Abstract
To clarify the role of serotonin (5-HT) in the prevention of stress urinary incontinence (SUI) during sneezing, we investigated the effect of intraperitoneal application of p-chlorophenylalanine (PCPA; a serotonin synthesis inhibitor) and intravenous application of CP-809101 (a 5-HT2C agonist) or LP44 (a 5-HT7 agonist) using female rats, in which the neurally evoked continence reflex during sneezing was examined. Amplitudes of urethral pressure response during sneezing (A-URS), urethral baseline pressure (UBP) at the middle urethra, and sneeze-induced leak point pressure (S-LPP) were measured in normal female adult rats with or without drug administration. PCPA decreased A-URS by 35.1 cmH2O and UBP by 13.3 cmH2O compared with normal rats. In PCPA-administrated rats, CP-809101 increased A-URS by 24.1 cmH2O and UBP by 15.1 cmH2O, and LP44 also increased A-URS by 20.6 cmH2O and UBP by 11.4 cmH2O compared with rats treated with PCPA alone. SUI was observed with S-LPP of 40.1 cmH2O in PCPA-administrated rats, in which CP-809101 and LP44 increased S-LPP by 28.0 and 15.2 cmH2O, respectively, compared with rats treated with PCPA alone. The effects of CP-809101 and LP44 were antagonized by SB-242084 (a selective 5-HT2C antagonist) and SB-269970 (a selective 5-HT7 antagonist), respectively. These results indicate that activation of 5-HT receptors enhances the active urethral closure reflex during sneezing, at least in part via 5-HT2C and 5-HT7 receptors.
Keywords: neural pathway, rats, serotonin, urinary incontinence
INTRODUCTION
Stress urinary incontinence (SUI) is the most common type of women’s urinary incontinence after middle age. SUI involves involuntary leakage of urine in response to abdominal pressure elevation during stress conditions including sneeze and cough. Urethral hypermobility and intrinsic sphincter deficiency are thought to be two main conditions found in patients with SUI (11).
The mechanisms to maintain urinary continence during abdominal pressure elevation include both passive and active closure of the urethra. A passive closure mechanism involves the essentially simultaneous transmission of intra-abdominal pressure to the urinary bladder and proximal urethra and has been considered to play an important role in urinary continence (3). Although the maximal urethral closure pressure value of the urethral pressure profile measurement corresponds to the urethral pressure at rest, the active, neurally mediated urethral closure during abdominal stress conditions acts as another important mechanism controlling the urethral continence function. First, urethral pressure reportedly increases before cough transmission. Second, the urethral pressure increases during coughing exceeds the increase in bladder pressure. Third, the urethral closure pressure during coughing is significantly reduced by bilateral pudendal nerve blockade (20–22). Our previous studies established a rat model that can be used to examine sneeze-induced active urethral closure mechanisms that are mediated by somatic nerve-induced reflex contractions of external urethral sphincter and pelvic floor striated muscles (10) and that these active urethral closure mechanisms during sneezing are impaired in a rat model of SUI induced by simulated birth trauma (9, 24).
The spinal serotonergic pathways have been reported to be involved in the control of urethral continence reflexes. We also previously demonstrated that serotonin (5-HT) receptor subtypes, 5-HT1A or 5-HT2C, respectively, reduce or enhance the urethral continence reflex during sneezing in rats (16). However, because there are other multiple excitatory and inhibitory 5-HT receptor subtypes, the overall effects of the 5-HT system or the subtype-specific mechanism in the control of the urethral function remain to be elucidated. Moreover, 5-HT7 receptors are shown to be expressed in the spinal cord and dorsal root ganglia of rats (4) although the role of 5-HT7 receptors in the urethral continence reflex is not well elucidated. Therefore, in this study, we examined the impacts of 5-HT depletion induced by p-chlorophenylalanine (PCPA) and the effects of activation of 5-HT receptor subtypes such as 5-HT2C and 5-HT7 on urethral baseline activity and reflex contractions during sneezing in PCPA-pretreated rats.
MATERIAL AND METHODS
Animals.
Sixty adult female Sprague-Dawley rats weighing 228–270 g were used. They were housed in temperature controlled conditions under a 12:12-h light-dark cycle (light on at 0700) with libitum access to water and standard food for 24 h. The experimental protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Experiments were performed in normal rats and rats with 5-HT-depletion induced by PCPA.
PCPA injection.
Saline (10 ml/kg body wt as a single daily dose, n = 12) or PCPA (200 mg/10 ml/kg body wt as a single daily dose, n = 48) was administered intraperitoneally for 2 days according to the previous report showing that systemic pretreatment of PCPA for 2 days resulted in 95% depletion in 5-HT in the brain (25).
Surgical procedures.
While rats were under isoflurane anesthesia, the urinary bladder was exposed through an abdominal incision, and ureters were cut bilaterally and their distal ends were ligated. The visceral branches of pelvic nerves were cut bilaterally near internal iliac vessels to prevent the bladder-to-urethra reflex induced by bladder distension (10, 13). Feces were removed from the distal colon through a small incision of the colon wall. A handmade small balloon catheter with a 1-cm diameter-size balloon connected to a pressure transducer (Transbridge 4M; World Precision Instruments, Sarasota, FL) was then inserted through the rectum into the abdominal cavity to record abdominal pressure (Pabd) during sneezing in experimental protocols 1 and 2. Additionally, a polyethylene catheter (PE-90; Clay Adams, Parsippany, NJ) connected to the pressure transducer was inserted into the bladder through the dome to record intravesical pressure to detect leak point pressure (LPP) during sneezing in the experimental protocol 2. Then, the abdomen was closed with sutures. A polyethylene catheter (PE-10; Clay Adams) was inserted into a jugular vein for drug injection in experimental protocols 1 and 2. After surgery, isoflurane anesthesia was turned off and replaced with urethane anesthesia (0.5 g/kg ip; Sigma, St. Louis, MO), and additional doses of anesthetic (0.02 g/kg per injection) were administered as required before starting sneeze reflex testing to obtain sufficient levels of anesthesia, which was confirmed by negative reflex responses to toe pinch. The final dose of urethane ranged from 0.6 to 0.7 g/kg among animals. Rats were then placed in a spine position for the experiments.
Sneeze reflex and sneeze-induced urethral continence reflex.
We induced the sneeze reflex by gently inserting a rat’s whisker into the nostril under urethane anesthesia. Sneeze induces a urethral pressure increase, which is elicited by reflex contractions of external urethral sphincter and pelvic floor muscles (10). We previously examined this active urethral closure mechanism during sneezing in rats and reported that the reflex is mediated by somatic nerves, occurs only at the middle portion of the urethra, and is not affected by abdominal opening (10).
Experimental protocol 1: effects of PCPA, CP-809101, or LP44 on middle urethral pressure responses and baseline pressure.
After abdominal and jugular catheters insertion, the bladder was emptied and a 3.5-Fr-size nylon catheter with a side-mounted microtransducer located 1 mm from the catheter tip (SPR-524; Millar Instruments, Houston, TX) was inserted into the urethra from the urethral orifice. The side-mounted sensor was positioned to face the inner urethral surface in the 3-o’clock direction, because measurement in a lateral orientation corresponds most closely to urethral pressure in human studies (1). The microtransducer-tipped catheter was connected to a pressure transducer (Transbridge 4M; World Precision Instruments), and urethral responses were recorded using data-acquisition software (sampling rate was 400 Hz; Chart; AD Instruments, Castle Hill, NSW, Australia) on a computer system equipped with an analog-to-digital converter (PowerLab; AD Instruments). By monitoring changes in urethral pressure on the computer screen, the tip of a microtransducer-tipped catheter was fixed at the middle urethra (inserted catheter length; 10–15 mm from the urethral orifice) where the highest urethral pressure was observed (10). Throughout the experiments, the catheter position was monitored to confirm that the location of the transducer had not changed. Sneezes were then induced by a whisker in the nostril, and the amplitude of urethral pressure responses during sneezing (A-URS) and urethral baseline pressure (UBP) were measured. A-URS represents the maximal pressure change in centimeters of water (H2O) from the baseline. The averaged UBP was obtained from a plateau section of pressure recording just before the sneeze response according to our previous reports (8, 15, 16). Representative traces of urethral pressure responses measured by a microtransducer-tipped catheter are shown in Fig. 1. Predrug sneeze-induced responses were measured before intravenous application of a 5-HT2C agonist (CP-809101; Tocris, Cookson, Ellisville, MO), a 5-HT2C antagonist (SB-242084; Sigma), a 5-HT1A antagonist (WAY-100635; Sigma), a 5-HT7 agonist (LP44; Cayman, Ann Arbor, MI), and a 5-HT7 antagonist (SB-269970; Sigma) in PCPA-pretreated rats. WAY-100635 was administered before LP44 administration to suppress the partial 5-HT1A effect of LP44. We induced sneeze reflexes again 30 min after drug application of each antagonist or agonist for evaluation of the effect of each drug on both A-URS and UBP, which required another 15 min. Therefore, the interval between antagonist and agonist applications was ~45 min. We evoked multiple sneeze reflexes to obtain at least 10 measurable responses before and after drugs administrations. To evaluate the intensity of the induced sneeze, which varied with each sneeze event, pressure increases in Pabd during sneezing were also measured during a sneeze event via an intra-abdominal balloon catheter inserted through the rectum. Sneeze-induced increases in Pabd were measured from the baseline to the peak of the pressure responses as reported in our previous reports (8, 15, 16).
Fig. 1.
Representative traces of urethral (top) and abdominal pressure (bottom) recordings during sneeze-induced urethral continence reflexes in a normal rat (A) and a p-chlorophenylalanine (PCPA)-pretreated rat (B). C: measuring methods of urethral baseline pressure (UBP), amplitudes of urethral pressure response during sneezing (A-URS), and abdominal pressure (Pabd). A-URS (a) and Pabd (b) represent the maximal pressure change in cmH2O from the baseline during sneezing. The averaged UBP was obtained from a plateau section of urethral pressure recording just before the sneeze event. Note that, in the normal rat (A), large urethral pressure elevations (top) were observed during low-intensity sneeze events, which are shown by relatively low elevations in abdominal pressure during sneezing (bottom), whereas urethral pressure elevations (top) are lower in the PCPA-pretreated rat (B) than in the normal rats even at high-intensity sneeze events with higher abdominal pressure elevations (bottom).
Experimental protocol 2: effects of PCPA, CP-809101, or LP44 on sneeze-induced LPP.
After the insertion of an abdominal catheter, a bladder catheter, and a jugular catheter, the bladder was emptied and 0.4 ml of saline solution containing Evans blue (100 μg/ml; Sigma) were injected into the bladder. We induced sneeze reflexes to examine whether fluid leakage from the urethral orifice was induced by sneezing. Intravesical pressure changes were recorded to monitor an increase in Pabd during sneezing using the Chart and PowerLab systems. We induced sneeze reflexes at least 50 times to obtain large sneezes with high intravesical pressures sufficient to induce fluid leakage from the urethral orifice. The maximal intravesical pressure was measured during each sneeze event, and the lowest pressure value that induced fluid leakage from the urethral orifice was defined as the sneeze-induced leak point pressure (S-LPP). Predrug sneeze-induced responses were measured before intravenous application of a 5-HT2C agonist (CP-809101; Tocris), a 5-HT2C antagonist (SB-242084; Sigma), a 5-HT1A antagonist (WAY-100635; Sigma), a 5-HT7 agonist (LP44; Cayman), and a 5-HT7 antagonist (SB-269970; Sigma) in PCPA-pretreated rats. We induced sneeze reflexes again 30 min after application of each antagonist or agonist for evaluation of the effect of each drug on sneeze-induced fluid leakage and S-LPP, which required another 15 min. Thus the interval between antagonist and agonist applications during S-LPP testing was ~45 min.
Drugs administrations.
PCPA (200 mg·10 ml−1·kg body wt−1 as a single daily dose) was administered intraperitoneally for 2 days based on a previous study (25). CP-809101 (0.3 mg/kg), SB-242084 (0.1 mg/kg), WAY-100635 (0.1 mg/kg), LP44 (0.3 mg/kg), and SB-269970 (0.1 mg/kg) were dissolved in distilled water and administered in doses based on results of previous reports (2, 5–7, 12, 19) and our own preliminary experiments. For intravenous application, 500 μl of drug solution were given via the implanted jugular catheter and flushed by 500 μl of saline. In preliminary experiments, we confirmed intravenous saline injection had no effect on urethral activity during sneezing. To examine the effects of intravenous drugs, sneeze was induced 30 min after each drug administration.
Statistical analysis.
Data are expressed as means ± SE. The values of the A-URS and UBP as well as increases in Pabd during sneezing were averaged in each rat. The means ± SE in a group of animals were then calculated from the averaged value in each rat. Unpaired t-test was used to compare the A-URS, UBP, and Pabd between normal and PCPA-pretreated rats. A paired t-test was also used to compare the values before and after CP-809101 administration. One-way ANOVA followed by Bonferroni’s multiple comparison tests were used to compare before and after administrations of multiple drugs. Statistical significance was set at P < 0.05.
RESULTS
Experimental protocol 1: effects of PCPA, CP-809101, or LP44 on middle urethral pressure responses and baseline pressure.
Two-day pretreatment of PCPA (n = 5) significantly decreased A-URS from 71.8 ± 7.1 to 36.8 ± 4.3 cmH2O (P < 0.01), and UBP from 31.1 ± 3.0 to 17.8 ± 2.2 cmH2O (P < 0.01) compared with normal rats (n = 6) (Figs. 1 and 2 and Table 1). In PCPA-pretreated rats, CP-809101 (n = 4) or LP44 with preadministration of WAY-100635 (5-HT1A antagonist) (n = 7) significantly increased A-URS from 42.1 ± 5.7 to 66.2 ± 6.5 cmH2O (P < 0.01) and 30.0 ± 2.7 to 50.5 ± 5.3 cmH2O (P < 0.01) and UBP from 17.3 ± 1.2 to 32.4 ± 2.7 cmH2O (P < 0.05) and 15.2 ± 1.6 to 26.6 ± 2.1 cmH2O (P < 0.01), respectively (Fig. 3 and Table 2). The effects of CP-809101 and LP44 were antagonized by antagonists of each receptor subtype (SB-242084, n = 4 and SB-269970, n = 6, respectively) (Table 2). The average values of sneeze-induced increases in Pabd measured by intra-abdominal catheters were not significantly different between normal and PCPA-pretreated rats or after injection of any drugs (Table 2).
Fig. 2.
Effects of 5-HT depletion induced by p-chlorophenylalanine (PCPA) on amplitudes of urethral pressure response during sneezing (A-URS), urethral baseline pressure (UBP), and abdominal pressure (Pabd). Data are expressed as means ± SE (normal, n = 6; PCPA, n = 5). **P < 0.01, compared with the normal group (unpaired t-test).
Table 1.
Comparison of UBP, A-URS, and Pabd between normal and PCPA-administered rats
| Normal (n = 6) | PCPA (n = 5) | |
|---|---|---|
| UBP, cmH2O | 31.1 ± 3.0 | 17.8 ± 2.2** |
| A-URS, cmH2O | 71.8 ± 7.1 | 36.8 ± 4.3** |
| Pabd, cmH2O | 18.2 ± 4.3 | 28.3 ± 5.5 |
Values are means ± SE. PCPA, p-chlorophenylalanine; UBP, urethral baseline pressure; A-URS, amplitudes of urethral pressure response during sneezing; Pabd, abdominal pressure.
P < 0.01, compared with normal (unpaired t-test).
Fig. 3.
Effects of CP-809101 (5-HT2C agonist) (A) and LP44 (5-HT7 agonist) with WAY-100635 (B) on amplitudes of urethral pressure response during sneezing (A-URS), urethral baseline pressure (UBP), and abdominal pressure (Pabd) in p-chlorophenylalanine (PCPA)-pretreated rats. In LP44 experiments (B), WAY-100635 was administered before LP44 application to inhibit its partial 5-HT1A agonistic effect of LP44. Data are expressed as means ± SE (CP-809101, n = 4; LP44, n = 7). *P < 0.05 and **P < 0.01, compared with the predrug condition (A, paired t-test; B, one-way ANOVA followed by Bonferroni's multiple comparison tests).
Table 2.
The changes of UBP, A-URS, and Pabd after drug administrations in PCPA-pretreated rats
|
Experimental Protocol 1 |
|||
|---|---|---|---|
| PCPA (n = 4) | PCPA + CP-809101 (n = 4) | ||
| UBP, cmH2O | 17.3 ± 1.2 | 32.4 ± 2.7* | |
| A-URS, cmH2O | 42.1 ± 5.7 | 66.2 ± 6.5** | |
| Pabd, cmH2O | 31.2 ± 5.4 | 28.0 ± 1.7 | |
| PCPA (n = 7) | PCPA + WAY-100635 (n = 4) | PCPA + WAY-100635 + LP44 (n = 6) | |
|---|---|---|---|
| UBP, cmH2O | 15.2 ± 1.6 | 16.8 ± 3.0 | 26.6 ± 2.1††,‡ |
| A-URS, cmH2O | 30.0 ± 2.7 | 32.2 ± 3.9 | 50.5 ± 5.3††,‡ |
| Pabd, cmH2O | 37.3 ± 5.1 | 33.4 ± 6.2 | 35.4 ± 3.3 |
| PCPA (n = 4) | PCPA + SB-242084 (n = 4) | PCPA + SB-242084 + CP-809101 (n = 4) | |
|---|---|---|---|
| UBP, cmH2O | 20.3 ± 0.9 | 16.5 ± 1.8 | 15.8 ± 1.2 |
| A-URS, cmH2O | 40.6 ± 5.1 | 38.6 ± 8.9 | 48.9 ± 4.8 |
| Pabd, cmH2O | 29.0 ± 5.8 | 28.3 ± 3.1 | 25.0 ± 3.0 |
| PCPA (n = 6) | PCPA + WAY-100635 + SB-269970 (n = 6) | PCPA + WAY-100635 + SB-269970 + LP44 (n = 6) | |
|---|---|---|---|
| UBP, cmH2O | 20.1 ± 2.5 | 20.6 ± 2.3 | 20.1 ± 2.7 |
| A-URS, cmH2O | 32.7 ± 4.4 | 36.4 ± 5.2 | 38.9 ± 8.2 |
| Pabd, cmH2O | 25.2 ± 2.7 | 32.1 ± 5.0 | 32.1 ± 3.0 |
Values are means ± SE. PCPA, p-chlorophenylalanine; UBP, urethral baseline pressure; A-URS, amplitudes of urethral pressure response during sneezing; Pabd, abdominal pressure.
P < 0.05,
P < 0.01, compared with PCPA (paired t-test).
P < 0.01, compared with PCPA,
P < 0.05, compared with PCPA + WAY-100635 (one-way ANOVA followed by Bonferroni's multiple comparison tests).
Experimental protocol 2: effects of PCPA, CP-809101, or LP44 on sneeze-induced LPP.
Although SUI was not observed even at the highest intravesical pressure during sneezing of 108.8 ± 5.6 cmH2O in normal rats (n = 6), fluid leakage from the urethra during sneezing was observed with S-LPP values of 40.1 ± 2.5 cmH2O in all PCPA-pretreated rats (n = 6). In these 5-HT-depleted rats, fluid leakage from the urethra was still observed during sneezing after the treatment with CP-809101(n = 4) or LP44 (n = 6), which, however, significantly increased S-LPP by 28.0 cmH2O; from 39.7 ± 4.5 cmH2O to 67.7 ± 3.1 cmH2O (P < 0.01), and 15.2 cmH2O; from 37.8 ± 1.4 cmH2O to 53.0 ± 3.4 cmH2O (P < 0.01), respectively, compared with rats treated with PCPA alone. The effects of CP-809101 and LP44 were blocked by antagonists of each receptor subtype (n = 3 each) (Fig. 4 and Table 3).
Fig. 4.
Effects of CP-809101 (5-HT2C agonist; A), LP44 (5-HT7 agonist) with WAY-100635 (B), CP-809101 with pretreatment of SB-242084 (5-HT2C antagonist; C), and LP44 and WAY-100635 with pretreatment of SB-269970 (5-HT7 antagonist; D) on S-LPP in p-chlorophenylalanine (PCPA)-pretreated rats. In LP44 experiments (B and D), WAY-100635 was administered before LP44 application to inhibit its partial 5-HT1A agonistic effect of LP44. Data are expressed as means ± SE (A, n = 4; B, n = 6; C, n = 3; and D; n = 3). **P < 0.01, compared with the predrug condition (A, paired t-test; B–D, one-way ANOVA followed by Bonferroni's multiple comparison tests).
Table 3.
The changes of S-LPP after drug administrations in PCPA-pretreated rats
|
Experimental Protocol 2 |
|||
|---|---|---|---|
| PCPA (n = 4) | PCPA + CP-809101 (n = 4) | ||
| S-LPP, cmH2O | 39.7 ± 4.5 | 67.7 ± 3.1** | |
| PCPA (n = 6) | PCPA + WAY-100635 (n = 6) | PCPA + WAY-100635 + LP44 (n = 6) | |
|---|---|---|---|
| S-LPP, cmH2O | 37.8 ± 1.4 | 39.5 ± 2.5 | 53.0 ± 3.4††,‡‡ |
| PCPA (n = 3) | PCPA + SB-242084 (n = 3) | PCPA + SB-242084 + CP-809101 (n = 3) | |
|---|---|---|---|
| S-LPP, cmH2O | 38.7 ± 4.6 | 37.1 ± 4.2 | 37.7 ± 2.8 |
| PCPA (n = 3)PCPA + WAY-100635 + SB-269970 (n = 3) | PCPA + WAY-100635 + SB-269970 + LP44 (n = 3) | ||
|---|---|---|---|
| S-LPP, cmH2O | 41.5 ± 2.7 | 40.6 ± 1.0 | 40.1 ± 1.8 |
Values present as mean ± SE. Values are means ± SE. PCPA, p-chlorophenylalanine; S-LPP; sneeze-induced leak point pressure.
P < 0.01 compared with PCPA (paired t-test),
P < 0.01 compared with PCPA,
P < 0.01 compared with PCPA + WAY-100635 (one-way ANOVA followed by Bonferroni's multiple comparison tests).
DISCUSSION
There are multiple excitatory and inhibitory 5-HT receptor subtypes; therefore, the overall effects of the 5-HT system or the subtype-specific mechanism controlling the urethral function remain to be elucidated. Previous clinical studies show the evidence for the efficacy of duloxetine, serotonin noradrenalin reuptake inhibitor, as a pharmacological treatment for SUI in women (14, 23). However, because duloxetine induces adverse events, such as nausea (17), receptor-specific therapeutic drugs may be necessary (26). Therefore, we examined the effects of 5-HT subtype-selective drugs on urethral baseline activity and reflex contractions during sneezing in rats.
In present study, we first examined the effect of PCPA because systemic pretreatment of PCPA for 2 days reportedly resulted in 95% depletion in brain 5-HT content (25). Using this method, we sought to investigate the overall 5-HT effect on urinary continence mechanisms. As shown in results, we clarified that PCPA treatment caused SUI during sneezing, indicating that the overall 5-HT system acts to maintain urinary continence. We previously reported intrathecal application of a selective serotonin reuptake inhibitor, fluoxetine, did not affect A-URS or S-LPP in normal rats and rats with vaginal distention (16). On the other hand, the present study indicate that the endogenous 5-HT system maintains A-URS and UBP to prevent SUI during sneezing. Additionally, application of a 5-HT2C agonist (CP-809101) after endogenous 5-HT depletion increased not only A-URS and S-LPP but also UBP in this study although the 5-HT2C agonist did not affect UBP in a previous study (16), in which endogenous 5-HT might have already modulated the 5-HT receptor activity. Thus we speculate that the different findings in the effects of 5-HT2C receptor activation between previous and present studies could be attributable to the presence or absence of endogenous 5-HT and that our results of this study performed under the 5-HT-depleted condition could provide the more precise information for an action of each 5-HT receptor subtype.
Second, we investigated the effects of a 5-HT7 agonist, LP44, a potent 5-HT7 receptor agonist endowed with 200-fold selectivity over 5-HT1A receptor (12), and we previously reported that intrathecal application of a 5-HT1A agonist, 8-OH-DPAT, decreased A-URS and S-LPP in rats (16). Therefore, to suppress the partial 5-HT1A agonistic effect of LP44, a 5-HT1A antagonist, WAY-100635, was administered before LP44 administration. As shown in results, we clarified that 5-HT7 receptor activation has excitatory effects on urethral baseline activity and reflex contractions during sneezing in PCPA-pretreated rats. The site of the action in the spinal cord is probably in the Onuf’s nucleus, where dense norepinephrine- and 5-HT-containing terminals and urethral rhabdosphincter motoneurons are located (18). In addition, immunocytochemical studies demonstrated that 5-HT7 receptors are localized in some laminae of the spinal cord dorsal horn and in dorsal root ganglion cells in rats (4). Taken together, our results indicate that the 5-HT7 receptor could be a pharmacological target for increasing the urethral resistance, possibly through activation of spinal 5-HT7 receptors.
Finally, when the effects of 5-HT2C and 5-HT7 agonists are compared, the effects of 5-HT2C agonist on S-LPP (28.0 cmH2O increase) were greater than those of 5-HT7 agonist (15.2 cmH2O increase) in this study. However, in this study, either 5-HT2C or 5-HT7 agonist alone did not completely prevent urinary incontinence although it increased S-LPP values; thus further studies will be planned to examine the additive effects of 5-HT2C and 5-HT7 receptor stimulation on urethral continence reflexes during sneezing. In addition, because we administered 5-HT2C and 5-HT7 agonists systemically, it is not possible to discriminate the effects in central and or peripheral nerve systems. Previous reports showed that intravenous administration of duloxetine, a 5-HT and norepinephrine reuptake inhibitor, increased both A-URS and UBP whereas intrathecal application of a 5-HT2C agonist did not affect UBP in rats (15, 16). Thus it is assumed that 5-HT2C or 5-HT7 agonist-induced increases in UBP values in this study could be attributable to the peripheral effects due to activation of these 5-HT receptor subtypes although further studies are needed to clarify this point.
In conclusion, the results of this study indicate that activation of 5-HT receptors as a whole enhances the active urethral closure reflex during sneezing and that 5-HT2C and 5-HT7 receptors have facilitatory roles in urethral continence mechanisms in rats. However, it is not known whether activation of these 5-HT receptor subtypes can lead to a treatment of human SUI. Further studies using animal models of SUI are planned to examine the efficacy of activation of excitatory 5-HT receptor subtypes such as 5-HT2C and 5-HT7 in the SUI condition.
GRANTS
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-107450.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
T. Suzuki and N.Y. conceived and designed research; T. Suzuki, T. Shimizu, J.K., S.Y., and Y.S. performed experiments; T. Suzuki, T. Shimizu, J.K., E.T., S.Y., and N.Y. analyzed data; T. Suzuki, T. Shimizu, J.K., E.T., S.Y., Y.S., T.K., M.M., H.M., and N.Y. interpreted results of experiments; T. Suzuki prepared figures; T. Suzuki drafted manuscript; T. Suzuki, T. Shimizu, Y.S., T.K., M.M., H.M., and N.Y. edited and revised manuscript; T. Suzuki, T. Shimizu, J.K., E.T., S.Y., Y.S., T.K., M.M., H.M., and N.Y. approved final version of manuscript.
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