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Fluorescent styryl dyes FM1‐43 and FM2‐10 are muscarinic receptor antagonists: intravital visualization of receptor occupancy
Author(s) -
Mazzone Stuart B.,
Mori Nanako,
Burman Miriam,
Palovich Michael,
Belmonte Kristen E.,
Canning Brendan J.
Publication year - 2006
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2006.106351
Subject(s) - muscarinic acetylcholine receptor , acetylcholine , cholinergic , chemistry , muscarinic acetylcholine receptor m5 , muscarinic acetylcholine receptor m2 , muscarinic acetylcholine receptor m3 , receptor , muscarinic acetylcholine receptor m1 , acetylcholine receptor , pharmacology , biochemistry , biology , endocrinology
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 n m ) 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 (IC 50 : FM1‐43, 0.4 ± 0.1; FM2‐10, 1.6 ± 0.1 μ m ; 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 μ m ). 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 μ m 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.