Sana-Ur-Rehman Hafiz, Markus Irit, Moalem-Taylor Gila, Moore Kate H, Mansfield Kylie J, Liu Lu
Department of Pharmacology, School of Biomedical Sciences, University of New South Wales, Sydney, New South Wales, NSW 2052, Australia.
Translational Neuroscience Facility, School of Biomedical Sciences, University of New South Wales, Sydney, New South Wales, NSW 2052, Australia.
Bladder (San Franc). 2025 Aug 21;12(3):e21200056. doi: 10.14440/bladder.0125. eCollection 2025.
Connexin (Cx) proteins form gap junctions between adjacent cells to facilitate intercellular communication and also assemble into hemichannels that release small molecules, including adenosine triphosphate (ATP), into the extracellular microenvironment, where ATP acts on purinergic receptors.
This study investigated the roles of Cx43 and Cx45 as ATP release channels in the urinary bladder.
Porcine bladder tissues and cultured cells were stained for Cx43 and Cx45 using immunofluorescence. Cx43- and Cx45-mediated ATP release in response to hypotonic stretch and extracellular Ca depletion was assessed in porcine urothelial, suburothelial, and detrusor muscle cells, as well as in the human RT4 cell line.
The expression of Cx43 and Cx45 was Immunohistochemically confirmed in porcine bladder tissue, cultured porcine bladder urothelial cells, suburothelial myofibroblasts, detrusor muscle cells, and the human urothelial RT4 cell line. Hypotonic stretch increased ATP release in all four cell types, with porcine urothelial cells exhibiting a 3.8 ± 1.3-fold and RT4 cells a 2.0 ± 0.5-fold increase relative to control levels. Similarly, depletion of extracellular calcium ions (Ca) stimulated ATP release from porcine urothelial cells and RT4 cells, yielding 5.4 ± 2.9-fold and 2.4 ± 0.8-fold increases, respectively. Blockade of Cx43 channels with a Cx43 mimetic peptide (peptide 5) and Cx45 channels with a Cx45 mimetic peptide reduced ATP release induced by stretch and Ca depletion in porcine urothelial cells by 50% and 67%, respectively. These blockers also reduced ATP release in RT4 cells. The contributions of Cx43 and Cx45 to ATP release were less prominent in suburothelial and detrusor muscle cells compared to urothelial cells.
These findings highlighted ATP's role as an autocrine/paracrine signaling molecule acting on purinergic receptors during bladder distension and suggested that Cx hemichannels regulate ATP release through mechanotransduction and Ca-sensitive pathways, providing new insights into bladder sensory mechanisms.
连接蛋白（Cx）形成相邻细胞间的间隙连接以促进细胞间通讯，还能组装成半通道，将包括三磷酸腺苷（ATP）在内的小分子释放到细胞外微环境中，ATP在其中作用于嘌呤能受体。
本研究探讨Cx43和Cx45作为膀胱中ATP释放通道的作用。
使用免疫荧光法对猪膀胱组织和培养细胞进行Cx43和Cx45染色。在猪尿路上皮细胞、尿路上皮下层细胞、逼尿肌细胞以及人RT⁴细胞系中，评估Cx43和Cx45介导的ATP释放对低渗牵张和细胞外钙耗竭的反应。
免疫组织化学证实Cx43和Cx45在猪膀胱组织、培养的猪膀胱尿路上皮细胞、尿路上皮下层肌成纤维细胞、逼尿肌细胞以及人尿路上皮RT⁴细胞系中均有表达。低渗牵张增加了所有四种细胞类型中的ATP释放，相对于对照水平，猪尿路上皮细胞增加了3.8±1.3倍，RT⁴细胞增加了2.0±0.5倍。同样，细胞外钙离子（Ca）耗竭刺激了猪尿路上皮细胞和RT⁴细胞释放ATP，分别增加了5.4±2.9倍和2.4±0.8倍。用Cx43模拟肽（肽5）阻断Cx43通道和用Cx45模拟肽阻断Cx45通道，可使猪尿路上皮细胞中由牵张和钙耗竭诱导的ATP释放分别减少50%和67%。这些阻滞剂也减少了RT⁴细胞中的ATP释放。与尿路上皮细胞相比，Cx43和Cx45对尿路上皮下层和逼尿肌细胞中ATP释放的贡献不那么显著。
这些发现突出了ATP在膀胱扩张过程中作为作用于嘌呤能受体的自分泌/旁分泌信号分子的作用，并表明Cx半通道通过机械转导和钙敏感途径调节ATP释放，为膀胱感觉机制提供了新的见解。
