Aequorin chimeras as valuable tool in the measurement of Ca2+ concentration during cadmium injury.
The ability of cadmium to disrupt calcium homeostasis has been known since a long time, but the precise cellular targets of its toxic action are still debated. A great problem in the interpretation of data has been associated with the ability of cadmium to strongly bind traditional calcium probes. Aequorin, the well-characterized calcium-sensitive photoprotein, was used as intracellular calcium indicator during cadmium injury in NIH 3T3 murine fibroblasts. NIH 3T3 cells were transfected with a cDNA construct containing aequorin fused to a truncated glutamate receptor, which directs the probe to the outer surface of intracellular membranes. At first, we tested if different cadmium concentrations were able to modify the rate of light emission by aequorin showing that cadmium concentrations <15 microM were ineffective on aequorin luminescence. Hence, aequorin chimeras revealed as a useful tool in the analyses of Cd2+/Ca2+ interference. To directly investigate the role of Cd2+ in Ca2+ homeostasis, we have started to selectively measure the free Ca2+ concentration in different cell compartments. Here, we report that cadmium reduces the transient free calcium signal after stimulation of cells with bradykinin. Further studies are in progress to clarify the role of mitochondria and endoplasmic reticulum in cadmium-induced alterations of Ca2+ homeostasis in order to link signal transduction modifications with the onset of apoptosis induced by cadmium exposure.
Biagioli
M
M
Department of Physiology and Biochemistry, Laboratory of Cellular and Development Biology, University of Pisa, Italy.
Pinton
P
P
Scudiero
R
R
Ragghianti
M
M
Bucci
S
S
Rizzuto
R
R
D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002104:Cadmium / Q000378:metabolism / Q000633:toxicity*; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D049408:Luminescent Agents / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008858:Microscopy, Phase-Contrast; D041681:NIH 3T3 Cells; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D013997:Time Factors
D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D000331:Aequorin; D002118:Calcium