Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Neurourol Urodyn. 2020 Jun;39(5):1321-1329. doi: 10.1002/nau.24377. Epub 2020 May 6.
While 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.
The 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).
Human 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.
Stimulation 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.
These 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.
尽管许多研究已经证实了 ATP 在膀胱生理学/病理生理学中的重要性，但关于从尿路上皮细胞释放 ATP 的机制，文献仍然存在争议。已经确定了多种机制，包括通过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 储备，可以对外界刺激（如 Toll 样受体（TLR）刺激）释放到细胞外。
本研究的目的是确定 TLR4 激动剂细菌脂多糖（LPS）刺激是否会引起尿路上皮细胞发生溶酶体胞吐。
用人永生化细胞系（TRT-HU1）的尿路上皮细胞用细菌 LPS（100 μg/ml）或烟碱激动剂 cytisine（100 μM）处理，并测量细胞外 ATP 和溶酶体酸性磷酸酶的释放。使用 Brilliant Blue FCF 抑制连接蛋白通道，用 Glycyl-l-phenylalanine-β-naphthylamide（GPN）破坏溶酶体来区分连接蛋白介导的 ATP 释放和溶酶体 ATP 释放。使用 LysoSensor 染料测量溶酶体 pH 值和 Fura-2 测量细胞内钙信号来检查控制溶酶体胞吐的机制。
LPS 刺激 TRT-HU1 细胞可显著增加 ATP 释放，该释放被 GPN 抑制，但不被 Brilliant Blue FCF 抑制。相反，用 cytisine 刺激诱导的 ATP 释放对 Brilliant Blue FCF 敏感，但对 GPN 不敏感。LPS 刺激还诱导了溶酶体酸性磷酸酶的释放。LPS 增加了溶酶体 pH 值，使用氯喹或巴弗洛霉素 A1 直接碱化溶酶体 pH 值可诱导 ATP 和酸性磷酸酶释放，表明 pH 值在溶酶体胞吐中起重要作用。此外，刺激溶酶体瞬时受体电位 mucolipin 1 钙通道可引发细胞内钙瞬变和 ATP 释放。
这些数据表明，LPS 诱导的尿路上皮细胞 ATP 释放是由溶酶体胞吐介导的，这是一种与通过连接蛋白通道的非囊泡释放明显不同的囊泡机制。
