LPS-mediated release of ATP from urothelial cells occurs by lysosomal exocytosis.
LPS-mediated release of ATP from urothelial cells occurs by lysosomal exocytosis.
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LPS 介导的尿路上皮细胞 ATP 释放是通过溶酶体胞吐作用发生的。
DOI：
10.1002/nau.24377
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发表时间：
2020
影响因子：
2
通讯作者：
Beckel,JonathanM
中科院分区：
文献类型：
--
作者：
Silberfeld,Andrew;Chavez,Brittany;Obidike,Chinonso;Daugherty,Stephanie;deGroat,WilliamC;Beckel,JonathanM
背景虽然许多研究已经证实了ATP在膀胱生理/病理生理学中的重要性，但关于ATP从膀胱释放的机制，文献仍然存在争议。已经确定了多种机制，包括通过泛连接蛋白通道的非囊泡释放以及通过肉毒杆菌毒素阻断的机制的囊泡释放。最近，已经表明溶酶体含有大量的ATP储存，其可以响应于Toll样受体（TLR）刺激而在细胞外释放。目前研究的目标是确定在尿路上皮细胞中是否响应于其激动剂的TLR 4刺激而发生溶酶体胞吐，材料和方法用细菌脂多糖（LPS）处理来自永生化细胞系（TRT-HU 1）的人尿路上皮细胞，（100 μg/ml）或烟碱激动剂金雀花碱（100 μM）的作用，并测定细胞外ATP和溶酶体酸性磷酸酶的释放。使用亮蓝FCF抑制泛连接蛋白通道和甘氨酰-l-苯丙氨酸-β-萘胺（GPN）破坏溶酶体，区分泛连接蛋白介导的ATP释放和溶酶体ATP释放。结果LPS刺激TRT‐ HU 1细胞后，TRT‐ HU 1细胞ATP释放明显增加，GPN可抑制ATP释放，而Brilliant Blue FCF对ATP释放无明显抑制作用。相反，用野靛碱刺激诱导ATP释放，其对亮蓝FCF敏感，但对GPN不敏感。LPS刺激还诱导溶酶体酸性磷酸酶的释放。LPS增加溶酶体pH和直接碱化溶酶体pH使用氯喹或巴弗洛霉素A1诱导的ATP和酸性磷酸酶释放，表明pH在溶酶体胞吐中的重要作用。此外，刺激溶酶体瞬时受体电位mucolipin 1钙通道诱发细胞内钙瞬变以及ATP release.ConclusionThese数据表明，LPS诱导的ATP释放从尿路上皮细胞是介导的溶酶体胞吐，囊泡机制明显独立于非囊泡释放通过泛连接蛋白通道。
BackgroundWhile numerous studies have confirmed ATP's importance in bladder physiology/pathophysiology, the literature is still conflicted regarding the mechanism of ATP release from the urothelium. Multiple mechanisms have been identified including non‐vesicular release via pannexin channels as well as vesicular release via a mechanism blocked by botulinum toxin. Recently, it has been shown that lysosomes contain significant stores of ATP which can be released extracellularly in response to Toll‐like receptor (TLR) stimulation.ObjectiveThe goal of the current study was to determine if lysosomal exocytosis occurs in urothelial cells in response to TLR4 stimulation by its agonist, bacterial lipopolysaccharide (LPS).Materials and MethodsHuman urothelial cells from an immortalized cell line (TRT‐HU1) were treated with bacterial LPS (100 μg/ml) or the nicotinic agoinist cytisine (100 μM) and extracellular release of ATP and lysosomal acid phosphatase were measured. Pannexin‐mediated ATP release and lysosomal ATP release were differentiated using Brilliant Blue FCF to inhibit pannexin channels and glycyl‐l‐phenylalanine‐β‐naphthylamide (GPN) to destroy lysosomes. The mechanisms controlling lysosomal exocytosis were examined using lysosomal pH measurements using LysoSensor dye and intracellular calcium signaling using Fura‐2.ResultsStimulation of TRT‐HU1 cells with LPS significantly increased ATP release, which was inhibited by GPN, but not by Brilliant Blue FCF. Conversely, stimulation with cytisine induced ATP release that was sensitive to Brilliant Blue FCF but not GPN. LPS stimulation also induced the release of the lysosomal acid phosphatases. LPS increased lysosomal pH and direct alkalization of lysosomal pH using chloroquine or bafilomycin A1 induced ATP and acid phosphatase release, indicating an important role for pH in lysosomal exocytosis. Additionally, stimulation of lysosomal transient receptor potential mucolipin 1 calcium channels evoked intracellular calcium transients as well as ATP release.ConclusionThese data indicate that LPS‐induced ATP release from urothelial cells is mediated by lysosomal exocytosis, a vesicular mechanism distinctly separate from non‐vesicular release via pannexin channels.