Effects of Hg²+ on Ca²⁺ dynamics in the scallop sarcoplasmic reticulum (Pecten jacobaeus): Protective role of glutathione

Scallop (Pecten jacobaeus) sarcoplasmic reticulum (SR) vesicles were functionally characterized and tested for Hg²⁺ effects. An SR-containing 20,000 g supernatant from adductor muscle homogenate was incubated with fluo-3, allowing free Ca²⁺ variations to be spectrofluorimetrically followed. Data showed an ATP-dependent thapsigargin-sensitive Ca²⁺ uptake, revealing the activity of the SR Ca²⁺ ATPase. Treatment with ryanodine elicited Ca²⁺ release, showing the presence of ryanodine-sensitive Ca²⁺ channels, whereas InsP₃ caused negligible effects. Exposure to different concentrations of Hg²⁺ (1-50 μM) produced a dose-dependent Ca²⁺ release from SR vesicles, which was shown to depend on both Ca²⁺ ATPase inhibition and Ca²⁺ channel opening. Hg²⁺ binding to sulfhydryls was pointed out by incubation with Thiolyte, whereas an involvement of sulfhydryls in Ca²⁺ release was assessed by treatment with the sulfhydryl reagent N-ethylmaleimide (NEM). Yet, conversely to Hg²⁺, NEM seemed unable to open Ca²⁺ channels, suggesting that the latter effect occurs via some specific heavy metal interaction, possibly involving sulfhydryls nor available to larger molecules or even components other than sulfhydryls. Pre-incubation of SR with reduced glutathione (GSH) largely prevented Hg²⁺ effects, whereas a certain reduction of metal injury also occurred by adding GSH after Hg²⁺ exposure, thus confirming the role of GSH as a first line of defense against heavy metals.