Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake.

Abstract

Cellular uptake of Cd2+ has been monitored using intracellularly trapped dyes, Fura 2 and Quin 2, which bind Cd2+ with extremely high affinity, and digital fluorescence imaging has been used to visualize intracellular free Cd2+. The excitation spectrum of the Cd2+ complex of Fura 2 is similar to that of the Ca2+ complex, whereas Cd2+ displaces Ca2+ from Quin 2 and reduces fluorescence. Fluorescence of Fura 2-loaded cells increased when 50 microM extracellular Cd2+ was added and fluorescence of Quin 2-loaded cells decreased. Cd2+ uptake by GH3 pituitary cells, which occurs in part via voltage-sensitive L-type calcium channels, was increased by BAY K8644 and depolarization and decreased by nimodipine. When Fura 2 and Quin 2 were used to measure Cd2+ uptake by glial C6 cells, which have no L-channel activity, high K+ and BAY K8644 did not change the apparent rate of Cd2+ uptake. GH3 and C6 cells were incubated with Cd2+ for 24 h and loaded with Fura 2, and fluorescence was measured before and after addition of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a membrane permeant chelator with extremely high affinity for metals. TPEN had little effect on fluorescence of Fura 2-loaded GH3 and C6 cells not exposed to Cd2+ but decreased fluorescence of cells that had been incubated with 1-10 microM Cd2+. Fluorescence ratio imaging of Fura 2-loaded cells was used to image intracellular free Cd2+ for both GH3 and C6 cells. Cd2+ uptake over 30-180 min could be followed by the increase in 340/380 fluorescence ratio and the increase in fluorescence ratio was reversed within 5 min by TPEN. The results provide further evidence for the importance of voltage-gated calcium channels to Cd2+ uptake of certain cells.