Abstract
The mechanism of cellular uptake of cadmium, a highly toxic metal ion, is not known. We have studied cadmium uptake and toxicity in an established secretory cell line, GH4C1, which has well characterized calcium channels. Nimodipine, an antagonist of voltage-sensitive calcium channels, protected cells against cadmium toxicity by increasing the LD50 for CdCl2 from 15 to 45 microM, whereas the calcium channel agonist BAY K8644 decreased the LD50. Organic calcium channel blockers of three classes protected cells from cadmium toxicity at concentrations previously shown to block high K+-induced 45Ca2+ influx and secretion. Half-maximal protective effects were obtained at 20 nM nifedipine, 4 microM verapamil, and 7 microM diltiazem. Increasing the extracellular calcium concentration from 20 microM to 10 mM also protected cells from cadmium by causing a 5-fold increase in the LD50 for CdCl2. Neither the calcium channel antagonist nimodipine nor the agonist BAY K8644 altered intracellular metallothionein concentrations, while cadmium caused a 9-20-fold increase in metallothionein over 18 h. Cadmium was a potent blocker of depolarization-stimulated 45Ca2+ uptake (IC50 = 4 microM), and the net uptake of cadmium measured with 109Cd2+ was less than 0.3% that of calcium. Although the rate of cadmium uptake was low relative to that of calcium, entry via voltage-sensitive calcium channels appeared to account for a significant portion of cadmium uptake; 109Cd2+ uptake at 30 min was increased 57% by high K+/BAY K8644, which facilitates entry through channels. Furthermore, calcium channel blockade with 100 nM nimodipine decreased total cell 109Cd2+ accumulation after 24 h by 63%. These data indicate that flux of cadmium through dihydropyridine-sensitive, voltage-sensitive calcium channels is a major mechanism for cadmium uptake by GH4C1 cells, and that pharmacologic blockade of calcium channels can afford dramatic protection against cadmium toxicity.
Highlights
From the Demrtment of Pharmacology and the Cancer Center, Uniuersity of Rochester School of Medicine and Dentistry, "
Tory cellline, GH4C1, which has well characterized Cadmium is highly reactive with thiolgroups of proteins, can calcium channels
Cad- Little is known about how cadmium and other metal ions mium was a potent blocker of depolarization-stimu- permeate cells
Summary
Cells were incubated for 24 h in complete medium with or without 40 pM CdClz, 100nM nitrendipine, and 1pM BAY K8644. In order to measure therate of protein synthesis, 1 pCi/ml [3H]leucine was included in the medium of some dishes, and [3H]leucineincorporated into trichloroacetic acid-insoluble material was measured at the end of the experiment. Trypan blue exclusion was determined in a separate experiment in which cadmium caused a 59% reduction in adherent cell protein. Trypan blue uptake [3H]Leucine in the legends. In order to measure the rate of incorporation of [3H]leucineor [3H]. Thymidine into trichloroacetic acid-insoluble material, cells were incubated with the [3H]leucineor [3H]thymidineas noted in the text, washed three timesin ice-cold 0.15 M NaCl, and incubated at 0 "C in 10%trichloroacetic acid for 60 min.
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