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Assessment of Extracellular ATP Concentrations

  • Lucia Seminario-Vidal
  • Eduardo R. Lazarowski
  • Seiko F. Okada
Part of the Methods in Molecular Biology™ book series (MIMB, volume 574)

Abstract

Most cells release ATP to the extracellular milieu. Extracellular ATP plays important signaling roles by activating a score of broadly distributed cell surface purinergic receptors (purinoceptors). Biological responses regulated by purinergic receptors include neurotransmission, smooth muscle relaxation and contraction, epithelial cell ion transport, inflammation, platelet activation, immune responses, cardiac function, endocrine and exocrine secretion, glucose transport, and cell proliferation. ATP concentrations at the cell surface, and consequently the magnitude of purinergic receptor stimulation, reflect a well-controlled balance between rates of ATP release and extracellular metabolism. Given the broad spectrum of responses triggered by extracellular ATP, there is a growing interest in accurately assessing the concentrations of this nucleotide at the cell surface. In this chapter, we discuss the use of the luciferin/luciferase-based reaction to measure extracellular ATP concentrations with high sensitivity. Protocols are adapted to assess ATP levels either in sampled extracellular fluids or in situ at the cell surface. Although our focus is on studies of ATP release from epithelial cells, protocols described here are applicable to practically all cell types.

Key words

ATP release extracellular ATP ecto-ATPase luciferase protein A-luciferase luciferin 

Abbreviations:

6 × His

hexa-histidine

ALU

arbitrary light unit

ARL-67156

6-N-N-diethyl-β,γ-dibromomethylene-d-ATP

β,γ-metATP

β,γ-methyleneadenosine 5′-triphosphate

BSA

bovine serum albumin

DMEM

Dulbecco’s modified eagle’s medium

ebselen

2-phenyl-1,2-benzisoselenazol-3(2H)-one

FBS

fetal bovine serum

HBSS

Hank’s balanced salt solution

HEPES

4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

MEM

minimum essential medium

PBS

phosphate-buffered saline

RT

room temperature

SPA-luc

Staphylococcus protein A-fused luciferase.

References

  1. 1.
    Burnstock, G. (2006) Purinergic signalling. Br J Pharmacol 147 (Supple 1), S172–S181.PubMedGoogle Scholar
  2. 2.
    North, R. A. (2002) Molecular physiology of P2X receptors. Physiol Rev 82, 1013–1067.PubMedGoogle Scholar
  3. 3.
    Zimmermann, H. (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362, 299–309.PubMedCrossRefGoogle Scholar
  4. 4.
    Okada, S. F., Nicholas, R. A., Kreda, S. M., Lazarowski, E. R., and Boucher, R. C. (2006) Physiological regulation of ATP release at the apical surface of human airway epithelia. J Biol Chem 281, 22992–23002.PubMedCrossRefGoogle Scholar
  5. 5.
    Lazarowski, E. R., Shea, D. A., Boucher, R. C., and Harden, T. K. (2003) Release of cellular UDP-glucose as a potential extracellular signaling molecule. Mol Pharmacol 63, 1190–1197.PubMedCrossRefGoogle Scholar
  6. 6.
    Gatof, D., Kilic, G., and Fitz, J. G. (2004) Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells. Am J Physiol Gastrointest Liver Physiol 286, G538–G546.PubMedCrossRefGoogle Scholar
  7. 7.
    Boudreault, F., and Grygorczyk, R. (2004) Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations. J Physiol 561, 499–513.PubMedCrossRefGoogle Scholar
  8. 8.
    Button, B., Picher, M., and Boucher, R. C. (2007) Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia. J Physiol 580, 577–592.PubMedCrossRefGoogle Scholar
  9. 9.
    Kreda, S. M., Seminario-Vidal, L., Heusden, C. V., and Lazarowski, E. R. (2008) Thrombin-promoted release of UDP-glucose from human astrocytoma cells. Br J Pharmacol 153, 1528–1537PubMedCrossRefGoogle Scholar
  10. 10.
    Joseph, S. M., Buchakjian, M. R., and Dubyak, G. R. (2003) Colocalization of ATP release sites and ecto-ATPase activity at the extracellular surface of human astrocytes. J Biol Chem 278, 23331–23342.PubMedCrossRefGoogle Scholar
  11. 11.
    Kreda, S. M., Okada, S. F., van Heusden, C. A., O'Neal, W., Gabriel, S., Abdullah, L., Davis, C. W., Boucher, R. C., and Lazarowski, E. R. (2007) Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells. J Physiol 584, 245–259.PubMedCrossRefGoogle Scholar
  12. 12.
    Beigi, R., Kobatake, E., Aizawa, M., and Dubyak, G. R. (1999) Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am J Physiol 276, C267–C278.PubMedGoogle Scholar
  13. 13.
    Lundin, A. (2000) Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Methods Enzymol 305, 346–370.PubMedCrossRefGoogle Scholar
  14. 14.
    Taylor, A. L., Kudlow, B. A., Marrs, K. L., Gruenert, D. C., Guggino, W. B., and Schwiebert, E. M. (1998) Bioluminescence detection of ATP release mechanisms in epithelia. Am J Physiol 275, C1391–C1406.PubMedGoogle Scholar
  15. 15.
    DeLuca, M., and McElroy, W. D. (1978) Purification and properties of firefly luciferase. In Methods Enzymol 57, 3–15.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Lucia Seminario-Vidal
    • 1
  • Eduardo R. Lazarowski
    • 1
  • Seiko F. Okada
    • 1
  1. 1.Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel HillChapel HillUSA

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