title: Intact soluble P-glycoprotein is secreted by sinonasal epithelial cells.
abstract: BACKGROUND
  P-glycoprotein (P-gp) is a 170 kDa transmembrane efflux pump, which is upregulated in chronic rhinosinusitis. Studies of leukemia demonstrated that P-gp may also be secreted in an intact soluble form. The purpose of this study was to explore whether sinonasal epithelial cells were capable of secreting soluble P-gp and whether P-gp has any functional role.
  
  
  METHODS
  Soluble and cytoplasmic P-gp were quantified in vehicle and lipopolysaccharide exposed cultures by enzyme-linked immunosorbent assay. The molecular weight of the soluble P-gp was determined by Western blot. Naive cultures were exposed to recombinant human P-gp at 0-2000 ng/mL. The degree of membranous interpolation was determined by quantitative fluorescent immunocytochemistry and function was determined by a calcein acetoxymethyl ester assay.
  
  
  RESULTS
  Soluble P-gp was secreted intact at 170 kDa. Mean (standard deviation) secretion was detected within vehicle wells at 55.43 ± 26.26 ng/mL, which significantly increased to 333.27 ± 305.98 ng/mL (p < 0.001) after lipopolysaccharide stimulation. Soluble P-gp strongly and significantly correlated with cytoplasmic P-gp (r = 0.57, p = 0.000001). Exposure to 2000 ng/mL of recombinant P-gp significantly increased corrected total cell fluorescence (1.34 ± 1.85) relative to vehicle control 0.29 ± 0.26 (p = 0.01) and significantly reduced calcein acetoxymethyl ester fluorescence (82.03 ± 43.69) relative to 100 ng/mL recombinant P-gp exposed cells (123.11 ± 42.16, p = 0.001).
  
  
  CONCLUSIONS
  Cultured sinonasal epithelial cells were able to both secrete intact P-gp and could functionally interpolate soluble P-gp into their cell membrane. These in vitro findings indicated that soluble P-gp may be present in nasal mucus as a biomarker and could participate in the maintenance of P-gp overexpression in chronic rhinosinusitis and associated inflammation.
authors.affiliation: Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Angela L
authors.initials: AL
authors.lastname: Nocera
authors.forename: Ana T
authors.initials: AT
authors.lastname: Meurer
authors.forename: Amy
authors.initials: A
authors.lastname: Singleton
authors.forename: Christopher
authors.initials: C
authors.lastname: Simons
authors.forename: Joseph
authors.initials: J
authors.lastname: BuSaba
authors.forename: Nicole
authors.initials: N
authors.lastname: Tara Gass
authors.forename: Xue
authors.initials: X
authors.lastname: Han
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Bleier
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D009297:Nasal Mucosa / Q000166:cytology / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008070:Lipopolysaccharides