Analysis of Pb2+ entry into cultured astroglia.
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abstract
Astroglia serve as a presumptive lead (Pb) sink in the brain; therefore, this study examined Pb entry into cultured rat astroglia utilizing the Ca2+ fluorophore indo-1 as a tool for detecting Pb2+ entry during acute exposure. The interactions of Pb2+ with indo-1 were analyzed by fluorescence spectrophotometry in a cell-free system. The emission spectrum of Pb2+/indo-1 was substantially different from that of Ca2+/indo-1 due to suppression of indo-1 fluorescence emission intensity. Next, we established the presence of L-type Ca2+ channels in astroglial cultures and demonstrated that Pb accumulation is enhanced under serum-free conditions and by the application of Bay-K 8644. Because acute exposure is of less toxicologic relevance than repeated low-level exposure, we then examined Pb uptake in cultures treated for up to 1 week with Pb. AAS revealed that Pb accumulation was accompanied by an increase in total cellular [Ca]. In addition, differences in basal indo-1 fluorescence levels and differences in responsiveness to ionomycin were observed. Ionomycin induced an increase in the fluorescence ratio in untreated cells but cells treated for 1 day with Pb showed no response to ionomycin. However, cells treated for 3 and 7 days showed a partial response to ionomycin. TPEN was used to evaluate the interactions of Pb2+ with indo-1 and only cells treated for 7 days showed a response to TPEN. Thus, the present study characterizes Pb2+ entry into astroglia via L-type Ca2+ channels and presents the possibility of using indo-1 for analysis of Pb2+ uptake and the subsequent neurotoxic events in astroglia.
