Nanomolar concentrations of inorganic lead increase Ca2+ efflux and decrease intracellular free Ca2+ ion concentrations in cultured rat hippocampal neurons by a calmodulin-dependent mechanism

Inorganic lead (Pb2+) activates calmodulin, which in turn may stimulate man y other cellular processes. The plasma membrane Ca(2+)ATPase is a calmoduli n-stimulated enzyme that plays the major role in regulating the "resting" i ntracellular free Ca2+ ion concentration, [Ca2+](i). We hypothesized that e xposing neurons to low levels of Pb2+ would cause Pb2+ to enter the cytopla sm, and that intracellular Pb2+, by activating calmodulin, would stimulate plasma membrane Ca(2+)ATPase activity, thereby increasing Ca2+ extrusion an d reducing [Ca2+](i) We used the ratiometric Ca2+ indicator fura-2 to estim ate changes in [Ca2+](i). In vitro calibrations of fura-2 with solutions of defined free Ca2+ and free Pb2+ concentrations showed that, at free Ca2+ c oncentrations from 10 nM to 1000 nM, adding Pb2+ caused either no significa nt change in the F-340/F-380 ratio (free Pb2+ concentrations from 100 fM to 1 pM) or increased the F-340/F-380 ratio (free Pb2+ concentrations from 5 to 50 pM). Therefore, fura-2 should De suitable for estimating Pb2+-induced decreases in [Ca2+](i) but not increases in [Ca2+](i). We exposed cultured embryonic rat hippocampal neurons to 100 nM Pb2+ for periods from 1 hour t o 2 days and measured the F-340/F-380 ratio; the ratio decreased significan tly by 9 to 16% at all time points, indicating that Pb2+ exposure decreased [Ca2+](i) In neurons loaded with Ca-45, Pb2+ exposure increased Ca2+ efflu x for at least two hours; by 24 hours, Ca2+ efflux returned to control leve ls. Influx of Ca-45 was not altered by Pb2+ exposure. Low concentrations (2 50 nM) of the calmodulin inhibitor calmidazolium had no effect on either Ca -45 efflux or on the F-340/F-380 ratio in iura-loaded control neurons, but completely eliminated the increase in Ca-45 efflux and decrease in F-340/F- 380 ratio in Pb2+-exposed neurons. Zaldoride, another calmodulin inhibitor, also eliminated the decrease in F-340/F-380 ratio in Pb2+-exposed neurons. We conclude that Pb2+ exposure decreases [Ca2+](i), and increases Ca2+ eff lux in cultured hippocampal neurons by a calmodulin-dependent mechanism, pr obably by stimulating Ca2+ extrusion by the plasma membrane Ca2+-ATPase. (C ) 2000 Intox Press, Inc.