Increasing Effects of Selective 5-Hydroxytryptamine Type 2C Receptor Stimulation on Evoked Momentary Urethral Closure in Female Rats and Humans.

Under healthy conditions, more than one urethra-closing reflex, including both bladder afferent-independent and -dependent actions, function during momentary elevation of intravesical (bladder) pressure to prevent urinary incontinence. In the current study, the effects of a novel selective 5-hydroxytryptamine type 2C (5-HT2C) receptor agonist, TAK-233, on evoked momentary urethra-closing functions were investigated in female rats and humans to elucidate 5-HT2C receptor functions. In anesthetized female rats, TAK-233 dose-dependently and significantly increased urethral resistance during sneezing in rats with distended vaginas and bilaterally transected pelvic nerves. The drug also dose-dependently and significantly increased urethral resistance during momentary intravesical pressure elevation by electrical stimulation of abdominal muscles in rats with a transected spinal cord at the T8-T9 level and intact pelvic nerves. The increased effects observed during electrical stimulation were abolished by either an intravenously administered selective 5-HT2C receptor antagonist, SB 242084, or bilateral transection of the pelvic nerves or somatic nerves innervating the external urethral sphincter and pelvic floor muscles. In the spinal cord-transected and pelvic nerve-intact rats, TAK-233 enlarged the urethra-closing responses induced by both passive and abrupt intravesical pressure elevation, measured by a microtip transducer located in the middle urethra. Additionally, the effects of TAK-233 on the stimulus threshold of urethral contractile responses induced by transcranial magnetic stimulation were investigated in healthy female volunteers. The drug dose-dependently and significantly lowered this stimulus threshold, indicating an increased sensitivity of the response. These results demonstrate that 5-HT2C receptor stimulation enhances the evoked momentary urethra-closing functions in both female rats and humans. SIGNIFICANCE STATEMENT: 5-hydroxytryptamine (serotonin) type 2C (5-HT2C) receptor stimulation by TAK-233 enhanced urethral resistance in rats during an evoked momentary event in which the bladder afferent-independent or -dependent reflex functions via striated muscle-mediated mechanisms. The increases in sensitivity of transcranial magnetic stimulation-evoked urethral contractile responses in healthy female subjects indicates that this mechanism also functions in humans. The evoked momentary conditions activating these reflexes provide a suitable model to demonstrate the effects of 5-HT2C receptor stimulation.

Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors-Part 2.

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood-brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H(3))methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.

Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors - Part 3.

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein 1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We observed that the MDR1 efflux ratio was correlated with the pKa value of the acidic moiety. The resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic acid hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral administration, increased urethral resistance in a dose-dependent manner.

Discovery of 5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide (TAK-259) as a Novel, Selective, and Orally Active α1D Adrenoceptor Antagonist with Antiurinary Frequency Effects: Reducing Human Ether-a-go-go-Related Gene (hERG) Liabilities.

A novel structural class of iminopyridine derivative 1 was identified as a potent and selective human α1D adrenoceptor (α1D adrenergic receptor; α1D-AR) antagonist against α1A- and α1B-AR through screening of an in-house compound library. From initial structure-activity relationship studies, we found lead compound 9m with hERG K(+) channel liability. To develop analogues with reduced hERG K(+) channel inhibition, a combination of site-directed mutagenesis and docking studies was employed. Further optimization led to the discovery of (R)-9s and 9u, which showed antagonistic activity by a bladder strip test in rats with bladder outlet obstruction, as well as ameliorated cystitis-induced urinary frequency in rats. Ultimately, 9u was selected as a clinical candidate. This is the first study to show the utility of iminopyridine derivatives as selective α1D-AR antagonists and evaluate their effects in vivo.

Design, synthesis and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitor.

Centrally acting noradrenaline reuptake inhibitor (NRI) is reportedly effective for patients with stress urinary incontinence (SUI) by increasing urethral closure in the clinical Phase IIa study with esreboxetine. Noradrenaline transporters are expressed in both central and peripheral nervous systems and the contribution of each site to efficacy has not been clarified. This report describes the development of a series of peripheral-selective 7-phenyl-1,4-oxazepane NRIs to investigate the contribution of the peripheral site to increasing urethral resistance in rats. (6S,7R)-1,4-Oxazepane derivative 7 exhibited noradrenaline transporter inhibition with high selectivity against inhibitions of serotonin and dopamine transporters. A replacement of hydroxyl with acetamide group contributed to enhancement of peripheral selectivity by increasing molecular polarity. Compound 12, N-{[(6S,7R)-7-(3,4-dichlorophenyl)-1,4-oxazepan-6-yl]methyl}acetamide 0.5 fumarate, which showed effectively no brain penetration in rats, increased urethral resistance in a dose-dependent manner and exhibited a maximal effect on par with esreboxetine. These results demonstrate that the urethral resistance-increasing effects of NRI in rats are mainly caused by the inhibition of noradrenaline transporters in the peripheral sites.

Novel 3-phenylpiperidine-4-carboxamides as highly potent and orally long-acting neurokinin-1 receptor antagonists with reduced CYP3A induction.

The synthesis and biological evaluation of a series of novel 3-phenylpiperidine-4-carboxamide derivatives are described. These compounds are generated by hybridization of the substructures from two types of tachykinin NK(1) receptor antagonists. Compound 42 showed high metabolic stability and excellent efficacy in the guinea-pig GR-73637-induced locomotive activity assay at 1 and 24h after oral administration. It also exhibited good pharmacokinetic profiles in four animal species, and a low potential in a pregnane X receptor induction assay.

Design, structure-activity relationship, and highly efficient asymmetric synthesis of 3-phenyl-4-benzylaminopiperidine derivatives as novel neurokinin-1 receptor antagonists.

We synthesized a series of novel 3-phenyl-4-benzylaminopiperidine derivatives that were identified as potent tachykinin NK(1) receptor antagonists by structural modification of the 3-benzhydrylpiperidone derivative through high-throughput screening. N-{2-[(3R,4S)-4-({2-Methoxy-5-[5-(trifluoromethyl)-1H-tetrazol-1-yl]benzyl}amino)-3-phenyl-1-piperidinyl]-2-oxoethyl}acetamide ((+)-39) was found to be one of the most potent tachykinin NK(1) receptor antagonists with high metabolic stability. Highly efficient asymmetric synthesis of (+)-39 was achieved via dynamic kinetic resolution.

Increased urethral resistance by periurethral injection of low serum cultured adipose-derived mesenchymal stromal cells in rats.

OBJECTIVES: To evaluate the effects of a periurethral injection of low serum cultured adipose tissue-derived mesenchymal stromal cells (LASC) and to develop a new autologous cell therapy for stress urinary incontinence. METHODS: F344 rats were divided into three groups as based on the periurethral injection of LASC, GAX collagen or vehicle (control). At 2 and 4 weeks after injection, leak point pressure (LPP) was measured before and after transection of the pelvic nerves. For cell tracking, LASC of green fluorescent protein transgenic rats were injected into nude rats. RESULTS: At 2 weeks, both the LASC and collagen groups showed significantly higher LPP than the control group. At 4 weeks, the increase in LPP in the LASC group remained, whereas LPP in the collagen group decreased to baseline levels. In the absence of the urethral closure reflex after transection of the pelvic nerves, LPP in the LASC group was significantly higher than that in the other two groups. Histologically, the size of the urethral lumen was smaller in the LASC group than the collagen group. At 4 weeks, most of the LASC were positive for myogenic antigens including α-smooth muscle actin, desmin and calponin I. CONCLUSIONS: Periurethral injection of autologous LASC capable of myogenic differentiation made a greater contribution to the increase in urethral resistance than did the conventional collagen bulk injection. Thus, its use for treatment of stress urinary incontinence can be postulated.

Role of vasopressin V(1A) receptor in the urethral closure reflex in rats.

An enhanced urethral closure reflex via the spinal cord is related to urethral resistance elevation during increased abdominal pressure. However, with the exception of monoamines, neurotransmitters modulating this reflex are not understood. We investigated whether the vasopressin V(1A) receptor (V(1A)R) is involved in the urethral closure reflex in urethane-anesthetized female rats. V(1A)R mRNA was highly expressed among the vasopressin receptor family in the total RNA purified from lamina IX in the spinal cord L6-S1 segment. In situ hybridization analysis of the spinal L6-S1 segment confirmed that these positive signals from the V(1A)Rs were only detected in lamina IX. Intrathecally injected Arg8-vasopressin (AVP), an endogenous ligand, significantly increased urethral resistance during an intravesical pressure rise, and its effect was blocked by the V(1A)R antagonist. AVP did not increase urethral resistance in rats in which the pelvic nerves were transected bilaterally. Urethral closure reflex responses to the intravesical pressure rise increased by up to threefold compared with the baseline response after AVP administration in contrast to no increase by vehicle. In addition, intravenously and intrathecally injected V(1A)R antagonists decreased urethral resistance. These results suggest that V(1A)R stimulation in the spinal cord enhances the urethral closure reflex response, thereby increasing urethral resistance during an abdominal pressure rise and that V(1A)R plays a physiological role in preventing urine leakage.

Role of spinal serotonergic pathways in sneeze-induced urethral continence reflex in rats.

To clarify the role of spinal serotonergic mechanisms in preventing stress urinary incontinence (SUI) during sneezing, we investigated the effect of intrathecal (it) application of 8-OH-DPAT (a 5-HT(1A) agonist), mCPP (a 5-HT(2B/2C) agonist), and fluoxetine (a serotonin reuptake inhibitor) using a rat model that can examine the neurally evoked continence reflex during sneezing. Amplitudes of urethral pressure responses during sneezing (A-URS), urethral baseline pressure (UBP) at the midurethra, and sneeze-induced leak point pressure (S-LPP) were measured in normal female adult rats and rats with SUI induced by vaginal distention (VD). In normal rats, 8-OH-DPAT decreased A-URS by 48.9%, whereas mCPP increased A-URS by 33.6%. However, A-URS was not changed after fluoxetine. 8-OH-DPAT, mCPP, or fluoxetine did not alter UBP. The effect of 8-OH-DPAT and mCPP was antagonized by WAY-100635 (it), a selective 5-HT(1A) antagonist, and RS-102221 (it), a selective 5-HT(2C) antagonist, respectively. Fluoxetine in the presence of WAY-100635 did not change either A-URS or UBP, but fluoxetine in the presence of RS-102221 decreased A-URS. In VD rats, S-LPP was decreased by 14.6 cmH2O after 8-OH-DPAT, whereas it was increased by 12.8 cmH2O after mCPP. However, S-LPP was not changed after fluoxetine. These results indicate that activation of 5-HT(2C) receptors enhances the active urethral closure reflex during sneezing at the spinal level, whereas 5-HT(1A) inhibits it and that no apparent changes in the sneeze-induced continence reflex after fluoxetine treatment are due to coactivation of excitatory 5-HT(2C) receptors and inhibitory 5-HT receptors other than the 5-HT(1A) subtype. Thus, activation of excitatory 5-HT receptor subtypes such as 5-HT(2C) could be effective for the treatment of SUI.

Two kinds of urinary continence reflexes during abrupt elevation of intravesical pressure in rats.

Urethral closure mechanisms during abrupt elevation of intravesical pressure (P(ves)) were investigated. During sneezing, the middle urethral closing response was observed and it still remained after opening the abdomen. The middle urethral response was almost completely abolished after bilateral transection of somatic nerves innervating the external urethral sphincter and the pelvic floor muscles, while bilateral transection of both pelvic nerves and hypogastric nerves had no effects. Somatic nerve transection resulted in fluid leakage from the urethral orifice during sneezing. Passive increments of P(ves) for 120 seconds by elevating a saline reservoir connected to the bladder also induced the middle urethral closing response in rats with spinal cord transection at T8-T9. The response was totally abolished by cutting pelvic nerves bilaterally, and partially reduced after bilateral transection of pudendal nerves, nerves to pelvic floor muscles or hypogastric nerves. Electrical stimulation of abdominal muscles (ESAM) for 1 second elevated P(ves) in a stimulus-dependent manner in the spinal cord-transected rats, and the P(ves) rise was almost lost when the abdomen was opened. The P(ves) inducing fluid leakage from the urethral orifice was lowered in rats when pelvic nerves or somatic nerves were cut bilaterally, while transection of bilateral hypogastric nerves showed smaller effects. These results indicate that at least two kinds of urinary continence reflexes close the middle urethra during abrupt elevation of P(ves); one reflex observed during sneeze is preprogrammed so as to close the urethra automatically irrespective of bladder afferent activity, and the other reflex is triggered by bladder afferent excitation. During momentary stress events such as sneezing (<0.15 seconds) and ESAM (1 second), the striated muscles mainly contribute to the urethral closure, while during events for a relatively long period like passive P(ves) elevation for 120 seconds, both striated and smooth muscles are involved in the prevention of stress urinary incontinence.

Effect of duloxetine, a norepinephrine and serotonin reuptake inhibitor, on sneeze-induced urethral continence reflex in rats.

We investigated the effect of duloxetine, a norepinephrine (NE) and serotonin (5-HT) reuptake inhibitor, on the neurally evoked urethral continence reflex induced by sneezing in rats. To clarify the role of noradrenergic and serotonergic mechanisms in preventing stress urinary incontinence (SUI) during sneezing, we examined the effect of duloxetine followed by intrathecal (it) methiothepin maleate (5-HT receptor and alpha1-adrenoceptor antagonist) or terazosin or idazoxan (selective alpha1- and alpha2-adrenoceptor antagonists, respectively). Amplitude of urethral pressure responses during sneezing (A-URS), urethral baseline pressure (UBP) at the midurethra, and sneeze-induced leak point pressure (S-LPP) were measured in normal adult female rats and rats with SUI induced by vaginal distension (VD). In normal and VD rats, intravenous application of duloxetine (1 mg/kg) increased A-URS by 35% and 34% and UBP by 21% and 34%, respectively. Sneezing-induced fluid leakage from the urethral orifice was observed in VD rats but not in normal rats. S-LPP was increased from 39.1 to 92.2 cmH2O by intravenous duloxetine in incontinent VD rats. Duloxetine-mediated enhancement of A-URS was inhibited by terazosin but not methiothepin maleate (it). In addition, simultaneous intrathecal application of methiothepin and terazosin induced a reduction in A-URS during sneezing, which was not increased by intravenous duloxetine. However, the reduced A-URS after intrathecal application of methiothepin and terazosin returned to the control level when duloxetine (iv) was applied after intrathecal idazoxan administration. These results indicate that duloxetine can prevent SUI by facilitating noradrenergic and serotonergic systems in the spinal cord to enhance the sneeze-induced active urethral closure mechanism.

Involvement of reflex urethral closure mechanisms in urethral resistance under momentary stress condition induced by electrical stimulation of rat abdomen.

A novel method for evaluating the urethral resistance during abrupt elevation of abdominal pressure was developed in spinalized female rats under urethane anesthesia. Electrical stimulation of abdominal muscles for 1 s induced increases in both the intra-abdominal and the intravesical pressure in a stimulus-dependent manner, and the bladder response was almost lost when the abdomen was opened. The lowest intravesical pressure during electrical stimulation that induced fluid leakage from the urethral orifice (leak point pressure) and the maximal intravesical pressure without urine leakage below the leak point pressure were evaluated as the indexes of urethral resistance. Lower urethral resistance was obtained in the rats whose pelvic nerves or somatic nerves containing pudendal nerves and nerves to iliococcygeus/pubococcygeus muscles were transected bilaterally. In contrast, transection of bilateral hypogastric nerves showed smaller effects. Duloxetine, a drug for stress urinary incontinence, enlarged the reflex urethral closing contractions that were induced by an increase in intravesical pressure and measured using a microtip transducer catheter in the middle urethra. This drug also increased the urethral resistance (leak point pressure), whereas it did not show any effect in the rats whose pelvic nerves were bilaterally transected, showing that the augmentation of the reflex urethral closure by the drug resulted in the elevation of the urethral resistance. From these findings, it was concluded that during momentary elevation of abdominal pressure, the reflex urethral closure mechanisms via bladder-spinal cord-urethral sphincter and pelvic floor muscles greatly contribute to the increase in the urethral resistance to prevent the urinary incontinence.

Role of noradrenergic pathways in sneeze-induced urethral continence reflex in rats.

To clarify the role of noradrenergic pathways in preventing stress urinary incontinence (SUI) during sneezing, we investigated the effect of the norepinephrine reuptake inhibitor nisoxetine and alpha-adrenoceptor antagonists phentolamine (nonspecific blocker) and prazosin (alpha(1)-receptor-selective blocker) on the neurally evoked urethral continence reflex induced by sneezing in rats. The amplitude of urethral pressure responses during sneezing (A-URS), urethral baseline pressure (UBP) at the midurethra, and sneeze-induced leak point pressure (S-LPP) were measured in normal female adult rats and rats with SUI induced by vaginal distention (VD). In normal rats, intrathecal (it) phentolamine (0.02 nmol) and prazosin (0.02 nmol) decreased A-URS by 11.9 and 15.7%, respectively, without affecting UBP. In both normal and VD rats, intravenous (iv) application of nisoxetine (1 mg/kg) increased A-URS by 17.2 and 18.3% and UBP by 23.7 and 32.7%, respectively. Phentolamine or prazosin (both it) eliminated nisoxetine-induced increases in A-URS, but not the increases in UBP, which were, however, suppressed by iv phentolamine (5 mg/kg) or prazosin (1 mg/kg). Sneezing induced fluid leakage from the urethral orifice in VD rats, but not in normal rats. In VD rats, S-LPP was increased by 30.2% by iv nisoxetine. Application of phentolamine and prazosin (both it) decreased S-LPP by 15.7 and 20.6%, respectively, and nisoxetine induced increases in S-LPP to 13.2 and 12.3%, respectively. These results indicate that activation of the noradrenergic system by a norepinephrine reuptake inhibitor can prevent SUI via alpha(1-)adrenoceptors by enhancing the sneeze-induced active urethral closure mechanism at the spinal level and augmenting UBP at the periphery.

Functional analysis of active urethral closure mechanisms under sneeze induced stress condition in a rat model of birth trauma.

PURPOSE: We evaluated changes in the urethral closure mechanism under a sneeze induced stress condition in a rat model of birth trauma. MATERIALS AND METHODS: Four days after vaginal distention induced by balloon catheter inflation in the vagina sneezing was induced while recording intravesical pressure with the rat under urethane anesthesia to evaluate sneeze induced leak point pressure, defined as the lowest pressure inducing fluid leakage from the urethral meatus during sneezing. Sneeze induced responses in the bladder and proximal or mid urethra were also measured using microtip transducer catheters. RESULTS: In 5 sham operated rats no leakage was observed from the urethral meatus during sneezing, which produced an increase in intravesical pressure of up to 34 cm H(2)O. However, in 5 of 6 rats with vaginal distention leakage during sneezing was observed with a sneeze leak point pressure of 26.2 cm H(2)O. In the mid urethra microtip transducer catheters revealed that pressure increases during sneezing were greater than those in the bladder but they were significantly decreased in the 5 incontinent vaginal distention rats. However, sneeze induced responses at the proximal urethra, which were similar to those in the bladder, were not different in sham operated and incontinent vaginal distention rats. CONCLUSIONS: Sneeze induced stress urinary incontinence in a rat model of birth trauma was caused by decreased active closure mechanisms at the mid urethra without affecting the passive transmission of abdominal pressure in the proximal urethra.

Comparison of leak point pressure methods in an animal model of stress urinary incontinence.

We compared three different methods of testing leak point pressure (LPP) in rats with or without the pudendal nerves and nerves to the iliococcygeus/pubococcygeus muscles transected: (1) sneeze induced with a whisker in the nostril (sneeze LPP), (2) manually increased abdominal pressure (Crede LPP), and (3) increased intravesical pressure using the vertical tilt table method (vertical tilt table LPP). In sham rats, passive intravesical pressure rises in Crede and vertical tilt table methods induced active urethral closure mechanisms that contributed to high LPPs (41.4 and 35.5 cm H2O, respectively), which were significantly reduced by nerve transection. During sneezing, leakage was observed in nerve-transected rats, but not in sham rats, indicating that sneezing can activate an additional urethral closure mechanism. Measuring LPP during sneezing or passive intravesical pressure rises in the vertical tilt table and Crede method seems to be useful for assessing the continence mechanisms under different stress conditions in rats.

A tissue-engineered suburethral sling in an animal model of stress urinary incontinence.

OBJECTIVE: To create and evaluate the functional effects of a tissue-engineered sling in an animal model of stress urinary incontinence (SUI). MATERIALS AND METHODS: Twenty female Sprague-Dawley rats were divided into four equal groups: a control group (C) had no intervention before the leak-point pressure (LPP) was measured; a denervated group (D) had bilateral proximal sciatic nerve transection (PSNT) and periurethral dissection with no sling placed; group S had concomitant bilateral PSNT and a suburethral sling of small intestinal submucosa (SIS) placed; and group (M) had concomitant bilateral PSNT with implantation of a tissue-engineered sling. The suburethral sling was placed via a transabdominal approach with the sling sutured to the pubic bone. Tissue-engineered slings were prepared with muscle-derived cells obtained via the pre-plate technique and subsequently seeded for 2 weeks on a SIS scaffold. Suburethral slings were implanted 2 weeks before LPP testing, using the vertical-tilt method. RESULTS: Surgically placing a suburethral sling is feasible in the female rat, with few complications. LPPs from both sling groups (S and M) were not significantly different from untreated controls (C). The S, M and C groups all had significantly higher LPPs than group D. Importantly, no rat from either sling group (S and M) had signs of urinary retention. CONCLUSIONS: Placing tissue-engineered slings in an animal model of SUI resulted in LPP values that were not significantly different from those in untreated control or SIS (S) groups. These data show that incorporating muscle stem cells into SIS slings does not adversely alter the advantageous mechanical properties of the SIS sling in a model of SUI, and provide the basis for future functional studies of tissue-engineered sling materials with long-term retention.

Differential effects of activation of peripheral and spinal tachykinin neurokinin(3) receptors on the micturition reflex in rats.

PURPOSE: We clarified the roles of tachykinin neurokinin (NK)3 receptors in the bladder or spinal cord for control of the micturition reflex in rats. MATERIALS AND METHODS: In female rats under urethane anesthesia repetitive bladder contractions were elicited by saline infusion into the bladder through intravesical bladder catheters. The effects of peripheral receptor activation were first examined by topical application of the tachykinin NK3 receptor agonist [MePhe]-NKB (Calbiochem, Darmstadt, Germany) in normal rats and rats pretreated with capsaicin (Sigma Chemical Co., St. Louis, Missouri) 4 days before the experiments. Subsequently the effects of spinal NK3 receptor activation were examined by intrathecal administration of [MePhe]-NKB via implanted intrathecal catheters. The effects of the tachykinin NK3 receptor antagonist SB235375 and the opioid receptor antagonist naloxone on changes in bladder activity induced by [MePhe]-NKB were also investigated. RESULTS: Topical application of [MePhe]-NKB onto the bladder surface decreased intercontraction intervals and bladder capacity, and increased baseline bladder pressure in dose dependent fashion. [MePhe]-NKB induced bladder overactivity was inhibited by simultaneous topical administration of SB235375 or by capsaicin pretreatment. In contrast, intrathecal injection of [MePhe]-NKB increased intercontraction intervals in dose dependent fashion and at a high dose it induced overflow incontinence or inefficient voiding. These inhibitory effects of [MePhe]-NKB in the spinal cord were antagonized by the intrathecal injection of SB235375 or naloxone. CONCLUSIONS: These results indicate that the tachykinin NK3 receptor mediated neural control of the micturition reflex has dual actions depending on the location of receptor activation. Activation of tachykinin NK3 receptors located in the bladder can induce bladder overactivity at least in part via the activation of capsaicin sensitive C-fiber afferents, while tachykinin NK3 receptor activation in the spinal cord can inhibit the micturition reflex through an opioid mechanism.

Differential roles of peripheral and spinal endothelin receptors in the micturition reflex in rats.

PURPOSE: We investigated the effects of endothelin (ET) receptor activation in the bladder and the spinal cord on the micturition reflex in urethane anesthetized rats. MATERIALS AND METHODS: The effects of ET receptor activation on bladder activity were examined during continuous infusion cystometrograms. ET-1 was administered intrathecally or intravesically in normal rats or rats pretreated with capsaicin. The effects of intravenous injection of the selective ETA receptor antagonist ABT-627, or selective ETB receptor antagonist A-192621 (Abbott Laboratories, Abbott Park, Illinois) intrathecal injection of the opioid receptor antagonist naloxone hydrochloride on changes in bladder activity induced by intravesical or intrathecal ET-1 administration were investigated. RESULTS: Intravesical injection of ET-1 (0.1 to 10 microM) induced detrusor overactivity, as evidenced by a decrease in intercontraction intervals, in a dose dependent manner. ET-1 induced detrusor overactivity was suppressed by intravenous application of ABT-627 (0.1 mg/kg) as well as capsaicin pretreatment but not by A-192621. In contrast, intrathecal injection of ET-1 (0.5 to 50 fmol) increased intercontraction intervals dose dependently and ET-1 at a higher dose (50 fmol) induced urinary retention. These inhibitory effects were antagonized by ABT-627 (10 mg/kg) and also by intrathecal application of naloxone but not by A-192621. CONCLUSIONS: These results indicate that the activation of ETA receptors in capsaicin sensitive C-fiber afferents in the bladder can induce detrusor overactivity, while ETA receptor activation in the spinal cord can inhibit the micturition reflex via activation of a spinal opioid mechanism. Thus, targeting peripheral ETA receptors could be an effective treatment for bladder overactivity and/or painful conditions.

The role of bladder-to-urethral reflexes in urinary continence mechanisms in rats.

Urethral closure mechanisms during passive increments in intravesicular pressure (P(ves)) were investigated using microtip transducer catheters in urethane-anesthetized female rats. After a block of reflex bladder contractions by spinal cord transection at T8-T9, abruptly raising P(ves) to 20, 40, or 60 cmH(2)O for 2 min induced a bladder pressure-dependent contractile response in a restricted portion of the middle urethra (12.5-15 mm from the urethral orifice) that was abolished by cutting the pelvic nerves bilaterally. In pelvic nerve-intact rats, the bilateral transection of either the pudendal nerves, the nerves to the iliococcygeous/pubococcygeous muscles, or the hypogastric nerves significantly reduced (49-74%) the urethral reflex response induced by passive P(ves) increases, and combined transection of these three sets of nerves totally abolished the urethra-closing responses. In spinal cord-intact rats, similar urethral contractile responses were elicited during P(ves) elevation (20 or 40 cmH(2)O) and were also eliminated by bilateral pelvic nerve transection. After spinal cord and pelvic nerve transection, leak point pressures, defined as the pressure inducing fluid leakage from the urethral orifice during passive P(ves) elevation by either bladder pressure clamping in 2.5-cmH(2)O steps or direct compression of the bladder, were significantly lowered by 30-35% compared with sham-operated (spinal cord-transected and pelvic nerve-intact) rats. These results indicate that 1) passive elevation of P(ves) can elicit pelvic afferent nerve-mediated contractile reflexes in the restricted portion of the urethra mediated by activation of sympathetic and somatic nerves and 2) bladder-to-urethral reflexes induced by passive P(ves) elevation significantly contribute to the prevention of stress urinary incontinence.