Müller cell Ca2+ waves evoked by purinergic receptor agonists in slices of rat retina

We have measured agonist evoked Ca2+ waves in Müller cells in situ within freshly isolated retinal slices. Using an eye cup dye loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca2+ waves in Müller cells, which propagate along their processes. These Ca2+ signals were insensitive to tetrodotoxin (TTX, 1.0 microM) pretreatment. Cells were readily identified as Müller cells by their unique morphology and by subsequent immunocytochemical labeling with glial fibrillary acidic protein antibodies. While cells never exhibited spontaneous Ca2+ oscillations, purinoreceptor agonists, ATP, 2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited Ca2+ waves. These waves persisted in the absence of [Ca2+]o but were abolished by thapsigargin pretreatment, suggesting that the purinergic agonists tested act by releasing Ca2+ from intracellular Ca2+ stores. The rank order of potency of different purines and pyrimidines for inducing Ca2+ signals was 2 MeSATP = 2MeSADP > ADP > ATP >> alphabetameATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca2+ signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 microM) and suramin (200 microM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 microM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or 8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we concluded that Müller cells express the P1A2 and P2Y1 subtypes of purinoceptors. Analysis of Ca2+ responses showed that, similar to glial cells in culture, wave propagation occurred by regenerative amplification at specialized Ca2+ release sites (wave amplification sites), where the rate of Ca2+ release was significantly enhanced. These data suggest that Müller cells in the retina may participate in signaling, and this may serve as an extra-neuronal signaling pathway.