Multiple pathways of Pb2+ permeation in rat cerebellar granule neurones

Abstract

The pathways of lead (Pb2+) uptake were studied in fura-2-loaded cerebellar granule cells from 8-day-old rats. In a nominal Ca-free external bath, Pb2+ (5-50 μM) determined an increase of the fluorescence emission ratio (R = E340/E380) even in the absence of any specific stimulus. This rise was dose-dependent, was not significantly affected by mM Mg2+ or Ca2+, but it was readily reversed by the membrane-permeant heavy metal chelator tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 100 μM), indicating that it was due to Pb2+ influx. The rate of rise, dR/dt, was increased up to a factor of 5 by depolarizing high-KCl solution, indicating a sizeable permeation through voltage-dependent channels. This effect was neither antagonized by nimodipine, nor enhanced by BayK8644, but it was slackened by ω-agatoxin IVA (200 nM), suggesting an involvement of non-L-type calcium channels. Pb2+ influx was also stimulated by glutamic acid or NMDA in the presence of 10-30 μM glycine, but only in Mg-free solution, suggesting that glutamate channels of the NMDA type are an additional pathway of Pb2+ uptake. Pb2+ caused a time-, dose- and stimulus-dependent saturation of the dye, whose intracellular concentration is ∼10 μM, indicating that intracellular Pb2+ can readily reach a concentration in the micromolar range. These results indicate that the particular vulnerability of neurones to Pb2+ poisoning is linked to the presence of specific transport systems, which mediate the rapid uptake of Pb2+ into the neurone.