Oscillatory cytosolic calcium waves independent of stimulated inositol 1,4,5-trisphosphate formation in hepatocytes

T. A. Rooney, D. C. Renard, E. J. Sass, A. P. Thomas

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The mechanisms underlying agonist-induced oscillations in intracellular free calcium ion concentration ([Ca2+](i)) in hepatocytes were investigated by utilizing tert-butyl hydroperoxide (TBHP) as a tool to perturb hepatocyte Ca2+ homeostasis independent of receptor activation. In permeabilized hepatocytes, TBHP inhibited Ca2+ uptake into the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool and increased the sensitivity to InsP3 for Ca2+ release. The effects of TBHP could be mimicked by addition of oxidized glutathione (GSSG) and reversed by pretreatment with dithiothreitol. TBHP and GSSG had no effect on the metabolic degradation of [3H]InsP3 in permeabilized cells. The effect of TBHP on [Ca2+](i) in intact cells was investigated by digital imaging fluorescence microscopy of Fura-2-loaded primary cultured hepatocytes. TBHP treatment initiated a series of [Ca2+](i) oscillations similar to those caused by Ca2+-mobilizing hormones. Moreover, in common with the actions of hormones in these cells (Rooney, T.A., Sass, E., and Thomas, A.P. (1990) J. Biol. Chem. 265, 10792-10796), the [Ca2+](i) oscillations induced by TBHP propagated through the cell as Ca2+ waves, originating from a discrete subcellular locus identical to that for phenylephrine-induced [Ca2+](i) oscillations. The Ca2+ waves induced by TBHP had similar rates of progress (24-27 μm·s-1) to those generated by phenylephrine. Removal of extracellular Ca2+ increased the initial latency of the TBHP responses, but had no effect on the amplitude or rate of propagation of the Ca2+ waves. Addition of TBHP to cells in the presence of phenylephrine converted the oscillatory phenylephrine [Ca2+](i) response into a sustained [Ca2+](i) increase. The effects of TBHP in intact cells occurred in the absence of any stimulated inositol polyphosphate formation as measured in populations of [3H]inositol-labeled hepatocytes. The data indicate that spatially organized [Ca2+](i) oscillations in intact hepatocytes can occur without any requirement for phospholipase C activation. Furthermore, for agents that act by mobilizing Ca2+ from the InsP3-sensitive pool, the kinetics of the Ca2+ release phase of the [Ca2+](i) oscillations appears to be independent of the nature of the stimulus.

Original languageEnglish (US)
Pages (from-to)12272-12282
Number of pages11
JournalJournal of Biological Chemistry
Issue number19
StatePublished - 1991

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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