Abstract Aims To determine the role of opioid and β‐adrenergic receptors in bladder underactivity induced by prolonged pudendal nerve stimulation (PNS). Methods In α‐chloralose anesthetized cats, 30‐min PNS was applied repeatedly for 3–9 times to induce poststimulation or persistent bladder underactivity. Then, naloxone (opioid receptor antagonist, 1 mg/kg, IV) or propranolol (β‐adrenergic receptor antagonist, 3 mg/kg, IV) was given to reverse the bladder underactivity. After the drug treatment, an additional 30‐min PNS was applied to counteract the drug effect. Repeated cystometrograms were performed by slowly (1–2 mL/min) infusing the bladder with saline via a urethral catheter to determine the bladder underactivity and the treatment effects. Results Prolonged (2–4.5 h) PNS induced bladder underactivity evident as a large bladder capacity (169 ± 49% of control) and a reduced amplitude of bladder contraction (59 ± 17% of control). Naloxone fully reversed the bladder underactivity by reducing bladder capacity to 113 ± 58% and increasing the amplitude of bladder contraction to 104 ± 34%. After administration of naloxone an additional 30‐min PNS temporarily increased the bladder capacity to the underactive bladder level (193 ± 74%) without changing the amplitude of the bladder contraction. Propranolol had no effect on bladder underactivity. Conclusions A tonic enkephalinergic inhibitory mechanism in the CNS plays a critical role in the bladder underactivity induced by prolonged PNS, while the peripheral β‐adrenergic receptor mechanism in the detrusor is not involved. This study provides basic science evidence consistent with the clinical observation that comorbid opioid usage may contribute to voiding dysfunction in patients with Fowler's syndrome.
Mechanical strain due to increased pressure or swelling activates inflammatory responses in many neural systems. As cytokines and chemokine messengers lead to both pro-inflammatory and neuroprotective actions, understanding the signaling patterns triggered by mechanical stress may help improve overall outcomes. While cytokine signaling in neural systems is often associated with glial cells like astrocytes and microglia, the contribution of neurons themselves to the cytokine response is underappreciated and has bearing on any balanced response. Mechanical stretch of isolated neurons was previously shown to trigger ATP release through pannexin hemichannels and autostimulation of P2X7 receptors (P2X7Rs) on the neural membrane. Given that P2X7Rs are linked to cytokine activation in other cells, this study investigates the link between neuronal stretch and cytokine release through a P2X7-dependent pathway. Cytokine assays showed application of a 4% strain to isolated rat retinal ganglion cells (RGCs) released multiple cytokines. The P2X7R agonist BzATP also released multiple cytokines; Interleukin 3 (IL-3), TNF-α, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1Rα, MIP and CCL20 were released by both stimuli, with the release of IL-3 greatest with either stimuli. Stretch-dependent IL-3 release was confirmed with ELISA and blocked by P2X7R antagonists A438079 and Brilliant Blue G (BBG), implicating autostimulation of the P2X7R in stretch-dependent IL-3 release. Neuronal IL-3 release triggered by BzATP required extracellular calcium. The IL-3Rα receptor was expressed on RGCs but not astrocytes, and both IL-3Rα and IL-3 itself were predominantly expressed in the retinal ganglion cell layer of adult retinal sections, implying autostimulation of receptors by released IL-3. While the number of surviving ganglion cells decreased with time in culture, the addition of IL-3 protected against this loss of neurons. Expression of mRNA for IL-3 and IL-3Rα increased in rat retinas stretched with moderate intraocular pressure (IOP) elevation; BBG blocked the rise in IL-3, implicating a role for the P2X7R in transcriptional regulation in vivo. In summary, mechanical stretch triggers release of cytokines from neurons that can convey neuroprotection. The enhancement of these signals in vivo implicates P2X7R-mediated IL-3 signaling as an endogenous pathway that could minimize damage following neuronal exposure to chronic mechanical strain.
Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated. P2X7R stimulation triggered IL-6 release from primary mouse RPE, human iPS-RPE and human ARPE-19 cells. IL-6 release was polarized, with predominant rise across apical membranes. IL-6 release was inhibited by P2X7R antagonists A438079, A839977, and AZ10606120, but not the NRTI lamivudine (3TC), P2X1R antagonist NF279, or P2Y1R antagonist MRS2179. P2X7R-mediated IL-6 release required extracellular Ca2+ and was blocked by Ca2+ chelator BAPTA. IL-6 release and Ca2+ elevation occurred rapidly, consistent with vesicular IL-6 staining in unstimulated cells. P2X7R stimulation did not trigger IL-1β release in these unprimed cells. P2X7R-mediated IL-6 release was enhanced in RPE cells from the ABCA4−/− mouse model of retinal degeneration. In summary, P2X7R stimulation triggers rapid Ca2+-dependent IL-6 release across the apical membrane of RPE cells.
Antimuscarinic agents are the main treatment for overactive bladder. Recently muscarinic receptors (MRs) have been identified in urothelium, a tissue critically involved in bladder sensory mechanisms (review Birder, 2005). However, their properties and their role in bladder functions are presently unknown. This study investigates the expression and functionality of MRs in rat urothelial cells. RT-PCR revealed the expression of all five MR subtypes, M1 to M5. In voltage clamp, the muscarinic agonist Oxotremorine M (Oxo M 20 μM) reduces whole cell currents by 68.4±4.6% at -80mV and by 55.1±4.4% at +80mV in 50% of cells (5/10). The membrane resistance increases by 286.8±34.4% indicating inhibition of ion channels. Using a luciferin-luciferase assay, ACh (100 nM) triggered ATP release (n=5) which was reduced to 46% by the M2 antagonist AFDX-116 (50 μM; n=3), to 30% by the M3 antagonist 4-DAMP (50 μM; n=3) and to 18% by both antagonists (n=2). Using Fura-2 Ca2+ imaging, Oxo M elicited transient Ca2+ increases in 46.8% of cells (128/273). Responses were blocked by the muscarinic antagonist atropine (10 μM; n=12). In 36% of muscarine-responsive cells (24/65) Ca2+ responses had two components: influx of Ca2+ from the extracellular milieu and release of Ca2+ from the internal stores. In summary, urothelium expresses MRs whose activation modulates ion channels and results in ATP release possibly through mechanisms involving intracellular calcium increases. Understanding the mechanisms of urothelial MR activation may be important for treatment of overactive bladder. This work was supported by NIH DK R01 54824, NIH DK 049430 and AUA award to FAN.
Spinal cord injury (SCI) profoundly alters bladder function. Chronically, it induces urothelial hyperplasia, smooth muscle hypertrophy and detrusor hyperactivity, all of which prevent the bladder from properly storing and releasing urine. Transient receptor potential melastatin 4 (TRPM4) is a non‐selective cation channel activated by intracellular Ca 2+ and modulated by ATP. This channel has been implicated in various pathologies including SCI, inflammation, axonal degeneration and cardiac myocyte hypertrophy. We hypothesize that TRPM4 may play a role in bladder dysfunction after SCI. Here, we evaluated the expression and function of TRPM4 in the bladder urothelium and smooth muscle after SCI. Bladders were removed from control and spinal cord transected C57Bl6 female mice (T8‐T9; 1‐42 days post SCI). The mucosa and smooth muscle were separated surgically and used for RT‐PCR, western blot analysis, urothelial cell culture and muscle strips contractility studies. TRPM4 mRNA and protein were expressed in the mucosa and detrusor and were upregulated after SCI. Expression levels peaked at 3‐7 days post SCI and remained elevated for the duration of the study (6 weeks post SCI). Peak levels coincided with urothelial hyperplasia and smooth muscle hypertrophy following SCI. The TRPM4 antagonist, 9‐Phenanthrol (0.1‐30 µM), relaxed detrusor strips pre‐contracted with prostaglandin‐E2 (1µM) or carbachol (0.1µM) and reduced spontaneous activity. After SCI, the magnitude of these effects was increased. Our data reveal that TRPM4 is expressed in the bladder mucosa and detrusor and is upregulated after SCI, supporting the hypothesis that TRPM4 is involved in the remodeling processes and/or development of detrusor overactivity following SCI.
Introduction While it is well known that the sensory arm of the micturition reflex is mediated by A-δ afferents in the pelvic nerve, the urinary bladder is also innervated by afferents in the hypogastric nerve (HGN), whose role in micturition is less well understood. We have recently determined that stimulation of HGN afferents can facilitate nociceptive bladder activity in the cat induced by intravesical instillation of acetic acid. The aim of the present study, then, was to determine if activation of HGN afferents could similarly facilitate normal bladder activity in the cat, evoked by saline distension. Additional experiments examined if HGN stimulation could modulate the inhibitory effects of tibial or pudendal neuromodulation on bladder capacity. Methods Continuous infusion cystometry was performed in 9 (4 male, 5 female) α-chloralose anesthetized cats in which the HGNs were transected bilaterally. Bipolar electrodes were used to stimulate the central end of the cut HGNs at varying intensities (1-15 V) and frequencies (1-40 Hz) during bladder filling with saline (1-2 ml/min) until a micturition contraction occurred. Tripolar cuff electrodes were also placed on the left pudendal and left tibial nerve to examine the effect of neuromodulation on bladder capacity during cystometry. Results Hypogastric nerve stimulation (HGNS) at 30 Hz and 15 V significantly reduced bladder capacity to 80.01 ± 3.86% of control. Tibial or pudendal nerve stimulation (5 Hz, 2 T) significantly increased bladder capacity to 187.6 ± 20.2% or 193.8 ± 19.0% of control, respectively. Simultaneous HGNS significantly reduced the efficacy of tibial nerve neuromodulation (reduced to 158.7 ± 18.7% of control) and but only partially suppressed the bladder inhibition induced by pudendal neuromodulation (reduced to 161.9 ± 18.0% of control). Conclusion These results suggest that HGN afferents (possibly C-fiber nociceptors) from the bladder or other pelvic organs may facilitate normal bladder activity to cause bladder overactivity. Additionally, HGN afferents may influence the efficacy of tibial or pudendal neuromodulation therapy in treating OAB.
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