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 solut ion, 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 r ole in living cells. Digital imaging of living mussel haemocytes loaded wit h Fura-2/AM or Fluo-3/AM showed that Hg2+, Cu2+ and Cd2+ induced a rise in probe fluorescence, whereas up to 200 muM Zn2+ had no effect. In particular , Cd2+ produced the strongest probe signal rise in free solution, but the l owest fluorescence increase in cells. Probe calibration yielded [Ca2+](i) v alues characteristic of resting levels in control and Zn2+-exposed cells, a nd, as expected, indicated Ca2+ homeostasis impairment in cells exposed to Cd2+, Cu2+ and Hg2+. Our results show that Ca2+ probe responses to heavy me tals in living cells are completely different from those obtained in free s olution, indicating that fluorescent probes can be a suitable tool to recor d the effects of heavy metals on [Ca2+](i). (C) 2000 Harcourt Publishers Lt d.