Timothy F. Walseth

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.

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.

CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness

We previously demonstrated that cyclic ADP‐ribose (cADPR) elicits Ca2+ release in airway smooth muscle (ASM) cells through ryanodine receptor channels. CD38 is a cell surface protein that catalyzes the synthesis and degradation of cADPR. In inflammatory diseases such as asthma, augmented Ca2+ responses and Ca2+ sensitivity contribute to increased ASM contractility in response to agonists. In this study, we investigated the regulation of CD38 expression and the role of cADPR‐mediated Ca2+ release in airway inflammation. Human ASM cells in culture between the second and fifth passages were exposed to tumor necrosis factor α (TNF‐α), interleukin 1β, or interferon γ, or bovine serum albumin (controls). CD38 expression was measured by reverse transcriptase‐polymerase chain reaction (RT‐PCR), real‐time PCR, and Western blot analysis, and ADP‐ribosyl cyclase activity was assayed with nicotinamide guanine dinucleotide as the substrate. Ca2+ responses to acetylcholine, bradykinin, and thrombin were measured in fura‐2AM‐loaded cells by fluorescence microscopy. Cytokines caused significant augmentation of CD38 expression, ADP‐ribosyl cyclase activity, and Ca2+ responses to the agonists, compared with the control. TNF‐α effects were greater than those of the other two cytokines. The cADPR antagonist 8‐bromo‐cADPR attenuated the Ca2+ responses to the agonists in control and cytokine‐treated cells, with the magnitude of inhibition correlating with the level of CD38. This study provides the first demonstration of a role for CD38‐cADPR signaling in a model of inflammatory airway disease.

Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle

The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with β-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM-10 μM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 μM) and by the RyR blockers ruthenium red (10 μM) and ryanodine (10 μM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]ioscillations induced by acetylcholine (ACh), addition of 100 nM and 1 μM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]ioscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]iresponse to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]ioscillations.The purpose of the present study was to determine whether cyclic ADP-ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with beta-escin, and real-time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+]i). cADPR (10 nM-10 microM) induced a dose-dependent increase in [Ca2+]i, which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 microM) and by the RyR blockers ruthenium red (10 microM) and ryanodine (10 microM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+]i oscillations induced by acetylcholine (ACh), addition of 100 nM and 1 microM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+]i oscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR did not block the [Ca2+]i response to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+]i oscillations.

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.

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) activates two-pore channels (TPCs) to release Ca2+ from intracellular acidic Ca2+ stores. Results: A photoactivatable probe based on NAADP labels proteins distinct from TPCs. Conclusion: NAADP may bind to an accessory protein within a larger TPC complex. Significance: First evidence that TPCs act as NAADP-activated Ca2+ release channels, but not NAADP receptors. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca2+ from intracellular acidic Ca2+ stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca2+ channels that release organellar Ca2+ in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([32P-5N3]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca2+ signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N3-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca2+ release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.

Pharmacological properties of the Ca2+‐release mechanism sensitive to NAADP in the sea urchin egg

The sea urchin egg homogenate is an ideal model to characterize Ca2+‐release mechanisms because of its reliability and high signal‐to‐noise‐ratio. Apart from the InsP3‐ and ryanodine‐sensitive Ca2+‐release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+‐releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. NAADP‐induced Ca2+‐release was blocked by L‐type Ca2+‐channel blockers and by Bay K 8644, while InsP3‐ and cyclic ADPR‐induced Ca2+‐release were insensitive to these agents. L‐type Ca2+‐channel blockers did not displace [32P]‐NAADP binding, suggesting that their binding site was different. Moreover, stopped‐flow kinetic studies revealed that these agents blocked NAADP in a all‐or‐none fashion. Similarly, a number of K+‐channel antagonists blocked NAADP‐induced Ca2+‐release selectively over InsP3‐ and cyclic ADPR‐induced Ca2+‐release. Radioligand studies showed that these agents were not competitive antagonists. As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+‐release mechanisms. The presence of K+ was not essential for NAADP‐induced Ca2+‐release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3‐sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP‐induced Ca2+‐release. While the alkaline condition permitted InsP3 and cyclic ADPR‐induced Ca2+‐release, the acidic condition completely hampered both Ca2+‐release mechanisms. The present results characterize pharmacologically and biochemically the novel Ca2+‐release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle.

Cyclic ADP‐ribose (cADPR) induces intracellular Ca2+ ([Ca2+]i) release in airway smooth muscle, and the cADPR antagonist, 8‐amino‐cADPR, abolishes [Ca2+]i oscillations elicited by acetylcholine (ACh), suggesting that cADPR is involved during muscarinic receptor activation. Whether the cADPR signaling pathway is common to agonists acting through different G protein‐coupled receptors is not known. Using digital video imaging of Fura2‐AM loaded porcine airway smooth muscle cells, we examined the effects of the membrane‐permeant cADPR antagonist, 8‐bromo‐cADPR (8Br‐cADPR), on the [Ca2+]i responses to ACh, histamine and endothelin‐1 (ET‐1). In cells preincubated with 100 μM 8Br‐cADPR, the [Ca2+]i responses to ACh and ET‐1 were significantly attenuated, whereas responses to histamine were not, suggesting agonist specificity of cADPR signaling. The effects of 8Br‐cADPR were concentration dependent. We further examined whether muscarinic receptor subtypes specifically couple to this pathway, because in porcine airway smooth muscle cells, ACh activates both M2 and M3 muscarinic receptors coupled to Gαi and Gαq, respectively. Methoctramine, an M2‐selective antagonist, attenuated the [Ca2+]i responses to Ach, and there was no further attenuation by 8Br‐cADPR. In airway smooth muscle, the CD38/cADPR signaling pathway is involved in [Ca2+]i responses to contractile agonists in an agonist‐specific manner.

Chemotaxis of Mouse Bone Marrow Neutrophils and Dendritic Cells Is Controlled by ADP-Ribose, the Major Product Generated by the CD38 Enzyme Reaction

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD+. Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD+ analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.