Hon Cheung Lee

Synthesis and characterization of antagonists of cyclic-ADP-ribose-induced Ca2+ release.

Cyclic ADP-ribose (cADPR) is a naturally-occurring metabolite of NAD+ that is as effective as inositol trisphosphate in mobilizing intracellular Ca2+. A series of analogs modified at the 8-position of the adenine group were synthesized for the investigation of the relationship between the structure of the metabolite and its Ca(2+)-mobilizing activity. Substitution with an amino group at the 8-position of the adenine ring produced an antagonist. The 1H-NMR spectrum of 8-amino-cADPR showed characteristics of that of cADPR and confirmed the replacement of the 8-proton. By itself, 8-amino-cADPR (150 nM) did not induce Ca2+ release from sea-urchin-egg homogenates but totally blocked cADPR (135 nM) from doing so. The effect was reversible, since high concentrations of cADPR could overcome the inhibition. Addition of 8-amino-cADPR to egg homogenates during the cADPR-induced Ca2+ release blocked the release immediately, demonstrating the effectiveness of the antagonist. Measurements of [32P]cADPR binding to its microsomal binding site showed that 8-amino-cADPR was as effective as cADPR itself in competing for the binding site. In addition to blocking cADPR from releasing Ca2+, 8-amino-cADPR also inhibited cADPR from potentiating Ca(2+)-release induced by either divalent cations or by caffeine. Two other 8-substituted analogs were also synthesized. Both 8-Br- and 8-azido-cADPR were also antagonists, although with less potency than 8-amino-cADPR. These results show that alterations at the 8-position of the adenine group do not inhibit cADPR from binding to its receptor but do eliminate the ability of the metabolite to activate the Ca(2+)-release mechanism.

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pHi) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na+-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pHi is more acidic than the external medium (pHo = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na+ to sperm suspended in Na+-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pHi by 0.4-0.5 pH units. That this pHi change is the causal trigger of motility was suggested by experiments using NH4Cl and nigericin, which increased the pHi and resulted in activation of motility in the absence of Na+. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pHi was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca2+ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pHi by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca2+ uptake. Either mitochondrial agents or the removal of external Ca2+ could also block this pHi change suggesting a similar mechanism is involved.

Activation and inactivation of Ca2+ release by NAADP+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently identified metabolite of NADP that is as potent as inositol trisphosphate (IP) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2 in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C.(1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R.(1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca release activated by NAADP and the Ca stores it acts on are different from those of IP and cADPR. In this study we show that photolyzing caged NAADP in intact sea urchin eggs elicits long term Ca oscillations. On the other hand, uncaging threshold amounts of NAADP produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP can fully inactivate subsequent binding to the receptor in a time-dependent manner. Thus, the NAADP-sensitive Ca release process has novel regulatory characteristics, which are distinguishable from Ca release mediated by either IP or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca mobilization.

Wide distribution of an enzyme that catalyzes the hydrolysis of cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as effective as inositol trisphosphate in mobilizing intracellular-Ca2+ stores. Its synthesizing enzyme, ADP-ribosyl cyclase, has been shown to be present in mammalian and invertebrate tissues. In this study we identify another widely-distributed enzyme that can hydrolyze cADPR to ADP-ribose. Incubation of cADPR with brain extracts resulted in progressive decrease in its Ca2+ mobilizing activity. The degradation of cADPR was catalyzed by a heat-labile protein factor in the brain extracts. Analysis by HPLC indicated a single degradation product was produced in equal molar quantity and that it has identical elution time as ADP-ribose. Proton NMR confirmed that the product was ADP-ribose. The degradation enzyme had a Michaelis constant of 0.16 mM and a broad pH maximum around neutrality. Centrifugation studies of the total brain extracts showed that the degradation activity was membrane-bound. Survey of tissues from various animals established that both the degradation and the synthesizing enzyme of cADPR were widely distributed from mammals to invertebrates. Since the degradation enzyme hydrolyzes an unique linkage between the adenine group and the terminal ribosyl moiety of cADPR, we propose to call it cyclic ADP-ribose hydrolase.

NAADP: A Universal Ca2+ Trigger

NAADP elicits an initial release of calcium, which is subsequently amplified through the action of other calcium messengers. Cells possess multiple calcium ion (Ca2+) stores and multiple messenger molecules to mobilize them. These include d-myo-inositol 1,4,5-trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and the most recently identified Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), which acts on a wide spectrum of cells, from plant cells to mammalian cells. Accumulating evidence indicates that NAADP targets both acidic (lysosome-like) Ca2+ stores and endoplasmic reticular stores. Recent studies in invertebrate and mammalian cells suggest that NAADP provides an initiating Ca2+ signal, which is amplified by cADPR- or IP3-dependent mechanisms (or both) through Ca2+-induced Ca2+ release. Diverse stimuli activate a rapid rise of endogenous NAADP concentration, resulting in severalfold increases of NAADP over basal values within seconds. The enzyme CD38 can catalyze both the synthesis and hydrolysis of NAADP, making it ideal for effecting the rapid metabolism of NAADP. The crystal structure of CD38 and the structures of its various substrate complexes have now been determined, clarifying the mechanism of its multifunctional catalysis. We anticipate that these advances will lead to the unmasking of all the key components of the Ca2+ signaling pathway mediated by NAADP. Intracellular signal transduction is fundamental to the ability of multicellular organisms to convert incoming signals into meaningful cellular responses. Calcium ions (Ca2+) released from intracellular stores transduce numerous physiological functions, from fertilization (at the beginning of life) to muscle contraction, induction of cell differentiation and proliferation, and finally apoptotic cell death. The free cytosolic Ca2+ concentration is much lower than that in the extracellular space or in intracellular stores, such as the endoplasmic reticulum (ER). Moreover, the free cytosolic Ca2+ concentration is tightly controlled through the activity of ion carrier proteins and adenosine 5′-triphosphate (ATP)–driven Ca2+ pumps, and through the open probability of Ca2+ channels. The latter is modulated by various mechanisms, including regulation by small signaling molecules generated in response to incoming extracellular signals, which are called second messengers. NAADP is the most recently identified and also the most potent messenger molecule known to mobilize Ca2+ stores in cells. Here, we summarize recent advances concerning NAADP, including the Ca2+ stores it targets, the receptor(s) with which it interacts, its Ca2+-signaling functions, and its metabolism, thereby unraveling a universal cellular signaling pathway.

Calcium Signaling by Cyclic ADP-ribose, NAADP, and Inositol Trisphosphate Are Involved in Distinct Functions in Ascidian Oocytes

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+releasing molecules, cyclic ADP-ribose (cADPR) from β-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from β-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca2+ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, β-NAD, into unfertilized oocytes. The changes induced by β-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca2+ chelator, indicating the effects were due to Ca2+ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca2+ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca2+ oscillation while cADPR did not. A similar Ca2+ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP3 and NAADP could likewise inhibit such an InsP3-induced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocyte.

Determination of endogenous levels of cyclic ADP-ribose in rat tissues.

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10 degrees C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 +/- 0.64 pmol/mg, in heart is 1.04 +/- 0.08 pmol/mg and in brain is 2.75 +/- 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.

Cyclic ADP-Ribose: Metabolism and Calcium Mobilizing Function

Publisher Summary This chapter describes the discovery of cyclic adenosine diphosphate–ribose (cADPR) as a novel endogenous Ca 2+ -mobilizing agent, and the way by which it fulfills most of the criteria necessary for it to be considered a second messenger. The enzymatic pathways for the synthesis and degradation of the metabolite are also summarized. The metabolic pathway of cADPR consists of synthesis from nicotinamide adenine dinucleotide (NAD + ) by ADP–ribosyl cyclase and degradation by the cADPR hydrolase to ADP-ribose. CD38-like bifunctional enzymes are responsible for regulating the cellular concentration of cADPR. CD38 is an ecto-enzyme catalyzing the synthesis and the degradation of cADPR raises the possibility that cADPR may have extracellular functions. The properties of its intracellular receptor and the mechanism of its Ca 2+ -mobilizing activity are discussed. The physiological roles of cADPR in two specific cellular systems are reviewed in the chapter: the sea urchin egg, an invertebrate cell; and the pancreatic β cell, a mammalian system.

Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) as Messengers for Calcium Mobilization

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate were discovered >2 decades ago. That they are second messengers for mobilizing Ca2+ stores has since been firmly established. Separate stores and distinct Ca2+ channels are targeted, with cyclic ADP-ribose acting on the ryanodine receptors in the endoplasmic reticulum, whereas nicotinic acid adenine dinucleotide phosphate mobilizes the endolysosomes via the two-pore channels. Despite the structural and functional differences, both messengers are synthesized by a ubiquitous enzyme, CD38, whose crystal structure and catalytic mechanism have now been well elucidated. How this novel signaling enzyme is regulated remains largely unknown and is the focus of this minireview.

Nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated calcium signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP)2 is a metabolite of NADP that was first identified as the most potent Ca2 stores mobilizing molecule in sea urchin egg homogenates more than a decade ago (1, 2). It has since been shown to be effective in a wide variety of cells, from plant to animal, including human (reviewed in Refs. 3–5). Its mechanism of action is distinct from those of cyclic ADP-ribose (cADPR) and inositol trisphosphate (IP3), and the stores it targets are separate as well (reviewed in Ref. 6). Recent evidence establishes that NAADP fulfills the criteria of being a second messenger for mobilizing Ca2 stores (reviewed in Ref. 7). This article reviews the structure, functions, and the enzymatic synthesis of this new addition to the list of Ca2 messengers.