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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on April 20, 2005; DOI: 10.1124/jpet.104.079822

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Received for publication November 16, 2004.
Revised April 8, 2005.
Accepted for publication April 11, 2005.

Cigarette Smoke Extract Increases C5a Receptor Expression in Human Bronchial Epithelial Cells

Diane S. Allen-Gipson 1*, Anthony A. Floreani 1, Art J. Heires 1, Sam D. Sanderson 1, Richard G. MacDonald 1, Todd A. Wyatt 1

1 University of Nebraska Medical Center

* Address correspondence to: E-mail: dallengipson{at}unmc.edu

Abstract

We have shown that exposing human bronchial epithelial cells (HBECs) to 5% cigarette smoke extract (CSE) upregulates C5aR expression as determined by flow cytometry (FACS) and immunohistochemistry. In this study, we conducted whole-cell saturation studies to quantitate receptor number. After exposing a human bronchial epithelial cell line (BEAS-2B) to CSE, radiolabeled C5a bound saturably with Kd = 2.71 ± 1.03 nM; n=4 and Bmax = 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 x 10-16 M; Ki2=1.60 x 10-9 M). BEAS-2Bs were transfected with wild-type (WT) or mutant dominant-negative (DN) protein kinase C-alpha (PKC-{alpha}) to investigate the relationship between PKC-a and C5aR availability and affinity. Western blot analysis revealed a 75 kDa lysate band from cells expressing both WT and DN PKC-{alpha}, but DN cells exposed to 5% CSE had no functional PKC activity. Pretreatment with Gö6976 (PKC-{alpha} inhibitor) had no effect on DN but significantly decreased WT PKC activity. Competitive binding studies conducted on either WT or DN PKC-{alpha} 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 exposure to CSE increases the availability of C5a binding sites. The data also indicate that PKC-{alpha} may play an important role in modulating C5aR binding.


Key words: C5a, G protein-coupled receptor family, bronchial epithelial cells, cigarette smoke extract, ligand binding, receptors


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[Abstract] [Full Text] [PDF]


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