Abstract

We have shown that exposing human bronchial epithelial cells (HBECs) to 5% cigarette smoke extract (CSE) up-regulates C5a anaphylatoxin receptor (C5aR) expression as determined by flow cytometric analysis and immunohistochemistry. In this study, we conducted whole-cell saturation studies to quantitate the receptor number. After exposing an HBEC line (BEAS-2B) to CSE, radiolabeled C5a bound saturably with Kd = 2.71 ± 1.03 nM (n = 4) and B max = 15,044 ± 5702 receptors/cells. Without 5% CSE, no C5a binding was detected. Competitive binding studies revealed two classes of sites with distinct affinities for C5a (Ki1 = 3.28 × 10-16 M; Ki2 = 1.60 × 10-9 M). BEAS-2Bs were transfected with wild-type (WT) or mutant dominant-negative (DN) protein kinase C-α (PKC-α) to investigate the relationship between PKC-α and C5aR availability and affinity. Western blot analysis revealed a 75-kDa lysate band from cells expressing WT and DN PKC-α, but DN cells exposed to 5% CSE had no functional PKC activity. Pretreatment with Gö6976 [12-(2-cyanoethyl)-6, 7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (PKC-α inhibitor) had no effect on DN but significantly decreased WT PKC activity. Competitive binding studies conducted on either WT or DN PKC-α-transfected cells also revealed two classes of binding sites for C5a having different affinities. There was a significant rightward shift of the binding curve when WT cells were pretreated with Gö6976. These data suggest that C5aR is detectable on bronchial epithelial cells exposed to CSE and that exposure to CSE increases the availability of C5a binding sites. The data also indicate that PKC-α may play an important role in modulating C5aR binding.

Original languageEnglish (US)
Pages (from-to)476-482
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume314
Issue number1
DOIs
StatePublished - Jul 1 2005

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Anaphylatoxin C5a Receptor
Smoke
Tobacco Products
Protein Kinase C
Epithelial Cells
Competitive Binding
Binding Sites
Protein C Inhibitor
Protein Kinase Inhibitors
Up-Regulation
Western Blotting
Immunohistochemistry
Cell Line

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

Cigarette smoke extract increases C5a receptor expression in human bronchial epithelial cells. / Allen-Gipson, Diane S.; Floreani, Anthony A.; Heires, Art J.; Sanderson, Sam D.; MacDonald, Richard G; Wyatt, Todd A.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 314, No. 1, 01.07.2005, p. 476-482.

Research output: Contribution to journalArticle

Allen-Gipson, Diane S. ; Floreani, Anthony A. ; Heires, Art J. ; Sanderson, Sam D. ; MacDonald, Richard G ; Wyatt, Todd A. / Cigarette smoke extract increases C5a receptor expression in human bronchial epithelial cells. In: Journal of Pharmacology and Experimental Therapeutics. 2005 ; Vol. 314, No. 1. pp. 476-482.
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abstract = "We have shown that exposing human bronchial epithelial cells (HBECs) to 5{\%} cigarette smoke extract (CSE) up-regulates C5a anaphylatoxin receptor (C5aR) expression as determined by flow cytometric analysis and immunohistochemistry. In this study, we conducted whole-cell saturation studies to quantitate the receptor number. After exposing an HBEC line (BEAS-2B) to CSE, radiolabeled C5a bound saturably with Kd = 2.71 ± 1.03 nM (n = 4) and B max = 15,044 ± 5702 receptors/cells. Without 5{\%} CSE, no C5a binding was detected. Competitive binding studies revealed two classes of sites with distinct affinities for C5a (Ki1 = 3.28 × 10-16 M; Ki2 = 1.60 × 10-9 M). BEAS-2Bs were transfected with wild-type (WT) or mutant dominant-negative (DN) protein kinase C-α (PKC-α) to investigate the relationship between PKC-α and C5aR availability and affinity. Western blot analysis revealed a 75-kDa lysate band from cells expressing WT and DN PKC-α, but DN cells exposed to 5{\%} CSE had no functional PKC activity. Pretreatment with G{\"o}6976 [12-(2-cyanoethyl)-6, 7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (PKC-α inhibitor) had no effect on DN but significantly decreased WT PKC activity. Competitive binding studies conducted on either WT or DN PKC-α-transfected cells also revealed two classes of binding sites for C5a having different affinities. There was a significant rightward shift of the binding curve when WT cells were pretreated with G{\"o}6976. These data suggest that C5aR is detectable on bronchial epithelial cells exposed to CSE and that exposure to CSE increases the availability of C5a binding sites. The data also indicate that PKC-α may play an important role in modulating C5aR binding.",
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AU - Floreani, Anthony A.

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AU - MacDonald, Richard G

AU - Wyatt, Todd A

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