FIGURE 3.

Inhibition of InsP₇ formation in TNP-treated HeLa cells. A, HPLC separation of inositol phosphates extracted from HeLa cells stimulated with DMSO (filled circles) or TNP at 100 nm (open circles), 1 μm (filled squares), or 10 μm (open squares) for 2 h at 37 °C. The cells were grown in inositol-free medium supplemented with 10% (v/v) dialyzed fetal calf serum and 50 μCi of [2-³H]inositol for 3 days, and inositol phosphates were extracted (see “Experimental Procedures”). Since the InsP₆ peak did not change by more than 10% between all of the conditions (average in control cells = 564,651 dpm and average in cells treated to 10 μm TNP = 599,011 dpm), the dpm in the InsP₆ peak was used to normalize levels of other inositol phosphates. In control HeLa cells, the percentages of InsP₃, InsP₄, InsP₇, and InsP₈ with respect to InsP₆ are 12, 4, 6, and 0.7%, respectively. Similar values for InsP₇ and InsP₈ have been reported in DDT₁-MF2 cells and HEK cells (30). B, change in InsP₅ (filled triangles), InsP₃ (filled squares), and InsP₄ (open squares) normalized to InsP₆ in the presence of increasing TNP. In each case, the total radioactivity under the corresponding peak (base line-subtracted) was divided by the total radioactivity under the InsP₆ peak (base line-subtracted) and expressed as a percentage. Data are the mean of two different experiments, each an average of triplicate assays, and error bars represent the S.D. of the data. C, TNP-induced dose-dependent decrease in InsP₇. For each concentration of TNP, radioactivity in the [³H]InsP₇ peak was normalized to that in the [³H]InsP₆ peak. Data are the average of triplicate experiments, and error bars represent the S.D. of the data. Sigma Plot software was used to carry out curve fitting to the InsP₇ data, and the IC₅₀ calculation was carried out from the equation used to fit the curve.