The thiol reagent, thimerosal, evokes Ca²⁺ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor

Bootman, Martin D.; Taylor, Colin W. and Berridge, Michael J. (1992). The thiol reagent, thimerosal, evokes Ca²⁺ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor. Journal of Biological Chemistry, 267(35) pp. 25113–25119.

URL: http://www.jbc.org/content/267/35/25113.full.pdf

Abstract

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in several cell types, and to cause Ca²⁺ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca²⁺ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca²⁺ spikes occurred in the absence of extracellular (Ca²⁺_o), suggesting that they result from mobilization of Ca²⁺ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca²⁺ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca²⁺ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP₃). The effects of thimerosal on Ca²⁺ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca²⁺]_i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca²⁺ accumulation, which was entirely due to inhibition of Ca²⁺ uptake into stores because thimerosal did not affect unidirectional ⁴⁵Ca²⁺ efflux from stores preloaded with ⁴⁵Ca²⁺.Thimerosal also caused a concentration-dependent sensitization of InsP₃-induced Ca²⁺ mobilization: half-maximal mobilization of Ca²⁺ stores occurred with 161 ± 20 nM InsP₃ in control cells and with 62 ± 5 nM InsP₃ after treatment with 10 μM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca²⁺ spiking without stimulating the formation of InsP₃ and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP₃ levels.

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