Interference of heavy metal cations with fluorescent Ca2+ probes does not affect Ca2+ measurements in living cells.
In studies about the effects of heavy metals on intracellular Ca2+, the use of fluorescent probes is debated, as metal cations are known to affect the probe signal. In this study, spectrofluorimetric experiments in free solution, using Fluo-3 and Fura-2, showed that Zn2+ and Cd2+ enhanced the probe signal, Cu2+ quenched it, and Hg2+ had no effect. Addition of GSH prevented most of these effects, suggesting the occurrence of a similar protective role in living cells. Digital imaging of living mussel haemocytes loaded with Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 microM Zn2+ had no effect. In particular, Cd2+ produced the strongest probe signal rise in free solution, but the lowest fluorescence increase in cells. Probe calibration yielded [Ca2+]i values characteristic of resting levels in control and Zn2+-exposed cells, and, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy metals in living cells are completely different from those obtained in free solution, indicating that fluorescent probes can be a suitable tool to record the effects of heavy metals on [Ca2+]i.
Marchi
B
B
Dip. di Biologia Sperimentale Applicata e Ambientale, Università di Genova, Genova, Italy.
Burlando
B
B
Panfoli
I
I
Viarengo
A
A
D000814:Aniline Compounds; D000818:Animals; D049872:Bivalvia; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006458:Hemolymph / Q000166:cytology / Q000378:metabolism; D007089:Image Enhancement; D066298:In Vitro Techniques; D008628:Mercury / Q000494:pharmacology; D019216:Metals, Heavy / Q000494:pharmacology*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014966:Xanthenes; D015032:Zinc / Q000494:pharmacology
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D019216:Metals, Heavy; D012996:Solutions; D014966:Xanthenes; D002104:Cadmium; C059715:Fluo-3; D003300:Copper; D008628:Mercury; D015032:Zinc; D002118:Calcium; D016257:Fura-2