DETERMINATION OF INOSITOL POLYPHOSPHATES FROM HUMAN LYMPHOCYTE-T CELL-LINES BY ANION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND POSTCOLUMN DERIVATIZATION

The intracellular amounts of several inositol tris-, tetrakis- and pentakisphosphates and inositol hexakisphosphate were determined in resting and stimulated cells from human T-lymphocyte lines. The inositol polyphosphates were separated by anion-exchange high-performance liquid chromatography and were detected on-line by a recently developed post-column dye system. In the human T-lymphocyte cell line Jurkat, basal intracellular concentrations ranged between 25 +/- 10 pmol per 10(9) cells for inositol 1,4,5-trisphosphate to 6380 +/- 355 pmol per 10(9) cells for inositol hexakisphosphate. Similar basal concentrations were observed in the human T-lymphocyte cell line HPB.ALL, with the exception that inositol hexakisphosphate was approximately 665 +/- 10 pmol per 10(9) cells. Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor by a monoclonal antibody directed against the T-cell receptor-CD3 complex induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate isomers, including inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate, an as yet unidentified inositol tetrakisphosphate isomer, inositol 1,3,4,5,6-pentakisphosphate, inositol 1,2,3,4,6-pentakisphosphate and DL-inositol 1,2,4,5,6-pentakisphosphate. Inositol 1,4,5-trisphosphate increased only transiently after 5 min, whereas DL-inositol 1,4,5,6-tetrakisphosphate (determined as the enantiomeric mixture) increased after 20 min.