Role of calcium channels in heavy metal toxicity

Abstract

The role of voltage-dependent Ca channels (VDCC) in the membrane permeation of two toxic metals, lead (Pb) and cadmium (Cd), was studied in mammalian cells. Both metals interact with Ca-binding sites, but, while Cd influx appears to occur mainly through the same pathways as Ca, Pb is also rapidly taken up by different passive transport systems. Furthermore, I compared the effect of Cd in two Chinese hamster ovary (CHO) cell lines, a wild-type and a modified cell line, which were permanently transfected with an L-type VDCC. When cultures were subjected to a brief (30-60 min) exposure to 50-100 μ M Cd, apoptotic features, metal accumulation, and death were comparable in both cell lines although, in transfected cells, the effect of Cd treatment was partially prevented by nimodipine (VDCC antagonist) and enhanced by BayK8644 (VDCC agonist). Thus, expression of L-type Ca channels is not sufficient to modify Cd accumulation and sensitivity to a toxicological significant extent and while both Cd and Pb can take advantage of VDCC to permeate the membrane, these transport proteins are not the only, and frequently not the most important, pathways of permeation.

Figure 1

Uptake of Cd and Pb in cerebellar granule neurons measured by fluorescent dyes. (A) Confocal microscopy images of Cd (a, b) and Pb (c, d) uptake by cerebellar granule cells preloaded with the divalent metal-sensitive dye Oregon Green. In (a) and (c), neurons were bathed in a physiological saline. In (b) they have been superfused with a solution containing 0 Ca, 30 mM KCl and 100 μM Cd Cl₂, which caused the dye fluorescence to increase significantly. In (d), external solution contained 0 Ca and Pb 15 μM, which permeates through the neuron membrane even in the absence of a depolarizing stimulus and also caused an increase in the dye fluorescence. Bar = 10 μM. (B) Real-time recording of the influx of Cd and Pb in Fura-2 loaded cerebellar granule neuron and the effect of membrane depolarization. Cells were treated with the metals in nominal Ca-free solution. From left to right: time course of the fluorescence ratio, R = E340/E380, following application of 50 μM Cd and membrane depolarization (25 mM KCl) and application of 15–50 μM Pb in resting conditions and increasing depolarizations (25 and 75 mM KCl). (C) Summary of the results obtained in basal and in depolarizing solutions (25 and 75 mM KCl) with different doses of Pb. The time course of R(t) was approximated by a straight line and the slope dR/dt was calculated in each case, as a measure of Pb influx. Data are mean ±  SEM in 4 experiments and *indicates significantly different from basal (no depolarization) for each dose of Pb (P < 0.05).

Figure 2

CHOCα cells express L-type VDCC. (a) Representative microphotographs of monolayer cell cultures of permanently transfected CHOCα cells (left) and wild-type CHO cells (right). Bar = 35 μM. (b) Representative current traces evoked by depolarizing voltage steps of 40 ms duration from −60 to +80 mV from a holding potential of −80 mV in a CHOCα cell (left) and a wild-type CHO cell (right). The external solution contained 5 mM CaCl₂. This protocol evoked voltage-dependent calcium current in the permanently transfected cell, while no current was present in CHO cell of the wild-type. (c) Characterization of the Ca current in CHOCα cell. The current was increased by more than 50% when the external solution was changed from 5 mM CaCl₂ to 5 mM BaCl₂, and it was reversibly blocked by 10 μM nimodipine. Current traces evoked by 50 ms depolarizing steps from −80 mV (holding potential) to +10 mV in the three conditions are shown on the left. The graph on the right shows the time course of the experiment.

Figure 3

Effect of pulse treatment with Cd in CHOCα VDCC-expressing and wild-type CHO cells. Microphotographs of control (a, b) and Cd-treated (c, d) cells: (a, c) VDCC-expressing CHOCα cells and (b, d) wild-type CHO cells. Both cell types were incubated in 100 μM Cd for 60 minutes in the presence of 30 mM KCl. Pictures were taken 24 h after wash of the metal. Bar = 35 μM. The graph (e) shows the effect of a 30 min pulse treatment with Cd on cell adhesion, as a function of concentration. Trypan-blue excluding adherent cells were counted 24 h after wash of the metal. Points are average ±  sem of 3 experiments in the same condition in CHOCα (filled circles) and wild-type CHO (empty circles) and were best fitted to the function. N_adher/N_{adher(control)} = 1/(1 + ([Cd]/ED₅₀)), where N_adher/N_{adher(control)} is the number of adherent cells after Cd treatment normalized to the number of adherent cells in control culture; [Cd] is the concentration of Cd and ED₅₀ is the concentration of Cd that causes detachment from the substrate of 50% of cells. The best fit yielded ED₅₀ = 40 μM for CHOCα and 43 μM for wild-type CHO. The two curves are overlapped. In contrast with the different appearance, the two cell types were similarly affected by Cd treatment.

Figure 4

Effect of dihydropyridines on Cd cytotoxicity in VDCC-expressing CHOCα cells. Cells were treated for 30 min in (a) control medium containing 30 mM KCl, (b) 100 μM  Cd + 30 mM  KCl, (c) 100 μM  Cd + 30 mM  KCl + 1 μM nimodipine, (d) 100 μM  Cd + 30 mM  KCl + 1 μM  BayK8644. Note the partial recovery in shape of the cells treated in the presence of nimodipine and definitive lost of adhesion when the treatment was performed in the presence of BayK. Bar = 25 μM. The graph (e) represents counts of viable adherent cells in the same experiment (average ±  sem in 3 samples) in control (basal), following for a 30 min treatment with 100 μM Cd (Cd), Cd + 30 mM  KCl (K), Cd + 30 mM  KCl and 1 μM nimodipine (N), and Cd + 30 mM  KCl and 1 μM BayK (B). * indicates significantly different from control with P < 0.05, and ** with P < 0.0001.

Figure 5

Evidence of Cd-induced apoptosis: change in cell capacitance. Cells were incubated with 100 μM Cd for 60 minutes, and electrical measurements were performed 24 h after wash. Membrane capacitance was estimated from transient compensation (see Section 2.4). Bars represent mean ±  sem. Treatment with Cd caused shrinkage of all cell, as revealed by reduction of the cell capacitance in both wild-type (n = 15 in control and n = 6 with Cd treatment) and CHOCα cells (n = 17 in control and n = 39 with Cd treatment). Nimodipine protected CHOCα cells from Cd-induced shrinkage (n = 10). * indicates significantly different from control with P < 0.05, and ** with P < 0.001.

Figure 6

Total Cd accumulation measured by FAAS in CHOCα and wild-type CHO cells. Both cell types were incubated for 1 hour in medium containing 0, 50, or 100 μM Cd and other modifiers. Cd determination was normalized to the cell volume (see text). Treatments were as follows: control 0 Cd (basal), 60 min Cd (Cd), Cd + 30 mM  KCl (K), Cd + 30 mM  KCl, and 1 μM BayK (B), Cd + 30 mM  KCl and 1 μM nimodipine (N). Depolarization (treatment K) did not increase significantly the intracellular concentration of Cd (P > 0.05). In CHOCα cells, but not in wild-type CHO, BayK significantly enhanced and nimodipine significantly reduced Cd accumulation, both with P < 0.001 (*), with respect to the same treatments in depolarization. Bars represent mean ±  sem in at least 10 experiments in each group.