title: P2X antagonists inhibit styryl dye entry into hair cells.
abstract: The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X(2). Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 microM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating "tip links" of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS], suramin, 2',3'-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 microM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 microM). At a concentration that also blocks P2X(2) receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.
authors.affiliation: Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan Medical School, 4605 MS II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5616, USA. crumling@umich.edu
authors.forename: M A
authors.initials: MA
authors.lastname: Crumling
authors.forename: M
authors.initials: M
authors.lastname: Tong
authors.forename: K L
authors.initials: KL
authors.lastname: Aschenbach
authors.forename: L Qian
authors.initials: LQ
authors.lastname: Liu
authors.forename: C M
authors.initials: CM
authors.lastname: Pipitone
authors.forename: R K
authors.initials: RK
authors.lastname: Duncan
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D002645:Chickens; D017630:Connexins / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006198:Hair Cells, Auditory / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D058919:Purinergic P2 Receptor Antagonists; D011726:Pyridinium Compounds / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: C507005:AM1-43 compound; D002614:Chelating Agents; D017630:Connexins; C073804:FM1 43; D005456:Fluorescent Dyes; D058919:Purinergic P2 Receptor Antagonists; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D018048:Receptors, Purinergic P2; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D004533:Egtazic Acid; D011732:Pyridoxal Phosphate; D013498:Suramin; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014403:Tubocurarine