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Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging

  • Kirstan A. VesseyEmail author
  • Tracy Ho
  • Andrew I. Jobling
  • Anna Y. Wang
  • Erica L. Fletcher
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)

Abstract

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

Key words

Retina VNUT Purine P2X7 Adenosine Purinergic Solute carrier 17, member 9 (SLC17A9Dopamine Dopaminergic 

References

  1. 1.
    Abbracchio MP, Burnstock G, Verkhratsky A, Zimmermann H (2009) Purinergic signalling in the nervous system: an overview. Trends Neurosci 32(1):19–29.  https://doi.org/10.1016/j.tins.2008.10.001CrossRefPubMedGoogle Scholar
  2. 2.
    Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 87(2):659–797.  https://doi.org/10.1152/physrev.00043.2006CrossRefPubMedGoogle Scholar
  3. 3.
    Taruno A (2018) ATP release channels. Int J Mol Sci 19(3).  https://doi.org/10.3390/ijms19030808
  4. 4.
    Burnstock G, Satchell DG, Smythe A (1972) A comparison of the excitatory and inhibitory effects of non-adrenergic, non-cholinergic nerve stimulation and exogenously applied ATP on a variety of smooth muscle preparations from different vertebrate species. Br J Pharmacol 46(2):234–242CrossRefGoogle Scholar
  5. 5.
    Lazarowski ER (2012) Vesicular and conductive mechanisms of nucleotide release. Purinergic Signal 8(3):359–373.  https://doi.org/10.1007/s11302-012-9304-9CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Loiola EC, Ventura AL (2011) Release of ATP from avian Muller glia cells in culture. Neurochem Int 58(3):414–422.  https://doi.org/10.1016/j.neuint.2010.12.019CrossRefPubMedGoogle Scholar
  7. 7.
    Reigada D, Mitchell CH (2005) Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport. Am J Physiol Cell Physiol 288(1):C132–C140.  https://doi.org/10.1152/ajpcell.00201.2004CrossRefPubMedGoogle Scholar
  8. 8.
    Santos PF, Caramelo OL, Carvalho AP, Duarte CB (1999) Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons. J Neurobiol 41(3):340–348CrossRefGoogle Scholar
  9. 9.
    Sawada K, Echigo N, Juge N, Miyaji T, Otsuka M, Omote H, Yamamoto A, Moriyama Y (2008) Identification of a vesicular nucleotide transporter. Proc Natl Acad Sci U S A 105(15):5683–5686.  https://doi.org/10.1073/pnas.0800141105CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Geisler JC, Corbin KL, Li Q, Feranchak AP, Nunemaker CS, Li C (2013) Vesicular nucleotide transporter-mediated ATP release regulates insulin secretion. Endocrinology 154(2):675–684.  https://doi.org/10.1210/en.2012-1818CrossRefGoogle Scholar
  11. 11.
    Haanes KA, Kowal JM, Arpino G, Lange SC, Moriyama Y, Pedersen PA, Novak I (2014) Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells. Purinergic Signal 10(3):431–440.  https://doi.org/10.1007/s11302-014-9406-7CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Harada Y, Hiasa M (2014) Immunological identification of vesicular nucleotide transporter in intestinal L cells. Biol Pharm Bull 37(7):1090–1095CrossRefGoogle Scholar
  13. 13.
    Iwatsuki K, Ichikawa R, Hiasa M, Moriyama Y, Torii K, Uneyama H (2009) Identification of the vesicular nucleotide transporter (VNUT) in taste cells. Biochem Biophys Res Commun 388(1):1–5.  https://doi.org/10.1016/j.bbrc.2009.07.069CrossRefPubMedGoogle Scholar
  14. 14.
    Larsson M, Sawada K, Morland C, Hiasa M, Ormel L, Moriyama Y, Gundersen V (2012) Functional and anatomical identification of a vesicular transporter mediating neuronal ATP release. Cereb Cortex 22(5):1203–1214.  https://doi.org/10.1093/cercor/bhr203CrossRefPubMedGoogle Scholar
  15. 15.
    Oya M, Kitaguchi T, Yanagihara Y, Numano R, Kakeyama M, Ikematsu K, Tsuboi T (2013) Vesicular nucleotide transporter is involved in ATP storage of secretory lysosomes in astrocytes. Biochem Biophys Res Commun 438(1):145–151.  https://doi.org/10.1016/j.bbrc.2013.07.043CrossRefPubMedGoogle Scholar
  16. 16.
    Sathe MN, Woo K, Kresge C, Bugde A, Luby-Phelps K, Lewis MA, Feranchak AP (2011) Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles. J Biol Chem 286(28):25363–25376.  https://doi.org/10.1074/jbc.M111.232868CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Sesma JI, Kreda SM, Okada SF, van Heusden C, Moussa L, Jones LC, O’Neal WK, Togawa N, Hiasa M, Moriyama Y, Lazarowski ER (2013) Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules. Am J Physiol Cell Physiol 304(10):C976–C984.  https://doi.org/10.1152/ajpcell.00371.2012CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ziogas J, Vessey K (2001) Angiotensin-induced enhancement of excitatory junction potentials evoked by periarteriolar nerve stimulation and vasoconstriction in rat mesenteric arteries are both mediated by the angiotensin AT1 receptor. Pharmacology 63(2):103–111.  https://doi.org/10.1159/000056120CrossRefPubMedGoogle Scholar
  19. 19.
    Bodin P, Burnstock G (2001) Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol 38(6):900–908CrossRefGoogle Scholar
  20. 20.
    Ho T, Jobling AI, Greferath U, Chuang T, Ramesh A, Fletcher EL, Vessey KA (2015) Vesicular expression and release of ATP from dopaminergic neurons of the mouse retina and midbrain. Front Cell Neurosci 9:389.  https://doi.org/10.3389/fncel.2015.00389CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mitchell CH, Carre DA, McGlinn AM, Stone RA, Civan MM (1998) A release mechanism for stored ATP in ocular ciliary epithelial cells. Proc Natl Acad Sci U S A 95(12):7174–7178CrossRefGoogle Scholar
  22. 22.
    Sorensen CE, Novak I (2001) Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy. J Biol Chem 276(35):32925–32932.  https://doi.org/10.1074/jbc.M103313200CrossRefPubMedGoogle Scholar
  23. 23.
    Dou Y, Wu HJ, Li HQ, Qin S, Wang YE, Li J, Lou HF, Chen Z, Li XM, Luo QM, Duan S (2012) Microglial migration mediated by ATP-induced ATP release from lysosomes. Cell Res 22(6):1022–1033.  https://doi.org/10.1038/cr.2012.10CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Irvin JL, Irvin EM (1954) The interaction of quinacrine with adenine nucleotides. J Biol Chem 210(1):45–56PubMedGoogle Scholar
  25. 25.
    Menendez-Mendez A, Diaz-Hernandez JI, Ortega F, Gualix J, Gomez-Villafuertes R, Miras-Portugal MT (2017) Specific temporal distribution and subcellular localization of a functional vesicular nucleotide transporter (VNUT) in cerebellar granule neurons. Front Pharmacol 8:951.  https://doi.org/10.3389/fphar.2017.00951CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Alund M, Olson L (1979) Depolarization-induced decreases in fluroescence intensity of gastro-intestinal quinacrine-binding nerves. Brain Res 166(1):121–137CrossRefGoogle Scholar
  27. 27.
    Moriyama S, Hiasa M (2016) Expression of vesicular nucleotide transporter in the mouse retina. Biol Pharm Bull 39(4):564–569.  https://doi.org/10.1248/bpb.b15-00872CrossRefPubMedGoogle Scholar
  28. 28.
    Neal M, Cunningham J (1994) Modulation by endogenous ATP of the light-evoked release of ACh from retinal cholinergic neurones. Br J Pharmacol 113(4):1085–1087CrossRefGoogle Scholar
  29. 29.
    Rodriguez PC, Pereira DB, Borgkvist A, Wong MY, Barnard C, Sonders MS, Zhang H, Sames D, Sulzer D (2013) Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain. Proc Natl Acad Sci U S A 110(3):870–875.  https://doi.org/10.1073/pnas.1213569110CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Kirstan A. Vessey
    • 1
    Email author
  • Tracy Ho
    • 1
  • Andrew I. Jobling
    • 1
  • Anna Y. Wang
    • 1
  • Erica L. Fletcher
    • 1
  1. 1.Visual Neuroscience Laboratory, Department of Anatomy and NeuroscienceThe University of MelbourneParkvilleAustralia

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