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首页> 外文期刊>Journal of the Association for Research in Otolaryngology: JARO >Permeation of fluorophore-conjugated phalloidin into live hair cells of the inner ear is modulated by P2Y receptors
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Permeation of fluorophore-conjugated phalloidin into live hair cells of the inner ear is modulated by P2Y receptors

机译：P2Y受体调节荧光团缀合的鬼笔环肽渗透到内耳的活毛细胞中

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摘要

Phalloidin, a toxin isolated from the death cap mushroom, Amanita phalloides, binds to filamentous actin with high affinity, and this has made fluorophore-conjugated phalloidin a useful tool in cellular imaging. Hepatocytes take up phalloidin via the liver-specific organic anion transporting polypeptide 1b2, but phalloidin does not permeate most living cells. Rapid entry of styryl dyes into live hair cells has been used to evaluate function, but the usefulness of those fluorescence dyes is limited by broad and fixed absorption spectra. Since phalloidin can be conjugated to fluorophores with various spectra, we investigated whether it would permeate living hair cells. When we incubated mouse utricles in 66 nM phalloidin-CF488A and followed that by washes in phalloidin-free medium, we observed that it entered a subset of hair cells and labeled entire hair bundles fluorescently after 20 min. Incubations of 90 min labeled nearly all the hair bundles. When phalloidin-treated utricles were cultured for 24 h after washout, the label disappeared from the hair cells and progressively but heterogeneously labeled filamentous actin in the supporting cells. We investigated how phalloidin may enter hair cells and found that P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid and suramin, blocked phalloidin entry, while the P2Y receptor ligands, uridine-5'-diphosphate and uridine-5'-triphosphaste, stimulated uptake. Consistent with that, the P2Y6 receptor antagonist, MRS 2578, decreased phalloidin uptake. The results show that phalloidin permeates live hair cells through a pathway that requires metabotropic P2Y receptor signaling and suggest that phalloidin can be transferred from hair cells to supporting cells in culture.
机译：鬼笔环肽（一种从死菌蘑菇伞形毒蕈中分离出的毒素）以高亲和力与丝状肌动蛋白结合，这使荧光团缀合的鬼笔环肽成为细胞成像中的有用工具。肝细胞通过肝脏特异性有机阴离子转运多肽1b2吸收鬼笔环肽，但鬼笔环肽不会渗透到大多数活细胞中。苯乙烯基染料快速进入活毛细胞已被用于评估功能，但这些荧光染料的实用性受到宽而固定的吸收光谱的限制。由于鬼笔环肽可以与各种光谱的荧光团结合，因此我们研究了它是否会渗透到活的毛细胞中。当我们在66 nM鬼笔环肽-CF488A中孵育小鼠尿囊，然后在不含鬼笔环肽的培养基中洗涤时，我们观察到它进入了毛细胞的一部分，并在20分钟后荧光标记了整个发束。 90分钟的孵育几乎标记了所有的发束。当冲洗后用鬼笔环肽处理的细胞培养24小时时，标记从毛细胞中消失，在支持细胞中逐渐但异质地标记丝状肌动蛋白。我们研究了鬼笔环肽可能如何进入毛细胞，发现P2受体拮抗剂，吡ido醛磷酸盐-6-偶氮苯基2'，4'-二磺酸和苏拉明能阻止鬼笔环肽进入，而P2Y受体配体，尿苷5'-二磷酸和尿苷-5'-triphosphaste，刺激摄取。与此一致的是，P2Y6受体拮抗剂MRS 2578降低了鬼笔环肽的摄取。结果表明，鬼笔环肽通过需要代谢型P2Y受体信号传导的途径渗透到活的毛细胞中，这表明鬼笔环肽可以从毛细胞转移到培养的支持细胞中。

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