Urethral dysfunction contributes to conditions such as stress urinary incontinence (SUI), yet current treatments remain suboptimal (1). While urethral smooth muscle contraction is known to depend on intracellular calcium and calcium sensitisation pathways (2), the contribution of these mechanisms within the urethral mucosa remains unclear. While the functional role of mucosal layers in the bladder is well recognized (3), the contribution of the urethral mucosa to contractile function remains poorly defined and has received limited mechanistic investigation.  This study aimed to determine the role of extracellular and intracellular calcium and Rho-kinase–mediated calcium sensitisation on 5-HT–induced contraction of the urethral mucosa.

Isolated porcine urethral mucosa tissue strips were mounted in organ baths containing Krebs-bicarbonate solution, maintained at 37°C, and gassed with 95% O2 and 5% CO2. Tissue strips were allowed to equilibrate for 45 minutes with washout every 15 minutes. After a stable pattern of contractile activity was generated, contractile responses of isolated porcine urethral mucosal strips to cumulative concentrations of 5-HT (10 nmol/L – 100 μmol/L) were recorded. To assess underlying mechanisms, the strips were incubated in the absence and presence of inhibitors targeting calcium influx through L-type calcium channels (nifedipine, 1 μmol/L), intracellular calcium release (cyclopiazonic acid (CPA) and ryanodine, 10 μmol/L), and Rho-kinase (fasudil and Y-27632, 10 μmol/L). The tissue responses were recorded using isometric transducers via a Powerlab recording system and Labchart® software. Paired Student’s t-tests were performed, with p-values <0.05 considered statistically significant.

When subjected to 5-HT, the urethral mucosal strips generated a concentration-dependent increase in tone and frequency of phasic spontaneous contractions. Following inhibition of extracellular calcium influx with nifedipine the 5-HT-mediated tonic contractions were significantly attenuated in the urethral mucosa (p < 0.05). Additionally, the spontaneous contractions of the urethral mucosa were also completely abolished. When subjected to ryanodine or CPA (10 μmol/L), to inhibit intracellular calcium release, there was no effect on either tonic or phasic 5-HT-induced contractions of the urethral mucosa. In the presence of Rho-kinase inhibitors fasudil or Y-27632, there was a significant decrease in 5-HT mediated tonic contractions (p < 0.05) at higher concentrations of the agonist (100 μmol/L), while spontaneous contraction rate also decreased.

5-HT–induced contraction of the urethral mucosa is primarily dependent on extracellular calcium influx via L-type channels and is further modulated by Rho-kinase–mediated calcium sensitisation. Intracellular calcium release appears to play a minimal role.

To our knowledge, this is the first study to characterise intracellular signaling mechanisms underlying urethral mucosal contraction. Urethral mucosal contraction is dependent on L-type calcium influx and Rho-kinase–mediated sensitisation, but not intracellular calcium release, distinguishing it from classical smooth muscle regulation. These findings reveal a previously uncharacterised regulatory role of 5-HT in the urethral mucosa and highlight novel therapeutic targets for urethral dysfunction.

Yang B et al (2022) Medicina (Kaunas) 59:5
Rembetski B et al (2020) Am J Physiol Renal Rhysiol 318:496-505
Chau et al (2016) Auton Neurosci 200:49-56

Continence 19S (2026) 102657
DOI: 10.1016/j.cont.2026.102657