July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Signal transduction mechanism underlying H+ efflux from retinal Müller glial cells induced by extracellular ATP.
Author Affiliations & Notes
  • Robert Paul Malchow
    Biological Sciences & Ophthalmology, University of Illinois at Chicago, Chicago, Illinois, United States
  • Boriana K Tchernookova
    Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Michael Gongwer
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Brock Goeglein
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Lexi Shepherd
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Hannah Caringal
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Thomas Leuschner
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Lech Kiedrowski
    Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Matt Kreitzer
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Footnotes
    Commercial Relationships   Robert Malchow, None; Boriana Tchernookova, None; Michael Gongwer, None; Brock Goeglein, None; Lexi Shepherd, None; Hannah Caringal, None; Thomas Leuschner, None; Lech Kiedrowski, None; Matt Kreitzer, None
  • Footnotes
    Support  NSF grants 1557820 & 1557725
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3106. doi:
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      Robert Paul Malchow, Boriana K Tchernookova, Michael Gongwer, Brock Goeglein, Lexi Shepherd, Hannah Caringal, Thomas Leuschner, Lech Kiedrowski, Matt Kreitzer; Signal transduction mechanism underlying H+ efflux from retinal Müller glial cells induced by extracellular ATP.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3106.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Small alterations in extracellular acidity (H+) have profound effects on synaptic transmission and signal processing in the retina. Here we provide an outline of the signal transduction pathway by which Müller glial cells acidify the extracellular fluids surrounding retinal neurons.

Methods : Isolated Müller glial cells from tiger salamander were obtained using a papain-based dissociation protocol. Self-referencing H+-selective electrodes measured changes in extracellular H+ flux. BCECF was used to monitor changes in intracellular H+ and Oregon Green and Fura-2 reported changes in intracellular calcium. Experiments were conducted in 1 mM extracellular HEPES with no bicarbonate or CO2 added.

Results : Low micromolar levels of extracellular ATP induce a significant increase in extracellular H+ flux from isolated Müller cells. H+ efflux is reduced by suramin and PPADS, suggesting activation of metabotropic G-protein-coupled ATP receptors. Extracellular ATP induces an increase in intracellular calcium via release from internal stores by a PLC pathway; increases in intracellular calcium are reduced by thapsigargin and U73122. The rise in intracellular calcium leads to Müller glial intracellular acidification as measured by changes in fluorescence BCECF fluorescence. Surprisingly, even without bicarbonate or CO2 in the extracellular solution bathing cells, the carbonic anhydrase inhibitors methazolamide and acetazolamide significantly reduce extracellular H+ flux. Based on this, we hypothesize that the rise in intracellular calcium leads to stimulation of mitochondria, which produce CO2 that is converted to H+ and HCO3 via the large amount of the enzyme carbonic anhydrase known to be present in Müller glial cells. A large fraction of the intracellular acid appears to be exported via a Na+/H+ exchanger, as the extracellular H+ flux induced by extracellular ATP is significantly reduced when extracellular sodium is replaced with choline or when amiloride or other compounds known to block Na+/H+ exchange are added to the bath.

Conclusions : Our results show the molecular mechanisms mediating ATP-induced H+ flux from Müller glial cells. This ATP-mediated extracellular acidification is likely to play a key role in modulating neurotransmitter release from retinal neurons, significantly shaping the processing of signals in the retina.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

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