Ukr.Biochem.J. 2014; Volume 86, Issue 5, Sep-Oct, pp. 65-73


NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

S. V. Bychkova1, T. I. Chorna2

1Ivan Franko National University of Lviv, Ukraine;
2National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India;

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide that is potent to release calcium from intracellular stores in different cell types. NAADP was shown to target specific type of intracellular store namely endolysosomal system or acidic store. Despite intense studies, its effect on endoplasmatic reticulum (ER) still remains to be elucidated. The main aim of our work was to investigate NAADP-sensitive store in permeabilized rat hepatocytes monitoring the level of Ca2+ inside intracellular organelles using chlorotetracycline (CTC). We have shown that NAADP triggered changes of stored Ca2+ in rat hepatocytes are dependent on concentration of EGTA-Ca2+-buffer in cell incubation medium, i.e. the higher is the EGTA concentration in incubation medium the smaller or absent is the effect of NAADP. Besides, the effect of NAADP was more pronounced upon cells pretreatment with the inhibitory concentration of ryanodine (100 µM). This might suggest that the effect of NAADP is dependent on ER luminal calcium. We have also found that NAADP-evoked Ca2+ release in permeabilized hepatocytes is sensitive to nigericin, bafilomycin A and thapsigargin. Additionally, NAADP triggered changes in stored Ca2+ were completely abolished by NED-19 as antagonist of NAADP.

Keywords: , , , , ,


  1. Galione A, Morgan AJ, Arredouani A, Davis LC, Rietdorf K, Ruas M, Parrington J. NAADP as an intracellular messenger regulating lysosomal calcium-release channels. Biochem Soc Trans. 2010 Dec;38(6):1424-31. PubMed, CrossRef
  2. Lee HC, Aarhus R. Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose. J Cell Sci. 2000 Dec;113 Pt 24:4413-20. PubMed
  3. Gerasimenko JV, Maruyama Y, Yano K, Dolman NJ, Tepikin AV, Petersen OH, Gerasimenko OV. NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors. J Cell Biol. 2003 Oct 27;163(2):271-82. PubMed, PubMedCentral, CrossRef
  4. Yamasaki M, Thomas JM, Churchill GC, Garnham C, Lewis AM, Cancela JM, Patel S, Galione A. Role of NAADP and cADPR in the induction and maintenance of agonist-evoked Ca2+ spiking in mouse pancreatic acinar cells. Curr Biol. 2005 May 10;15(9):874-8. PubMed, CrossRef
  5. Davis LC, Morgan AJ, Chen JL, Snead CM, Bloor-Young D, Shenderov E, Stanton-Humphreys MN, Conway SJ, Churchill GC, Parrington J, Cerundolo V, Galione A. NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing. Curr. Biol. 2012 Dec;22(24):2331-7. PubMed, PubMedCentral, CrossRef
  6. Guse AH. Linking NAADP to ion channel activity: a unifying hypothesis. Sci Signal. 2012 Apr 24;5(221):pe18. Review. PubMed, CrossRef
  7. Mándi M, Tóth B, Timár G, Bak J. Ca2+ release triggered by NAADP in hepatocyte microsomes. Biochem J. 2006 Apr 15;395(2):233-8. PubMed, PubMedCentral, CrossRef
  8. Zhang F, Li PL. Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats. J Biol Chem. 2007 Aug 31;282(35):25259-69. PubMed, CrossRef
  9. Blaauboer BJ, Boobis AR, Castell J V. The practical applicability of hepatocyte cultures in routine testing. The report and recommendations of ECVAM Workshop 1. ATLA. 1994;22:231-241.
  10. Jacob J, Chandran D, Sasidharan R, Kuruvila L, Madhusudan UK. Chlortetracycline, a fluo­rescent probe for pH of calcium stores in cells. Curr. Sci. 2003;84(5):671-674.
  11. Cerella C, Mearelli C, De Nicola M, D’Alessio M, Magrini A, Bergamaschi A, Ghibelli L. Analysis of calcium changes in endoplasmic reticulum during apoptosis by the fluorescent indicator chlortetracycline.  Ann N Y Acad Sci. 2007 Mar;1099(1):490-3. PubMed, CrossRef
  12. Genazzani AA, Galione A. Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca2+ from a thapsigargin-insensitive pool. Biochem J. 1996 May 1;315 (Pt 3):721-5. PubMed, PubMedCentral, CrossRef
  13. Boittin FX, Galione A, Evans AM. Nicotinic acid adenine dinucleotide phosphate mediates Ca2+ signals and contraction in arterial smooth muscle via a two-pool mechanism. Circ Res. 2002 Dec 13;91(12):1168-75. PubMed, CrossRef
  14. Kinnear NP, Boittin FX, Thomas JM, Galione A, Evans AM. Lysosome-sarcoplasmic reticulum junctions. A trigger zone for calcium signaling by nicotinic acid adenine dinucleotide phosphate and endothelin-1. J Biol Chem. 2004 Dec 24;279(52):54319-26. PubMed, CrossRef
  15. Brailoiu E, Hoard JL, Filipeanu CM, Brailoiu GC, Dun SL, Patel S, Dun NJ. Nicotinic acid adenine dinucleotide phosphate potentiates neurite outgrowth. J Biol Chem. 2005 Feb 18;280(7):5646-50. PubMed, CrossRef
  16. Morgan AJ, Davis LC, Wagner SK, Lewis AM, Parrington J, Churchill GC, Galione A. Bidirectional Ca2+ signaling occurs between endoplasmic reticulum and acidic organelles. J Cell Biol. 2013 Mar 18;200(6):789-805. PubMed, PubMedCentral, CrossRef
  17. Morgan AJ, Platt FM, Lloyd-Evans E, Galione A. Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease. Biochem J. 2011 Nov 1;439(3):349-74. Review. PubMed, CrossRef
  18. Patel S, Muallem S. Acidic Ca(2+) stores come to the fore. Cell Calcium. 2011 Aug;50(2):109-12. PubMed, CrossRef
  19. Camello C, Pariente JA, Salido GM, Camello PJ. Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells. Curr Biol. 2000 Feb 10;10(3):161-4. PubMed, CrossRef
  20. Petersen OH, Gerasimenko OV, Tepikin AV, Gerasimenko JV. Aberrant Ca(2+) signalling through acidic calcium stores in pancreatic acinar cells. Cell Calcium. 2011 Aug;50(2):193-9. Review. PubMed, CrossRef
  21. Sutko JL, Airey JA, Welch W, Ruest L. The pharmacology of ryanodine and related compounds. Pharmacol Rev. 1997 Mar;49(1):53-98. Review. PubMed
  22. Chaudhuri D, Sancak Y, Mootha VK, Clapham DE. MCU encodes the pore conduc­ting mitochondrial calcium currents. Elife (Cambridge). 2013 Jun 4;2:e 00704. PubMed, PubMedCentral, CrossRef
  23. Kosterin SO, Babich LH, Shlykov SH, Titus OV, Borisova LA. Kinetic model of spermine effect on the Mg2+, ATP-dependent Ca2+ transport in smooth muscle mitochondria. Ukr Biokhim Zhurn. 2005 May-Jun;77(3):76-86. (In Ukrainian). PubMed
  24. Calcraft PJ, Ruas M, Pan Z, Cheng X, Arredouani A, Hao X, Tang J, Rietdorf K, Teboul L, Chuang KT, Lin P, Xiao R, Wang C, Zhu Y, Lin Y, Wyatt CN, Parrington J, Ma J, Evans AM, Galione A, Zhu MX. NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature. 2009 May 28;459(7246):596-600. PubMed, PubMedCentralCrossRef
  25. Rosen D, Lewis AM, Mizote A, Thomas JM, Aley PK, Vasudevan SR, Parkesh R, Galione A, Izumi M, Ganesan A, Churchill GC. Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19 indicate two binding sites on the NAADP receptor. J Biol Chem. 2009 Dec 11;284(50):34930-4. PubMed, PubMedCentral, CrossRef
  26. Walseth TF, Lin-Moshier Y, Jain P, Ruas M, Parrington J, Galione A, Marchant JS, Slama JT. Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. J Biol Chem. 2012 Jan 20;287(4):2308-15. PubMed, PubMedCentral, CrossRef
  27. López J, Dionisio N, Berna-Erro A, Galan C, Salido GM, Rosado JA. Two-pore channel 2 (TPC2) modulates store-operated Ca2+ entry. Biochim Biophys Acta. 2012 Oct;1823(10):1976-83. PubMedCrossRef
  28. Ramos I, Wessel GM. Calcium pathway machinery at fertilization in echinoderms. Cell Calcium. 2013 Jan;53(1):16-23. Review. PubMed, CrossRef

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.