Formation of phosphatidylinositol‐4‐phosphate in human peripheral blood eosinophils

Abstract

To evaluate the presence of phosphatidylinositol-4-phosphate in peripheral blood eosinophils, venous blood was drawn from normal healthy volunteers. The eosinophils were isolated on a Percoll gradient and were incubated with [gamma32P]ATP in the presence of Mg2+. After stopping the reaction, lipid extraction was performed with acidic medium and phospholipids were separated by thin-layer chromatography on 1% (w/v) oxalic acid- and potassium oxalate-impregnated silica gel plates. Considerable amounts of radioactivity were found to be incorporated into phosphatidylinositol-4-phosphate on both plates. This reaction requires ATP and Mg2+, and maximal response is seen at 10 microM ATP and 20 mM Mg2+. The reaction is dependent upon the time and temperature of the assay system. No significant superoxide anion generation from the eosinophils incubated with ATP at the concentrations used in the study was observed. These results suggest the possible presence of phosphatidylinositol kinase which catalyses the formation of phosphatidylinositol-4-phosphate from endogenous phosphatidylinositol in human peripheral blood eosinophils.

Incorporation of Radioactive Phosphate from Adenosine Triphosphate into Phosphatidylinositol-4-Phosphate in Human Peripheral Blood Hypodense Eosinophils Is Less than in the Normodense Eosinophils

“…Each tube con tained 7 x KP purified eosinophils. Incubations were carried out at 37°C for 15 min in a total volume of 100 pi, because these conditions were shown to be optimal for the reaction based on recent studies under various conditions for time, tem perature, and so on [4]. The reaction was stopped by the addition of methanol and chloroform in the acidic condition to the entire eosinophil suspension as described below.…”

“…A series of studies using guinea-pig peritoneal eosinophils [2,3] indicated the presence of stimulus-re sponse coupling in eosinophils, such as accumulation of inositol trisphosphate and subsequent mobilization of Ca2+, suggesting the presence of PtdIns(4,5)P2 in eosi nophils. However, to our knowledge, no information is available about the presence of PtdIns(4,5)P2 in human eosinophils, and very recently we reported that human peripheral blood eosinophils can produce phosphatidylinositol-4-phosphate [PtdIns(4)P] [4], Most PtdIns(4)P in cells is thought to be produced en zymatically from phospatidylinositol (Ptdlns) by Ptdlns kinase and the reaction requires ATP and Mg2+ [5]: Ptdlns + ATP-^ PtdIns(4)P + ADP. Ptdlns kinase appears to be widely distributed among mammalian tissues [6][7][8] and PtdIns (4,5)P2 is synthesized by stepwise phosphorylation of Ptdlns by Ptdlns kinase and PtdIns(4)P kinase.…”

“…Increasing evidence suggests that the most rapid re sponse to receptor-mediated stimulus in cell membranes is the breakdown of phosphatidylinositol-4,5-bisphosphatc [PtdIns (4,5)P2] to inositol-1,4,5-trisphosphate and diacylglycérol [1], and much interest has been paid to these two lipids in intracellular signalling systems. Al though considerable progress has been made toward un derstanding the mechanisms of signal transduction in a variety of cell types, not enough information is available on eosinophils which may be involved in the activation process.…”

Signal transduction mechanisms in human eosinophils

“…The difficulty in isolating normal human eosinophils from peripheral blood in sufficient number, yet without contamination by other cell types, for the investigation of signal transduction mechanisms has hampered progress in this area of research. Consequently, while there are a few studies using normal human eosinophils, for example see the article by Kurosawa et al in this issue [1], others have used cells isolated from patients with hypereosinophilic syndrome (HES) [2]. The altered density, metabolism and surface receptors of these cells indicate that activated eosinophils circulate in HES [3].…”

“…PAF receptors are coupled to the pertussis toxin-sensitive Gi protein which, it is suggested, transduces ligand binding to effector systems, possibly phospholipase C [37]. The presence of phosphatidylinositol kinase in normal human eosinophils [1] indicates the machinery is in place for receptormediated phosphoinositide turnover and subsequent generation ofthe short-lived intracellular second messengers, diacylglycerol and inositol trisphosphate. Diacylglycerol produced in the plasma membrane, either as a direct result of phospholipase C activation or indirectly via the action of phospholipase D on cholinephospholipids [38], induces tbe translocation and activation of PK-C, whilst inositol trisphosphate raises intracellular Ca^+ levels by inducing Ca^+ release from endoplasmic reticulum.…”