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 μM 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). (C) 2000 Harcourt Publishers Ltd.