Inhibition of mitochondrial Ca2+ release diminishes the effectiveness of methyl mercury to release acetylcholine from synaptosomes

The interaction of methyl mercury (MeHg) with nerve-terminal mitochondria as a potential mechanism for its effects on the release of acetylcholine (ACh) was studied using rat brain synaptosomes. The primary goal was to assess the relative contribution of extracellular Ca2+ and Ca2+ released from nerve-terminal mitochondria to the previously described stimulatory effects of MeHg on spontaneous release of ACh. A secondary goal was to address possible mechanisms by which MeHg might interact with nerve-terminal mitochondria to elicit Ca2+ discharge and subsequent release of ACh. MeHg depressed the high-affinity uptake of [3H]choline into synaptosomes by approximately 25 and 45% when synaptosomes were incubated with [3H]choline in the presence of 10 and 100 microM MeHg, respectively. In Ca(2+)-containing solutions, 10 and 100 microM MeHg increased the release of [3H]ACh from [3H]choline-loaded synaptosomes by 10 and 30%, respectively; this effect was maximal at 10 sec. Excluding Ca2+ from the reaction medium diminished the effectiveness of both 10 and 100 microM MeHg for inducing [3H]ACh release by about 30 and 25%, respectively, from that of Ca(2+)-containing solutions; however, significant increases still occurred in nominally Ca(2+)-free solutions. Ruthenium red (RR), an inhibitor of mitochondrial Ca2+ transport, was tested for its ability to disrupt MeHg-induced release. RR alone increased [3H]ACh release by 8-10 and 10-13% at 20 and 60 microM, respectively. RR-induced release was attenuated only slightly in Ca(2+)-free solutions. Preincubation of [3H]choline-loaded synaptosomes with RR reduced the stimulatory effect of MeHg on release of [3H]ACh both in the presence and in the absence of Ca2+. The fluorescent potentiometric carbocyanine dye diS-C2(5) was used to assess the ability of RR to prevent MeHg-induced depolarization of intrasynaptosomal mitochondria. RR (20 microM) itself did not depolarize the mitochondrial membrane potential, nor did it prevent MeHg from depolarizing the mitochondria. The results indicate that extracellular Ca2+ contributes only partially to MeHg-induced spontaneous release of ACh. The results with RR suggest that MeHg interacts with mitochondria to induce release of bound intraterminal Ca2+ stores, resulting ultimately in stimulated release of ACh. The ability of RR to prevent release of mitochondrial Ca2+ and, subsequently, ACh is not due to prevention of access of MeHg to the mitochondria, nor to stabilization of the mitochon