Connexins 43 and 45 hemichannels mediate ATP release in the urinary bladder.

Abstract

Background: 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.

Objective: This study investigated the roles of Cx43 and Cx45 as ATP release channels in the urinary bladder.

Methods: 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 Ca²⁺ depletion was assessed in porcine urothelial, suburothelial, and detrusor muscle cells, as well as in the human RT4 cell line.

Results: 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 (Ca²⁺) 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 Ca²⁺ 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.

Conclusion: 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 Ca²⁺-sensitive pathways, providing new insights into bladder sensory mechanisms.