Abstract
The level of inositol phosphates was measured in rat hepatocytes treated with 2-methyl-1,4-naphthoquinone (menadione) or tert-butyl hydroperoxide, which cause Ca2+ mobilization from intracellular stores and an increase in cytosolic free Ca2+ concentration. Although neither agent produced any apparent changes in the resting level of inositol phosphates, pretreatment of hepatocytes with either menadione or tert-butyl hydroperoxide, as well as with several sulfhydryl reagents, markedly inhibited the increase in inositol phosphates induced by both hormonal and nonhormonal stimuli. Addition of dithiothreitol to menadione- or tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness to extracellular stimuli. Our findings suggest that the inhibition of the inositol phosphate response by menadione and tert-butyl hydroperoxide occurs through the modification of critical sulfhydryl group(s) and that the alterations in intracellular Ca2+ homeostasis occurring during the metabolism of menadione and tert-butyl hydroperoxide in hepatocytes are not mediated by inositol phosphates.
Highlights
From the Department of Toxicology, Karolinska Institutet, S-104-01 Stockholm, Sweden and the SDipartimento di Medicina Interna e Terapiu Medica, Clinica MedicaI, Uniuersity of Pavia, 27100 Pauia, Italy
Incubation of isolated rat hepatocytes with 0.2 mM menadione or 1mM tert-butyl hydroperoxide (t-BH), concentrations which cause a depletion of the endoplasmic reticular Ca2+pool [7] and a sustained increase in cytosolic Ca2+ concentration [17, 18], had no apparent effect on the levels of inositol phosphates (Table I)
When NaF was used, hepatocytes were incubated in a modified Krebs-Henseleit-Hepes buffer containing 100PM CaCl, to prevent the formation of CaFz precipitate; this modification did not affect the normal inositol phosphate response elicited by the hormones
Summary
None (menadione)or tert-butyl hydroperoxide, which In hepatocytes, several hormones cause Ca2+release from cause Ca2+mobilization from intracellular stores and the endoplasmic reticulum and a transient increase in cytoan increase in cytosolic free Ca2+concentration. Addition ofdithiothreitol to menadioneor tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness nisms underlying hormone-induced Ca2+mobilization have been shown to involve the generation of second messengers arising from the breakdown of phosphatidylinositol [24,25,26,27]. More inhibition of the inositol phosphate response by mena- recently, phosphatidylinositol 4,5-bisphosphate or one of its dione and tert-butyl hydroperoxide occurs through the hydrolysis products has been proposed to be responsible for modification of critical sulfhydryl group(s) and that the inhibition of the plasma membrane Ca2+ translocase the alterations in intracellular Ca2+homeostasisoccur- which occurs followingadministration of Ca2+-mobilizing ring during the metabolism of menadione and tert- agents [31]. We report that inositol phosphates are notinvolved in themobilization of Ca2+induced by either menadione or t-
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