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Am J Physiol Cell Physiol 297: C886-C897, 2009. First published March 18, 2009; doi:10.1152/ajpcell.00416.2008
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RECEPTORS AND SIGNAL TRANSDUCTION

Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils

Phillip C. Eckels,1 Anirban Banerjee,2 Ernest E. Moore,1,2 Nathan J. D. McLaughlin,3 Lynn M. Gries,2 Marguerite R. Kelher,2 Kelly M. England,3 Fabia Gamboni-Robertson,2 Samina Y. Khan,1 and Christopher C. Silliman2,3,4

1Department of Surgery, Denver Health Medical Center; Departments of 2Surgery and 3Pediatrics, School of Medicine, University of Colorado Denver; and 4Bonfils Blood Center, Denver, Colorado

Submitted 12 September 2007 ; accepted in final form 11 March 2009

Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to >98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET+)] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans.

G protein-coupled receptor; clathrin-coated vesicle; polymorphonuclear neutrophils


Address for reprint requests and other correspondence: C. C. Silliman, Associate Medical Director, Research Dept., Bonfils Blood Center, 717 Yosemite St., Denver, CO 80230 (e-mail: christopher.silliman{at}ucdenver.edu).






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