Surfactant Releases Internal Calcium Stores in Neutrophils by G Protein–Activated Pathway

Abstract

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca⁺⁺ stores and that PS+SAP induces depletion through release of Ca⁺⁺ stores and through inhibition of Ca⁺⁺ influx. Our model predicts that PS+SAP releases Ca⁺⁺ stores through insertion of channels, depolarization of neutrophils, and activation of a G protein–dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin—a channel protein with properties similar to those of PS+SAP—is expected to mimic these effects. Human neutrophils were monitored for [Ca⁺⁺] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca⁺⁺] responses were reexamined following preexposure to inhibitors of internal Ca⁺⁺ release or the G protein pathway. We observed that (i) 1% PS+SAP—but not PS-SAP—causes transient increase of neutrophil [Ca⁺⁺] within seconds of exposure; (ii) 1% PLG—but not gramicidin alone—closely mimics the effect of PS+SAP on Ca⁺⁺ response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca⁺⁺ stores releases or of G protein activation suppresses Ca⁺⁺ responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca⁺⁺ influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca⁺⁺ from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca⁺⁺ stores and by inhibiting Ca⁺⁺ influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.