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Keywords:

  • Zn2+;
  • Methylmercury;
  • Synaptosomes;
  • 19F-NMR;
  • Neurotoxicity;
  • Heavy metal

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca2+ ([Ca2+]i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca2+]i was strictly dependent on extracellular Ca2+ (Ca2+e); similarly, in NG108-15 cells, a component of the elevations in [Ca2+]i was Ca2+e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca2+e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn2+. Using 19F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn2+ concentration ([Zn2+]i). In buffer containing 200 µM EGTA to prevent the Ca2+e-dependent elevations in [Ca2+]i, the [Zn2+]i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn2+]i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn2+]i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn2+]i to 3.99 ± 0.68 nM. No Zn2+ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca2+]i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca2+]i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca2+]i differed significantly from either pretreatment levels or buffer-treated controls.