Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells.
Absolute concentrations of inositol phosphate isomers (InsP(s] were quantified in the myeloid cell line HL-60 using the metal-dye detection technique. Stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) led to distinct alterations in at least seven different inositol phosphate species. Whereas the intracellular concentrations of the tetrakisphosphate isomers (InsP4(s] were found below the micromolar range, inositol 1,3,4,5,6-pentakis- and hexakisphosphate levels were about two orders of magnitude higher (36 and 54 +/- 2 microM (mean +/- S.D.), respectively). The three InsP4(s) showed distinct kinetic pattern upon receptor activation, the transient elevation of inositol 1,3,4,5-tetrakisphosphate being faster both in onset and in redecrease than inositol 1,3,4,6-tetrakisphosphate. Whereas the two latter isomers reached maximally 2.75 and 2.9 +/- 0.2 microM, respectively, 1 min after stimulation, inositol 3,4,5,6-tetrakisphosphate remained elevated (3.5 +/- 0.4 microM) up to 5 min after fMLP. Unexpected changes in highly phosphorylated InsP(s) were observed, notably a rise in inositol 1,3,4,5,6-pentakisphosphate and in inositol hexakisphosphate to 52 +/- 3 and 60 +/- 1 microM, respectively. In terms of mass, the increases in highly phosphorylated inositols are by far highest among all InsP(s). Combining radiotracer method with mass determination it was observed that the specific radioactivity of various InsP(s) was different and changed markedly upon fMLP stimulation, in spite of a prolonged labeling period leading to apparent isotopic steady state. The data presented demonstrate agonist-induced elevations of highly phosphorylated InsP(s) and suggest that inositol 1,4,5-trisphosphate, product of receptor-activated phospholipase C, is metabolized rather via phosphorylation than only by dephosphorylation pathways.
Pittet
D
D
Infectious Diseases Division, University Hospital, Genève, Switzerland.
Schlegel
W
W
Lew
D P
DP
Monod
A
A
Mayr
G W
GW
D002851:Chromatography, High Pressure Liquid; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000502:physiology; D012118:Resorcinols; D014407:Tumor Cells, Cultured
D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D012118:Resorcinols; C081655:inositol-1,3,4,5-tetrakisphosphate; C081653:inositol-1,3,4,6-tetrakisphosphate; C081652:inositol-3,4,5,6-tetrakisphosphate; C013571:inositol pentaphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; C028398:4-(2-pyridylazo)resorcinol