THE INTRACELLULAR-DISTRIBUTION OF INOSITOL POLYPHOSPHATES IN HL-60 PROMYELOID CELLS

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP(5) and InsP(6) was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at similar to 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP(5) and InsP(6), but only slight release of the intracellular enzymes. Multiple discharges released similar to 80-90 % of total InsP(5) and InsP(6). In the absence of bivalent-cation chelators, InsP(5) and InsP(6) were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus cc-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only similar to 50-70 % of InsP(2), InsP(3) and InsP(4) and less than or equal to 20% of InsP(5) and InsP(6) were released, indicating that the high charge or large hydrated radius of InsP(5) and InsP(6) might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP(5) and InsP(6) (less than or equal to 10-20 %) might be in some intracellular bound form.