Ca2+ release triggered by NAADP in hepatocyte microsomes

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.     

Differential effect of glycolytic intermediaries upon cyclic ADP-ribose-, inositol 1',4',5'-trisphosphate-, and nicotinate adenine dinucleotide phosphate-induced Ca(2+) release systems.

We investigated the effect of glycolytic pathway intermediaries upon Ca(2+) release induced by cyclic ADP-ribose (cADPR), inositol 1',4', 5-trisphosphate (IP(3)), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6, -diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca(2+) release by itself in sea urchin egg homogenate. However, FDP potentiates Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca(2+) release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca(2+) release induced by IP(3) or NAADP had no effect upon Ca(2+) release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca(2+) release induced by both IP(3) and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca(2+) homeostases by switching preferential effects from one intracellular Ca(2+) release channel to another.     

Transformation of local Ca2+ spikes to global Ca2+ transients: the combinatorial roles of multiple Ca2+ releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.     

Nicotinic acid adenine dinucleotide phosphate triggers Ca2+ release from brain microsomes.

Mobilization of Ca2+ from intracellular stores is an important mechanism for generating cytoplasmic Ca2+ signals [1]. Two families of intracellular Ca(2+)-release channels - the inositol-1,4, 5-trisphosphate (IP3) receptors and the ryanodine receptors (RyRs) - have been described in mammalian tissues [2]. Recently, nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule derived from NADP+, has been shown to trigger Ca2+ release from intracellular stores in invertebrate eggs [3] [4] [5] [6] and pancreatic acinar cells [7]. The nature of NAADP-induced Ca2+ release is unknown but it is clearly distinct from the IP3- and cyclic ADP ribose (cADPR)-sensitive mechanisms in eggs (reviewed in [8] [9]). Furthermore, mammalian cells can synthesize and degrade NAADP, suggesting that NAADP-induced Ca2+ release may be widespread and thus contribute to the complexity of Ca2+ signalling [10] [11]. Here, we show for the first time that NAADP evokes Ca2+ release from rat brain microsomes by a mechanism that is distinct from those sensitive to IP3 or cADPR, and has a remarkably similar pharmacology to the action of NAADP in sea urchin eggs [12]. Membranes prepared from the same rat brain tissues are able to support the synthesis and degradation of NAADP. We therefore suggest that NAADP-mediated Ca2+ signalling could play an important role in neuronal Ca2+ signalling.     

Ca2+ release via ryanodine receptors and Ca2+ entry: major mechanisms in NAADP-mediated Ca2+ signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.     

NAADP-induced calcium release in sea urchin eggs

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores described. It acts on a mechanism distinct from inositol trisphosphate and ryanodine receptors, the two major Ca2+ release channels characterised. NAADP-gated Ca2+ release channels do not appear to be regulated by Ca2+ and may be better suited for triggering Ca2+ signals rather than propagating them. They exhibit a remarkable pharmacology for a putative intracellular Ca2+ release channel in that they are selectively blocked by potassium and L-type Ca2+ channel antagonists. Furthermore, in contrast to microsomal Ca2+ stores expressing IP3Rs and RyRs, those sensitive to NAADP are thapsigargin-insensitive, suggesting that they may be expressed on a different part of the endoplasmic reticulum. Perhaps the most unusual feature of the NAADP-gated Ca2+ release mechanisms is its inactivation properties. Unlike the mechanisms regulated by IP3 and cADPR in sea urchin eggs which after induction of Ca2+ release appear to become refractory to subsequent activation, very low concentrations of NAADP are able to inactivate NAADP-induced Ca2+ release fully at concentrations well below those required to activate Ca2+ release. The mechanism and physiological significance of this most unusual desensitisation phenomenon are unclear. More recently, NAADP has been shown to mobilise Ca2+ in ascidian oocytes, brain microsomes and pancreatic acinar cells suggesting a more widespread role in Ca2+ signalling. A possible role for this novel Ca2+ release mechanism in sea urchin egg fertilisation is discussed.     

NAADP induces Ca2+ oscillations via a two-pool mechanism by priming IP3- and cADPR-sensitive Ca2+ stores

In sea urchin eggs, Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) potently self-inactivates but paradoxically induces long-term Ca2+ oscillations. We investigated whether NAADP-induced Ca2+ oscillations arise from the recruitment of other Ca2+ release pathways. NAADP, inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) all mobilized Ca2+ from internal stores but only NAADP consistently induced Ca2+ oscillations. NAADP-induced Ca2+ oscillations were partially inhibited by heparin or 8-amino-cADPR alone, but eliminated by the presence of both, indicating a requirement for both IP3- and cADPR-dependent Ca2+ release. Thapsigargin completely blocked IP3 and cADPR responses as well as NAADP-induced Ca2+ oscillations, but only reduced the NAADP-mediated Ca2+ transient. Following NAADP-mediated release from this Ca2+ pool, the amount of Ca2+ in the Ca2+-induced Ca2+ release stores was increased. These results support a mechanism in which Ca2+ oscillations are initiated by Ca2+ release from NAADP-sensitive Ca2+ stores (pool 1) and perpetuated through cycles of Ca2+ uptake into and release from Ca2+-induced Ca2+ release stores (pool 2). These results provide the first direct evidence in support of a two-pool model for Ca2+ oscillations.     

Effect of luminal and extravesicular Ca2+ on NAADP binding and release properties

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be a powerful Ca2+ release agent in numerous systems, including echinoderms, plants, and mammalian cells. NAADP has been shown to release Ca2+ via a separate mechanism to IP3 and ryanodine receptors, and specific binding sites have recently been characterised. However, functional studies have shown that there is a functional interplay between the NAADP-sensitive mechanism and the other two. In particular, it appears that activation of the NAADP receptor might act as a trigger to facilitate responses from IP3 and ryanodine receptors. To further characterise this interplay, we have investigated the effects of luminal and cytosolic Ca2+ on the NAADP receptor in sea urchin egg homogenates. We report that neither cytosolic nor luminal Ca2+ appears to influence NAADP binding. Conversely, emptying of stores significantly amplifies NAADP-induced fractional Ca2+-release, providing a mechanism of self-adjustment independent of store loading.     

Nicotinic acid adenine dinucleotide phosphate (NAADP) and Ca2+ mobilization

Many physiological processes are controlled by a great diversity of Ca2+ signals that depend on Ca2+ entry into the cell and/or Ca2+ release from internal Ca2+ stores. Ca2+ mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca2+ release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP3 and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca2+ stores. Activation of the NAADP-sensitive Ca2+ channels evokes complex changes in cytoplasmic Ca2+ levels by means of channel chatter with other intracellular Ca2+ channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca2+ signaling.     

Interactions between calcium release pathways: multiple messengers and multiple stores

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles.In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.     

Roles of cADPR and NAADP in pancreatic cells

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are Ca(2+)-mobilizing nucleotides that were discovered in the late 1980s. Two decades of investigations have built up a considerable understanding about these two molecules that are related because both are derived from pyridine nucleotides and known to be generated by CD38/ADP-ribosyl cyclases. cADPR has been shown to target the ryanodine receptors in the endoplasmic reticulum whereas NAADP stimulates the two-pore channels in the endo-lysosomes. Accumulating results indicate that cADPR and NAADP are second messenger molecules mediating Ca(2+) signaling activated by a wide range of agonists. This article reviews what is known about these two molecules, especially regarding their signaling roles in the pancreatic cells.     

Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

NAADP induces pH changes in the lumen of acidic Ca2+ stores

NAADP (nicotinic acid-adenine dinucleotide phosphate)-induced Ca2+ release has been proposed to occur selectively from acidic stores in several cell types, including sea urchin eggs. Using fluorescence measurements, we have investigated whether NAADP-induced Ca2+ release alters the pH(L) (luminal pH) within these acidic stores in egg homogenates and observed their prompt, concentration-dependent alkalinization by NAADP (but not beta-NAD+ or NADP). Like Ca2+ release, the pH(L) change was desensitized by low concentrations of NAADP suggesting it was secondary to NAADP receptor activation. Moreover, this was a direct effect of NAADP upon the acidic stores and not secondary to increases in cytosolic Ca2+ as it was not mimicked by IP3 (inositol 1,4,5-trisphosphate), cADPR (cyclic adenine diphosphoribose), ionomycin, thapsigargin or by direct addition of Ca2+, and was not blocked by EGTA. The results of the present study further support acidic stores as targets for NAADP and for the first time reveal an adjunct role for NAADP in regulating the pH(L) of intracellular organelles.     

NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing messenger. First described in the sea urchin egg, it has been shown to mobilize Ca2+ from intracellular stores. It is a remarkably potent molecule, and recent reports show that its cellular levels change in response to a variety of agonists confirming its role as a Ca2+ mobilizing messenger. In many cases NAADP interacts with other Ca2+ mobilizing messengers such as inositol 1,4,5 trisphosphate (IP3 and cyclic adenosine diphosphate ribose (cADPR) in shaping cytosolic Ca2+ signals. What is not clear is the molecular nature of the NAADP-sensitive Ca2+ release mechanism and its sub-cellular localization. In this review we focus on the recent progress made in sea urchin eggs, which indicates that NAADP activates a novel Ca2+ release channel distinct from the relatively well-characterized IP3 and ryanodine receptors. Furthermore, in the sea urchin egg, the NAADP-sensitive store appears to be separate from the endoplasmic reticulum (ER) and is most likely an acidic store. These findings have also been reinforced by similar findings by some in mammalian cells. Finally, we discuss ongoing strategies to characterise NAADP-binding proteins which will greatly enhance our understanding of NAADP-mediated Ca2+ signalling, and lead to the development of more selective tools to probe the role of this messenger.     

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.     

Regulation of calcium signaling by the second messenger cyclic adenosine diphosphoribose (cADPR)

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will focus on the effects of cADPR in different cells and tissues, the mechanisms of cADPR-mediated Ca2+ release and Ca2+ entry, extracellular effects of cADPR, and the role of cADPR in a cell system studied in detail, human T-lymphocytes.     

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP): novel regulators of Ca2+-signaling and cell function

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review concentrates on (i) receptor-mediated formation of cADPR by ADP-ribosyl cyclases, (ii) intracellular and extracellular effects of cADPR in a variety of cell types, and (iii) cADPR in the nucleus. Though our understanding of the role of NAADP is still unclear in many aspects, important recent findings are reviewed, e.g. Ca2+ release activity and binding studies in mammalian cell types.     

Functional ryanodine receptor expression is required for NAADP-mediated local Ca2+ signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581-588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between approximately 30 and 100 nM and diameters of approximately 0.5 microM, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the D-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.     

Nicotinic acid adenine dinucleotide phosphate-induced Ca(2+) release. Interactions among distinct Ca(2+) mobilizing mechanisms in starfish oocytes

An intracellular mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP(+)) contributes to intracellular Ca(2+) release alongside inositol 1,4,5-trisphosphate (Ins-P(3)) and ryanodine receptors. The NAADP(+)-sensitive mechanism has been shown to be operative in sea urchin eggs, ascidian eggs, and pancreatic acinar cells. Furthermore, most mammalian cell types can synthesize NAADP(+), with nicotinic acid and NADP(+) as precursors. In this contribution, NAADP(+)-induced Ca(2+) release has been investigated in starfish oocytes. Uncaging of injected NAADP(+) induced Ca(2+) mobilization in both immature oocytes and in oocytes matured by the hormone 1-methyladenine (1-MA). The role of extracellular Ca(2+) in NAADP(+)-induced Ca(2+) mobilization, which was minor in immature oocytes, was instead essential in mature oocytes. Thus, the NAADP(+)-sensitive Ca(2+) pool, which is known to be distinct from those sensitive to inositol 1,4,5-trisphosphate or cyclic ADPribose, apparently migrated closer to (or became part of) the plasma membrane during the maturation process. Inhibition of both Ins-P(3) and ryanodine receptors, but not of either alone, substantially inhibited NAADP(+)-induced Ca(2+) mobilization in both immature and mature oocytes. The data also suggest that NAADP(+)-induced Ca(2+) mobilization acted as a trigger for Ca(2+) release via Ins-P(3) and ryanodine receptors.     

Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors

The calcium release channels/ryanodine receptors (RyRs) are potential/putative targets of cADPR (cyclic ADP-ribose) action in many tissue systems. In striated muscles, where RyRs predominate, cADPR action on these channels is controversial. Here cADPR modulation of cardiac and skeletal muscle RyR channels was tested. We considered factors reported as necessary for cADPR action, such as the presence of calmodulin and/or FK binding proteins (FKBPs). We found: 1) The RyR channel isoforms were insensitive to cADPR (or its metabolite NAADP [nicotinic acid adenine dinucleotide phosphate]) under all conditions examined, as studied by: 1a) single channel recordings in planar lipid bilayers; 1b) macroscopic behavior of the RyRs in sarcoplasmic reticulum (SR) microsomes (including crude microsome preparations likely to retain putative cADPR cofactors) at room temperature and at 37 degrees C (net energized Ca2+ uptake or passive Ca2+ leak); 2) [32P]cADPR did not bind significantly to SR microsomes; 3) cADPR did not affect FKBP association to SR membranes. We conclude that cADPR does not interact directly with RyRs or RyR-associated SR proteins. Our results under in vitro conditions suggest that c ADPR effects on Ca2+ signaling observed in vivo in mammalian striated muscle cells may reflect indirect modulation of RyRs or RyR-independent Ca2+ release systems.