Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

A E Traynor-Kaplan,B L Thompson,A L Harris,P Taylor,G M Omann,L A Sklar

doi:10.1016/s0021-9258(19)84884-6

Abstract

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.

Highlights

We recently showed that phosphatidylinositol tris- cated in the transductionof a variety of intracellular signals phosphate (PIP3)was present in a unique lipid fractiomnediating pleiotropic cell functions (1, 2)
Inadditiontheinitial ing the rise in intracellular calcium did not diminish analysis of the TLC band containing PIP3 suggested that the increase inPIPS.The elevation of PIP3appears to occur during activation withphysiological agonists, its level varying with the degree of activation
By minimizing experimental manipulations to quantitate the TLC band containing PIP3, we show that it is predominantly a singleInsP,containing phospholipid of which atrace is found in resting neutrophils

Summary

Lipid Analysis

Phospholipid Labeling--Elutriated cells were washed in buffer A containing 1.53 mM Ca'', sedimented, and resuspended in buffer A without Ca'+ with 2mg/mlbovine serumalbumin, pH 7.4, to a concentration of 5 X 10' cells/ml. 3'P0, (HC1 free 500 pCi/ml) was added, and the cells were incubated a t 37 "C for 60 min. Phospholipid Labeling--Elutriated cells were washed in buffer A containing 1.53 mM Ca'', sedimented, and resuspended in buffer A without Ca'+ with 2mg/mlbovine serumalbumin, pH 7.4, to a concentration of 5 X 10' cells/ml. 3'P0, (HC1 free 500 pCi/ml) was added, and the cells were incubated a t 37 "C for 60 min. The cells were washed threetimesin bufferA without bovine serum albumin and were resuspended a t 2 X lO'/ml in buffer A with Ca'+ (1.53 mM). Labelingwithmyoinositolwas essentiallythesameas labeling with "Po, except that the incubation was carried out for 4 h instead of 1 h. Theconcentration of [3H]myoinositol (12.8 Ci/ mmol, NEN) was 50 pCi/'ml. Phospholipid Extraction-Following the labeling procedure, cells were stimulated as described in the figure legends.

RESULTS

TO nM FlPEP h e lsecandsl

DISCUSSION