Iron promotes cadmium binding to citrate.

Iron-cadmium interactions are important in cadmium toxicity. Dietary iron supplements may decrease cadmium retention after oral cadmium exposure but the underlying mechanism is not known. Using a CdS/AgS ion selective electrode to measure [Cd2+] in physiological saline solution at pH 7.4, we show that Fe2+ promotes Cd2+ binding to citrate thereby decreasing the availability of free Cd2+. This suggests the formation of high molecular weight Cd2+-Fe2+-citrate complexes. We confirm this suggestion by showing that 109Cd2+ is retained by 1 kDa cut off filters when present with total 50 microM Fe2+ plus 1 mM citrate but not when present with citrate alone. The formation of high molecular weight complexes may prevent Cd2+ absorption. As citrate is part of the diet, we suggest that these iron-cadmium interactions may contribute to the protective effect of iron against cadmium toxicity.     

Iron promotes cadmium binding to citrate

Ironcadmium interactions are important in cadmium toxicity. Dietary iron supplements may decrease cadmium retention after oral cadmium exposure but the underlying mechanism is not known. Using a CdS/AgS ion selective electrode to measure [Cd²⁺] in physiological saline solution at pH 7.4, we show that Fe²⁺ promotes Cd²⁺ binding to citrate thereby decreasing the availability of free Cd²⁺. This suggests the formation of high molecular weight Cd²⁺Fe²⁺citrate complexes. We confirm this suggestion by showing that ¹⁰⁹Cd²⁺ is retained by 1 kDa cut off filters when present with total 50 M Fe²⁺ plus 1 mM citrate but not when present with citrate alone. The formation of high molecular weight complexes may prevent Cd²⁺ absorption. As citrate is part of the diet, we suggest that these ironcadmium interactions may contribute to the protective effect of iron against cadmium toxicity.     

On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca(2+)-dependent and Ca(2+)-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca(2+) with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe(2+) to NTA with and without Ca(2+). We observed Ca(2+) activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe(2+) to NTA with or without Ca(2+). However, Ca(2+) did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca(2+) and Fe(2+) by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe(2+), and total Ca(2+) concentrations). In solutions based on these constants and taking Ca(2+) chelation into account, Ca(2+) did not affect NTBI uptake over a range of free Fe(2+) concentrations. Thus, the Ca(2+) activation of NTBI uptake observed using the constant total Fe(2+) to NTA ratio was because of Ca(2+)-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca(2+) dependence of NTBI uptake be re-evaluated.     

Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.     

Ascorbate uptake in pig coronary artery endothelial cells

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When ¹⁴C-ascorbate uptake in endothelial cells was examined as ¹⁴C or by HPLC, the two values did not differ from each other. ¹⁴C-ascorbate uptake was Na⁺-dependent, stereoselective for l-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: K_m = 27± 3 M (Hill coefficient = 1) for ascorbate and K_m = 73± 14 mM (Hill coefficient = 2) for Na⁺. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect. (Mol Cell Biochem 271: 43–49, 2005)