Psychological stress is a known precipitant of lower urinary tract (LUT) dysfunction and can exacerbate pelvic pain, yet the underlying mechanisms remain incompletely understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel expressed in LUT tissues and associated afferent circuits, is positioned to couple stress-related signaling to changes in voiding function. We tested the hypothesis that TRPV4 is required for repeated variate stress (RVS)-evoked bladder dysfunction and contributes to stress-associated somatic hypersensitivity and anxiety-like behavior. Male and female wild-type (WT) and Trpv4 knockout (KO) mice underwent RVS and were assessed using conscious cystometry, void spot assay (VSA), mechanical sensitivity testing (pelvic and hindpaw), and behavioral assays (open field, elevated plus maze). RVS produced sex- and tissue-specific regulation of Trpv4 transcripts LUT tissues. In WT mice, RVS induced bladder overactivity in both sexes, characterized by increased voiding frequency and reduced intermicturition interval. These stress-induced changes were absent in Trpv4 KO mice, demonstrating that TRPV4 is required for stress-evoked bladder dysfunction. Bladder pressure endpoints revealed additional sex-dependent effects in WT but not Trpv4 KO mice. VSA identified sex-specific spontaneous voiding changes that did not uniformly parallel cystometric measures, indicating complementary sensitivity of provoked and unprovoked assays. Despite protection from stress-associated bladder dysfunction, Trpv4 KO mice exhibited increased pelvic and hindpaw mechanical sensitivity following RVS. Behavioral testing revealed sex-dependent differences in stress resilience. These findings identify TRPV4 as a key mediator of stress-induced bladder overactivity and demonstrate a dissociation between referred mechanical pain outcomes in Trpv4 KO mice.

The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. Transient receptor potential vanilloid 1 (TRPV1) channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+ nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (P ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild-type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1 knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell-deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression, whereas control (no stress) mast cell-deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia (DRG) in WT mice exposed to RVS, but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction.NEW & NOTEWORTHY Using pharmacological tools and transgenic mice in a repeated variate stress (RVS) model in female mice, we demonstrate that transient receptor potential vanilloid 1 (TRPV1) and mast cells contribute to the increased voiding frequency observed following RVS. TRPV1 and mast cells should continue to be considered as targets to improve bladder function in stress-induced bladder dysfunction.

IC/BPS is a chronic inflammatory pelvic pain syndrome characterized by lower urinary tract symptoms including unpleasant sensation (pain, pressure, or discomfort) in the suprapubic or bladder area, as well as increased urinary frequency and urgency, and decreased bladder capacity. While its etiology remains unknown, increasing evidence suggests a role for changes in nerve growth factor (NGF) signaling. However, NGF signaling is complex and highly context dependent. NGF activates two receptors, TrkA and p75NTR, which activate distinct but overlapping signaling cascades. Dependent on their coexpression, p75NTR facilitates TrkA actions. Here, we show effects of CYP treatment and pharmacological inhibition of p75NTR (via LM11A-31) and TrkA (ARRY-954) on NGF signaling–related proteins: NGF, TrkA, phosphorylated (p)-TrkA, p75NTR, p-ERK1/2, and p-JNK. Cystitis conditions were associated with increased urothelial NGF expression and decreased TrkA and p75NTR expression as well as altering their co-expression ratio; phosphorylation of ERK1/2 and JNK were also altered. Both TrkA and p75NTR inhibition affected the activation of signaling pathways downstream of TrkA, supporting the hypothesis that NGF actions during cystitis are primarily TrkA-mediated. Our findings, in tandem with our recent companion paper demonstrating the effects of TrkA, TrkB, and p75NTR inhibition on bladder function in a mouse model of cystitis, highlight a variety of potent therapeutic targets and provide further insight into the involvement of NGF signaling in sustained conditions of bladder inflammation.

Symptom exacerbation due to stress is prevalent in many disease states, including functional disorders of the urinary bladder (e.g., overactive bladder (OAB), interstitial cystitis/bladder pain syndrome (IC/BPS)); however, the mechanisms underlying the effects of stress on micturition reflex function are unclear. In this study we designed and evaluated a stress-induced symptom exacerbation (SISE) mouse model that demonstrates increased urinary frequency and somatic (pelvic and hindpaw) sensitivity. Cyclophosphamide (CYP) (35 mg/kg; i.p., every 48 hours for a total of 4 doses) or 7 days of repeated variate stress (RVS) did not alter urinary bladder function or somatic sensitivity; however, both CYP alone and RVS alone significantly (p ≤ 0.01) decreased weight gain and increased serum corticosterone. CYP treatment when combined with RVS for 7 days (CYP+RVS) significantly (p ≤ 0.01) increased serum corticosterone, urinary frequency and somatic sensitivity and decreased weight gain. CYP+RVS exposure in mice significantly (p ≤ 0.01) increased (2.6-fold) voiding frequency as we determined using conscious, open-outlet cystometry. CYP+RVS significantly (p ≤ 0.05) increased baseline, threshold, and peak micturition pressures. We also evaluated the expression of NGF, BDNF, CXC chemokines and IL-6 in urinary bladder in CYP alone, RVS alone and CYP+RVS mouse cohorts. Although all treatments or exposures increased urinary bladder NGF, BDNF, CXC and IL-6 content, CYP+RVS produced the largest increase in all inflammatory mediators examined. These results demonstrated that CYP alone or RVS alone creates a change in the inflammatory environment of the urinary bladder but does not result in a change in bladder function or somatic sensitivity until CYP is combined with RVS (CYP+RVS). The SISE model of CYP+RVS will be useful to develop testable hypotheses addressing underlying mechanisms where psychological stress exacerbates symptoms in functional bladder disorders leading to identification of targets and potential treatments.

Psychological stress is associated with urinary bladder dysfunction (e.g., increased voiding frequency, urgency and pelvic pain); however, the mechanisms underlying the effects of stress on urinary bladder function are unknown. Transient receptor potential (TRP) channels (vanilloid family) may be potential targets for intervention due to their distribution in the LUT and role in pain. Here, we examine a model of repeated variate stress (RVS) of 2 week (wk) or 4 wk duration in female mice and its effects on bladder function, anxiety-like behavior, and TRPV transcript expression in urinary bladder and lumbosacral spinal cord and associated dorsal root ganglia (DRG). Using continuous infusion, open-outlet cystometry in conscious mice, RVS significantly (p ≤ 0.05) decreased infused volume and intermicturition interval. Bladder pressures (threshold, average, minimum, and maximum pressures) were unchanged with RVS. Quantitative PCR demonstrated significant (p ≤ 0.05) changes in TrpV1 and TrpV4 mRNA expression between control and RVS cohorts in the urothelium, lumbosacral spinal cord, and DRG. Future directions will examine the contribution of TRP channels on bladder function, somatic sensation and anxiety-like behavior following RVS.

Symptom exacerbation due to stress is prevalent in many disease states, including functional disorders of the urinary bladder (e.g., overactive bladder (OAB), interstitial cystitis/bladder pain syndrome (IC/BPS)); however, the mechanisms underlying the effects of stress on micturition reflex function are unclear. In this study we designed and evaluated a stress-induced symptom exacerbation (SISE) mouse model that demonstrates increased urinary frequency and somatic (pelvic and hindpaw) sensitivity. Cyclophosphamide (CYP) (35 mg/kg; i.p., every 48 hours for a total of 4 doses) or 7 days of repeated variate stress (RVS) did not alter urinary bladder function or somatic sensitivity; however, both CYP alone and RVS alone significantly (p ≤ 0.01) decreased weight gain and increased serum corticosterone. CYP treatment when combined with RVS for 7 days (CYP+RVS) significantly (p ≤ 0.01) increased serum corticosterone, urinary frequency and somatic sensitivity and decreased weight gain. CYP+RVS exposure in mice significantly (p ≤ 0.01) increased (2.6-fold) voiding frequency as we determined using conscious, open-outlet cystometry. CYP+RVS significantly (p ≤ 0.05) increased baseline, threshold, and peak micturition pressures. We also evaluated the expression of NGF, BDNF, CXC chemokines and IL-6 in urinary bladder in CYP alone, RVS alone and CYP+RVS mouse cohorts. Although all treatments or exposures increased urinary bladder NGF, BDNF, CXC and IL-6 content, CYP+RVS produced the largest increase in all inflammatory mediators examined. These results demonstrated that CYP alone or RVS alone creates a change in the inflammatory environment of the urinary bladder but does not result in a change in bladder function or somatic sensitivity until CYP is combined with RVS (CYP+RVS). The SISE model of CYP+RVS will be useful to develop testable hypotheses addressing underlying mechanisms where psychological stress exacerbates symptoms in functional bladder disorders leading to identification of targets and potential treatments.

Interstitial cystitis/bladder pain syndrome is a chronic inflammatory pelvic pain syndrome of unknown etiology characterized by a number of lower urinary tract symptoms, including increased urinary urgency and frequency, bladder discomfort, decreased bladder capacity, and pelvic pain. While its etiology remains unknown, a large body of evidence suggests a role for changes in neurotrophin signaling, particularly that of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Here, we evaluated the effects of pharmacological inhibition of the NGF receptor TrkA, BDNF receptor TrkB, and pan-neurotrophin receptor p75NTR on bladder function in acute (4-hour) and chronic (8-day) mouse models of cyclophosphamide (CYP)-induced cystitis. TrkA inhibition via ARRY-954 significantly increased intermicturition interval and bladder capacity in control and acute and chronic CYP-treatment conditions. TrkB inhibition via ANA-12 significantly increased intermicturition interval and bladder capacity in acute, but not chronic, CYP-treatment conditions. Interestingly, intermicturition interval and bladder capacity significantly increased following p75NTR inhibition via LM11A-31 in the acute CYP-treatment condition, but decreased in the chronic condition, potentially due to compensatory changes in neurotrophin signaling or increased urothelial barrier dysfunction in the chronic condition. Our findings demonstrate that these receptors represent additional potent therapeutic targets in mice with cystitis and may be useful in the treatment of interstitial cystitis and other inflammatory disorders of the bladder.

Lamina propria interstitial cells that express the tyrosine kinase receptor, platelet-derived growth factor receptor alpha (PDGFRα) may play a role in urinary sensory signaling. Imatinib mesylate, also referred to as imatinib, is a tyrosine kinase inhibitor that can inhibit PDGFRα and has been widely used in urological research. We evaluated the functional effects of imatinib administration (via oral gavage or intravesical infusion) with two different experimental designs (prevention and treatment), in a cyclophosphamide (CYP)-induced cystitis (acute, intermediate, and chronic), male and female rodent model using conscious cystometry and somatic sensitivity testing. Imatinib significantly (0.0001 ≤ p ≤ 0.05) decreased voiding frequency and increased bladder capacity in acute CYP-induced cystitis, by the prevention (females) and treatment (females and males) designs. Imatinib was not effective in preventing or treating intermediate or chronic CYP-induced cystitis in either sex. Interestingly, in the prevention experiments, imatinib administration increased (0.0001 ≤ p ≤ 0.01) voiding frequency and decreased bladder capacity in control mice. However, in the treatment experiments, imatinib administration decreased (0.01 ≤ p ≤ 0.05) voiding frequency and increased bladder capacity in control mice. Bladder function improvements observed with imatinib treatment in acute CYP-induced cystitis mice remained and additionally improved with a second dose of imatinib 24 hours after CYP treatment. Imatinib administration did not affect pelvic somatic sensitivity in female mice with acute CYP-induced cystitis. Our studies suggest that (1) imatinib improves bladder function in mice with acute CYP-induced cystitis with a prevention and treatment design and (2) interstitial cells may be a useful target to improve bladder function in cystitis.

Imatinib mesylate is a tyrosine kinase inhibitor that inhibits platelet-derived growth factor receptor (PDGFR)-α, -β, stem cell factor receptor (c-KIT), and BCR-ABL. PDGFRα is expressed in a subset of interstitial cells in the lamina propria (LP) and detrusor muscle of the urinary bladder. PDGFRα + interstitial cells may contribute to bladder dysfunction conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) or overactive bladder (OAB). We have previously demonstrated that imatinib prevention via oral gavage or treatment via intravesical infusion improves urinary bladder function in mice with acute (4 hour, h) cyclophosphamide (CYP)-induced cystitis. Here, we investigate potential underlying mechanisms mediating the bladder functional improvement by imatinib using a prevention or treatment experimental design. Using qRT-PCR and ELISAs, we examined inflammatory mediators (NGF, VEGF, BDNF, CCL2, IL-6) previously shown to affect bladder function in CYP-induced cystitis. We also examined the distribution of phosphorylated (p) ERK and pAKT expression in the LP with immunohistochemistry. Imatinib prevention significantly (0.0001 ≤ p ≤ 0.05) reduced expression for all mediators examined except NGF, whereas imatinib treatment was without effect. Imatinib prevention and treatment significantly (0.0001 ≤ p ≤ 0.05) reduced pERK and pAKT expression in the upper LP (U. LP) and deeper LP (D. LP) in female mice with 4 h CYP-induced cystitis. Although we have previously demonstrated that imatinib prevention or treatment improves bladder function in mice with cystitis, the current studies suggest that reductions in inflammatory mediators contribute to prevention benefits of imatinib but not the treatment benefits of imatinib. Differential effects of imatinib prevention or treatment on inflammatory mediators may be influenced by the route and frequency of imatinib administration and may also suggest other mechanisms (e.g., changes in transepithelial resistance of the urothelium) through which imatinib may affect urinary bladder function following CYP-induced cystitis.

In the urinary bladder, mechanosensitive ion channels (MSCs) underlie the transduction of bladder stretch into sensory signals that are relayed to the PNS and CNS. PIEZO1 is a recently identified MSC that is Ca2+ permeable and is widely expressed throughout the lower urinary tract. Recent research indicates that PIEZO1 is activated by mechanical stretch or by pharmacological agonism via Yoda1. Aberrant activation of PIEZO1 has been suggested to play a role in clinical bladder pathologies like partial bladder outlet obstruction and interstitial cystitis/bladder pain syndrome (IC/BPS). In the present study, we show that intravesical instillation of Yoda1 in female Wistar rats leads to increased voiding frequency for up to 16 hours after administration compared to vehicle treatment. In a cyclophosphamide (CYP) model of cystitis, we found that the gene expression of several candidate MSCs (Trpv1, Trpv4, Piezo1, and Piezo2) were all upregulated in the urothelium and detrusor following chronic CYP-induced cystitis, but not acute CYP-induced cystitis. Functionally with this model, we show that Ca2+ activity is increased in urothelial cells following PIEZO1 activation via Yoda1 in acute and intermediate CYP treatment, but not in naïve (no CYP) nor chronic CYP treatment. Lastly, we show that activation of PIEZO1 may contribute to pathological bladder dysfunction through the downregulation of several tight junction genes in the urothelium including claudin-1, claudin-8, and zona occludens-1. Together, these data suggest that PIEZO1 activation plays a role in dysfunctional voiding behavior and may be a future, clinical target for the treatment of pathologies like IC/BPS.

Stress causes symptom exacerbation in functional disorders of the urinary bladder. However, the potential mediators and underlying mechanisms of stress effects on micturition reflex function are unknown. We have characterized PACAP (Adcyap1) and PAC1 receptor (Adcyap1r1) signaling in stress-induced urinary bladder dysfunction in mice. We determined PACAP and PAC1 transcripts and protein expressions in the urinary bladder and lumbosacral dorsal root ganglia (DRG) and spinal cord in repeated variate stress (RVS) or control mouse (handling only) groups. RVS in mice significantly (p ≤ 0.01) increased serum corticosterone and urinary bladder NGF content and decreased weight gain. PACAP and PAC1 mRNA and protein were differentially regulated in lower urinary tract tissues with changes observed in lumbosacral DRG and spinal cord but not in urinary bladder. RVS exposure in mice significantly (p ≤ 0.01) increased (2.5-fold) voiding frequency as determined using conscious cystometry. Intrabladder administration of the PAC1 receptor antagonist, PACAP(6-38) (300 nM), significantly (p ≤ 0.01) increased infused volume (1.5–2.7-fold) to elicit a micturition event and increased the intercontraction interval (i.e., decreased voiding frequency) in mice exposed to RVS and in control mice, but changes were smaller in magnitude in control mice. We also evaluated the effect of PAC1 blockade at the level of the urinary bladder on pelvic sensitivity in RVS or control mouse groups using von Frey filament testing. Intrabladder administration of PACAP(6-38) (300 nM) significantly (p ≤ 0.01) reduced pelvic sensitivity following RVS. PACAP/receptor signaling in the CNS and PNS contributes to increased voiding frequency and pelvic sensitivity following RVS and may represent a potential target for therapeutic intervention.

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 μM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly ( P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly ( P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly ( P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

High expression of VEGF is associated with immature angiogenesis within the urinary bladder wall and bladder afferent nerve sensitization, leading to visceral hyperalgesia and pelvic pain. Research suggests a shift in VEGF alternative splice variant (VEGF-Axxxa and VEGF-Axxxb) expression with several pathologies (e.g., neuropathic pain and inflammation) as well as differing effects on pain. Translational studies have also demonstrated increased total VEGF expression in the bladders of women with interstitial cystitis/bladder pain syndrome. In the present study, we quantified VEGF alternative splice variant expression in lower urinary tract tissues under control conditions and with cyclophosphamide (CYP)-induced cystitis. Using conscious cystometry and intravesical instillation of a potent and selective VEGF receptor 2 (VEGFR2) tyrosine kinase inhibitor (Ki-8751, 1 mg/kg) in Wistar rats (male and female) with acute and chronic CYP-induced cystitis and control (no CYP) rats, we further determined the functional effects of VEGFR2 blockade on bladder function. With VEGFR2 blockade, bladder capacity increased ( P ≤ 0.01) in male and female control rats as well as in male and female rats with acute ( P ≤ 0.05) or chronic ( P ≤ 0.01 or P ≤ 0.05, respectively) CYP-induced cystitis. Void volume also increased in female control rats ( P ≤ 0.01) and female rats with acute ( P ≤ 0.05) or chronic ( P ≤ 0.05) CYP-induced cystitis as well as in male control rats ( P ≤ 0.05) and male rats with chronic CYP-induced cystitis ( P ≤ 0.01). These data suggest that VEGF may be a biomarker for interstitial cystitis/bladder pain syndrome and that targeting VEGF/VEGFR2 signaling may be an effective treatment.

Neural injury, inflammation, or diseases commonly and adversely affect micturition reflex function that is organized by neural circuits in the CNS and PNS. One neuropeptide receptor system, pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1), and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in the lower urinary tract. PACAP and associated receptors are expressed in the LUT and exhibit changes in expression, distribution, and function in preclinical animal models of bladder pain syndrome (BPS)/interstitial cystitis (IC), a chronic, visceral pain syndrome characterized by pain, and LUT dysfunction. Blockade of the PACAP/PAC1 receptor system reduces voiding frequency and somatic (e.g., hindpaw, pelvic) sensitivity in preclinical animal models and a transgenic mouse model that mirrors some clinical symptoms of BPS/IC. The PACAP/receptor system in micturition pathways may represent a potential target for therapeutic intervention to reduce LUT dysfunction following urinary bladder inflammation.

Changes in urinary bladder function and somatic sensation may be mediated, in part, by inflammatory changes in the urinary bladder including the expression of chemokines. Male and female C57BL/6 mice were treated with cyclophosphamide (CYP; 75 mg/kg, 200 mg/kg, i.p.) to induce bladder inflammation (4 h, 48 h, chronic). We characterized the expression of CXC chemokines (CXCL9, CXCL10 and CXCL11) in the urinary bladder and determined the effects of blockade of their common receptor, CXCR3, at the level urinary bladder on bladder function and somatic (hindpaw and pelvic) sensation. qRT-PCR and Enzyme-Linked Immunoassays (ELISAs) were used to determine mRNA and protein expression of CXCL9, CXCL10 and CXCL11 in urothelium and detrusor. In urothelium of female mice treated with CYP, CXCL9 and CXCL10 mRNA significantly (p ≤ 0.01) increased with CYP treatment whereas CXC mRNA expression in the detrusor exhibited both increases and decreases in expression with CYP treatment. CXC mRNA expression urothelium and detrusor of male mice was more variable with both significant (p ≤ 0.01) increases and decreases in expression depending on the specific CXC chemokine and CYP treatment. CXCL9 and CXCL10 protein expression was significantly (p ≤ 0.01) increased in the urinary bladder with 4 h CYP treatment in female mice whereas CXC protein expression in the urinary bladder of male mice did not exhibit an overall change in expression. CXCR3 blockade with intravesical instillation of AMG487 (5 mg/kg) significantly (p ≤ 0.01) increased bladder capacity, reduced voiding frequency and reduced non-voiding contractions in female mice treated with CYP (4 h, 48 h). CXCR3 blockade also reduced (p ≤ 0.01) hindpaw and pelvic sensitivity in female mice treated with CYP (4 h, 48 h). CXC chemokines may be novel targets for treating urinary bladder dysfunction and somatic sensitization resulting from urinary bladder inflammation.

Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels (Ih) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

Complex organization of CNS and PNS pathways is necessary for the coordinated and reciprocal functions of the urinary bladder, urethra and urethral sphincters. Injury, inflammation, psychogenic stress or diseases that affect these nerve pathways and target organs can produce lower urinary tract (LUT) dysfunction. Numerous neuropeptide/receptor systems are expressed in the neural pathways of the LUT and non-neural components of the LUT (e.g., urothelium) also express peptides. One such neuropeptide receptor system, pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in the LUT. Mice with a genetic deletion of PACAP exhibit bladder dysfunction and altered somatic sensation. PACAP and associated receptors are expressed in the LUT and exhibit neuroplastic changes with neural injury, inflammation, and diseases of the LUT as well as psychogenic stress. Blockade of the PACAP/PAC1 receptor system reduces voiding frequency in preclinical animal models and transgenic mouse models that mirror some clinical symptoms of bladder dysfunction. A change in the balance of the expression and resulting function of the PACAP/receptor system in CNS and PNS bladder reflex pathways may underlie LUT dysfunction including symptoms of urinary urgency, increased voiding frequency, and visceral pain. The PACAP/receptor system in micturition pathways may represent a potential target for therapeutic intervention to reduce LUT dysfunction.

The storage and periodic elimination of urine, termed micturition, requires a complex neural control system to coordinate the activities of the urinary bladder, urethra, and urethral sphincters. At the level of the lumbosacral spinal cord, lower urinary tract reflex mechanisms are modulated by supraspinal controls with mechanosensory input from the urothelium, resulting in regulation of bladder contractile activity. The specific identity of the mechanical sensor is not yet known, but considerable interest exists in the contribution of transient receptor potential (TRP) channels to the mechanosensory functions of the urothelium. The sensory, transduction, and signalling properties of the urothelium can influence adjacent urinary bladder tissues including the suburothelial nerve plexus, interstitial cells of Cajal, and detrusor smooth muscle cells. Diverse stimuli, including those that activate TRP channels expressed by the urothelium, can influence urothelial release of chemical mediators (such as ATP). Changes to the urothelium are associated with a number of bladder pathologies that underlie urinary bladder dysfunction. Urothelial receptor and/or ion channel expression and the release of signalling molecules (such as ATP and nitric oxide) can be altered with bladder disease, neural injury, target organ inflammation, or psychogenic stress. Urothelial receptors and channels represent novel targets for potential therapies that are intended to modulate micturition function or bladder sensation.