Novel Cycling Assays for cADPR and NAADP



An important criterion for a second messenger is that its cellular concentrations must be responsive to the first messenger. It is thus crucial that specific and sensitive assays for cADPR and NAADP be widely available for monitoring their endogenous levels under a variety of physiological conditions. The first assay for cADPR was a bioassay based on its Ca2+ releasing activity in sea urchin egg homogenates [1, 2]. Using this assay, it was demonstrated that cADPR was naturally occurring in many mammalian tissues [3]. Since then, a more sensitive radioimmunoassay (RIA) for cADPR has also been developed [4, 5]. With these assays, the cellular levels of cADPR have been shown to be modulated by various surface receptor agonists, including abscisic acid [6], a plant hormone, a T-cell receptor antibody [7] and acetylcholine [8]. Intriguingly, cell permeant first messengers, such as nitric oxide [9, 10] and retinoic acid [4], metabolic factors, such as glucose [11], vitamin B12 [12], and even a physical stimulus, such as heat shock [13], can elevate cADPR levels, indicating cADPR is involved in a very broad spectrum of signaling functions.


Alcohol Dehydrogenase Enzyme Treatment Nicotinic Acid Adenine Dinucleotide Phosphate NADH Level CD38 Knockout Mouse 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Clapper DL, Walseth TF, Dargie PJ and Lee HC. 1987. Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J. Biol. Chem. 262: 9561–9568.PubMedGoogle Scholar
  2. 2.
    Lee HC, Walseth TF, Bratt GT, Hayes RN and Clapper DL. 1989. Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J. Biol. Chem. 264: 1608–1615.PubMedGoogle Scholar
  3. 3.
    Walseth TF, Aarhus R, Zeleznikar RJ, Jr. and Lee HC. 1991. Determination of endogenous levels of cyclic ADP-ribose in rat tissues. Biochim. Biophys. Acta 1094: 113–120.PubMedCrossRefGoogle Scholar
  4. 4.
    Takahashi K, Kukimoto I, Tokita K, Inageda K, Inoue S, Kontani K, Hoshino S, Nishina H, Kanaho Y and Katada T. 1995. Accumulation of cyclic ADP-ribose measured by a specific radioimmunoassay in differentiated human leukemic HL-60 cells with all-trans-retinoic acid. FEBS Lett. 371: 204–208.PubMedCrossRefGoogle Scholar
  5. 5.
    Graeff RM, Walseth TF and Lee HC. 1997. A radio-immunoassay for measuring endogenous levels of cyclic ADP-ribose in tissues. Meth. Enzymol. 280: 230–241.PubMedCrossRefGoogle Scholar
  6. 6.
    Wu Y, Kuzma J, Marechal E, Graeff R, Lee HC, Foster R and Chua NH. 1997. Abscisic acid signaling through cyclic ADP-ribose in plants. Science 278: 2126–2130.PubMedCrossRefGoogle Scholar
  7. 7.
    Guse AH, da Silva CP, Berg I, Skapenko AL, Weber K, Heyer P, Hohenegger M, Ashamu GA, Schulze-Koops H, Potter BVL and Mayr GW. 1999. Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose. Nature 398: 70–73.PubMedCrossRefGoogle Scholar
  8. 8.
    Fukushi Y, Kato I, Takasawa S, Sasaki T, Ong BH, Sato M, Ohsaga A, Sato K, Shirato K, Okamoto H and Maruyama Y. 2001. Identification of cyclic ADP-ribose-dependent mechanisms in pancreatic muscarinic Ca2+ signaling using CD38 knockout mice. J. Biol. Chem. 276: 649–655.PubMedCrossRefGoogle Scholar
  9. 9.
    Willmott N, Sethi JK, Walseth TF, Lee HC, White AM and Galione A. 1996. Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP-ribose signaling pathway. J. Biol. Chem. 271: 3699–3705.PubMedCrossRefGoogle Scholar
  10. 10.
    Reyes-Harde M, Empson R, Potter BVL, Galione A and Stanton PK. 1999. Evidence of a role for cyclic ADP-ribose in long-term synaptic depression in hippocampus. Proc. Natl. Acad. Sci. USA 96: 4061–4066.PubMedCrossRefGoogle Scholar
  11. 11.
    Takasawa S, Akiyama T, Nata K, Kuroki M, Tohgo A, Noguchi N, Kobayashi S, Kato I, Katada T, Okamoto H, Takasawa S, Akiyama T, Nata K, Kuroki M, Tohgo A, Noguchi N, Kobayashi S, Kato I, Katada Tet al. 1998. Cyclic ADP-ribose and inositol 1,4,5-trisphosphate as alternate second messengers for intracellular Ca2+ mobilization in normal and diabetic beta-cells. J. Biol. Chem. 273: 2497–2500.PubMedCrossRefGoogle Scholar
  12. 12.
    Masuda W, Takenaka S, Inageda K, Nishina H, Takahashi K, Katada T, Tsuyama S, Inui H, Miyatake K and Nakano Y. 1997. Oscillation of ADP-ribosyl cyclase activity during the cell cycle and function of cyclic ADP-ribose in a unicellular organism, Euglena Gracilis. FEBSLett. 405: 104–106.CrossRefGoogle Scholar
  13. 13.
    Zocchi E, Carpaneto A, Cerrano C, Bavestrello G, Giovine M, Bruzzone S, Guida L, Franco L and Usai C. 2002. The temperature-signaling cascade in sponges involves a heat-gated cation channel, abscisic acid, and cyclic ADP-ribose. Proc. Natl. Acad. Sci. USA 98: 14859–14864.CrossRefGoogle Scholar
  14. 14.
    Graeff R and Lee HC. 2002. A novel cycling assay for cellular cyclic ADP-ribose with nanomolar sensitivity. Biochem. J. 361: 379–384.  PubMedGoogle Scholar
  15. 15.
    Inageda K, Takahashi K, Tokita K, Nishina H, Kanaho Y, Kukimoto I, Kontani K, Hoshino S and Katada T. 1995. Enzyme properties of Aplysia ADP-ribosyl cyclase -Comparison with NAD glycohydrolase of CD38 antigen. J. Biochem. 117: 125–131.    PubMedGoogle Scholar
  16. 16.
    Graeff R and Lee HC. 2002. A novel cycling assay for NAADP with nanomolar sensitivity. Biochem. J. (in press).Google Scholar
  17. 17.
    Lee HC. 1997. Mechanisms of calcium signaling by cyclic ADP-ribose and NAADP. Physiol. Rev. 11: 1133–1164.Google Scholar
  18. 18.
    Munshi C and Lee HC. 1997. High-level expression of recombinant Aplysia ADP-ribosyl cyclase in Pichia Pastoris by fermentation. Prot. Express. Purif. 11: 104–110.CrossRefGoogle Scholar
  19. 19.
    Lee HC and Aarhus R. 1991. ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite. Cell Regul. 2: 203–209.PubMedGoogle Scholar
  20. 20.
    Kato T, Berger SJ, Carter JA and Lowry OH. 1973. An enzymatic cycling method for nicotinamide adenine dinucleotide with malic and alcohol dehydrogenases. Anal. Biochem. 53: 86–97.PubMedCrossRefGoogle Scholar
  21. 21.
    Khym JX. 1975. An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. Clin. Chem. 21: 1245–1250.PubMedGoogle Scholar
  22. 22.
    Graeff R, Munshi C, Aarhus R, Johns M and Lee HC. 2001. A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities. J. Biol. Chem. 276: 12169–12173.PubMedCrossRefGoogle Scholar
  23. 23.
    Collins SJ. 1987. The HL-60 promyelocyte leukemia cell line: Proliferation, differentiation, and cellular oncogene expression. Blood 70: 1233–1244.PubMedGoogle Scholar
  24. 24.
    Jacobson EL, Shieh WM and Huang AC. 1999. Mapping the role of NAD metabolism in prevention and treatment of carcinogenesis. Mol. Cell. Biochem. 193: 69–74.PubMedCrossRefGoogle Scholar
  25. 25.
    Kato I, Yamamoto Y, Fujimura M, Noguchi N, Takasawa S and Okamoto H. 1998. CD38 disruption impairs glucose-induced increases in cyclic ADP-ribose, [Ca2+]i and insulin secretion. 1 Biol. Chem. 274: 1869–1872.Google Scholar
  26. 26.
    Partida-Sanchez S, Cockayne D, Monard S, Jacobson EL, Oppenheimer N, Garvy B, Kusser K, Goodricj S, Howard M, Harmsen A, Randall T and Lund FE. 2001. Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo. Nature Med. 7: 1209–1216.PubMedCrossRefGoogle Scholar
  27. 27.
    da Silva CP, Potter BVL, Mayr GW and Guse AH. 1998. Quantification of intracellular levels of cyclic ADP-ribose by high-performance liquid chromatography. J. Chromatogr. B 707: 43–50.CrossRefGoogle Scholar
  28. 28.
    Walseth TF, Wong L, Graeff RM and Lee HC. 1997. A bioassay for determining endogenous levels of cyclic ADP-ribose. Meth. Enzymol. 280: 287–294.PubMedCrossRefGoogle Scholar
  29. 29.
    Lee HC, Aarhus R, Gee KR and Kestner T. 1997. Caged nicotinic acid adenine dinucleotide phosphate - Synthesis and use. J. Biol. Chem. 272: 4172–4178.PubMedCrossRefGoogle Scholar
  30. 30.
    Magni G, Emanuelli M, Amici A, Raffaelli N and Ruggieri S. 1997. Purification of human nicotinamide-mononucleotide adenylyltransferase. Meth. Enzymol. 280: 241–255.PubMedCrossRefGoogle Scholar
  31. 31.
    Aarhus R, Graeff RM, Dickey DM, Walseth TF and Lee HC. 1995. ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP. J. Biol. Chem. 270: 30327–30333.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2002

Authors and Affiliations

  1. 1.Department of PharmacologyUniversity of MinnesotaMinneapolisUSA

Personalised recommendations