Clinically relevant osmolar stress inhibits priming-induced PMN NADPH oxidase subunit translocation
Forest R Sheppard1, Ernest E Moore, Nathan McLaughlin
1Department of Surgery, Denver Health Medical Center, University of Colorado School of Medicine, Denver, Colorado, USA.
Background:
The plasma membrane NADPH oxidase is responsible for the external generation of superoxide by neutrophils (polymorphonucleocytes [PMNs]). The oxidase is a multicomponent enzyme, active only when all subunits are translocated to and assembled at the membrane. We have recently demonstrated that platelet-activating factor (PAF) priming of PMNs translocates the cytosolic p67 subunit to the membrane position. Osmolar stress attenuates PAF priming of the oxidase. Consequently, we hypothesized that clinically relevant osmolar stress inhibits PAF priming-induced p67 translocation.
Methods:
Isolated human PMNs were incubated at 37 degrees C for 5 minutes in buffer or 180 mmol/L hypertonic saline (HTS) followed by 3 minutes of incubation with or without 2 mumol/L PAF (resting, PAF, HTS, and HTS-PAF). Digital microscopy was used to determine p67 location in whole PMNs. Subcellular fractions were prepared and membrane translocation of p67 determined by protein electrophoresis. Resting cytosol fractions were immunodepleted of p67 and NADPH oxidase activity measured using p67-deficient sodium dodecyl sulfate cell-free oxidase assays: resting, PAF, or HTS-PAF membrane (1 mug) was combined with immunodepleted resting cytosol (25 mug).
Results:
By all methodologies, PAF stimulated translocation of p67 to the PMN membrane and this translocation was prevented by osmolar stress (HTS-PAF). In cell-free oxidase assays, the membrane content of p67 after PAF stimulation was increased sufficiently to induce oxidase activity, whereas resting and HTS-PAF membrane did not (0.1 +/- 0.02, 0.23 +/- 0.04, and 0.14 +/- 0.04, respectively, p < 0.01) (resting versus HTS-PAF, no difference).
Conclusion:
PAF priming of the PMN oxidase involves translocation of p67 to the plasma membrane. Clinically relevant osmolar stress with hypertonic saline prevents this PAF-induced translocation of the p67 oxidase subunit. This finding provides new insight into the mechanisms responsible for osmolar control of PMN functional responses.
Insights
Osmolar stress, such as hypertonic saline, prevents platelet-activating factor (PAF) from moving the p67 subunit to the neutrophil plasma membrane. This finding explains how osmotic conditions regulate neutrophil oxidase function.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophil (polymorphonucleocyte [PMN]) superoxide generation relies on the plasma membrane NADPH oxidase complex.
- This enzyme requires translocation and assembly of its subunits at the membrane for activation.
- Platelet-activating factor (PAF) primes PMNs by translocating the p67 subunit to the membrane, but osmolar stress can impair this process.
Purpose of the Study:
- To investigate the hypothesis that clinically relevant osmolar stress inhibits PAF priming-induced p67 translocation.
- To elucidate the role of osmolar stress in regulating PMN oxidase activity.
Main Methods:
- Human PMNs were exposed to hypertonic saline (HTS) and/or PAF.
- Digital microscopy and subcellular fractionation with protein electrophoresis assessed p67 translocation.
- Cell-free oxidase assays were performed using p67-deficient cytosol and membrane fractions.
Main Results:
- PAF stimulation successfully translocated p67 to the PMN membrane.
- Osmolar stress (HTS) significantly prevented PAF-induced p67 translocation.
- Cell-free assays confirmed that HTS-treated membranes lacked sufficient p67 for oxidase activity.
Conclusions:
- PAF priming of the PMN oxidase is dependent on p67 translocation to the plasma membrane.
- Clinically relevant osmolar stress, specifically hypertonic saline, inhibits this crucial PAF-induced translocation.
- These findings offer novel insights into the mechanisms governing osmolar control of PMN functional responses.
