FIGURE 9.
The effects of elevated extracellular [Pi] upon phosphate homeostasis and inositol phosphate levels in HCT116 cells. HCT116 cells were labeled with [3H]inositol in medium containing 1 mm Pi and then an additional 5 mm Pi (as a NaH2PO4/Na2HPO4 mixture (pH 7.4)) was added for intervals of up to 60 min. Next, cells were quenched, and the levels of the indicated inositol phosphates (InsP5, InsP6, InsP7, and InsP8) were analyzed by HPLC (see “Experimental Procedures”). A and B show representative HPLC chromatographs for the 0- and 60- min time points; data for InsP7 and InsP8 are replotted on an expanded y axis scale. Data are representative of three biological replicates. C shows time course data (InsP5, InsP6, and InsP7; mean ± S.E., n = 3) from all experiments. D and E show time course data (error bars represent S.E.) for intracellular levels of ATP and Pi (malachite green/molybdate method), respectively, determined in parallel experiments with non-radiolabeled cells (n = 6–7) incubated for the indicated times with 6 mm extracellular [Pi]. The data for cell Pi content (nmol/mg of protein) at 0, 15, 30, 45, and 60 min (error bars represent S.E.; 56 ± 4.3, 59 ± 4.7, 58 ± 3.3, and 60 ± 1.8) closely match the Pi levels that we obtained by an independent enzymatic method (54 ± 3.5, 57 ± 3.7, 56 ± 2.7, and 62 ± 4.3; n = 6–7). F shows time course data for InsP8 (*, p < 0.05; **, p < 0.02; paired t test). G shows representative HPLC chromatographs from the region in which InsP8 elutes (as indicated by the elution profile (open circles) for an [3H]InsP8 standard from a parallel run); cell extracts (closed circles) were derived from [3H]inositol-labeled PPIP5K−/− HCT116 cells (see Ref. 27) that had been incubated for 1 h with either 1 or 6 mm Pi as indicated.