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

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.