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This Article Full Text Full Text (PDF) All Versions of this Article: 575/1/23    most recent jphysiol.2006.106351v2 jphysiol.2006.106351v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mazzone, S. B. Articles by Canning, B. J. Search for Related Content PubMed PubMed Citation Articles by Mazzone, S. B. Articles by Canning, B. J. Related Collections Cellular

¹ The Howard Florey Institute, University of Melbourne, Victoria 3010, Australia
² The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA
³ GlaxoSmithKline, Respiratory and Inflammation Centre of Excellence in Drug Discovery, 709 Swedeland Road, King of Prussia, PA 19406, USA

The fluorescent styryl dyes FM1-43 and FM2-10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular-independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1-43 and FM2-10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1-43 and FM2-10 bound with moderate affinity (23–220 nM) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1–M5). In functional studies in vitro, FM1-43 and FM2-10 inhibited electrical field stimulation (EFS) and acetylcholine-induced cholinergic contractions of guinea-pig tracheal strips (IC₅₀: FM1-43, 0.4 ± 0.1; FM2-10, 1.6 ± 0.1 µM; concentration of antagonist producing a 2-fold leftward shift in the acetylcholine concentration–response curve (K_b): FM1-43, 0.3 ± 0.1; FM2-10, 15.8 ± 10.1 µM). Neither compound inhibited EFS-evoked, non-adrenergic non-cholinergic nerve-mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole-mount preparations with 5 µM FM1-43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.

(Received 30 January 2006; accepted after revision 11 May 2006; first published online 25 May 2006)
Corresponding author B. J. Canning: Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA. Email: bjc{at}jhmi.edu