ATP can induce a P2Y1-mediated release of adenosine from Müller cells that inhibits their swelling in
tissues submitted to hypotonic conditions (Uckermann et al., 2006). Activation of P2Y1 receptors GSK1120212 manufacturer is also involved in Müller cell gliosis after ouabain-induced cell injury in the fish retina (Battista et al., 2009). Although ATP is released from Müller cells when calcium transients are induced in the retina (Newman, 2001), the mechanism by which these cells release this nucleotide is poorly understood. In the present study, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with bafilomycin A1, a potent inhibitor of vacuolar ATPases. Moreover, 50 mM KCl, glutamate and kainate were able to decrease quinacrine staining in the cells as well as to increase the extracellular levels of ATP in the medium. Glutamate-induced rise in extracellular ATP was completely blocked by the glutamatergic antagonists DNQX and MK-801, as well as by BAPTA-AM and bafilomycin A1, suggesting that glutamate, through activation of NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller find more cells through a calcium-dependent exocytotic mechanism.
Glutamine, penicillin-G, streptomycin sulfate, glutamate, kainate, 6,7-dinitroquinoxaline-2,3-dione (DNQX), dizocilpine maleate (MK-801), BAPTA-AM, EGTA, quinacrine, Evans blue were from Sigma (St. Louis, MO, USA); ATP determination kit, MEM, Fetal Bovine Serum, Life Technologies Inc.; trypsin, Worthington Biochemical (Freehold, NJ, USA); all other reagents were of analytical grade. The use of animals was in accordance with the “NIH guide for the Care and Use of Laboratory Animals” and approved by the department’s crotamiton commission for animal care. Glial cultures were obtained according to a previous published procedure (Cossenza and Paes De Carvalho, 2000). Retinas from White-Leghorn chick embryos
were used and monolayer retinal cultures enriched in glial cells prepared. Neuroretinas from 8-day-old embryos were dissected from other structures of the eye and immediately transferred to 1 mL of Ca2+ and Mg2+-free balanced salt solution (CMF). Trypsin, at a final concentration of 0.1%, was added and the suspension incubated at 37 °C for 20–25 min. Trypsin solution was removed and the retinas resuspended in MEM containing 5% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. The tissues were mechanically dissociated by successive aspirations of the medium. For quinacrine staining experiments, the cells were seeded at a density of 2.5 × 103 cells/mm2 on 40 mm plastic Petri dishes. For experiments measuring the extracellular levels of ATP, cells were seeded on 4-well dishes, at the same density.