Chloride-dependent mechanisms contribute to urinary bladder smooth muscle contractility: Pharmacological and molecular evidence from rat bladder tissue

A BSTRACT Objectives: To investigate the functional role of chloride-dependent signaling pathways in urinary bladder smooth muscle (SM) contractility and to evaluate the expression of ClC-3 and CLCA4 in rat bladder tissue. Materials and Methods: Expression of ClC-3 and CLCA4 in rat bladder tissue was examined using Western blotting and immunohistochemistry. Functional studies were conducted using isolated bladder SM strips (2 mm × 2 mm × 10 mm) from adult male Wistar rats mounted in organ bath chambers for isometric tension recording. Potassium chloride (KCl)-induced contractions were assessed under varying extracellular chloride concentrations (138–8 mM) and after substitution with Br − or I − . Norepinephrine (NE)-induced contractions were evaluated with pretreatment of chloride transport inhibitors (bumetanide, HEPES without bicarbonate, and ethacrynic acid) and chloride channel blockers (4,4’-diisothiocyano-2,2’- stilbene-disulfonic acid, anthracene-9-carboxylic acid, and niflumic acid) at different concentrations. Results: ClC-3 and CLCA4 were expressed in both urothelial and SM layers. Reduction of extracellular chloride concentration significantly attenuated KCl-induced contractions in a concentration-dependent manner ( P < 0.01) and substitution with bromide or iodide produced similar effects. Chloride transport inhibitors and channel blockers significantly and dose-dependently suppressed NE-induced contractions (all P < 0.01). The inhibitory effects were consistently observed across multiple pharmacological interventions. Conclusion: The present study provides pharmacological and molecular evidence supporting a contributory role of chloride-dependent signaling in bladder SM contractility under ex vivo conditions. Expression of ClC-3 and CLCA4 in urothelial and SM layers further suggests that chloride-dependent regulation may involve multiple bladder compartments. Although the precise molecular mechanisms remain incompletely defined, these findings support further investigation into chloride-dependent signaling pathways in bladder physiology and dysfunction.