Rapid-onset, short-duration induction of colorectal contractions in anesthetized, adult, male rats.

Substantial clinical and preclinical evidence indicates that transient receptor potential vanilloid 1 (TRPV1) receptors are expressed on terminals of colorectal chemoreceptors and mechanoreceptors and are involved in various rectal hypersensitivity disorders with common features of colorectal overactivity. These stimulatory properties of TRPV1 receptors on colorectal function suggested that brief stimulation of TRPV1 might provide a means of pharmacologically activating the colorectum to induce defecation in patients with an "unresponsive" colorectum. The current studies explored the basic features of TRPV1 receptor-induced contractions of the colorectum in anesthetized rats with and without acute spinal cord injury (aSCI). Cumulative concentration-response curves to intrarectal (IR) capsaicin (CAP) solutions (0.003% to 3.0%) were performed in anesthetized aSCI and spinal intact rats. CAP produced an "inverted U", cumulative concentration-response curve with a threshold for inducing colorectal contractions at 0.01% and a peak response at 0.1% and slight decreases in responses up to 3%. Decreases in responses with concentrations > 0.1% are due to a rapid desensitization (i.e. < 30 min) of TRPV1 receptors to each successive dose. Desensitization appeared fully recovered within 24 hours in spinal intact rats. Colorectal contractions were completely blocked by atropine, indicating a reflexogenic activation of parasympathetic neurons, and responses were completely unaffected by a neurokinin 2 receptor antagonist, indicating that release of neurokinin A (NKA) from afferent terminals and subsequent direct contractions of the smooth muscle was not involved. IR administration of 3 other TRPV1 receptor agonists produced similar results as CAP. Significance Statement Individuals with spinal cord injury often lose control of defecation. Time consuming and inconvenient bowel programs using digital stimulation of the rectum and manual extraction of stool are used to empty the bowel. We show that intrarectal administration of the TRPV1 receptor agonist, capsaicin, can induce rapid onset, short duration colorectal contractions capable of inducing defecation in spinal cord injured and intact rats. Therefore, TRPV1 agonists show promise as a potential therapeutics to induce defecation in individuals with neurogenic bowel.

Recent developments in on-demand voiding therapies.

One cannot survive without regularly urinating and defecating. People with neurological injury (spinal cord injury, traumatic brain injury, stroke) or disease (multiple sclerosis, Parkinson's disease, spina bifida) and many elderly are unable to voluntarily initiate voiding. The great majority of them require bladder catheters to void urine and "manual bowel programs" with digital rectal stimulation and manual extraction to void stool. Catheter-associated urinary tract infections frequently require hospitalization, while manual bowel programs are time-consuming (1-2 hours), stigmatizing, and cause rectal pain and discomfort. Laxatives and enemas produce defecation, but onset and duration are unpredictable, prolonged, and difficult to control, which can produce involuntary defecation and fecal incontinence. Patients with spinal cord injury (SCI) consider recovery of bladder and bowel function a higher priority than recovery of walking. Bladder and bowel dysfunction are a top reason for institutionalization of elderly. Surveys indicate that convenience, rapid onset and short duration, reliability and predictability, and efficient voiding are priorities of SCI individuals. Despite the severe, unmet, medical need; there is no literature regarding on-demand, rapid-onset, short-duration, drug-induced, voiding therapies. This article provides in depth discussion of recent discovery and development of two candidates for on-demand voiding therapies. The first, DTI-117, a neurokinin2 receptor agonist, induces both urination and defecation after systemic administration. The second, DTI-301, is a TRPV1 receptor agonist that induces defecation after intrarectal administration. The review also presents clinical studies of a combination drug therapy administered via iontophoresis and preclinical studies of neuromodulation devices that induce urination and defecation. Significance Statement Safe, effective, on-demand, rapid-onset, short-duration, drug-induced, voiding therapy could eliminate or reduce need for bladder catheters, manual bowel programs, and colostomies in patient populations that are unable to voluntarily initiate voiding. People with spinal injury place more importance on restoring bladder and bowel control than restoring their ability to walk. This paradigm-changing therapy would reduce stigmatism and healthcare costs while increasing convenience and quality of life.

Effect of GR205171 on autonomic dysreflexia induced by colorectal distension in spinal cord injured rats.

STUDY DESIGN: Preclinical pharmacology. OBJECTIVES: To determine whether blocking substance P signaling attenuates the hypertension and bradycardia evoked by colorectal distension (CRD) in spinal cord injured (SCI) rats. SETTING: University laboratory in Pennsylvania, U.S.A. METHODS: Tachykinin NK1 receptor antagonists were administered 30 min prior to CRD three weeks after complete spinal cord transection at the 4th thoracic (T4) level. The dose range, route of administration, and pretreatment time was based on published data demonstrating occupancy of brain NK1 receptors in rodents. RESULTS: Subcutaneous (SC) administration of 10-30 mg/kg GR205171 ((2S,3S)-N-[[2-methoxy-5-[5-(trifluoromethyl)tetrazol-1-yl]phenyl]methyl]-2-phenylpiperidin-3-amine dihydrochloride) reduced CRD-induced hypertension and bradycardia by 55 and 49%, respectively, compared with pretreatment values. There was no effect of GR205171 on resting blood pressure or heart rate. In contrast, the same dose range of CP-99,994 ((2S,3S)-N-[(2-methoxyphenyl)methyl]-2-phenyl-3-piperidinamine dihydrochloride) had no effect on CRD-induced cardiovascular responses. CONCLUSIONS: The effective dose range of GR205171 to alleviate autonomic dysreflexia is consistent with the blockade of NK1 receptors on pelvic sensory afferents in the lumbosacral spinal cord, which may in turn prevent the over-excitation of sympathetic preganglionic neurons (SPNs) that regulate blood pressure and heart rate. The findings provide preclinical support for the utility of NK1 receptor antagonists to treat autonomic dysreflexia in people with SCI. The difference in the effects of the two NK1 receptor antagonists may reflect the ~200-fold lower affinity of CP-99,994 than GR205171 for the rat NK1 receptor.

Colorectal and bladder prokinetic activity of [Lys5, MeLeu9, Nle10]-NKA(4-10) after intranasal or sublingual delivery in dogs.

The feasibility of eliciting defecation and urination after intranasal (IN) or sublingual (SL) delivery of a small peptide NK2 receptor agonist, [Lys5, MeLeu9, Nle10]-NKA(4-10), was examined using prototype formulations in dogs. In anesthetized animals, administration of 100 or 300 µg/kg IN or 2.0-6.7 mg/kg SL increased colorectal peak pressure and area under the curve. Peak bladder pressure was also increased at the same doses, and this was accompanied by highly efficient voiding at normal physiological bladder pressure. The onset of these effects was rapid (≤2.5 min), and the primary contractions lasted ∼25 min, returning to baseline in <60 min. Slight hypotension lasting a few minutes and causing <10% change from baseline was detected after higher doses and was statistically significant after only 100 µg/kg IN. In conscious dogs, there was a dose-related increase in voiding responses and reduction in the latency to urinate and defecate after 300 and 1000 µg/kg IN; emesis was also observed at these doses. SL administration of 6.7 mg/kg induced urination within 10 min, but not defecation or emesis. These findings support the feasibility of developing a convenient dosage form of small peptide NK2 receptor agonists as on-demand defecation or urination therapies.

Neurokinin 2 receptor-mediated bladder and colorectal responses in aged spinal cord injured rats.

STUDY DESIGN: Animal proof of principle study. OBJECTIVES: Bladder and bowel dysfunction are common after spinal cord injury (SCI) and in the elderly. Neurokinin 2 receptor agonists are known to produce on-demand urination and defecation in adult SCI rats. This study compared the ability of a neurokinin 2 receptor (NK2R) agonist to produce bladder and colorectal contractions in both young adult and aged SCI rats. SETTING: Dignify Therapeutics and Integrated Laboratory Systems, Durham, NC USA. METHODS: Bladder and colorectal pressure and voiding efficiency were measured in response to the NK2R agonist, [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA), in anesthetized animals. The potency and efficacy of LMN-NKA was examined in young adult and aged SCI (T3 or T9 transected) rats, with young adult and aged spinal intact rats included as controls. RESULTS: LMN-NKA (3-300 µg/kg i.v.) produced dose-dependent increases in bladder and colorectal pressure in all anesthetized rats. No differences in the bladder or colorectal pressure responses or voiding efficiency were observed with age or after SCI. The level of SCI did not change the pharmacodynamic responses to the agonist. CONCLUSIONS: An NK2R agonist produced similar responses in young adult and aged SCI rats, suggesting this class of agonists could be used as a potential therapy to induce on-demand urination and defecation in aged populations, with or without SCI.

Needs, priorities, and attitudes of individuals with spinal cord injury toward nerve stimulation devices for bladder and bowel function: a survey.

STUDY DESIGN: Survey. OBJECTIVES: To investigate the needs and priorities of people with spinal cord injury for managing neurogenic bladder and bowel function and to determine their willingness to adopt neuromodulation interventions for these functions. METHODS: Anonymous online survey. It was advertised by word-of-mouth by community influencers and social media, and by advertisement in newsletters of advocacy groups. RESULTS: Responses from 370 individuals (27% female, 73% male) were included. Bladder emptying without catheters was the top priority for restoring bladder function, and maintaining fecal continence was the top priority for restoring bowel function. The biggest concerns regarding external stimulation systems were wearing a device with wires connecting to electrodes on the skin and having to don and doff the system daily as needed. The biggest concerns for implanted systems were the chances of experiencing problems with the implant that required a revision surgery or surgical removal of the whole system. Respondents were willing to accept an external (61%) or implanted (41%) device to achieve improved bladder or bowel function. CONCLUSIONS: Bladder and bowel dysfunction remain important unmet challenges for individuals living with SCI who answered our survey. These individuals are willing to accept some potential risks of nerve stimulation approaches given potential benefits. Additional consumer input is critical for guiding both research and translation to clinical use and personalized medicine.

Chronic, Twice-Daily Dosing of an NK2 Receptor Agonist [Lys5,MeLeu9,Nle10]-NKA(4-10), Produces Consistent Drug-Induced Micturition and Defecation in Chronic Spinal Rats.

Acute administration of [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA) produces contractions of the detrusor and rectum with voiding in intact and acutely spinal cord injured (SCI) rats. In the current study, the ability of LMN-NKA (10 µg/kg or 100 µg/kg, subcutaneous [SC], twice a day [bid]) or vehicle to induce voiding and defecation in chronic SCI rats was examined across 30 days. After the last day of administration, voiding response rates and bladder pressure (BP) responses to LMN-NKA (intravenous [IV] and SC) were evaluated under anesthesia. In conscious rats, LMN-NKA (100 µg/kg) produced dose-dependent micturition within 5 min, with response rates >90%, and voiding efficiency >80% in males and >60% in females, which remained stable across the 1-month test period. Similarly, LMN-NKA administration rapidly induced defecation, which also remained stable. Under anesthesia, LMN-NKA increased BP, voiding efficiency, and voiding response rates, which reached 100% at 3 and 10 µg/kg IV in males and females, respectively. SC administration produced 100% response rates in males (30 µg/kg) but only 71% in females (100 µg/kg). Efficacy in rats chronically treated with LMN-NKA was similar to naïve and vehicle-treated rats, except for reduced voiding efficiency in chronically dosed female rats (100 µg/kg). No differences in bladder weights or collagen-to-smooth muscle ratios in histological sections were seen between the groups. Thus neither tolerance, nor sensitization, to LMN-NKA-induced micturition and defecation occurs with chronic administration in rats with chronic SCI. Efficacy was higher in male than in female rats.

Potency, efficacy, and selectivity of GR64349 at human recombinant neurokinin NK2 and NK1 receptors.

The affinity, potency, efficacy, and selectivity of the NK2 receptor agonist GR64349 ([Lys3,Gly8,-R-γ-lactam-Leu9]NKA(3-10)) at human recombinant NK2 and NK1 receptors was examined. In radioligand binding studies, GR64349 displaced [125I]-NKA binding to NK2 receptors with high affinity (pKi 7.77 + 0.10) but only weakly displaced [3H]-septide binding to NK1 receptors (pKi <5). In functional studies examining increases in intracellular inositol-1 phosphate (IP-1) accumulation, calcium levels, and cyclic AMP synthesis, GR64349 was a full agonist by reference to the endogenous agonists NKA (NK2 receptors) and substance P (NK1 receptors). GR64349 increased IP-1 accumulation with 1,400-fold greater potency in cells expressing NK2 receptors (pEC50 9.10 + 0.16) than cells expressing NK1 receptors (pEC50 5.95 + 0.80). For calcium responses, GR64349 was 500-fold more potent in the assay using NK2 receptors (pEC50 9.27 + 0.26) than NK1 receptors (pEC50 6.55 + 0.16). GR64349 also stimulated cyclic AMP synthesis in both cell lines, and was almost 900-fold more potent at NK2 receptors (pEC50 10.66 + 0.27) than NK1 receptors (pEC50 7.71 + 0.41). These findings confirm that GR64349 is the most selective NK2 receptor agonist described to date.

Prokinetic effects of the neurokinin NK2 receptor agonist [Lys5,MeLeu9,Nle10]-NKA(4-10) on bladder and colorectal activity in minipigs.

The effects of the neurokinin NK2 receptor agonist [Lys5,MeLeu9,Nle10]-NKA(4-10) (LMN-NKA) on bladder and colorectal function were examined in minipigs. In anesthetized animals, subcutaneous (SC) administration of 30-100 µg/kg increased peak bladder and colorectal pressures. Increases in bladder and colorectal pressure were inhibited by a 15 min pretreatment with the NK2 receptor antagonist GR 159897 (1 mg/kg intravenously (IV)). Bladder and colorectal pressures were also increased after IV (0.3 µg/kg), intranasal (IN; 100 µg/kg) and sublingual administration (SL; 5 mg/kg). There was a nonsignificant trend for hypotension (16 or 12% decrease in mean arterial pressure) after 100 µg/kg SC and 0.3 µg/kg IV, respectively, but not after 100 µg/kg IN or 5 mg/kg SL. In conscious minipigs, 30-300 µg/kg SC caused a dose-related increase in defecation that was accompanied by emesis in 38% of subjects receiving 300 µg/kg. Urination was increased after 100 µg/kg SC but not lower or higher doses. The peak plasma exposure (Cmax) after 100 µg/kg SC was 123 ng/mL, and area under the curve (AUC) was 1790 min * ng/mL. Defecation response rates (~82%) were maintained after SC administration of LMN-NKA (30 µg/kg) given 3 times daily over 5 consecutive days. Defecation rates were higher after a single dose of 100 µg/kg IN compared with vehicle, but this did not reach significance. After 7-10 mg/kg SL, 83% of animals urinated and defecated, and none had emesis. The data support the feasibility of developing a convenient and well-tolerated route of administration of LMN-NKA for human use. Minipigs may be a suitable species for toxicology studies with LMN-NKA due to the relatively low rate of emesis in this species.

Affinity, potency, efficacy, and selectivity of neurokinin A analogs at human recombinant NK2 and NK1 receptors.

A series of peptide NK2 receptor agonists was evaluated for affinity, potency, efficacy, and selectivity at human recombinant NK2 and NK1 receptors expressed in CHO cells to identify compounds with the greatest separation between NK2 and NK1 receptor agonist activity. Binding studies were performed using displacement of [125I]-NKA binding to NK2 receptors and displacement of [3H]-Septide binding to NK1 receptors expressed in CHO cells. Functional studies examining the increase in intracellular calcium levels and cyclic AMP stimulation were performed using the same cell lines. A correlation was demonstrated between binding affinities (Ki) and potency to increase intracellular calcium (EC50) for NK2 and NK1 receptors. Ranking compounds by their relative affinity (Ki) or potency (EC50) at NK2 or NK1 receptors indicated that the most selective NK2 agonists tested were [Lys5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 674; NK1/NK2 EC50 ratio = 105) and [Arg5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 561; NK1/NK2 EC50 ratio = 70). The endogenous peptide, NKA, lacked selectivity with an NK1/NK2 Ki ratio = 20 and NK1/NK2 EC50 ratio = 1. Of the compounds selected for evaluation in cyclic AMP stimulation assays, [ß-Ala8]-NKA(4-10) had the greatest selectivity for activation of NK2 over NK1 receptors (NK1/NK2 EC50 ratio = 244), followed by [Lys5,MeLeu9,Nle10]-NKA(4-10) (ratio = 74), and NKA exhibited marginal selectivity (ratio = 2.8).

Colorectal and cardiovascular effects of [Lys5,MeLeu9,Nle10]-NKA(4-10) in anesthetized macaques.

The effects of the tachykinin NK2 receptor agonist LMN-NKA ([Lys5,MeLeu9,Nle10]-NKA(4-10)) on colorectal and arterial blood pressure were examined in anesthetized macaques. Intravenous (IV) administration of 1-100 µg/kg caused dose-related increases in colorectal pressure up to 120 mmHg above baseline, and area under the curve (AUC) up to 24,987 mmHg*s. This was accompanied at all doses by transient hypotension, with up to 26% reduction in mean arterial pressure (MAP) from baseline. Hypotension, but not the increase in colorectal pressure, was inhibited by a 10-min pretreatment with the NK1 receptor antagonist CP-99,994. In a pilot experiment using subcutaneous (SC) injection, a similar dose range of LMN-NKA (3-100 µg/kg) again appeared to increase colorectal pressure with a similar AUC (up to 18,546 mmHg*s) to that seen after IV injection, but lower peak amplitude (up to 49 mmHg). Unlike the effects of IV injection, hypotension was only present after the highest SC dose (100 µg/kg) in one of two animals. Pharmacokinetic analysis revealed markedly lower plasma exposures after SC compared with IV administration. Cmax was 39.6 versus 1070 ng/mL, and AUCinf was 627 versus 2090 ng/mL*min, respectively. These findings are consistent with previous observations in anesthetized dogs and indicate that the prokinetic effects of LMN-NKA may be achieved without hypotension using a route of administration that avoids unnecessarily high plasma exposures.

[Lys5,MeLeu9,Nle10]-NKA(4-10) Elicits NK2 Receptor-Mediated Micturition and Defecation, and NK1 Receptor-Mediated Emesis and Hypotension, in Conscious Dogs.

Tachykinin neurokinin 2 (NK2) receptor agonists may have potential to alleviate clinical conditions associated with bladder and gastrointestinal underactivity by stimulating contraction of visceral smooth muscle. The ability of [Lys5,MeLeu9,Nle10]-neurokinin A(4-10) (LMN-NKA) to elicit micturition and defecation was examined after repeated administration in groups of 2-10 conscious dogs. Administration of 10-100 µg/kg, i.v., four times daily for six consecutive days, reliably elicited micturition after ≥90% of doses and defecation after ≥50% of doses. Voiding occurred <4 minutes after dosing and was short lasting (<10 minutes). LMN-NKA was well tolerated, with emesis after ∼25% of doses at 100 µg/kg, i.v. Hypotension was induced by 100 µg/kg, i.v., of LMN-NKA but not by lower doses. Administration of 30-300 µg/kg, s.c., twice daily for seven consecutive days, reliably elicited both urination and defecation after 88%-100% of doses, and was accompanied by a high rate of emesis (50%-100%). The onset of voiding was rapid (<7 minutes) but was more prolonged than after intravenous administration (30-60 minutes). Emesis induced by 30 or 300 µg/kg, s.c., of LMN-NKA was significantly reduced (from 58% to 8% and from 96% to 54%, respectively) by a 30-minute pretreatment with the neurokinin 1 (NK1) receptor antagonist, (2S,3S)-N-(2-methoxybenzyl)-2-phenylpiperidin-3-amine (CP-99,994; 1 mg/kg, s.c.). The ability of selective NK2 receptor agonists to elicit on-demand voiding could potentially address a major unmet need in people lacking voluntary control of micturition and/or defecation. LMN-NKA unexpectedly activated NK1 receptors at doses that stimulated voiding, causing emesis and hypotension that may limit the clinical utility of nonselective NK2 receptor agonists.

Prokinetic effects of neurokinin-2 receptor agonists on the bladder and rectum of rats with acute spinal cord transection.

The suitability of various neurokinin-2 (NK2) receptor agonists and routes of administration to elicit on-demand voiding of the bladder and bowel, as future therapy for individuals with spinal cord injury, was examined using a rat model. The current study examined the feasibility of alternative routes of administration, which are more practical for clinical use than intravenous (IV) administration. Voiding and isovolumetric cystometry were recorded in anesthetized, acutely spinalized, female rats after IV, subcutaneous (SC), intramuscular (IM), intranasal (IN), or sublingual (SL) administration of [Lys5,MeLeu9,Nle10]-NKA(4-10) (LMN-NKA). Administration of LMN-NKA (1-10µg/kg IV; 10-300µg/kg SC or IM; 15-1000µg/kg IN or 300-1500µg/kg SL) elicited rapid-onset, short-duration, dose-related increases in bladder pressure and voiding with the rank order for time of both onset and duration being IV < IN < SC = IM < SL. The incidence of voiding was dependent on the dose and route, with all routes resulting in a high voiding efficiency (~ 70%). Like LMN-NKA, neurokinin A (NKA 1-100µg/kg IV) and GR 64349 (0.1-30µg/kg IV or 1-300µg/kg SC) produced rapid-onset, short-duration increases in bladder pressure, as well as colorectal pressure. Administration of vehicle never produced bladder or rectal contractions or voiding. Transient hypotension was observed after IV injection of LMN-NKA, which was less pronounced after SC injection. Hypotension was not apparent with GR 64349. In conclusion, selective NK2 receptor agonists, administered through various non-IV routes of administration, may provide a safe, convenient, and efficacious method for inducing voiding.

NK2 and NK1 receptor-mediated effects of NKA and analogs on colon, bladder, and arterial pressure in anesthetized dogs.

Tachykinin NK2 receptor (NK2R) agonists have potential to alleviate clinical conditions associated with bladder and gastrointestinal under activity. The effects of agonists with differing selectivity for NK2R over NK1Rs on colorectal, bladder, and cardiovascular function were examined in anesthetized dogs. Intravenous (IV) administration of NKA, LMN-NKA ([Lys5,MeLeu9,Nle10]-NKA(4-10)), and [ß-Ala8]-NKA(4-10) caused a dose-related increase in colorectal pressure (up to 98 mmHg) that was blocked by pretreatment with the NK2R antagonist GR 159897 (1 mg/kg), and hypotension (decrease in mean arterial pressure of ~40 mmHg) that was blocked by the NK1R antagonist CP-99,994 (1 mg/kg). Despite the greater in vitro selectivity of LMN-NKA and [ß-Ala8]-NKA(4-10) for NK2R over NK1Rs compared with NKA, all 3 agonists increased colorectal pressure and caused hypotension within a similar dose range when administered as a bolus (0.1-300 µg/kg IV), or even as a slow IV infusion over 5 min (NKA; 0.02-0.6 µg/kg/min). In contrast, subcutaneous (SC) administration of LMN-NKA (3-10 µg/kg) increased colorectal pressure (up to 50 mmHg) and elicited micturition (≧ 85% voiding efficiency) without causing hypotension. NK2R agonists can produce rapid-onset, short-duration, colorectal contractions, and efficient voiding of urine without hypotension after SC administration, indicating that routes of administration that avoid the high plasma concentrations associated with IV dosing improve the separation between desired and unwanted pharmacodynamic effects. The potent hypotensive effect of NKA in dogs was unexpected based on published studies in humans in which IV infusion of NKA did not affect blood pressure at doses that increased gastrointestinal motility.

Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo.

AIMS: Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction. MATERIAL AND METHODS: In vivo cystometry experiments were performed in adult female Sprague-Dawley rats under urethane anesthesia. Diabetes was induced by streptozotocin (STZ; 65mg/kg, i.p.) injection. Experiments were performed 9 and 20weeks post STZ-treatment. Drugs included neuromedin B (NMB; BB1 receptor preferring agonist), and gastrin-releasing peptide (GRP; BB2 receptor preferring agonist). KEY FINDINGS: NMB and GRP (0.01-100µg/kg in sham rats; 0.1-300µg/kg in STZ-treated rats, i.v.) increased micturition frequency, bladder contraction amplitude and area under the curve dose dependently in both sham and STZ-treated rats. In addition, NMB (3, 10µg/kg i.v.) triggered voiding in >80% of STZ-treated rats when the bladder was filled to a sub-threshold voiding volume. NMB and GRP increased mean arterial pressure and heart rate at the highest doses, 100 and 300µg/kg. SIGNIFICANCE: Activation of bombesin receptors facilitated neurogenic bladder contractions in vivo. Single applications of agonists enhanced or triggered voiding in sham rats as well as in the STZ-treated rat model of diabetic voiding dysfunction. These results suggest that BB receptors may be targeted for drug development for conditions associated with poor detrusor contraction such as an underactive bladder condition.

Functional bombesin receptors in urinary tract of rats and human but not of pigs and mice, an in vitro study.

AIMS: Bombesin receptors (BB receptors) and/or bombesin related peptides are expressed in the lower urinary tract, though their function and distribution in different species is largely unknown. This study examines whether BB receptor agonists can contract bladder smooth muscle in rats, mice, pigs and humans. METHODS: Bladder strips were placed in tissue baths for in vitro contractility. Neuronally evoked contractions were elicited using electric field stimulation (EFS). Effects of the BB receptor agonists, neuromedin B (NMB; BB1 receptor agonist) and gastrin-releasing peptide (GRP; BB2 receptor agonist) on baseline tone and EFS-induced contractions were monitored. RESULTS: In rat and human bladder strips, NMB and GRP (10(-11)-10(-6)M) increased EFS-induced contractions in a concentration dependent manner. In these species, NMB and GRP also increased baseline tension. In mouse and pig bladder strips, NMB and GRP (10(-8)-3×10(-6)M) had no effects on either parameter. CONCLUSIONS: These data suggest that bombesin receptors BB receptor 1 and/or BB receptor 2 increase bladder contractions in rat and human. The site of action of these receptors may be pre- and/or post-synaptic, increasing release of transmitters or enhancing smooth muscle excitability, respectively. Thus, BB1 receptor and/or BB2 receptor may offer therapeutic targets for voiding dysfunction associated with impaired bladder contractility; however, species differences must be considered when studying these receptors. Part of this work was published in an abstract form at the SFN meeting New Orleans, 2012.

Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats.

Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min "quiet period" (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8(th). Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.

Effects of the 5-HT4 receptor agonist, cisapride, on neuronally evoked responses in human bladder, urethra, and ileum.

This study evaluated the effects of a 5-HT4 agonist, cisapride, on neuronally evoked smooth muscle responses in bladder, urethra and ileum and compared these effects with those of an acetylcholinesterase inhibitor, distigmine. Electrical field stimulation (EFS) was applied to human bladder and ileum smooth muscle strips from human organ transplant donors and to urethral strips from prostatectomy patients, to evoke neuronally mediated smooth muscle responses. EFS induced contractions in bladder and mixed responses, consisting of contractions and relaxations, in urethra and ileum. Relaxations were mediated by nitric oxide while contractions were partially cholinergic (i.e. atropine sensitive). This atropine sensitive component amounted to~95% in bladder and ~75% in ileum, and it was enhanced by distigmine in a concentration dependent manner (0.1-3 µM; ~100-600% increase in bladder and ~50-250% increase in ileum). Cisapride (0.0003-1 µM) also enhanced bladder contractions (~75-100% increase) but had no effect on urethral contractions or relaxations, and modestly enhanced ileum contractions (~10-40% increase). Facilitatory effects of cisapride were reversed by the specific 5-HT4 receptor antagonist, SB-203186 (3 µM), but were resistant to repeated washing in the bladder. These data indicate that 5-HT4 receptor agonists enhanced EFS-induced contractions in bladder and ileum without an effect on urethra and suggest that it may be possible to enhance bladder activity with a dose of cisapride that is at, or below, those producing gastrointestinal (GI) effects. Although distigmine's maximal facilitation of bladder and GI tract function was greater than that of cisapride, at clinically relevant concentrations cisapride showed much greater efficacy.

Exogenous activation of muscarinic receptors decreases subsequent non-muscarinic bladder contractions in vivo in the female rat.

AIMS: To determine if the muscarinic agonist, bethanechol, inhibits the non-cholinergic, atropine-resistant (i.e. putatively purinergic) component of naturally occurring (i.e. reflexogenic) bladder contractions in vivo in the rat, as previously described in vitro. Our second aim was to determine if elevation of endogenous acetylcholine (ACh) with distigmine, an acetylcholine esterase (AChE) inhibitor, could also inhibit non-cholinergic component of reflexogenic bladder contractions. MAIN METHODS: Cystometry was performed in urethane anesthetized adult female Sprague Dawley rats. The nonselective muscarinic receptor (mAChR) antagonist, atropine, was administered intravenously (i.v.) before and after i.v. administration of the non-selective mAChR agonist, bethanechol, the AChE inhibitor, distigmine or the neurokinin receptor 2 agonist, [ßAla(8)]-Neurokinin A(4-10). Intermicturition interval (IMI), bladder contraction amplitude (BCA), postvoid bladder pressure (PVBP), and voiding efficiency (VE) were measured. KEY FINDINGS: Atropine (0.4 mg/kg; n=11 rats) delivered as the first drug, had insignificant effects on BCA (~15% reduction) or PVBP (~15% increase) and weakly reduced IMI and VE by ~40% (p<0.05) relative to vehicle. Bethanechol and distigmine on their own produced excitatory effects on bladder activity, consistent with mAChR activation. Unexpectedly atropine, administered after bethanechol or after distigmine but not after [ßAla(8)]-Neurokinin A(4-10), completely blocked bladder activity for 3-10 min. Partial recovery of bladder activity occurred after that time, but BCA, IMI, and VE remained significantly reduced and PVBP remained significantly increased. SIGNIFICANCE: Activation of mAChRs by an exogenous agonist or elevation of endogenous ACh levels by an AChE inhibitor inhibits the non-cholinergic, atropine-resistant, component of reflexogenic bladder contractions in vivo.

Synaptic activation of bulbospongiosus motoneurons via dorsal gray commissural inputs.

Ejaculation is controlled by coordinated and rhythmic contractions of bulbospongiosus (BSM) and ischiocavernosus muscles. Motoneurons that innervate and control BSM contractions are located in the dorsomedial portion of the ventral horn in the L(5-6) spinal cord termed the dorsomedial (DM) nucleus. We characterized intrinsic properties of DM motoneurons as well as synaptic inputs from the dorsal gray commissure (DGC). Electrical stimulation of DGC fibers elicited fast inhibitory and excitatory responses. In the presence of glutamate receptor antagonists, both fast GABAergic as well as glycinergic inhibitory postsynaptic potentials (IPSPs) were recorded. No slow GABA(B)-mediated inhibition was evident. In the presence of GABA(A) and glycine receptor antagonists, DGC stimulation elicited fast glutamatergic excitatory responses that were blocked by application of CNQX. Importantly, a slow depolarization (timescale of seconds) was routinely observed that sufficiently depolarized the DM motoneurons to fire "bursts" of action potentials. This slow depolarization was elicited by a range of stimulus train frequencies and was insensitive to glutamate receptor antagonists (CNQX and d-APV). The slow depolarization was accompanied by an increase in membrane resistance with an extrapolated reversal potential near the K(+) Nernst potential. It was mediated by the combination of the block of a depolarization-activated K(+) current and the activation of a QX-314-sensitive cation current. These results demonstrate that fast synaptic responses in DM motoneurons are mediated primarily by glutamate, GABA, and glycine receptors. In addition, slow nonglutamatergic excitatory postsynaptic potentials (EPSPs), generated through DGC stimulation, can elicit burstlike responses in these neurons.