Polyphosphoinositide Synthesis in Human Neutrophils: Effects of a Low Metabolic Energy State

• Published in: Prostaglandins & other lipid mediators Vol. 55; no. 4; pp. 245 - 264
• Main Authors: Catz, Sergio D, Speziale, Emir H, Sterin-Speziale, Norma B
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.1998
• Subjects: Adenosine Triphosphate - metabolism; Adult; Antimetabolites - pharmacology; ATP depletion; Deoxyglucose - pharmacology; Diphosphates - metabolism; Energy Metabolism - drug effects; Genistein - pharmacology; Humans; Kinetics; Magnesium - pharmacology; Membrane Lipids - biosynthesis; Neomycin - pharmacology; Neutrophils - drug effects; Neutrophils - metabolism; Oligomycins - pharmacology; Phosphatidylinositol Phosphates - biosynthesis; Phosphatidylinositols - biosynthesis; Phosphoinositide synthesis; Phosphorylation; Protein-Tyrosine Kinases - antagonists & inhibitors; tyrosine kinase; ATP depletion; Phosphoinositide synthesis; tyrosine kinase
• ISSN: 1098-8823
• DOI: 10.1016/S0090-6980(98)00021-5

Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [ 32P]orthophosphate ([ 32P]Pi) or [ 3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns “de novo” synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [ 32P]Pi into PPIs. Of the “de novo” synthesized [ 3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [ 32P]Pi labeling, indicating that PPI formation mainly involves a no “de novo” synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [ 32P]Pi. Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occured in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [γ- 32P]-adenosine triphosphate (ATP) or [ 32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [ 32P]from [ 32P]Pi was incorporated more efficiently into PPIs than that from [γ- 32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [ 32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI syntesis. These data suggest that PPI formation is not necessarely a futile cycle in PMNs.