CB2 cannabinoid receptor agonist selectively inhibits the mechanosensitivity of mucosal afferents in the guinea pig bladder.

Bladder afferents play a pivotal role in bladder function such as urine storage and micturition as well as conscious sensations such as urgency and pain. Endocannabinoids are ligands of cannabinoid 1 and 2 (CB1 and CB2) receptors but can influence the activity of a variety of G protein-coupled receptors as well as ligand-gated and voltage-gated channels. It is still not known which classes of bladder afferents are influenced by CB1 and CB2 receptor agonists. This study aimed to determine the role of CB2 receptors in two major classes of afferents in the guinea pig bladder: mucosal and muscular-mucosal. The mechanosensitivity of these two classes was determined by an ex vivo extracellular electrophysiological recording technique. A stable analog of endocannabinoid anandamide, methanandamide (mAEA), potentiated the mechanosensitivity of mucosal bladder afferents in response to stroking. In the presence of a transient receptor potential vanilloid 1 antagonist (capsazepine), the effect of mAEA switched from excitatory to inhibitory. A selective CB2 receptor agonist, 4-quinolone-3-carboxyamide (4Q3C), significantly inhibited the mechanosensitivity of mucosal bladder afferents to stroking. In the presence of a CB2 receptor antagonist, the inhibitory effect of 4Q3C was lost. mAEA and 4Q3C did not affect responses to stretch and/or mucosal stroking of muscular-mucosal afferents. Our findings revealed that agonists of CB2 receptors selectively inhibited the mechanosensitivity of capsaicin-sensitive mucosal bladder afferents but not muscular-mucosal afferents. This may have important implications for understanding of the role of endocannabinoids in modulating bladder function and sensation in health and diseases.NEW & NOTEWORTHY This article describes, for the first time, to our knowledge, the direct inhibitory effect of cannabinoid 2 receptor agonists on guinea pig mucosal bladder afferents. The cannabinoid 2 receptor is involved in pain and inflammation, suggesting that this may be a viable target for treatment of bladder disorders such as cystitis.

Stimulation of the pelvic nerve increases bladder capacity in the PGE2 cat model of overactive bladder.

Selective electrical stimulation of the pudendal nerve exhibits promise as a potential therapy for treating overactive bladder (OAB) across species (rats, cats, and humans). More recently, pelvic nerve (PelN) stimulation was demonstrated to improve cystometric bladder capacity in a PGE2 rat model of OAB. However, PelN stimulation in humans or in an animal model that is more closely related to humans has not been explored. Therefore, our objective was to quantify the effects of PGE2 and PelN stimulation in the cat. Acute cystometry experiments were conducted in 14 α-chloralose-anesthetized adult, neurologically intact female cats. Intravesical PGE2 decreased bladder capacity, residual volume, threshold contraction pressure, and mean contraction pressure. PelN stimulation reversed the PGE2-induced decrease in bladder capacity and increased evoked external urethral sphincter electromyographic activity without influencing voiding efficiency. The increases in bladder capacity generated by PelN stimulation were similar in the rat and cat, but the stimulation parameters to achieve this effect differed (threshold amplitude at 10 Hz in the rat vs. twice threshold amplitude at 1 Hz in the cat). These results highlight the potential of PGE2 as a model of OAB and provide further evidence that PelN stimulation is a promising approach for the treatment of OAB symptoms.

The STK16 inhibitor STK16-IN-1 inhibits non-adrenergic and non-neurogenic smooth muscle contractions in the human prostate and the human male detrusor.

Mixed lower urinary tract symptoms (LUTS) (voiding symptoms suggestive of benign prostatic hyperplasia plus storage symptoms, which can be caused by overactive bladder) are common in men. Unwanted contraction of prostate and/or bladder smooth muscle has been implied in the pathophysiology of male LUTS. Here, we examined effects of the serine/threonine kinase 16 (STK16) inhibitor STK16-IN-1 on contraction of human tissues from the prostate and male detrusor. Tissues were obtained from radical prostatectomy and radical cystectomy. Contractions were studied in an organ bath and STK16 expressions by Western blot analyses and fluorescence staining. In prostate tissues, STK16-IN-1 (1 µM) inhibited contractions induced by endothelin-1 and the thromboxane A2 analog U46619. Contractions of prostate tissues induced by noradrenaline, the α1-agonists phenylephrine and methoxamine, or electric field stimulation (EFS) were not changed by STK16-IN-1. In male detrusor tissues, STK16-IN-1 inhibited contractions induced by the cholinergic agonists carbachol and metacholine, and contractions induced by U46619. EFS-induced contractions of detrusor tissues were not changed by STK16-IN-1. Western blot analyses of prostate and detrusor tissues revealed bands matching the molecular weight of STK16. Fluorescence staining of prostate tissues using STK16 antibodies resulted in immunoreactivity in smooth muscle cells. STK16-IN-1 selectively inhibits non-adrenergic/non-neurogenic smooth muscle contractions in the male prostate and to limited extent in the bladder. Because non-adrenergic contractions in the male LUTS may account for limited efficacy of α1-blockers and for α1-blocker-resistant symptoms, studies assessing add-on of STK16-IN-1 to α1-blockers in mixed LUTS appear feasible.

Association of innervation-adjusted alpha-synuclein in arrector pili muscles with cardiac noradrenergic deficiency in autonomic synucleinopathies.

BACKGROUND: Autonomic synucleinopathies feature deposition of the protein alpha-synuclein (AS) in neurons [e.g., Lewy body neurogenic orthostatic hypotension (nOH)] or glial cells (multiple system atrophy, MSA). AS in skin biopsies might provide biomarkers of these diseases; however, this approach would be complicated or invalidated if there were substantial loss of AS-containing nerves. We report AS content in arrector pili muscles in skin biopsies after adjustment for local innervation in patients with Lewy body nOH or MSA. Cardiac sympathetic neuroimaging by myocardial 18F-dopamine positron emission tomography (PET) was done to examine pathophysiological correlates of innervation-adjusted AS. METHODS: Thirty-one patients (19 Lewy body nOH, 12 MSA) underwent thoracic 18F-dopamine PET and skin biopsies. AS signal intensity analyzed by immunofluorescence microscopy was adjusted for innervation by the ratio of AS to protein gene product (PGP) 9.5, a pan-axonal marker (Harvard lab site), or the ratio of AS to tyrosine hydroxylase (TH), an indicator of catecholaminergic neurons (NIH lab site). RESULTS: The Lewy body nOH group had higher ratios of AS/PGP 9.5 or log AS/TH than did the MSA group (0.89 ± 0.05 vs. 0.66 ± 0.04, -0.13 ± 0.05 vs. -1.60 ± 0.33; p < 0.00001 each). All 19 Lewy body patients had AS/PGP 9.5 > 0.8 or log AS/TH > 1.2 and had myocardial 18F-dopamine-derived radioactivity < 6000 nCi-kg/cc-mCi, the lower limit of normal. Two MSA patients (17%) had increased AS/PGP or log AS/TH, and two (17%) had low 18F-dopamine-derived radioactivity. CONCLUSIONS: Lewy body forms of nOH are associated with increased innervation-adjusted AS in arrector pili muscles and neuroimaging evidence of myocardial noradrenergic deficiency.

Characterization of projections of longitudinal muscle motor neurons in human colon.

BACKGROUND: The enteric nervous system contains inhibitory and excitatory motor neurons which modulate smooth muscle contractility. Cell bodies of longitudinal muscle motor neurons have not been identified in human intestine. METHODS: We used retrograde tracing ex vivo with DiI, with multiple labeling immunohistochemistry, to characterize motor neurons innervating tenial and inter-tenial longitudinal muscle of human colon. KEY RESULTS: The most abundant immunohistochemical markers in the tertiary plexus were vesicular acetylcholine transporter, nitric oxide synthase (NOS), and vasoactive intestinal polypeptide (VIP). Of retrogradely traced motor neurons innervating inter-tenial longitudinal muscle, 95% were located within 6mm oral or anal to the DiI application site. Excitatory motor neuron cell bodies, immunoreactive for choline acetyltransferase (ChAT), were clustered aborally, whereas NOS-immunoreactive cell bodies were distributed either side of the DiI application site. Motor neurons had small cell bodies, averaging 438 + 18µm2 in cross-sectional area, similar for ChAT- and NOS-immunoreactive subtypes. Motor neurons innervating the tenia had slightly longer axial projections, with 95% located within 9mm. ChAT-immunoreactive excitatory motor neurons to tenia were clustered aborally, whereas NOS-immunoreactive inhibitory motor neurons had both ascending and descending projections. VIP immunoreactivity was rarely present without NOS immunoreactivity in motor neurons. CONCLUSIONS AND INFERENCES: Tenial and inter-tenial motor neurons innervating the longitudinal muscle have short projections. Inhibitory motor neurons have less polarized projections than cholinergic excitatory motor neurons. Longitudinal and circular muscle layers are innervated by distinct local populations of excitatory and inhibitory motor neurons. A population of human enteric neurons that contribute significantly to colonic motility has been characterized.

Optical recording reveals topological distribution of functionally classified colorectal afferent neurons in intact lumbosacral DRG.

Neuromodulation as a non-drug alternative for managing visceral pain in irritable bowel syndrome (IBS) may target sensitized afferents of distal colon and rectum (colorectum), especially their somata in the dorsal root ganglion (DRG). Developing selective DRG stimulation to manage visceral pain requires knowledge of the topological distribution of colorectal afferent somata which are sparsely distributed in the DRG. Here, we implemented GCaMP6f to conduct high-throughput optical recordings of colorectal afferent activities in lumbosacral DRG, that is, optical electrophysiology. Using a mouse ex vivo preparation with distal colorectum and L5-S1 DRG in continuity, we recorded 791 colorectal afferents' responses to graded colorectal distension (15, 30, 40, and 60 mmHg) and/or luminal shear flow (20-30 mL/min), then functionally classified them into four mechanosensitive classes, and determined the topological distribution of their somata in the DRG. Of the 791 colorectal afferents, 90.8% were in the L6 DRG, 8.3% in the S1 DRG, and only 0.9% in the L5 DRG. L6 afferents had all four classes: 29% mucosal, 18.4% muscular-mucosal, 34% low-threshold (LT) muscular, and 18.2% high-threshold (HT) muscular afferents. S1 afferents only had three classes: 19.7% mucosal, 34.8% LT muscular, and 45.5% HT muscular afferents. All seven L5 afferents were HT muscular. In L6 DRG, somata of HT muscular afferents were clustered in the caudal region whereas somata of the other classes did not cluster in specific regions. Outcomes of this study can directly inform the design and improvement of next-generation neuromodulation devices that target the DRG to alleviate visceral pain in IBS patients.

Impact of polypropylene prolapse mesh on vaginal smooth muscle in rhesus macaque.

BACKGROUND: The use of polypropylene prolapse mesh to treat pelvic organ prolapse has been limited by mesh-related complications. Gynemesh PS mesh, implanted via sacrocolpopexy in rhesus macaques, had a negative impact on the vagina with thinning of vaginal muscularis and decreased vaginal smooth muscle contractility. The negative effect was attenuated when a bioscaffold derived from urinary bladder extracellular matrix was used as a composite with Gynemesh PS. OBJECTIVE: The objective of the study was to further elucidate the impact of Gynemesh PS polypropylene mesh and MatriStem extracellular matrix bioscaffolds on the vaginal smooth muscle in terms of micromorphology of vaginal smooth muscle (muscle bundles and individual myocytes), innervation, and nerve-mediated contractile function following their implantations in a rhesus macaque model via sacrocolpopexy. STUDY DESIGN: Thirty-two middle-aged rhesus macaques were randomized to undergo either a sham surgery (sham, n = 8), or the implantation of Gynemesh PS alone (n = 8) vs composite mesh comprised of Gynemesh PS plus 2-ply MatriStem (n = 8) vs 6-ply MatriStem alone (n = 8) via sacrocolpopexy. The graft-vagina complexes were harvested 3 months later. Histomorphometrics of smooth muscle bundles and myocytes were performed by immunofluorescent labeling of alpha smooth muscle actin, caveolin-3 (membrane protein), and cell nuclei followed by confocal imaging. The cross-sectional diameters of smooth muscle bundles and individual myocytes were quantified using images randomly taken in at least 5 areas of each section of sample. Contractile proteins alpha smooth muscle actin and smoothelin were quantified by Western immunoblotting. Nerve density was measured by immunohistochemical labeling of a pan-neuron marker, PGP9.5. Nerve-mediated smooth muscle contractility was quantified using electrical field stimulation. One-way analysis of variance and appropriate post hoc tests were used for statistical comparisons. RESULTS: Compared with sham, the implantation of Gynemesh PS alone resulted in a disorganized smooth muscle morphology with the number of small muscle bundles (cross-sectional diameter less than 20 µm) increased 67% (P = .004) and the myocyte diameter decreased 22% (P < .001). Levels of contractile proteins were all decreased vs sham with alpha smooth muscle actin decreased by 68% (P = .009), low-molecular-weight smoothelin by 51% (P = .014), and high-molecular-weight smoothelin by 40% (P = .015). Nerve density was decreased by 48% (P = .03 vs sham) paralleled by a 63% decrease of nerve-mediated contractility (P = .02). Following the implantation of composite mesh, the results of measurements were similar to sham (all P > .05), with a 39% increase in the myocyte diameter (P < .001) and a 2-fold increase in the level of alpha smooth muscle actin relative to Gynemesh (P = .045). Following the implantation of MatriStem alone, the number of small muscle bundles were increased 54% vs sham (P = .002), while the other parameters were not significantly different from sham (all P > .05). CONCLUSION: The implantation of Gynemesh PS had a negative impact on the structural and functional integrity of vaginal smooth muscle evidenced by atrophic macro- and microscopic muscle morphology, decreased innervation, and impaired contractile property, consistent with a maladaptive remodeling response. The extracellular matrix bioscaffold (MatriStem), when used with Gynemesh PS as a composite (2 ply), attenuated the negative impact of Gynemesh PS; when used alone (6 ply), it induced adaptive remodeling as evidenced by an increased fraction of small smooth muscle bundles with normal contractility.

Influence of sildenafil on the purinergic components of nerve-mediated and urothelial ATP release from the bladder of normal and spinal cord injured mice.

BACKGROUND AND PURPOSE: PDE inhibitors such as sildenafil alleviate lower urinary tract symptoms; however, a complete understanding of their action on the bladder remains unclear. We are investigating the effects of sildenafil, on post and preganglionic nerve-mediated contractions of the mouse bladder, and neuronal and urothelial ATP release. EXPERIMENTAL APPROACH: Bladders were used from young (12 weeks), aged (24 months), and spinal cord transected (SCT), mice, for in vitro contractility experiments. An arterially perfused in situ whole mouse model was used to record bladder pressure. Nerve-mediated contractions were generated by electrical field stimulation (EFS) of postganglionic nerve terminals or the pelvic nerve. ATP release during EFS in intact detrusor strips, and during stretch of isolated mucosa strips, was measured using a luciferin-luciferase assay. KEY RESULTS: Sildenafil (20 µM) inhibited nerve-mediated contractions in young mice, with an increase in f1/2 values in force-frequency relationships, demonstrating a greater effect at low frequencies. Sildenafil reduced the atropine-resistant, purinergic component of nerve-mediated contractions, and suppressed neuronal ATP release upon EFS in vitro. Sildenafil reduced the preganglionic pelvic nerve stimulated bladder pressure recordings in situ; comparable to in vitro experiments. Sildenafil reduced stretch-induced urothelial ATP release. Sildenafil also relaxed nerve-mediated contractions in aged and SCT mice. CONCLUSION AND IMPLICATIONS: Sildenafil has a greater effect on the low-frequency, purinergic-mediated contractions and suppresses neuronal ATP release. In addition, sildenafil reduces stretch-induced urothelial ATP release. These results demonstrate a novel action of sildenafil to selectively inhibit ATP release from nerve terminals innervating detrusor smooth muscle and the urothelium.

Sonic hedgehog regulation of human rhabdosphincter muscle:Potential implications for treatment of stress urinary incontinence.

AIMS: Rhabdosphincter (RS) muscle injury occurs during prostatectomy, and is a leading cause of stress urinary incontinence (SUI). Current SUI treatments engender significant side effects, which negatively impact patient quality of life. Thus an unmet need exists to develop novel RS regeneration methods. We have shown that Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and we have developed novel peptide amphiphile nanofiber hydrogel delivery of SHH protein to the penis to regenerate smooth muscle after prostatectomy induced injury. If similar SHH signaling mechanisms regulate RS muscle homeostasis, this innovative technology may be adapted for RS regeneration post-prostatectomy. We examine the SHH pathway in human RS muscle. METHODS: Human RS obtained during radical cystoprostatectomy (n = 13), underwent SHH pathway analysis. Primary cultures were established (n = 5), and RS cells were treated with SHH protein, SHH inhibitor, or PBS (control). Immunohistochemical analysis for SHH pathway, skeletal muscle actin, and trichrome stain were performed. RS growth was quantified at 3 and 6 days. RESULTS: SHH, it is receptors patched and smoothened, and transcriptional activators, GLI proteins, were identified in human RS muscle. At 3 and 6 days, RS cells increased 62% and 78% (P = 0.0001) with SHH treatment and decreased 40% (P = 0.0001) and 18% (P = 0.039) with SHH inhibition. CONCLUSIONS: The SHH pathway was identified in human RS. RS growth increased with SHH treatment, indicating intervention may be possible to enhance RS regeneration, and impact SUI. Peptide amphiphile delivery of SHH may be applicable for RS regeneration and SUI prevention.

Determining integrity of bladder innervation and smooth muscle function 1 year after lower spinal root transection in canines.

AIMS: To assess bladder smooth muscle function and innervation after long-term lower spinal root transection in canines. METHODS: Thirteen female mixed-breed hound dogs underwent bladder decentralization, which included transection of all sacral dorsal and ventral roots caudal to L7 and hypogastric nerves, bilaterally (n = 3); all sacral roots and hypogastric nerves plus transection of L7 dorsal roots, bilaterally (n = 4); or a sham operation (n = 6). At a year after initial surgery, bladder function was assessed in vivo by stimulation of the pelvic plexus. The bladder tissue was harvested for ex vivo smooth muscle contractility studies. Remaining bladder was evaluated for nerve morphology immunohistochemically using neuronal marker PGP9.5, apoptotic activity using terminal deoxynucleotidyl transferase dUTP nick end labeling, and histopathology using a hematoxylin and eosin stain. RESULTS: Sacral root decentralization did not reduce maximum strength of pelvic plexus stimulation-induced bladder contraction, although long-term sacral dorsal and ventral root plus L7 dorsal root transection significantly decreased contraction strength. Electric field stimulation-induced contractions of the detrusor from all decentralized animals were preserved, compared to controls. Viable nerves and intramural ganglia were visualized in the bladder wall, regardless of group. There was no difference in amount of apoptosis in bladder smooth muscle between groups. CONCLUSION: Bladder smooth muscle cells maintain their function after long-term bladder decentralization. While pelvic plexus-induced bladder contractions were less robust at 1 year after lower spinal root transection, the absence of atrophy and preservation of at least some nerve activity may allow for successful surgical reinnervation after long-term injury.

Impaired neural pathway in gastric muscles of patients with diabetes.

To explore the pathogenic mechanism of diabetic gastropathy, we investigated differences in response to electrical field stimulation (EFS) of gastric muscles from diabetic and non-diabetic (control) patients. Gastric specimens were obtained from 34 patients and 45 controls who underwent gastrectomy for early gastric cancer. Using organ bath techniques, we examined peak and nadir values of contraction under EFS. To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine, MRS2500, and N-nitro-L-arginine (L-NNA) were added sequentially to the organ bath. Tetrodotoxin (TTX) was used to confirm that the responses to EFS were mediated via neural stimulation. In the absence of pharmacological agents, peak contraction amplitude was greater in non-diabetic controls compared to diabetics only in the distal longitudinal gastric muscles. However, the nadir was greater in controls than in patients in both proximal and distal gastric circular muscles. Addition of MRS2500 could not decrease the nadir in both controls and patients, both in the proximal and distal stomach. However, L-NNA completely reversed the relaxation. TTX had no further effect on nadir. In conclusion, impaired inhibitory nitrergic neural pathway in both proximal and distal stomach and impaired excitatory cholinergic neural pathway in the distal stomach may contribute to the pathogenic mechanism underlying diabetic gastropathy.

Muscularis macrophages: Key players in intestinal homeostasis and disease.

Macrophages residing in the muscularis externa of the gastrointestinal tract are highly specialized cells that are essential for tissue homeostasis during steady-state conditions as well as during disease. They are characterized by their unique protective functional phenotype that is undoubtedly a consequence of the reciprocal interaction with their environment, including the enteric nervous system. This muscularis macrophage-neuron interaction dictates intestinal motility and promotes tissue-protection during injury and infection, but can also contribute to tissue damage in gastrointestinal disorders such as post-operative ileus and gastroparesis. Although the importance of muscularis macrophages is clearly recognized, different aspects of these cells remain largely unexplored such their origin, longevity and instructive signals that determine their function and phenotype. In this review, we will discuss the phenotype, functions and origin of muscularis macrophages during steady-state and disease conditions. We will highlight the bidirectional crosstalk with neurons and potential therapeutic strategies that target and manipulate muscularis macrophages to restore their protective signature as a treatment for disease.

Stimulation of the pelvic nerve increases bladder capacity in the prostaglandin E2 rat model of overactive bladder.

Overactive bladder (OAB) syndrome is a highly prevalent condition that may lead to medical complications and decreased quality of life. Emerging therapies focusing on selective electrical stimulation of peripheral nerves associated with lower urinary tract function may provide improved efficacy and reduced side effects compared with sacral neuromodulation for the treatment of OAB symptoms. Prior studies investigating the effects of pelvic nerve (PelN) stimulation on lower urinary tract function were focused on promoting bladder contractions, and it is unclear whether selective stimulation of the PelN would be beneficial for the treatment of OAB. Therefore our motivation was to test the hypothesis that PelN stimulation would increase bladder capacity in the prostaglandin E2 (PGE2) rat model of OAB. Cystometry experiments were conducted in 17 urethane-anesthetized female Sprague-Dawley rats. The effects of intravesical PGE2 vs. vehicle and PelN stimulation after intravesical PGE2 on cystometric parameters were quantified. Intravesical infusion of PGE2 resulted in decreased bladder capacity and increased voiding efficiency without a change in bladder contraction area under the curve, maximum contraction pressure, or contraction duration. Bladder capacity was also significantly decreased compared with vehicle (1% ethanol in saline) confirming that the change in bladder capacity was mediated by PGE2 PelN stimulation reversed the PGE2-induced change in bladder capacity and increased the external urethral sphincter electromyogram activity at a specific stimulation condition (amplitude of 1.0 times threshold at 10 Hz). These results confirm that the urodynamic changes reported in conscious rats are also observed under urethane anesthesia and that PelN stimulation is a novel and promising approach for the treatment of the symptoms of OAB.

The effects of neuromodulation in a novel obese-prone rat model of detrusor underactivity.

Obesity is a global epidemic associated with an increased risk for lower urinary tract dysfunction. Inefficient voiding and urinary retention may arise in late-stage obesity when the expulsive force of the detrusor smooth muscle cannot overcome outlet resistance. Detrusor underactivity (DUA) and impaired contractility may contribute to the pathogenesis of nonobstructive urinary retention. We used cystometry and electrical stimulation of peripheral nerves (pudendal and pelvic nerves) to characterize and improve bladder function in urethane-anesthetized obese-prone (OP) and obese-resistant (OR) rats following diet-induced obesity (DIO). OP rats exhibited urinary retention and impaired detrusor contractility following DIO, reflected as increased volume threshold, decreased peak micturition pressure, and decreased voiding efficiency (VE) compared with OR rats. Electrical stimulation of the sensory branch of the pudendal nerve did not increase VE, whereas patterned bursting stimulation of the motor branch of the pudendal nerve increased VE twofold in OP rats. OP rats required increased amplitude of electrical stimulation of the pelvic nerve to elicit bladder contractions, and maximum evoked bladder contraction amplitudes were decreased relative to OR rats. Collectively, these studies characterize a novel animal model of DUA that can be used to determine pathophysiology and suggest that neuromodulation is a potential management option for DUA.

Effect of short-term androgen deficiency on bladder contractility and urothelial mediator release.

In men, testosterone levels decline by 1% per year after the age of 40. Reduced androgen levels may directly contribute to lower urinary tract symptoms and bladder dysfunction, although the mechanisms are unclear. This study examined the effect of low testosterone and testosterone replacement on key mechanisms involved in local bladder function. Intraluminal release of the mediators ATP and ACh in response to bladder distension was measured in whole bladders from rats 8 weeks following castration, whilst bladder contractility was assessed using isolated strips. Human urothelial cells were cultured under low, physiological and supra-physiological testosterone conditions for 24 h or 5 days, and stretch-induced release of ATP and ACh was measured. Phasic contractile activity of bladder strips, agonist-induced reponses to carbachol and isoprenaline and nerve-evoked contractions were unaffected by castration. The acetylcholinesterase inhibitor neostigmine significantly increased amplitude of phasic activity only in bladder strips following castration, and this was prevented by testosterone replacement. Intraluminal ACh release following bladder distension was significantly reduced following castration, whilst ATP release was unaffected. In contrast, stretch-induced ATP release from urothelial cells was significantly enhanced in low testosterone conditions, whilst ACh release was unaltered. Testosterone-replacement to physiological levels prevented these changes. Whilst androgen deficiency of 8 weeks does not directly affect contractility of bladder smooth muscle, urothelial mediator release is sensitive to changes in testosterone. These changes in mediator release may be an early effect of the decline in testosterone and could affect sensory pathways in the longer term, contributing to the urinary symptoms and bladder dysfunction seen in androgen-deficient men.

Optogenetic Demonstration of Functional Innervation of Mouse Colon by Neurons Derived From Transplanted Neural Cells.

BACKGROUND & AIMS: Cell therapy offers the potential to treat gastrointestinal motility disorders caused by diseased or absent enteric neurons. We examined whether neurons generated from transplanted enteric neural cells provide a functional innervation of bowel smooth muscle in mice. METHODS: Enteric neural cells expressing the light-sensitive ion channel, channelrhodopsin, were isolated from the fetal or postnatal mouse bowel and transplanted into the distal colon of 3- to 4-week-old wild-type recipient mice. Intracellular electrophysiological recordings of responses to light stimulation of the transplanted cells were made from colonic smooth muscle cells in recipient mice. Electrical stimulation of endogenous enteric neurons was used as a control. RESULTS: The axons of graft-derived neurons formed a plexus in the circular muscle layer. Selective stimulation of graft-derived cells by light resulted in excitatory and inhibitory junction potentials, the electrical events underlying contraction and relaxation, respectively, in colonic muscle cells. Graft-derived excitatory and inhibitory motor neurons released the same neurotransmitters as endogenous motor neurons-acetylcholine and a combination of adenosine triphosphate and nitric oxide, respectively. Graft-derived neurons also included interneurons that provided synaptic inputs to motor neurons, but the pharmacologic properties of interneurons varied with the age of the donors from which enteric neural cells were obtained. CONCLUSIONS: Enteric neural cells transplanted into the bowel give rise to multiple functional types of neurons that integrate and provide a functional innervation of the smooth muscle of the bowel wall. Circuits composed of both motor neurons and interneurons were established, but the age at which cells are isolated influences the neurotransmitter phenotype of interneurons that are generated.

[ROLE OF MAST CELLS IN BRONCHIAL CONTRACTION IN NONALLERGIC OBSTRUCTIVE LUNG PATHOLOGY].

In model of chronic obstructive pulmonary disease induced in rats by 60-day intermittent exposure to nitrogen dioxide mast cells participation in the mechanism of bronchial smooth muscle contractile activity patterns was evaluated. Since the 31st day, one group of rats was inhaled with sodium cromoglycate every day before the nitrogen dioxide exposure to stabilize the mast cell membrane. The other group (control) hasn't been treated. Isometric contraction of the bronchial isolated preparations in response to nerve or smooth muscle stimulation were determined. Inhibition of mast cell degranulation and the release of endogenous histamine by stabilizing cell membranes prevented the development of bronchial smooth muscle hyperactivity caused by prolonged inhalation of nitrogen dioxide. It is believed that a mechanism to increase the contractile activity of the bronchial wall smooth muscles is mediated by activation of the transmembrane adenosine receptor in resident mast cells, leading to their partial degranulation with release of histamine, acting on the histamine Hl-receptors with the launch of reflex pathways through intramural ganglion neurons.

Octodon degus, a new model to study the agonist and plexus-induced response in the urinary bladder.

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm2) at 60 mM. In the case of cumulative dose-response of bethanecol, the maximum response (37.42 mN/mm2) was reached at 3.2 × 10-4 M. The response to ATP was clearly smaller (3.8 mN/mm2). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans.

Cystic Fibrosis and the Nervous System.

Cystic fibrosis (CF) is a life-shortening autosomal recessive disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an anion channel that conducts bicarbonate and chloride across cell membranes. Although defective anion transport across epithelial cells is accepted as the basic defect in CF, many of the features observed in people with CF and organs affected by CF are modulated by the nervous system. This is of interest because CFTR expression has been reported in both the peripheral and central nervous systems, and it is well known that the transport of anions, such as chloride, greatly modulates neuronal excitability. Thus it is predicted that in CF, lack of CFTR in the nervous system affects neuronal function. Consistent with this prediction, several nervous system abnormalities and nervous system disorders have been described in people with CF and in animal models of CF. The goal of this special feature article is to highlight the expression and function of CFTR in the nervous system. Special emphasis is placed on nervous system abnormalities described in people with CF and in animal models of CF. Finally, features of CF that may be modulated by or attributed to faulty nervous system function are discussed.