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Updated: Jan 11, 2026
Disorders of Erythrocytes
Phosphatidic acid as a second messenger in human polymorphonuclear leukocytes. Effects on activation of NADPH oxidase
D E Agwu1, L C McPhail, S Sozzani
1Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Abstract:
Receptor-mediated agonists, such as FMLP, induce an early, phospholipase D (PLD)-mediated accumulation of phosphatidic acid (PA) which may play a role in the activation of NADPH oxidase in human PMN. We have determined the effect of changes in PA production on O2 consumption in intact PMN and the level of NADPH oxidase activity measured in a cell-free assay. Pretreatment of cells with various concentrations of propranolol enhanced (less than or equal to 200 microM) or inhibited (greater than 300 microM) PLD-induced production of PA (mass and radiolabel) in a manner that correlated with enhancement or inhibition of O2 consumption in PMN stimulated with 1 microM FMLP in the absence of cytochalasin B. The concentration-dependent effects of propranolol on FMLP-induced NADPH oxidase activation was confirmed by direct assay of the enzyme in subcellular fractions. In PA extracted from cells pretreated with 200 microM propranolol before stimulation with 1 microM FMLP, phospholipase A1 (PLA1)-digestion for 90 min, followed by quantitation of residual PA, showed that a minimum of 44% of PA in control (undigested) sample was diacyl-PA; alkylacyl-PA remained undigested by PLA1. Propranolol was also observed to have a concentration-dependent enhancement of mass of 1,2-DG formed in PMN stimulated with FMLP. DG levels reached a maximum at 300 microM propranolol and remained unchanged up to 500 microM propranolol. However, in contrast to PA levels, the level of DG produced did not correlate with NADPH oxidase activation. Exogenously added didecanoyl-PA activated NADPH oxidase in a concentration-dependent manner (1-300 microM) in a reconstitution assay using membrane and cytosolic fractions from unstimulated PMN. In addition, PA synergized with SDS for oxidase activation. Taken together, these results indicate that PA plays a second messenger role in the activation of NADPH oxidase in human PMN and that regulation of phospholipase D is a key step in the activation pathway.
Insights
Phosphatidic acid (PA) acts as a second messenger in activating NADPH oxidase in human neutrophils. Its production, regulated by phospholipase D (PLD), is crucial for this activation pathway.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Receptor-mediated agonists like FMLP trigger phospholipase D (PLD)-dependent phosphatidic acid (PA) accumulation.
- PA accumulation is hypothesized to play a role in activating NADPH oxidase in human neutrophils (PMN).
- Understanding the precise role of PA and its regulation is key to understanding neutrophil activation.
Purpose of the Study:
- To investigate the effect of altered PA production on oxygen consumption and NADPH oxidase activity in human PMN.
- To elucidate the role of PA as a second messenger in NADPH oxidase activation.
- To determine the significance of phospholipase D regulation in this pathway.
Main Methods:
- Human PMN were pretreated with varying concentrations of propranolol to modulate PLD-induced PA production.
- Oxygen consumption and NADPH oxidase activity were measured in intact PMN and cell-free assays.
- PA composition was analyzed using phospholipase A1 (PLA1) digestion; diacylglycerol (DG) formation was quantified.
Main Results:
- Propranolol modulated PA production and O2 consumption in a concentration-dependent manner, correlating with NADPH oxidase activation.
- Exogenously added PA activated NADPH oxidase in a cell-free reconstitution assay, indicating a direct role.
- While DG levels changed, they did not correlate with NADPH oxidase activation, unlike PA levels.
Conclusions:
- Phosphatidic acid (PA) functions as a critical second messenger in the activation of NADPH oxidase in human PMN.
- Regulation of phospholipase D (PLD) activity is a key regulatory step in the NADPH oxidase activation pathway.
- These findings provide insights into the molecular mechanisms underlying neutrophil respiratory burst.
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