Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

Abstract

Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1–10mM) showed that 5–10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.

A journey into the retina: Müller glia commanding survival and death

“…In addition, MGCs synthesize glutathione, a molecule involved in the reduction in reactive oxygen species (ROS). Recently, it was described that glutathione also activates calcium‐mediated signalling (Freitas et al., ). MGCs achieve hydroelectrolytic balance of retinal tissue through the regulation of the extracellular ion concentration and pH, thereby avoiding neuronal swelling in hypoosmotic stress. For this purpose, MGCs display ion channels, aquaporins and purinergic transporters, which facilitate water uptake and ion transport (Vogler et al., ). MGCs actively participate in the recycling of photopigments from photoreceptors, catalysing the conversion of all‐ trans ‐retinal in 11‐ cis ‐retinol by retinaldehyde‐binding protein.…”

Glutathione induces GABA release through P2X7R activation on Müller glia

“…Despite the flourishing literature on antioxidant systems in the retinal environment, our group was the first to examine the signaling role of GSH on both M€ uller glia and retinal neurons from the vertebrate (avian) eye. 18 Initial characterization of the [Ca 2C ]i dynamics showed that, while cultured neurons selectively respond to KCl (labeled as b III tubulin positive cells), large Ca 2C inwards were observed in GSH-stimulated M€ uller cells (labeled and identified as 2M6 positive cells). We have also observed that GSH selectively evokes abundant [Ca 2C ]i variations in M€ uller cells at the milimolar range, and that purified retinal neurons do not respond to GSH in a broad concentration gradient (10 mM -10 mM).…”

“…[ 3 H] GABA release induced by GSH was also observed in cultured M€ uller cells or in dense mixed neuron-glia cultures, but not in purified neuronal cells. 18 GSH might be interacting at the P2X 7 receptor, as the effect was blocked by BBG or A74003. Addition of BAPTA-AM, a calcium chelator, also blocked propidium iodine uptake induced by GSH (or ATP) in M€ uller glial cells.…”

“…Addition of BAPTA-AM, a calcium chelator, also blocked propidium iodine uptake induced by GSH (or ATP) in M€ uller glial cells. 18 It is known that a subset of amacrine neurons 29 releases GABA in the retina. In addition, extracellular GABA levels are regulated by activation of glial P2X receptors.…”

“…16 ATP induces membrane currents at millimolar levels mainly through activation of P2X 7 receptor (P2X 7 R), permeating Ca 2C influx through the emergence of a macro pore. 17 We have recently shown that GSH selectively induces Ca 2C shifts in the avian M€ uller glia 18 through activation of purinergic pathway, once such shifts were inhibited by brilliant blue G (BBG) antagonism of P2X 7 R. Also, GSH treatment induced observable GABA release from both retinal tissue and M€ uller cells in culture. Thus, it's tempting to elaborate on emerging communicative roles for antioxidants in the neuroglial environment.…”