Hypothesis / aims of study
Urinary incontinence (UI) is the loss of urine due to a disruption of the normal micturition mechanism and decreases quality of life and overall health.
The urethra contributes to continence by contracting during bladder filling and relaxing during the voiding phase to permit bladder emptying. While neurogenic contraction of the urethra plays a role in continence, urethral smooth muscle contracts in the absence of neural input but the mechanism is still not fully understood[1]. Whether non-neuronal urethral contractions contribute to continence is also not known.
We recently found that serotonin drives smooth muscle contraction in ex vivo preparations of adult female mouse urethra. Urethral neuroendocrine cells (UNECs) are the major serotonin producing cell type in the urethra and synthesize serotonin (5-HT) via tryptophan hydroxylase 1 (TPH1). While it is not known how serotonin drives urethral contraction, serotonin drives colonic contraction by activating 5-HT2B receptor (HTR2B) and opening anoctamin-1 (ANO1) chloride channels on Interstitial cells of Cajal (ICCs), and depolarizing smooth muscle myocytes to which ICCs are electrically coupled[2].
We hypothesize that urethral (UNECs) release serotonin, activate HTR2B and open ANO1 channels on urethral ICCs to drive urethral contraction, and that serotonin is required for urinary continence in female mice.
Study design, materials and methods
All mice were housed in clear plastic cages containing corn cob bedding and maintained on a 12 h light and dark cycle at 22 ± 2°C.
Voiding behaviors in six- to ten-week-old (adult) female C57BL/6J wild type (WT) and Tph1 knock out (KO) mice were examined during a four-hour monitoring period using the void spot assay. Void spot assay data for three mice per genotype were analyzed using Void Whizzard. Mice were then anesthetized, a catheter was placed across the bladder wall and saline was infused while intravesicular pressure was recording during a one hour period. Cystometrograms were recorded for three mice per genotype and analyzed using LabChart software (ADInstruments). Five consecutive voids were analyzed per animal. Statistical analysis was conducted using GraphPad Prism software version 9.2.
Urethras were collected from six- to ten-week-old (adult) female C57BL/6J WT mice, a 2 mm urethral ring was excised and mounted under a resting tension of 2 mN in a 5 mL wire myography chamber containing Krebs solution maintained at 37 °C (pH 7.4) and aerated with 95% O2,5% CO2. Urethral rings were equilibrated for 1 h and graded concentrations of the following drugs were added: serotonin (5-HT; 0.1-30 µM), HTR2B agonist BW723C86 hydrochloride (1-100 µM), and ANO1 agonist Eact (1-50 µM). Urethral rings were then washed with drug-free Krebs, tensioned with 5-HT (30 µM), and exposed to the HTR2B antagonist SB 204741 (0.1-100 µM), the ANO1 antagonist Ani9 (1-50 µM) and the ICC antagonist Imatinib (100 µM). The contractile force of urethral rings was collected using a PowerLab 400™ Data Acquisition System (Software Chart, version 6.0, AD Instrument, Milford, MA, USA) and analyzed using GraphPad Prism software version 9.2.
Results
The preliminary results showed that Tph1 KO adult female mice deposited significantly more urine spots in the center of the enclosure (31.73% ± 1.0 ) than WT mice (0.5872% ± 0.1, p<0.0001, Fig.1A). Tph1 KO mice also deposited significantly fewer urine spots in the corners of the enclosure (46.64% ± 4.2 ) than wild type mice (94.41% ± 1.2, p=0.0305, Fig.1B) .
Tph1 KO adult female mice had significantly more non-voiding contractions (2.6 ± 1.0) than wild type mice (0.4 ± 0.4, p=0.0395, Fig.1C)
5-HT (Fig. 2A), BW723C86 hydrochloride (Fig. 2B) and Eact (Fig. 2C) drove concentration-dependent contractions in adult female WT mouse urethra, while SB 204741 (Fig. 2D) and Ani9 (Fig. 2E) drove concentration dependent relaxation in adult female WT mouse urethral rings pre-tensioned with serotonin. Also, Imatinib mesylate (Fig. 2F) relax adult female WT mouse urethral rings pre-contracted with serotonin.
Interpretation of results
We have shown that serotonin synthesized by TPH1 is required for the normal voiding cycle and that adult female Tph1 KO mice void more frequently in the center and less frequently in the corners of their enclosures, a behavior consistent with urinary incontinence. Adult female Tph1 KO mice also generate significantly more non-voiding contractions during bladder filling than wild type mice. Non-voiding contractions usually are myogenic and a potential sign of detrusor overactivity, although the causes are not well understood. Detrusor overactivity is a primary mechanism of overactive bladder (OAB). A study [3] demonstrated that a deficiency in serum 5-HT is independently associated with OAB and that urethral instability (experienced by 42% of patients with OAB) may be associated with urge incontinence.
Using ex vivo tissue baths, we have also shown that 5-HT and agonists of HTR2B and ANO1 induce urethral contraction while antagonists of HTR2B and ANO1, as well as Imatinib mesylate relax adult female WT mouse urethras pre-contracted with serotonin. Our results are consistent with a mechanism in which UNECs secrete serotonin, which binds HTR2B and activates ANO1 in ICCs to drive urethral contraction.
Concluding message
The combined results, in this preliminary study, from the in vivo and in vitro experiments showed that serotonin is by itself sufficient to mediate contraction of the mouse urethra. Serotonin also appears to be required for continence in mice, and mice deficient in Tph1 (and thereby deficient in serotonin biosynthesis outside of the central nervous system) appear to have bladder overactivity and incontinence. Further study is warranted to clarify a possible causal relationship between urinary continence, 5-HT, and urethral tonus.