Intracellular dynamics of lead and calcium in lead-exposed cultured neurons of Aplysia californica (Mollusca: Opisthobranchia)
The neurotoxin lead (Pb2+) is a continuing health and environmental problem. However, its neurotoxic mechanism remains unclear. Since calcium (Ca2+) is vital in cellular processes and with Pb2+ reported to interfere with Ca2+-mediated processes, the hypothesis that Pb2+ subverts Ca2+ homeostasis by entering cells through Ca2+ transport pathways and displacing intracellular Ca2+ ([Ca2+] i) was investigated. A multi-technique approach tested this hypothesis. Using cultured Aplysia bag cell neurons, changes in transmembrane Ca2+ flux after Pb2+ exposure were directly measured with the Ca2+-selective self-referencing electrode. Cumulative concentrations of extracellular Pb2+ produced a corresponding and significant (P < 0.05) increase in Ca 2+ efflux, independent of time exposure. Ca2+ channel blockers Co2+, La3+, and verapamil inhibited Pb2+-induced flux. The sarco/endoplasmic reticulum Ca 2+-ATPase pump inhibitors, thapsigargin and cyclopiazonic acid, both elicited a Ca2+ efflux reflecting inhibition of ER pumps and [Ca2+]i release. Furthermore, Pb2+ evoked a moderate and sustained elevation in thapsigargin-stimulated Ca 2+ efflux. ^ For morphological evidence that Pb2+ can mobilize [Ca 2+]i, potassium pyroantimonate (KPA) cytochemistry of bag cells were analyzed. 2% KPA resulted in widespread electron-dense precipitates. 10 mM EGTA + KPA or 200 μM Pb2+ + KPA both produced marked reductions of precipitates in neurons. X-ray microanalysis confirmed antimony (Sb) in precipitates but could not separate the overlapping Ca2+-Sb spectral peaks. However, reduced precipitates in EGTA-incubated cells indicate presence of Ca2+ in intracellular precipitates observed. No apparent pathological changes were correlated to Pb2+ exposure. ^ The effects of Pb2+ on [Ca2+]i using the fluorescent Ca2+ indicator, chlortetracycline (CTC), was examined. Control neurons incubated in 10 μM CTC demonstrated extensive yellow-green fluorescence while reduced fluorescence intensities were seen in cells exposed to 200 μM Pb2+. Multi-sized punctate fluorescence were observed along the perimeter and sometimes, throughout the soma. No neuronal autofluorescence nor neuritic fluorescence in control and Pb2+-exposed cells were detected. Average fluorescence intensities between control and Pb2+-exposed neurons were statistically significant (P < 0.0005). ^ Collectively, these results are consistent with the hypothesis that Pb 2+ perturbs Ca2+ homeostasis via mobilization of [Ca 2+]i. Pb2+ operates through Ca2+ transport pathways as shown by changes in transmembrane Ca 2+ flux, in KPA precipitate reactions, and in CTC fluorescence. ^
Biology, Neuroscience|Biology, Cell|Biology, Animal Physiology
Catherine T Tamse,
"Intracellular dynamics of lead and calcium in lead-exposed cultured neurons of Aplysia californica (Mollusca: Opisthobranchia)"
Dissertations and Master's Theses (Campus Access).