Key pointsr ATP is released through pannexin channels into the lumen of the rat urinary bladder in response to distension or stimulation with bacterial endotoxins.r 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.r 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.r Intravesical instillation of small interfering RNA-containing liposomes decreased pannexin 1 expression in the rat urothelium in vivo and increased bladder capacity.r 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 cmH 2 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.

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 significant phenotype, characterized by decreased urothelial responses to mechanical stimulation, diminished ATP release, bladder hypoactivity in anesthetized Piezo1/2-KO females, but not male ones, 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 manner to link urothelial PIEZO1/2 channeldriven mechanotransduction to normal voiding function and behavior, and in the absence of these signals, bladder dysfunction ensues.

When capsaicin is applied repeatedly to dorsal root ganglion (DRG) neurons for brief periods (10-15 s) at short intervals (5-10 min), the evoked responses rapidly decline, a phenomenon termed tachyphylaxis. In addition to this phenomenon, the present study using Ca(2+) imaging revealed that repeated application of capsaicin to rat dissociated DRG neurons at longer intervals (20-40 min) or during multiple applications at short intervals elicited an enhancement of the responses, termed potentiation. The potentiation occurred in 50-60% of the capsaicin-responsive cells, on average representing a 20- to 30% increase in the peak amplitude of the Ca(2+) signal, and was maximal at a 40-min application interval. An analysis of the mechanisms underlying potentiation revealed that it was suppressed by block of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with 5 μM KN-93 or block of the activation of extracellular signal-regulated kinase (ERK) 1/2 with 2 μM U-0126. Lowering the extracellular Ca(2+) concentration from 2 to 1 mM or pretreatment with deltamethrin (1 μM), which blocks calcineurin and tachyphylaxis, enhanced potentiation. Potentiation was not affected by: 1) inhibition of protein kinase C or protein kinase A, 2) block of the three subtypes of neurokinin receptors, or 3) block of the trafficking of transient receptor potential V1 channel to the membrane. These results indicate that the potentiation is a slowly developing Ca(2+)-modulated process that is mediated by a complex intracellular signaling pathway involving activation of CaMKII and ERK1/2. Potentiation may be an important peripheral autosensitization mechanism that occurs independently of the pronociceptive effects of inflammatory mediators and neurotrophic factors.

Nitro-oleic acid (9- and 10-nitro-octadeca-9-enoic acid, OA-NO2) is an electrophilic fatty acid nitroalkene derivative that modulates gene transcription and protein function via post-translational protein modification. Nitro-fatty acids are generated from unsaturated fatty acids by oxidative inflammatory reactions and acidic conditions in the presence of nitric oxide or nitrite. Nitroalkenes react with nucleophiles such as cysteine and histidine in a variety of susceptible proteins including transient receptor potential (TRP) channels in sensory neurons of the dorsal root and nodose ganglia. The present study revealed that OA-NO2 activates TRP channels on afferent nerve terminals in the urinary bladder and thereby increases bladder activity. The TRPV1 agonist capsaicin (CAPS, 1 μM) and the TRPA1 agonist allyl isothiocyanate (AITC, 30 μM), elicited excitatory effects in bladder strips, increasing basal tone and amplitude of phasic bladder contractions (PBC). OA-NO2 mimicked these effects in a concentration-dependent manner (1 μM – 33 μM). The TRPA1 antagonist HC3-030031 (HC3, 30 μM) and the TRPV1 antagonist diaryl piperazine analogue (DPA, 1 μM), reduced the effect of OA-NO2 on phasic contraction amplitude and baseline tone. However, the non-selective TRP channel blocker, ruthenium red (30 μM) was a more effective inhibitor, reducing the effects of OA-NO2 on basal tone by 75% and the effects on phasic amplitude by 85%. In bladder strips from CAPS-treated rats, the effect of OA-NO2 on phasic contraction amplitude was reduced by 65% and the effect on basal tone was reduced by 60%. Pretreatment of bladder strips with a combination of neurokinin receptor antagonists (NK1 selective antagonist, CP 96345; NK2 selective antagonist, MEN 10,376; NK3 selective antagonist, SB 234,375, 1 μM each) reduced the effect of OA-NO2 on basal tone, but not phasic contraction amplitude. These results indicate that nitroalkene fatty acid derivatives can activate TRP channels on CAPS-sensitive afferent nerve terminals, leading to increased bladder contractile activity. Nitrated fatty acids produced endogenously by the combination of fatty acids and oxides of nitrogen released from the urothelium and/or afferent nerves may play a role in modulating bladder activity.

Nitric oxide (NO), a neurotransmitter in the lower urinary tract, stimulates soluble guanylyl cyclase (sGC) and in turn cGMP-dependent protein kinase G (PKG) to modulate a number of downstream targets. NO donors reduce bladder hyperactivity in some pathological models but do not affect normal bladder activity in the adult rat. In this study, the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 100 microM) decreased the amplitude and frequency of spontaneous and carbachol-enhanced contractions in neonatal rat bladder strips, which are intrinsically hyperactive. This effect was blocked by inhibition of sGC and mimicked by application of a membrane-permeable cGMP analog (8-bromo-cGMP, 100 microM). Inhibition of PKG prevented or reversed the inhibitory effects of 8-bromo-cGMP. A portion of the SNAP-mediated inhibition was also dependent upon PKG; however, a short-lasting, sGC-dependent inhibitory effect of SNAP was still present after PKG inhibition. Inhibition of NO synthase with L-NAME (100 microM) did not change the amplitude or frequency of contractions. However, inhibition of endogenous phosphodiesterase (PDE)-5 with zaprinast (25 microM) reduced the amplitude and frequency of phasic contractions and increased the magnitude of inhibition produced by maximal concentrations of SNAP, suggesting that endogenous PDEs are constitutively active and regulate cGMP production. These results suggest that the NO-cGMP-PKG pathway may be involved in inhibitory control of the neonatal rat bladder.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions. The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release. The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro-nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase-positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one.

Key pointsr Nitro-oleic acid (OA-NO 2 ) and related nitroalkenes are electrophilic fatty acid derivatives that function as metabolic and anti-inflammatory signalling mediators and contribute to resolution of inflammation.r OA-NO 2 is known to activate transient receptor potential (TRP) A1 and V1 channels in rat and mouse primary sensory neurons.r However, in guinea-pig dorsal root ganglion neurons OA-NO 2 activated TRPC channels and elicited a transient excitatory effect followed by a long-lasting inhibitory effect.r Our data challenge the prevailing view that electrophilic compounds act specifically on TRPA1 and suggest that OA-NO 2 can exert a modulatory effect on sensory mechanisms via activation of TRPC channels.Abstract Effects of nitro-oleic acid (OA-NO 2 ) on TRP channels were examined in guinea-pig dissociated dorsal root ganglia (DRG) neurons using calcium imaging and patch clamp techniques. OA-NO 2 increased intracellular Ca 2+ in 60-80% DRG neurons. 1-Oleoyl-2acetyl-sn-glycerol (OAG), a TRPC agonist, elicited responses in 36% of OA-NO 2 -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-NO 2 ; however, a reducing agent DTT (50 mM) or La 3+ (50 μM) completely abolished OA-NO 2 responses. OA-NO 2 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-NO 2 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-NO 2 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 X. Zhang and J. Beckel contributed equally to this paper.