Effects of Lead, Mercury, and Methyl Mercury on Gap Junctions and [Ca²⁺]_i in Bone Cells

Abstract.

Heavy metals such as lead (Pb), mercury (Hg), and methyl mercury (MeHg) impair cell functions. For bone it is known that Pb changes bone formation rates, which depend on intracellular free calcium concentration ([Ca²⁺]_i). Since heavy metals compete with Ca²⁺ at multiple sites and increased [Ca²⁺]_i reduces gap junctional coupling between bone cells, we analyzed the effects of extracellular (e) and intracellular (i) application of Pb, Hg, and MeHg on these channels. Using primary cultures of osteoblast-like cells, relative changes of [Ca²⁺]_i were studied in Fura-2/AM loaded cells. Parallel intracellular recordings of neighboring cells were obtained using a conventional and a patch electrode. Pb_(e) (5 μmol/liter; n = 3) and Hg_(e) (5 μmol/liter; n = 3) as well as Pb_(i) (25 μmol/liter; n = 7) did not change the coupling (ΔMP₂/ΔMP₁). In contrast, MeHg_(e) (1–10 μmol/liter; n = 6) and Hg_(i) (≥5 μmol/liter; n = 8) reduced the coupling to 79.5 ± 19.3% and 62.4 ± 15.3%, respectively, within 15–20 minutes. The reduction of coupling followed individual time courses, and in no case was a steady state of decoupling reached within 20 minutes. Extracellular application of Pb_(e) (5 μmol/liter, n = 74) for 20 minutes, linearly elevated the Fura emission ratio reflecting transmembrane Pb permeation rather than [Ca²⁺]_i increase. Hg_(e) (n = 48) slightly increased [Ca²⁺]_i from 100 to ≤200 nmol/liter, whereas MeHg_(e) (5 μmol/liter, n = 52) released Ca²⁺ from internal stores, thus increasing [Ca²⁺]_i up to 2 μmol/liter. In conclusion, Pb_(e), Pb_(i) and Hg_(e) do not affect gap junctional coupling per se. Since MeHg_(e) and Hg_(i) deplete calcium stores, the decrease of the electric coupling is attributable to increased [Ca²⁺]_i, which affects gap junction channels.