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 many other cellular processes. The plasma membrane Ca2+ ATPase is a calmodulin-stimulated enzyme that plays the major role in regulating the "resting" intracellular free Ca2+ ion concentration, [Ca2+]i. We hypothesized that exposing neurons to low levels of Pb2+ would cause Pb2+ to enter the cytoplasm, and that intracellular Pb2+, by activating calmodulin, would stimulate plasma membrane Ca2+ ATPase activity, thereby increasing Ca2+ extrusion and reducing [Ca2+]i. We used the ratiometric Ca2+ indicator fura-2 to estimate 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+ concentrations from 10 nM to 1000 nM, adding Pb2+ caused either no significant change in the F340/F380 ratio (free Pb2+ concentrations from 100 fM to 1 pM) or increased the F340/F380 ratio (free Pb2+ concentrations from 5 to 50 pM). Therefore, fura-2 should be 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 to 2 days and measured the F340/F380 ratio; the ratio decreased significantly by 9 to 16% at all time points, indicating that Pb2+ exposure decreased [Ca2+]i. In neurons loaded with 45Ca, Pb2+ exposure increased Ca2+ efflux for at least two hours; by 24 hours, Ca2+ efflux returned to control levels. Influx of 45Ca was not altered by Pb2+ exposure. Low concentrations (250 nM) of the calmodulin inhibitor calmidazolium had no effect on either 45Ca efflux or on the F340/F380 ratio in fura-loaded control neurons, but completely eliminated the increase in 45Ca efflux and decrease in F340/F380 ratio in Pb2+-exposed neurons. Zaldoride, another calmodulin inhibitor, also eliminated the decrease in F340/F380 ratio in Pb2+-exposed neurons. We conclude that Pb2+ exposure decreases [Ca2+]i and increases Ca2+ efflux in cultured hippocampal neurons by a calmodulin-dependent mechanism, probably by stimulating Ca2+ extrusion by the plasma membrane Ca2+ ATPase.
Ferguson
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C
Biology Department, University of Colorado at Denver, 80217-3364, USA.
Kern
M
M
Audesirk
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G
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006624:Hippocampus; D007093:Imidazoles / Q000494:pharmacology; D007854:Lead / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007854:Lead; C031938:calmidazolium; D000252:Calcium-Transporting ATPases; D002118:Calcium