Purinergic Signalling

, Volume 15, Issue 3, pp 403–420 | Cite as

High-resolution detection of ATP release from single cultured mouse dorsal horn spinal cord glial cells and its modulation by noradrenaline

  • Varen Eersapah
  • Sylain Hugel
  • Rémy SchlichterEmail author
Original Article


Human embryonic kidney 293 (HEK293) cells stably transfected with the rat P2X2 receptor subunit were preincubated with 200 nM progesterone (HEK293-P2X2-PROG), a potent positive allosteric modulator of homomeric P2X2 receptors, and used to detect low nanomolar concentrations of extracellular ATP. Fura-2-loaded HEK293-P2X2-PROG cells were acutely plated on top of cultured DH glial cells to quantify ATP release from single DH glial cells. Application of the α1 adrenoceptor agonist phenylephrine (PHE, 20 μM) or of a low K+ (0.2 mM) solution evoked reversible increases in the intracellular calcium concentration ([Ca2+]i) in the biosensor cells. A reversible increase in [Ca2+]i was also detected in half of the biosensor cells following the interruption of general extracellular perfusion. All increases in [Ca2+]i were blocked in the presence of the P2X2 antagonist PPADS or after preloading the glial cells with the calcium chelator BAPTA, indicating that they were due to calcium-dependent ATP release from the glial cells. ATP release induced by PHE was blocked by -l-phenylalanine 2-naphtylamide (GPN) that permeabilizes secretory lysosomes and bafilomycin A1 (Baf A1), an inhibitor of the H+-pump of acidic secretory vesicles. By contrast, ATP release induced by application of a low-K+ solution was abolished by Baf A1 but not by GPN. Finally, spontaneous ATP release observed after interrupting general perfusion was insensitive to both GPN and Baf A1 pretreatment. Our results indicate that ATP is released in a calcium-dependent manner from two distinct vesicular pools and one non-vesicular pool coexisting in DH glial cells and that noradrenaline and PHE selectively target the secretory lysosome pool.


Astrocyte Exocytosis Secretory lysosome Vesicular release Non-vesicular release 



We would like to thank Mrs. Catherine Moreau and Mrs. Chantal Fitterer for excellent technical assistance. This work was supported by the Centre National de la Recherche Scientifique, Université de Strasbourg, and the French National Research Agency (ANR) contract ANR-17-EURE-0022.

V.E. was the recipient of an Initiative d’Excellence (IdEx) fellowship in the framework of the Investissement d’Avenir program of the Université de Strasbourg.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and institutional guidelines for care and use of animals were followed.

Supplementary material

11302_2019_9673_MOESM1_ESM.pdf (199 kb)
ESM 1 (PDF 199 kb)


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© Springer Nature B.V. 2019

Authors and Affiliations

  • Varen Eersapah
    • 1
  • Sylain Hugel
    • 1
  • Rémy Schlichter
    • 1
    Email author
  1. 1.Centre National de la Recherche Scientifique (UPR3212), Institut des Neurosciences Cellulaires et IntégrativesUniversité de StrasbourgStrasbourgFrance

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