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Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca ++ ) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca ++ chelation impaired ATP-induced senescence. We also find that Ca ++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca ++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: We examined sex differences of lower urinary tract function and molecular mechanisms in mice with and without spinal cord injury (SCI)., Methods: SCI was induced by Th8-9 spinal cord transection in male and female mice. We evaluated cystometrograms (CMG) and electromyography (EMG) of external urethral sphincter (EUS) at 6 weeks after SCI in spinal intact (SI) and SCI mice. The mRNA levels of Piezo2 and TRPV1 were measured in L6-S1 dorsal root ganglia (DRG). Protein levels of nerve growth factor (NGF) in the bladder mucosa was evaluated using an enzyme-linked immunosorbent assay., Results: Sex differences were found in the EUS behavior during voiding as voiding events in female mice with or without SCI occurred during EUS relaxation periods without EUS bursting activity whereas male mice with or without SCI urinated during EUS bursting activity in EMG recordings. In both sexes, SCI decreased voiding efficiency along with increased tonic EUS activities evident as reduced EUS relaxation time in females and longer active periods of EUS bursting activity in males. mRNA levels of Piezo2 and TRPV1 of DRG in male and female SCI mice were significantly upregulated compared with SI mice. NGF in the bladder mucosa showed a significant increase in male and female SCI mice compared with SI mice. However, there were no significant differences in Piezo2 or TRPV1 levels in DRG or NGF protein levels in the bladder mucosa between male and female SCI mice., Conclusions: We demonstrated that female and male mice voided during EUS relaxation and EUS bursting activity, respectively. Also, upregulation of TRPV1 and Piezo2 in L6-S1 DRG and NGF in the bladder could be involved in SCI-induced lower urinary tract dysfunction in both sexes of mice., (© 2023 Wiley Periodicals LLC.)

Objective: To determine the role of ion concentrations and ion pump activity in conduction block of myelinated axon induced by a long-duration direct current (DC)., Methods: A new axonal conduction model for myelinated axons based on the classical Frankenhaeuser-Huxley (FH) equations is developed that includes ion pump activity and allows the intracellular and extracellular Na + and K + concentrations to change with axonal activity., Results: Action potential generation, propagation, and acute DC block occurring within a short period (milliseconds) that do not significantly change the ion concentrations or trigger ion pump activity are successfully simulated by the new model in a similar way as the classical FH model. Different from the classical model, the new model also successfully simulates the post-stimulation block phenomenon, i.e., the axonal conduction block occurring after terminating a long-duration (30 seconds) DC stimulation as observed recently in animal studies. The model reveals a significant K + accumulation outside the axonal node as the possible mechanism underlying the post-DC block that is slowly reversed by ion pump activity during the post-stimulation period., Conclusion: Changes in ion concentrations and ion pump activity play an important role in post-stimulation block induced by long-duration DC stimulation., Significance: Long-duration stimulation is used clinically for many neuromodulation therapies, but the effects on axonal conduction/block are poorly understood. This new model will be useful for better understanding of the mechanisms underlying long-duration stimulation that changes ion concentrations and triggers ion pump activity.

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder. In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

The mechanisms that link visceral mechanosensation to the perception of internal organ status (i.e., interoception) remain elusive. In response to bladder filling, the urothelium releases ATP, which is hypothesized to stimulate voiding function by communicating the degree of bladder fullness to subjacent tissues, including afferent nerve fibers. To determine if PIEZO channels function as mechanosensors in these events, we generated conditional urothelial Piezo1-, Piezo2-, and dual Piezo1/2-knockout (KO) mice. While functional PIEZO1 channels were expressed in all urothelial cell layers, Piezo1-KO mice had a limited phenotype. Piezo2 expression was limited to a small subset of superficial umbrella cells, yet male Piezo2-KO mice exhibited incontinence (i.e., leakage) when their voiding behavior was monitored during their active dark phase. Dual Piezo1/2-KO mice had the most affected phenotype, characterized by decreased urothelial responses to mechanical stimulation, diminished ATP release, bladder hypoactivity in anesthetized Piezo1/2-KO females but not males, and urinary incontinence in both male and female Piezo1/2-KO mice during their dark phase but not inactive light one. Our studies reveal that the urothelium functions in a sex- and circadian rhythm-dependent manner to link urothelial PIEZO1/2 channel-driven mechanotransduction to normal voiding function and behavior, and in the absence of these signals, bladder dysfunction ensues.

Aims: The transient receptor potential melastin-8 (TRPM8) channel is a "cooling" receptor expressed in primary sensory neurons and can be activated by compounds like menthol or icilin. TRPM8 is involved in the regulation of urinary bladder sensory function and contraction, but the role of TRPM8 in the ureter, particularly in the human ureter, is poorly understood. The aim of this study is to examine the effects of TRPM8 activation on human ureter contraction., Methods: Human ureters were acquired from 20 patients undergoing radical nephrectomy. Contractions of ureter strips were recorded by an isometric transducer in the organ bath. Ureteral TRPM8 expression in the human ureter was examined by immunofluorescence and western blot., Results: The two TRPM8 agonists menthol and icilin both reduced the frequency of spontaneous, electrical field stimulation, or neurokinin A-evoked ureteral contractions in a dose-dependent manner. The inhibitory effects were decreased by 10-fold in mucosa-denuded strips. The inhibitory effects of TRPM8 agonists were mimicked by calcitonin gene-related peptide (CGRP), and were blocked by KRP2579 (a TRPM8 antagonist), tetrodotoxin (a sodium channel blocker), olcegepant (BIBN, a CGRP receptor antagonist), SQ22536 (an adenylate cyclase antagonist), or H89 (a nonspecific cAMP-dependent protein kinase A inhibitor). TRPM8 was coexpressed with CGRP on the nerves located in the suburothelial and intermuscular regions and was not expressed in the urothelium., Conclusions: The TRPM8 channel expressed on sensory nerve terminals of the human ureter is involved in the inhibitory sensory neurotransmission and modulate ureter contraction via the CGRP-adenylyl cyclase-protein kinase A pathway. TRPM8 may be involved in stone-induced changes in ureter contraction or pain., (© 2021 Wiley Periodicals LLC.)

Activation of TRP channels expressed in urinary bladder afferent nerves and urothelium releases neurotransmitters that influence bladder function. Experiments were undertaken to examine the mechanisms underlying effects of TRPA1 (allyl isothiocyanate, AITC), TRPV1 (capsaicin, CAPS), and TRPC (oleoyl-2-acetyl-sn-glycerol, OAG) agonists on guinea pig bladder activity. Effects of these agonists were compared with effects of nitro-oleic acid (OA-NO 2 ), an electrophilic nitro-fatty acid, known to activate TRPV1, TRPA1 or TRPC channels in sensory neurons. AITC (100 μM) increased (231%) area of spontaneous bladder contractions (SBCs) an effect reduced by a TRPA1 antagonist (HC3-03001, HC3, 10 μM) and reversed to inhibition by indomethacin (INDO, 500 nM) a cyclooxygenase inhibitor. The post-INDO inhibitory effect of AITC was mimicked (39% depression) by calcitonin gene-related peptide (CGRP, 100 nM) and blocked by a CGRP antagonist (BIBN, 25 μM). CAPS (1 μM) suppressed SBCs by 30% in 81% of strips, an effect blocked by a TRPV1 antagonist (diarylpiperazine, 1 μM) or BIBN. SBCs were suppressed by OA-NO 2 (30 μM, 21% in 77% of strips) or by OAG (50 μM, 30%) an effect blocked by BIBN. OA-NO 2 effects were not altered by HC3 or diarylpiperazine. OA-NO 2 also induced excitation in 23% of bladder strips. These observations raise the possibility that guinea pig bladder is innervated by at least two types of afferent nerves: [1] Type A express TRPA1 receptors that induce the release of prostaglandins and excite the detrusor, [2] Type B express TRPV1, TRPA1 and TRPC receptors and release CGRP that inhibits the detrusor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Daugherty, Beckel, Kim, Freeman, Liu, Wang, de Groat and Zhang.)

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 Ca 2+ and was blocked by Ca 2+ chelator BAPTA. IL-6 release and Ca 2+ 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 Ca 2+ -dependent IL-6 release across the apical membrane of RPE cells.

The goal of this study in anesthetized cats was to identify silent hypogastric nerve (HGN) afferent fibers that do not respond to bladder distention but become responsive after chemical irritation of the bladder. The HGN was split into multiple filaments small enough for recording action potentials from single or multiple afferent fibers. The bladder was distended by infusion of either saline or 0.5% acetic acid (AA) through a urethral catheter while recording intravesical pressure. A total of 90 HGN filaments from 17 cats responded to bladder distention with saline or AA. Three types of HGN afferents were identified. The first type was non-nociceptive mechano-sensitive that responded to bladder distention at normal physiological pressures (10-40 cmH 2 O). The second type was nociceptive mechano-sensitive that only responded to high-pressure (50-80 cmH 2 O) bladder distention with saline but responded to low-pressure bladder distention after sensitization with AA. The third type was chemo-sensitive nociceptive that was silent even during high-pressure bladder distention but after sensitization with AA did respond to low-pressure bladder distention. These results indicate that HGN afferents as well as pelvic nerve afferents may play a role in bladder nociception. The HGN afferent fibers that are silent during bladder distention at normal physiological pressures but become responsive after chemical irritation are important for understanding the possible pathophysiological mechanism underlying bladder allodynia in painful bladder syndrome., Competing Interests: Declaration of Competing Interest No conflicts of interests are declared by the authors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: While numerous studies have confirmed ATP's importance in bladder physiology/pathophysiology, the literature is still conflicted regarding the mechanism of ATP release from the urothelium. Multiple mechanisms have been identified including non-vesicular release via pannexin channels as well as vesicular release via a mechanism blocked by botulinum toxin. Recently, it has been shown that lysosomes contain significant stores of ATP which can be released extracellularly in response to Toll-like receptor (TLR) stimulation., Objective: The goal of the current study was to determine if lysosomal exocytosis occurs in urothelial cells in response to TLR4 stimulation by its agonist, bacterial lipopolysaccharide (LPS)., Materials and Methods: Human urothelial cells from an immortalized cell line (TRT-HU1) were treated with bacterial LPS (100 μg/ml) or the nicotinic agoinist cytisine (100 μM) and extracellular release of ATP and lysosomal acid phosphatase were measured. Pannexin-mediated ATP release and lysosomal ATP release were differentiated using Brilliant Blue FCF to inhibit pannexin channels and glycyl-l-phenylalanine-β-naphthylamide (GPN) to destroy lysosomes. The mechanisms controlling lysosomal exocytosis were examined using lysosomal pH measurements using LysoSensor dye and intracellular calcium signaling using Fura-2., Results: Stimulation of TRT-HU1 cells with LPS significantly increased ATP release, which was inhibited by GPN, but not by Brilliant Blue FCF. Conversely, stimulation with cytisine induced ATP release that was sensitive to Brilliant Blue FCF but not GPN. LPS stimulation also induced the release of the lysosomal acid phosphatases. LPS increased lysosomal pH and direct alkalization of lysosomal pH using chloroquine or bafilomycin A1 induced ATP and acid phosphatase release, indicating an important role for pH in lysosomal exocytosis. Additionally, stimulation of lysosomal transient receptor potential mucolipin 1 calcium channels evoked intracellular calcium transients as well as ATP release., Conclusion: These data indicate that LPS-induced ATP release from urothelial cells is mediated by lysosomal exocytosis, a vesicular mechanism distinctly separate from non-vesicular release via pannexin channels., (© 2020 Wiley Periodicals LLC.)

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs ∗ 81 and p.Ser340 ∗ led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Transient receptor potential cation channel subfamily M member 4 (TRPM4) has been shown to play a key role in detrusor contractility under physiological conditions. In this study, we investigated the potential role of TRPM4 in detrusor overactivity following spinal cord transection (SCT) in mice. TRPM4 expression and function were evaluated in bladder tissue with or without the mucosa from spinal intact (SI) and SCT female mice (T8-T9 vertebra; 1-28 days post SCT) using PCR, western blot, immunohistochemistry, and muscle strip contractility techniques. TRPM4 was expressed in the urothelium (UT) and detrusor smooth muscle (DSM) and was upregulated after SCT. Expression levels peaked 3-7 days post SCT in both the UT and DSM. Pharmacological block of TRPM4 with the antagonist, 9-Phenanthrol (30 μM) greatly reduced spontaneous phasic activity that developed after SCT, regardless of the presence or absence of the mucosa. Detrusor overactivity following spinal cord injury leads to incontinence and/or renal impairment and represents a major health problem for which current treatments are not satisfactory. Augmented TRPM4 expression in the bladder after chronic SCT supports the hypothesis that TRPM4 channels play a role in DSM overactivity following SCT. Inhibition of TRPM4 may be beneficial for improving detrusor overactivity in SCI.

Lysosomal dysfunction is associated with a number of age-related pathologies that affect all organ systems. While much research has focused on neurodegenerative diseases and aging-induced changes in neurons, much less is known about the impact that aging has on lower urinary tract function. Our studies explored age-dependent changes in the content of endo-lysosomal organelles (i.e., multivesicular bodies, lysosomes, and the product of their fusion, endolysosomes) and age-induced effects on lysosomal degradation in the urothelium, the epithelial tissue that lines the inner surface of the bladder, ureters, and renal pelvis. When examined by transmission electron microscopy, the urothelium from young adult rats (~3 months), mature adult rats (~12 months), and aged rats (~26 months old) demonstrated a progressive age-related accumulation of aberrantly large endolysosomes (up to 7μm in diameter) that contained undigested content, likely indicating impaired degradation. Stereological analysis confirmed that aged endolysosomes occupied approximately 300% more volume than their younger counterparts while no age-related change was observed in multivesicular bodies or lysosomes. Consistent with diminished endolysosomal degradation, we observed that cathepsin B activity was significantly decreased in aged versus young urothelial cell lysates as well as in live cells. Further, the endolysosomal pH of aged urothelium was higher than that of young adult (pH 6.0 vs pH 4.6). Our results indicate that there is a progressive decline in urothelial endolysosomal function during aging. How this contributes to bladder dysfunction in the elderly is discussed., Competing Interests: The authors have declared that no competing interests exist.

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and "non-targeting" scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1 -/- cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca 2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.

Nitro-oleic acid (NO 2 -OA) and related nitroalkenes are electrophilic fatty acid derivatives that are present in normal tissues at nanomolar concentrations and can increase significantly during inflammation. These substances can suppress multiple intracellular signaling pathways contributing to inflammation by reversible Michael addition reactions with nucleophilic residues such as cysteine and histidine leading to post-translational modification of proteins. NO 2 -OA also can influence inflammation and pain by acting on transient receptor potential (TRP) channels in primary sensory neurons. TRPV1, TRPA1 and TRPC can respond to electrophilic fatty acids because they have ankyrin-like repeats in their N terminus that are rich in cysteine residues that react with electrophiles and other thiol modifying species. NO 2 -OA acts on TRP channels to initially depolarize and induce firing in sensory neurons followed by desensitization and suppression of firing. In vivo experiments revealed that pretreatment with NO 2 -OA reduces nociceptive behavior evoked by local administration of a TRPA1 agonist (AITC) to the rat hind paw. These results raise the possibility that NO 2 -OA might be useful clinically to reduce neurogenic inflammation and certain types of painful sensations by desensitizing TRPA1 expressing nociceptive afferents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Inflammatory responses play a key role in many neural pathologies, with localized signaling from the non-immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. The NLRP3 inflammasome requires two stages to contribute: priming and activation. The priming stage involves upregulation of inflammasome components while the activation stage results in the assembly and activation of the inflammasome complex. The priming step can be rate limiting and can connect insult to chronic inflammation, but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammation is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non-ischemic elevation of intraocular pressure increased mRNA for inflammasome components IL-1 β , NLRP3, ASC , and CASP1 in rat and mouse retinas. The elevation was greater 1 day after the insult, with the rise in IL-1β most pronounced. The P2X7 receptor was implicated in the mechanosensitive priming of IL-1β mRNA in vivo , as the antagonist Brilliant Blue G (BBG) blocked the increased expression, the agonist BzATP mimicked the pressure-dependent rise in IL-1β, and the rise was absent in P2X7 knockout mice. In vitro measurements from optic nerve head astrocytes demonstrated an increased expression of IL-1 β following stretch or swelling. This increase in IL-1 β was eliminated by degradation of extracellular ATP with apyrase, or by the block of pannexin hemichannels with carbenoxolone, probenecid, or 10panx1 peptide. The rise in IL-1 β expression was also blocked by P2X7 receptor antagonists BBG, A839977 or A740003. The rise in IL-1 β was prevented by blocking transcription factor NFκB with Bay 11-7082, while the swelling-dependent fall in NFκB inhibitor IκB-α was reduced by A839977 and in P2X7 knockout mice. In summary, mechanical trauma to the retina primed NLRP3 inflammasome components, but only if there was ATP release through pannexin hemichannels, and autostimulation of the P2X7 receptor. As the P2X7 receptor can also trigger stage two of inflammasome assembly and activation, the P2X7 receptor may have a central role in linking mechanical strain to neuroinflammation.

Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) in vivo, and stretch or swelling in vitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60 mmHg for 4 h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7 -/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response., (© 2017 International Society for Neurochemistry.)

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.

Key Points: ATP is released through pannexin channels into the lumen of the rat urinary bladder in response to distension or stimulation with bacterial endotoxins. Luminal ATP plays a physiological role in the control of micturition because intravesical perfusion of apyrase or the ecto-ATPase inhibitor ARL67156 altered reflex bladder activity in the anaesthetized rat. The release of ATP from the apical and basolateral surfaces of the urothelium appears to be mediated by separate mechanisms because intravesical administration of the pannexin channel antagonist Brilliant Blue FCF increased bladder capacity, whereas i.v. administration did not. Intravesical instillation of small interfering RNA-containing liposomes decreased pannexin 1 expression in the rat urothelium in vivo and increased bladder capacity. These data indicate a role for pannexin-mediated luminal ATP release in both the physiological and pathophysiological control of micturition and suggest that urothelial pannexin may be a viable target for the treatment of overactive bladder disorders., Abstract: ATP is released from the bladder epithelium, also termed the urothelium, in response to mechanical or chemical stimuli. Although numerous studies have described the contribution of this release to the development of various bladder disorders, little information exists regarding the mechanisms of release. In the present study, we examined the role of pannexin channels in mechanically-induced ATP release from the urothelium. PCR confirmed the presence of pannexin 1 and 2 mRNA in rat urothelial tissue, whereas immunofluorescence experiments localized pannexin 1 to all three layers of the urothelium. During continuous bladder cystometry in anaesthetized rats, inhibition of pannexin 1 channels using carbenoxolone (CBX) or Brilliant Blue FCF (BB-FCF) (1-100 μm, intravesically), or by using intravesical small interfering RNA, increased the interval between voiding contractions. Intravenous administration of BB-FCF (1-100 μg kg(-1) ) did not alter bladder activity. CBX or BB-FCF (100 μm intravesically) also decreased basal ATP concentrations in the perfusate from non-distended bladders and inhibited increases in ATP concentrations in response to bladder distension (15 and 30 cmH2 O pressure). Intravesical perfusion of the ATP diphosphohydrolase apyrase (2 U ml(-1) ), or the ATPase inhibitor ARL67156 (10 μm) increased or decreased reflex bladder activity, respectively. Intravesical instillation of bacterial lipopolysaccharides (LPS) (Escherichia coli 055:B5, 100 μg ml(-1) ) increased ATP concentrations in the bladder perfusate, and also increased voiding frequency; these effects were suppressed by BB-FCF. These data indicate that pannexin channels contribute to distension- or LPS-evoked ATP release into the lumen of the bladder and that luminal release can modulate voiding function., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Effects of nitro-oleic acid (OA-NO2) on TRP channels were examined in guinea-pig dissociated dorsal root ganglia (DRG) neurons using calcium imaging and patch clamp techniques. OA-NO2 increased intracellular Ca(2+) in 60-80% DRG neurons. 1-Oleoyl-2acetyl-sn-glycerol (OAG), a TRPC agonist, elicited responses in 36% of OA-NO2-sensitive neurons while capsaicin (TRPV1 agonist) or allyl-isothiocyanate (AITC, TRPA1 agonist) elicited responses in only 16% and 10%, respectively, of these neurons. A TRPV1 antagonist (diarylpiperazine, 5 μm) in combination with a TRPA1 antagonist (HC-030031, 30 μm) did not change the amplitude of the Ca(2+) transients or percentage of neurons responding to OA-NO2; however, a reducing agent DTT (50 mm) or La(3+) (50 μm) completely abolished OA-NO2 responses. OA-NO2 also induced a transient inward current associated with a membrane depolarization followed by a prolonged outward current and hyperpolarization in 80% of neurons. The reversal potentials of inward and outward currents were approximately -20 mV and -60 mV, respectively. Inward current was reduced when extracellular Na(+) was absent, but unchanged by niflumic acid (100 μm), a Cl(-) channel blocker. Outward current was abolished in the absence of extracellular Ca(2+) or a combination of two Ca(2+)-activated K(+) channel blockers (iberiotoxin, 100 nm and apamin, 1 μm). BTP2 (1 or 10 μm), a broad spectrum TRPC antagonist, or La(3+) (50 μm) completely abolished OA-NO2 currents. RT-PCR performed on mRNA extracted from DRGs revealed the expression of all seven subtypes of TRPC channels. These results support the hypothesis that OA-NO2 activates TRPC channels other than the TRPV1 and TRPA1 channels already known to be targets in rat and mouse sensory neurons and challenge the prevailing view that electrophilic compounds act specifically on TRPA1 or TRPV1 channels. The modulation of sensory neuron excitability via actions on multiple TRP channels can contribute to the anti-inflammatory effect of OA-NO2., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma., (© 2014 Wiley Periodicals, Inc.)

Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TcFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TcFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimer's disease. While the absolute rise in pH is often small in these disorders, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these disorders and identifies potential approaches to treat diseases of lysosomal accumulation like AMD and Alzheimer's disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mutation in presenilin 1 (PS1) is one of the leading causes of familial Alzheimer's disease (fAD). PS1 mutation exacerbates the autophagic and lysosomal pathology in AD patients, leading to accumulation of partially degraded material in bloated lysosomes and autophagosomes - a pathology that bears some resemblance to other diseases characterized by elevated lysosomal pH, like age-related macular degeneration. In this study, we examined the effect of the PS1-fAD mutation A246E on lysosomal pH and lysosomal function, and asked whether restoration of lysosomal pH could reverse some of these changes. Lysosomal pH was elevated by 0.2-0.3 pH units in human fibroblasts with the PS1-fAD mutation. The lysosomal alkalization in PS1-fAD fibroblasts was supported by a reduction in the pH-dependent cleavage of cathepsin D and by a reduction in binding of boron-dipyrromethene (BODIPY) FL-pepstatin A to the cathepsin D active site. PS1-fAD cells had increased LC3B-II/-I ratios and p62 levels, consistent with impaired lysosomal degradation and analogous to changes induced by lysosomal alkalinization with chloroquine. PS1-fAD fibroblasts had increased expression of ATP6V1B2, ATG5, BECN1 TFEB mRNA, and of ATP6V1B2, ATG5 and beclin at the protein level, consistent with chronic impairment of autophagic and lysosomal functions in the mutant cells. Critically, cyclic adenosine monophosphate (cAMP) treatment reacidified lysosomal pH in mutant PS1-fAD; cAMP also increased the availability of active cathepsin D and lowered the LC3B-II/-I ratio. These results confirm a small elevation in the lysosomal pH of human PS1-fAD fibroblasts, demonstrate that this lysosomal alkalization is associated with chronic changes in autophagy and degradation, and suggest that treatment to reacidify the lysosomes with cAMP can reverse these changes., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca(2+); Ca(2+) influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4(-/-) mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.

Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide (10)panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin-dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X(7) receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X(7) receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X(7) receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X(7) receptor, is neuroprotective for stretched neurons.

It has been previously determined that the epithelial lining of the urinary bladder, or urothelium, expresses two subtypes of nicotinic acetylcholine receptors (nAChRs) that mediate distinct physiological effects in vivo. These effects include inhibition of bladder reflexes through α7 receptors and an excitation of bladder reflexes through α3-containing (α3*) receptors. It is believed that urothelial receptors mediate their effects through modulating the release of neurotransmitters such as ATP that subsequently influence bladder afferent nerve excitability. Therefore, we examined the distribution of nAChRs in the urothelium, as well as their ability to influence the release of the neurotransmitter ATP. Immunofluorescent staining of both whole bladder tissue and primary urothelial cultures from the rat demonstrated that the urothelium contains both α3* and α7 receptors. In primary urothelial cultures, α7 stimulation with choline (10 μM to 1 mM) caused a decrease in basal ATP release while α3* stimulation with cytisine (1–100 μM) caused a concentration-dependent, biphasic response, with low concentrations (1–10 μM) inhibiting release and higher concentrations (50–100 μM) increasing release. These responses were mirrored in an in vitro, whole bladder preparation. In vivo, excitation of bladder reflexes in response to intravesical cytisine (100 μM) is blocked by systemic administration of the purinergic antagonist PPADS (1 or 3 μg kg(−1)). We also examined how each receptor subtype influenced intracellular Ca2+ levels in cultured urothelial cells. nAChR stimulation increased [Ca2+]i through distinct mechanisms: α7 through a ryanodine-sensitive intracellular mechanism and α3* through extracellular influx. In addition, our findings suggest interactions between nAChR subtypes whereby activation of α7 receptors inhibited the response to a subsequent activation of α3* receptors, preventing the increase in [Ca2+]i previously observed. This inhibitory effect appears to be mediated through protein kinase A- or protein kinase C-mediated pathways.

The lower urinary tract (LUT), which consists of the urinary bladder and its outlet, the urethra, is responsible for the storage and periodic elimination of bodily waste in the form of urine. The LUT is controlled by a complex set of peripheral autonomic and somatic nerves, which in turn are controlled through neural pathways in the spinal cord and brain. This influence of the central nervous system allows for the conscious control of the bladder, allowing the individual to choose an appropriate place to urinate. Defects in the CNS pathways that control the LUT can lead to incontinence, an embarrassing condition that affects over 200 million people worldwide. As a first step in understanding the neural control of the bladder, we will discuss the neuroanatomy of the LUT, focusing first on the peripheral neural pathways, including the sensory pathways that transmit information on bladder filling and the motoneurons that control LUT muscle contractility. We will also discuss the organization of the central pathways in the spinal cord and brainstem that are responsible for coordinating bladder activity, promoting continuous storage of urine except for a few short minutes per day when micturition takes place. To conclude, we will discuss current studies underway that aim to elucidate the higher areas of the brain that control the voluntary nature of micturition in higher organisms.

The lower urinary tract (LUT) has two functions: (1) the storage of waste products in the form of urine and (2) the elimination of those wastes through micturition. The LUT operates in a simple "on-off" fashion, either storing urine or releasing it during voiding. While this activity may seem simple, micturition is controlled by a complex set of peripheral neurons that are, in turn, coordinated by cell groups in the spinal cord, brainstem, and brain. When this careful coordination is interrupted, the control of the bladder is lost, resulting in incontinence or retention of urine. The purpose of this chapter is to review how the neural systems coordinating the activity of the lower urinary tract form neural circuits that are responsible for either maintaining continence (the storage reflex) or inducing micturition (the voiding reflex). We will also discuss the brain centers that enable higher organisms to voluntarily choose the time and place for voiding. Finally, we will discuss how defects in the pathways controlling micturition can lead to urinary incontinence and which treatments may normalize LUT function.

The control and regulation of the lower urinary tract are partly mediated by purinergic signaling. This study investigated the distribution and function of P2Y receptors in the rat urinary bladder. Application of P2Y agonists to rat urothelial cells evoked increases in intracellular calcium; the rank order of agonist potency (pEC(50) +/- SE) was ATP (5.10 +/- 0.07) > UTP (4.91 +/- 0.14) > UTPgammaS (4.61 +/- 0.16) = ATPgammaS (4.70 +/- 0.05) > 2-methylthio adenosine 5'-diphosphate = 5'-(N-ethylcarboxamido)adenosine = ADP (<3.5). The rank order potency for these agonists indicates that urothelial cells functionally express P2Y(2)/P2Y(4) receptors, with a relative lack of contribution from other P2Y or adenosine receptors. Real-time PCR, Western blotting, and immunocytochemistry confirmed the expression of P2Y(2) and to a lesser extent P2Y(4) in the urothelium. Immunocytochemical studies revealed expression of P2Y(2) staining in all layers of the urothelium, with relative absence of P2Y(4). P2Y(2) staining was also present in suburothelial nerve bundles and underlying detrusor smooth muscle. Addition of UTP and UTPgammaS was found to evoke ATP release from cultured rat urothelial cells. These findings indicate that cultured rat urothelial cells functionally express P2Y(2)/P2Y(4) receptors. Activation of these receptors could have a role in autocrine and paracrine signaling throughout the urothelium. This could lead to the release of bioactive mediators such as additional ATP, nitric oxide, and acetylcholine, which can modulate the micturition reflex by acting on suburothelial myofibroblasts and/or pelvic afferent fibers.

Non-neuronal release of acetylcholine (ACh) has been proposed to play a role in urinary bladder function. These studies investigated the expression and function of the non-neuronal cholinergic system in cultured urothelial cells isolated from the rat urinary bladder. Our findings have revealed that urothelial cells express the high-affinity choline transporter (CHT1) and acetylcholine-synthesizing enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). In contrast to neurons, urothelial cells do not express the vesicular acetylcholine transporter (VAChT) but do express OCT3, a subtype of polyspecific organic cation transporter (OCT) that is thought to be involved in the release of acetylcholine from non-neuronal cells. Following exposure of cultured urothelial cells to (3)H-choline, radioactivity was detected in the cells and increased release of radioactivity into the eternal media was evoked by mechanical stimulation (exposure of the cells to 50% hypotonic Krebs) or chemical stimulation of purinergic receptors by 100 muM ATP. The present experiments did not establish if the evoked release of radioactivity (termed (3)H-ACh release in this paper) was due to release of acetylcholine or choline. (3)H-ACh release was not evoked by application of acetylcholine alone, however pretreatment with the non-selective muscarinic receptor antagonist atropine prior to application of acetylcholine facilitated (3)H-ACh release, suggesting that the acetylcholine released from urothelial cells may participate in a negative feedback mechanism by acting on muscarinic receptors to inhibit its own release in the urothelium. Brefeldin, an agent which disrupts vesicular exocytosis, did not block hypotonic-evoked (3)H-ACh release. These observations indicate that acetylcholine release from urothelial cells is mediated by different mechanisms than those such as vesicular storage and exocytosis that underlie the release of neurotransmitters from nerves.

Although nicotinic acetylcholine receptors in both the central and peripheral nervous systems play a prominent role in the control of urinary bladder function, little is known regarding expression or function of nicotinic receptors in the bladder epithelium, or urothelium. Nicotinic receptors have been described in epithelial cells lining the upper gastrointestinal tract, respiratory tract, and the skin. Thus the present study examined the expression and functionality of nicotinic receptors in the urothelium, as well as the effects of stimulation of nicotinic receptors on the micturition reflex. mRNA for the alpha3, alpha5, alpha7, beta3, and beta4 nicotinic subunits was identified in rat urothelial cells using RT-PCR. Western blotting also confirmed urothelial expression of the alpha3- and alpha7-subunits. Application of nicotine (50 nM) to cultured rat urothelial cells elicited an increase in intracellular Ca2+ concentration, indicating that at least some of the subunits form functional channels. These effects were blocked by the application of the nicotinic antagonist hexamethonium. During in vivo bladder cystometrograms in urethane-anesthetized rats, intravesical administration of nicotine, choline, or the antagonists methyllycaconitine citrate and hexamethonium elicited changes in voiding parameters. Intravesical nicotine (50 nM, 1 microM) increased the intercontraction interval. Intravesical choline (1-100 microM) also affected bladder reflexes similarly, suggesting that alpha7 nicotinic receptors mediate this effect. Intravesical administration of hexamethonium (1-100 microM) potentiated the nicotine-induced changes in bladder reflexes. Methyllycaconitine citrate, a specific alpha7-receptor antagonist, prevented nicotine-, choline-, and hexamethonium-induced bladder inhibition. These results are the first indication that stimulation of nonneuronal nicotinic receptors in the bladder can affect micturition.

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium. B2R immunoreactivity was localized throughout the bladder, including the urothelium and detrusor smooth muscle. Bradykinin-evoked activation of this receptor elevated intracellular calcium (EC(50) = 8.4 nM) in a concentration-related manner and evoked ATP release from control cultured rat urothelial cells. In contrast, B1R mRNA was not detected in control rat urinary bladder; however, following acute (24 h) and chronic (8 day) CYP-induced cystitis in the rat, B1R mRNA was detected throughout the bladder. Functional B1Rs were demonstrated by evoking ATP release and increases in [Ca(2+)](i) in CYP (24 h)-treated cultured rat urothelial cells with a selective B1 receptor agonist (des-Arg(9)-bradykinin). Cystometry performed on control anaesthetized rats revealed that intravesical instillation of bradykinin activated the micturition pathway. Attenuation of this response by the P2 receptor antagonist PPADS suggests that bradykinin-induced micturition facilitation may be due in part to increased purinergic responsiveness. CYP (24 h)-treated rats demonstrated bladder hyperactivity that was significantly reduced by intravesical administration of either B1 (des-Arg(10)-Hoe-140) or B2 (Hoe-140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.

The afferent limb of the vesicovascular reflex (VV-R) evoked by distension or contraction of the urinary bladder (UB) was studied in urethane-anesthetized female rats by examining the changes in VV-R after administration of C-fiber afferent neurotoxins [capsaicin and resiniferatoxin (RTX)]. Systemic arterial blood pressure increased parallel (5.1 to 53.7 mmHg) with graded increases in UB pressure (20 to 80 cm H(2)O) or during UB contractions. The arterial pressor response to UB distension was significantly reduced (60-85%) by acute or chronic (4 days earlier) intravesical administration of RTX (100-1,000 nM) or by capsaicin (125 mg/kg sc) pretreatment (4 days earlier). Chronic neurotoxin treatments also increased the volume threshold (>100%) for eliciting micturition in anesthetized rats but did not change voiding pressure. Acute RTX treatment (10-50 nM) did not alter the arterial pressor response during reflex UB contractions, whereas higher concentrations of RTX (100-1,000 nM) blocked reflex bladder contractions. It is concluded that VV-R is triggered primarily by distension- and contraction-sensitive C-fiber afferents located, respectively, near the luminal surface and deeper in the muscle layers of the bladder.