Interference of cadmium with ATP-stimulated nuclear calcium uptake.

The spatial and temporal regulation of intracellular free Ca2+ serves as a modulator of signal transduction pathways involved in cell growth and differentiation. Thus, interference of metals with intracellular Ca2+ homeostasis has been considered as a target of toxic action. We used the fluorescence indicator fura-2 to monitor the level of free Ca2+ in isolated bovine liver nuclei. Nuclei accumulated Ca2+ by an ATP-stimulated Ca2+ uptake system, which is sensitive to inhibition by thapsigargin, a specific inhibitor of intracellular Ca(2+)-ATPases. Preincubation of nuclei with nanomolar concentrations of free Cd2+ resulted in a dose-dependent inhibition of ATP-dependent nuclear Ca2+ uptake. We conclude that impairment of nuclear Ca2+ regulation caused by Cd2+ provokes alterations in nuclear events related to gene expression and cell proliferation.


Introduction
Beside direct genotoxic actions, epigenetic mechanisms have gained considerable attention for the interpretation of carcinogenic action of metals. Therefore, the interaction of metal ions with cellular signaling is of particular interest (1)(2)(3). Transient changes in intracellular free Ca2+ concentration induced by hormones, neurotransmitters, or growth factors modulate a variety of physiologic processes such as cell proliferation and differentiation. Thereby, it seems obvious that the toxic action of metal ions may be mediated by interference with Ca2+-modulated signal transduction pathways.
Cd2+ has been shown to inhibit Ca2+ transport at several cellular sites with different consequences. Cd2+ blocks Ca2+ influx via the plasma membrane by displacing Ca2+ from its transport site in Ca channels (4) or by interaction with the receptoroperated Ca + influx system (5). However, Cd2+ inhibits Ca2+ extrusion from cells mediated by the plasma membrane Ca2+ pump (6,7), resulting in a disturbed intracellular Ca2+ homeostasis and increasing cytosolic free Ca2+ levels. The same consequences could be evoked by interaction of Cd2+ with Ca2+ transport into intracellular stores, such as interference with hepatic microsomal Ca2+ sequestration (8) or inhibition of sarcoplasmic reticulum Ca2+-ATPase (9). Several lines of evidence suggest that the level of free Ca2+ in the nucleus is important for the regulation of nuclear functions such as DNA replication, gene transcription, phosphorylation and the dephosphorylation of nuclear proteins, and DNA fragmentation (10). Recently, two systems for the regulation of nuclear free Ca2' have been descibed in isolated rat liver nuclei: Ca2+ can be accumulated by an ATP-stimulated uptake system (11) and released in an IP3-induced manner (12,13). The ATP-dependent Ca2+ uptake is mediated by a Ca2+ transporting pump closely related to the endoplasmic/sarcoplasmic reticulum Ca2+-ATPase (14).
The present study was undertaken to evaluate the effect of Cd2+ on the ATPstimulated Ca2+ transport system of isolated bovine liver nuclei.

Preparation ofNudei
Pieces of bovine liver were washed with icecold buffer and blotted on filter paper to remove contaminating blood. Homogenization and differential centrifugation were carried out by the method of Nicotera et al. (11). The final pellet of highly purified nuclei was resuspended in standard incubation medium (125 mM KCl, 2 mM K2HPO4, 25 mM Hepes, 4 mM MgCl2, 0.5 mM EGTA, 0.5 mM EDTA, 2 mM NTA, pH 7.0).
The contamination of isolated nuclei by plasma membranes, microsomes, and mitochondria was checked by the determination of marker enzyme activities. The total activities of 5'-nucleotidase, alkaline phosphodiesterase I, glucose-6-phosphatase, and succinate-INT-reductase found in the nuclear fraction were less than 0.5% of those found in the initial homogenate.
Buffering of Ca2+ and Cd2+ Concentrations Free ion concentrations were adjusted in standard incubation medium. The appropriate total concentrations of Ca2+ or Cd2+ required to give the desired free ion concentrations were calculated by the SPECS computer program of Fabiato (15). Absolute stability constants were taken from Smith and Martell (16).

Fura-2 Loading ofNudei and Fluorescence Measurements of Intranudear Free Ca2+ Concentration
Loading of isolated nuclei with fura-2 acetoxymethyl ester and fluorescence measurements were carried out as described previously (17).

Results
Isolated bovine liver nuclei loaded with n ofIntranudear Total fura-2 showed a marked increase in Ca2+-   Cd2+ on ATP-stimulated Ca2+ uptake in fura-2-loaded nuclei directly. Therefore, it was necessary to preincubate the nuclear suspension with various concentrations of free Cd2+ and to wash the nuclei with chelator-containing medium before starting the Ca2+ sensitive fluorescence measurement. Figure 3 shows that preincubation of the nuclear suspension with Cd2+ decreased the ATP-stimulated Ca2+ uptake. The inhibitory effect of Cd2+ depends on the concentration of free metal ion employed during the preincubation procedure ( Figure 4). Half-maximal inhibition of nuclear Ca2+ accumulation is reached at about 10 nM free Cd2+. Additionally, we studied the uptake of Cd2+ in isolated nuclei in the concentration range, where Ca2+ uptake is inhibited. The data in Figure 5, obtained from determinations by atomic absorption spectroscopy, demonstrate that significant amounts of intranuclear bound Cd were detectable from about 1 pM free Cd2+ in the incubation medium. The level of total Cd in nuclei is elevated with increasing concentrations of extranuclear free Cd2+.

Discussion
In agreement with previous reports (11,,17), incubation of isolated liver nuclei with ATP led to an accumulation of intranuclear free Ca2+ concentration. The ATP-stimulated Ca2+ uptake system is sensitive to the inhibition by thapsigargin, a specific inhibitor of intracellular Ca2+ ATPases. This result confirms recent findings that the nuclear membrane contains a Ca2 -transporting pump that is closely related to that of the endoplasmic reticu-lum (14). The active Ca2+ transport system provides a mechanism for the regulation of intranuclear free Ca2+ and may be critical for the control of Ca2+ modulated nuclear functions. The results obtained after preincubation of nuclear suspension with Cd2+ show that ATP-stimulated nuclear Ca2+ uptake is sensitive to inhibition by the heavy metal ion. Significant inhibition is already observed at low nanomolar concentrations of free Cd2+. The inhibitory range of free Cd2+ is comparable to that reported for the inhibition of plasma membrane Ca2+ ATPase (6,7). Since Cd2+ has an ionic radius similar to Ca2 , it is able to replace Ca2+ from its intracellular transport and binding sites. Therefore, the inhibitory action of Cd2+ on ATP-stimulated nuclear Ca2+ transport may be a result of blocking the transport binding site of the nuclear Ca2+ pump. Furthermore, the data from determination of total Cd demonstrate that the toxic metal ion is able to enter the nucleus itself, depending on the concentration of free Cd + adjusted in the incubation medium. As a consequence, Cd2+ occupies binding sites of intranuclear Ca2+ stores, so that the capacity of the nucleus to sequester Ca2+ is reduced.
Alterations in nuclear Ca2+ homeostasis caused by Cd2+ would disturb the Ca2+-dependent regulation of DNA-replication and gene transcription. It is known that Ca2+ controls the expression of growth-and transformation-related genes, such as c-fos and c-jun (22). Therefore, the impairment of nuclear Ca2+ regulation by Cd + may have consequences for the regulation of cell proliferation and tumorigenesis.