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.
Mwanjewe
J
J
Departments of Medicine, Chemistry, and Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
Martinez
R
R
Agrawal
P
P
Samson
S E
SE
Coughlin
M D
MD
Brassard
P
P
Grover
A K
AK
D000818:Animals; D002104:Cadmium / Q000378:metabolism; D002118:Calcium / Q000502:physiology*; D007501:Iron / Q000378:metabolism*; D009571:Nitrilotriacetic Acid / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D014168:Transferrin / Q000378:metabolism*
D014168:Transferrin; D002104:Cadmium; D007501:Iron; D009571:Nitrilotriacetic Acid; D002118:Calcium