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S. Mukherjee, A. Giddabasappa, W. Xiao, J. E. Johnson, Jr., D. A. Fox; Developmental Changes in Retinal Calcium (Ca2+) Signaling Mediate Increased Retinal Progenitor Cell (RPC) Proliferation During Gestational Lead Exposure (GLE). Invest. Ophthalmol. Vis. Sci. 2010;51(13):5948.
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GLE in mice increased and prolonged RPC proliferation that resulted in an increased number of late-born neurons (rods and bipolar cells). Microarray, Western blot and confocal/IHC studies showed that GLE promoted cell cycle activation. Since the mechanism of intracellular Ca2+ concentration ([Ca2+]i) increase acts as a differential regulatory switch of RPC proliferation, we investigated extracellular and intracellular Ca2+ signaling pathways in developing control and GLE retinas.
C57BL/6 female mice were exposed to water or 55 ppm lead throughout gestation and until postnatal day (PN)10: equivalent to human gestation period. Dissociated cells from PN2-PN10 control and GLE retinas were incubated in buffers with or without 1.5 mM Ca2+, loaded with Fluo4-AM, and exposed to Ca2+ toolbox drugs or ligands in a 96-well plate. The baseline, kinetics of rise/decay of and maximum fluorescence of [Ca2+]i were determined. To determine the number of RPCs, dissociated retinal cells were co-labeled with Ki67 and DRAQ5, and counted. Since Pb2+ can act as a Ca2+-mimetic, leadmium assays measured free retinal [Pb2+].
The GLE-induced increase in RPCs was maximal from PN2-PN6. In PN2-10 control and GLE retinas, basal [Ca2+]i was 75-100 nM and [Pb2+] was <5 nM. Relative to controls at PN2 and PN4, ionomycin or caffeine without added Ca2+ produced dose-dependent increases in the rise of and maximum [Ca2+]i in GLE retinas. Similarly, ATP with or without added Ca2+ produced dose-dependent increases in the rise of and maximum [Ca2+]i in GLE retinas. Relative to controls at PN2 and PN4, glutamate, KCl or BayK8644 with added Ca2+ produced dose-dependent decreases in the rise of and maximum [Ca2+]i in GLE retinas. Dose-dependent responses and differences between control and GLE retinas declined from PN6 to PN10.
In response to selective pharmacological agents, isolated retinal cells from developing GLE mice increased Ca2+ release from intracellular stores and decreased Ca2+ entry through ligand and voltage-gated Ca2+ channels, relative to controls. These results suggest that the differential changes in [Ca2+]i contribute to G1/S-phase cell cycle activation and decreased cell cycle exit, respectively. Together, these increased RPC proliferation in GLE mice retinas. The effects appear unrelated to the free [Pb2+]. The quantitative changes in Ca2+ mobilization correlate with our in vivo and Ki67 ex vivo RPC proliferation results in developing GLE mice.
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