Signal transduction pathway of ATP-induced H+ efflux from retinal Müller glial cells: dependence upon calmodulin and PKC.

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.