Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca²⁺ release by thimerosal in permeabilized A7r5 smooth-muscle cells

Abstract

There is no consensus about the different types of Ca²⁺ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca²⁺ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional ⁴⁵Ca²⁺ flux technique. Thimerosal up to a concentration of 32 μM did not have an effect on the passive ⁴⁵Ca²⁺ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca²⁺ pump with an EC⁵⁰ of 9 μM. Thimerosal exerted a biphasic effect on the Ca²⁺ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] with a stimulation of the release at thimerosal concentrations below 10 μM, and an inhibitory effect at higher concentrations. Thimerosal (2.5–250 μM) did not exert an effect on the specific binding of [³H]Ins(1,4,5)P ₃ to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [³H]Ins(1,4,5)P ₃ binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 μM can stimulate the Ins(1,4,5)P ₃-induced Ca²⁺ release without inhibiting the Ca²⁺ pumps or without increasing the passive Ca²⁺ permeability of the endoplasmic reticulum.