Developmental Changes in Retinal Calcium (Ca2+) Signaling Mediate Increased Retinal Progenitor Cell (RPC) Proliferation During Gestational Lead Exposure (GLE)

Abstract

Purpose: : 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.

Methods: : 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⁺].

Results: : 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.

Conclusions: : 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.

Keywords: retinal development • calcium • retina: distal (photoreceptors, horizontal cells, bipolar cells)