title: 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.
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Ramón y Cajal 7, E-47005 Valladolid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Montero
authors.forename: M J
authors.initials: MJ
authors.lastname: Barrero
authors.forename: F
authors.initials: F
authors.lastname: Torrecilla
authors.forename: C D
authors.initials: CD
authors.lastname: Lobatón
authors.forename: A
authors.initials: A
authors.lastname: Moreno
authors.forename: J
authors.initials: J
authors.lastname: Alvarez
mesh_terms: D000331:Aequorin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology; D004357:Drug Synergism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010084:Oxidation-Reduction; D021381:Protein Transport; D013849:Thimerosal / Q000494:pharmacology*
chemical_list: D013849:Thimerosal; D008345:Manganese; D000331:Aequorin; D004533:Egtazic Acid; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D005978:Glutathione; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D004229:Dithiothreitol