The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells

J. A. Stuart, K. L. Anderson, P. J. French, C. J. Kirk, R. H. Michell*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)

Abstract

1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates. notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate InsP6). 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 InsP5 and InsP6 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 ~ 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released ~ 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus α-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 α-toxin, only ~ 50-70% of InsP2, InsP3 and InsP4 and ≤ 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 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 InsP5 and InsP6 ( ≤ 10-20%) might be in some intracellular bound form.

Original languageEnglish
Pages (from-to)517-525
Number of pages9
JournalBiochemical Journal
Volume303
Issue number2
DOIs
Publication statusPublished - 1 Jan 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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