Stimulation by thimerosal of histamine-induced Ca(2+) release in intact HeLa cells seen with aequorin targeted to the endoplasmic reticulum

Abstract

The oxidizing thiol reagent, thimerosal, has been shown to activate reversibly the inositol 1,4,5-trisphosphate (InsP(3)) receptor in several cell types. We have studied here the effects of thimerosal by monitoring the [Ca(2+)] inside the endoplasmic reticulum (ER) of intact HeLa cells with targeted aequorin. We show that thimerosal produced little effects on the ER-Ca(2+)-pump and only slightly increased the ER-Ca(2+)-leak in intact cells. Instead, thimerosal increased the sensitivity to histamine of ER-Ca(2+)-release by about two orders of magnitude, made the response much more prolonged at saturating histamine concentrations and enhanced both cytosolic and mitochondrial [Ca(2+)] responses to histamine. Moreover, inhibition of ER-Ca(2+)release by cytosolic [Ca(2+)] microdomains was fully preserved and sensitive to BAPTA-loading, and histamine-induced Ca(2+) release remained quantal in the presence of both thimerosal and intracellular BAPTA. The effects of thimerosal were reversible in the presence of dithiotreitol, suggesting the possible presence of a physiological redox regulatory mechanism. However, in permeabilized cells thimerosal potentiated InsP(3)-induced Ca(2+) release but oxidized glutathione had no effect. In addition, thimerosal increased the [Ca(2+)](ER) steady-state level in permeabilized cells. Thimerosal partially inhibited also plasma membrane Ca(2+)extrusion and increased Ca(2+)(Mn(2+)) entry through the plasma membrane, both phenomena contributing to increase the steady-state cytosolic [Ca(2+)]. Thimerosal-induced Ca(2+) entry was additive to that induced by emptying of the ER, suggesting that store-operated Ca(2+) channels may not be involved. These results provide new insights on the mechanisms of activation and inactivation of InsP(3) receptors.