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ORIGINAL RESEARCH ARTICLE

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

Mark E. Boston^*, G. Christopher Frech, Enrique Chacon-Cruz, E. Stephen Buescher^, and David G. Oelberg^,,1

^* Pediatric Otolaryngology Department, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, and Center for Pediatric Research and Department of Pediatrics, Children’s Hospital of The King’s Daughters and Eastern Virginia Medical School, Norfolk, Virginia 23510

To whom requests for reprints should be addressed at ¹ Children’s Hospital of The King’s Daughters, Division of Neonatal Medicine, 601 Children’s Lane, Norfolk, VA 23507. E-mail: doelberg{at}chkd.com

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.

Key Words: surfactant • neutrophils • calcium • gramicidin • G proteins