June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Signal transduction pathway of ATP-induced H+ efflux from retinal Müller glial cells: dependence upon calmodulin and PKC.
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
  • Alyssa Powell
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Adam Schantz
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Brock Goeglein
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Thomas Leuschner
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Michael Gongwer
    Biology, Indiana Wesleyan University, Marion, Indiana, United States
  • Rachel McKuras
    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; Alyssa Powell, None; Adam Schantz, None; Brock Goeglein, None; Thomas Leuschner, None; Michael Gongwer, None; Rachel McKuras, None; Lech Kiedrowski, None; Matt Kreitzer, None
  • Footnotes
    Support  NSF grants 1557820 & 1557725
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4514. doi:
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      Robert Paul Malchow, Boriana K. Tchernookova, Alyssa Powell, Adam Schantz, Brock Goeglein, Thomas Leuschner, Michael Gongwer, Rachel McKuras, Lech Kiedrowski, Matt Kreitzer; Signal transduction pathway of ATP-induced H+ efflux from retinal Müller glial cells: dependence upon calmodulin and PKC.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4514.

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

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Abstract

Purpose : Changes in extracellular acidity (H+) profoundly impact synaptic transmission in the retina. Here we examine the mechanisms mediating H+ efflux and the role of calmodulin and protein kinase C in regulating H+ efflux from retinal Muller cells induced by extracellular ATP.

Methods : Isolated Müller glial cells from tiger salamander were prepared using a papain 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 : Extracellular ATP induces a prominent extracellular H+ flux from isolated Müller cells which is reduced by the metabotropic G-protein receptor blockers suramin and PPADS. Extracellular ATP increases intracellular calcium via release from internal stores by a PLC pathway which leads to Müller glial intracellular acidification as measured by changes in BCECF fluorescence. Carbonic anhydrase inhibitors significantly reduce extracellular H+ flux, suggesting that intracellular calcium stimulates mitochondria, producing CO2 that is converted via intracellular carbonic anhydrase to H+ and HCO3. The intracellular acid is exported largely via Na+/H+ exchange, as H+ efflux is reduced by amiloride and replacement of extracellular sodium with choline. H+ efflux is also significantly reduced by chlorpromazine, trifluoperazine and W7, which block calmodulin activation, and also by chelerythrine, which blocks activation of PKC.

Conclusions : Our results suggest that extracellular ATP activates metabotropic ATP receptors in Müller glial cells which promotes an increase in intracellular calcium from intracellular stores and stimulation of CO2 production by mitochondria. The CO2 is converted to H+ and HCO3 via carbonic anhydrase and extruded largely via Na+/H+ exchange, which is stimulated by calcium-dependent activation of calmodulin and protein kinase C. 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 is a 2020 ARVO Annual Meeting abstract.

 

Signal transduction pathway for ATP-induced H+ efflux from retinal Muller cells.

Signal transduction pathway for ATP-induced H+ efflux from retinal Muller cells.

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