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Abstract：
We aimed to investigate the expression and motor modulatory roles of several mechano-sensitive channels (MSCs) in human ureter. Human proximal ureters were obtained from eighty patients subjected to nephrectomy. Expression of MSCs at mRNA, protein and functional levels were examined. Contractions of longitudinal ureter strips were recorded in organ bath. A fluorescent probe Diaminofluoresceins was used to measure nitric oxide (NO). RT-PCR analyses revealed predominant expression of Piezo1 and TRPV2 mRNA in intact ureter and mucosa. Immunofluorescence assays indicate proteins of MSCs (Piezo1/Piezo2, TRPV2 and TRPV4) were mainly distributed in the urothelium. Ca2+ imaging confirmed functional expression of TRPV2, TRPV4 and Piezo1 in cultured urothelial cells. Specific agonists of Piezo1 (Yoda1, 3-300 μM) and TRPV2 (cannabidiol, 3-300 μM) attenuated the frequency of ureteral contractions in a dose-dependent manner while the TRPV4 agonist GSK1016790A (100 nM-1 μM) exerted no effect. The inhibitory effects of Piezo1 and TRPV2 agonists were significantly blocked by the selective antagonists (Dooku 1 for Piezo1, Tranilast for TRPV2), removal of the mucosa, and pretreatment with NO synthase inhibitor L-NAME (10 μM). Yoda1 (30 μM) and cannabidiol (50 μM) increased production of NO in cultured urothelial cells. Our results suggest that activation of Piezo1 or TRPV2 evokes NO production and release from mucosa that may mediate mechanical stimulus-induced reduction of ureter contractions. Our findings support the idea that targeting Piezo1 and TRPV2 channels may be a promising pharmacological strategy for ureter stone passage or colic pain relief.
摘要：
我们旨在研究几种机械敏感通道（MSCs）在人输尿管中的表达和运动调节作用。从80例接受肾切除术的患者中获得了人近端输尿管。MSCsmRNA的表达，蛋白质和功能水平进行了检查。在器官浴中记录纵向输尿管条的收缩。使用荧光探针二氨基荧光素来测量一氧化氮(NO)。RT-PCR分析显示，完整输尿管和粘膜中Piezo1和TRPV2mRNA的主要表达。免疫荧光分析表明，MSCs的蛋白质（Piezo1/Piezo2，TRPV2和TRPV4）主要分布在尿路上皮中。Ca2+成像证实TRPV2、TRPV4和Piezo1在培养的尿路上皮细胞中的功能性表达。Piezo1（Yoda1，3-300μM）和TRPV2（大麻二酚，3-300μM）以剂量依赖性方式减弱了输尿管收缩的频率，而TRPV4激动剂GSK1016790A（100nM-1μM）没有作用。Piezo1和TRPV2激动剂的抑制作用被选择性拮抗剂（Dooku1用于Piezo1，Tranilast用于TRPV2）明显阻断，去除粘膜，并用NO合酶抑制剂L-NAME(10μM)预处理。Yoda1（30μM）和大麻二酚（50μM）增加了培养的尿路上皮细胞中NO的产生。我们的结果表明，Piezo1或TRPV2的激活会引起粘膜产生和释放NO，这可能会介导机械刺激引起的输尿管收缩减少。我们的发现支持这样的观点，即靶向Piezo1和TRPV2通道可能是输尿管结石通道或绞痛疼痛缓解的有希望的药理学策略。
