PACAP/PAC1 expression and function in micturition pathways

Abstract

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.

Introduction

Micturition involves the filling and storage of urine in the bladder, and the periodic voiding of urine. Micturition is a fundamental, physiological process that is taken for granted until this behavior is altered. Storage and elimination functions involve the coordination of the structural features of the urinary bladder and complex neural pathways organized in the central nervous system (CNS) and peripheral nervous system (PNS) (Andersson and Arner, 2004; Merrill et al., 2016). It is perhaps because of the complexity of the neural circuitry underlying micturition that urinary bladder dysfunction often results following neural injury, inflammation and diseases that affect the nervous system and/or the urinary bladder. This mini-review addresses the involvement of the neuropeptide, PACAP and the PACAP selective receptor, PAC1, in micturition reflex function and plasticity following urinary bladder inflammation using preclinical models that mimic some of the symptoms of the chronic urologic pain syndrome, bladder pain syndrome (BPS)/interstitial cystitis (IC).

Lower Urinary Tract Pathways Supporting Micturition

The storage and elimination of urine is necessary to daily life. This is made possible by intricate neural signaling pathways that coordinate the functions of the urinary bladder and urethra (Andersson and Arner, 2004). The mature micturition reflex is a spinobulbospinal reflex pathway activated by mechanoreceptors in the urinary bladder wall (Beckel and Holstege, 2011). During the storage phase, solely the sympathetic system is active, which causes relaxation of the detrusor and contraction of the bladder neck and urethra; this leaves the bladder free to elongate and to accommodate increased volume. During the voiding phase, parasympathetic activation excites and contracts the detrusor muscle, while release from sympathetic activation relaxes the urethral sphincter (de Groat, 1990; 1993). The switch between the storage and voiding phases of the micturition reflex occurs when mechanoreceptor activity in the urinary bladder exceeds a threshold. Micturition can become compromised following injury or inflammation. For example, the barrier of the urinary bladder (i.e., the urothelium) can break down, allowing toxic substances to penetrate and to contribute to LUT symptoms (e.g., urinary urgency, frequency, voiding discomfort) (Andersson and Arner, 2004).

Bladder pain syndrome/interstitial cystitis

Background

Bladder pain syndrome (BPS)/interstitial cystitis (IC) is an urologic chronic pelvic pain syndrome characterized by pelvic-perceived pain, pressure or discomfort with at least one urinary symptom (Hanno and Sant, 2001). Other symptoms include nocturia, exaggeration of normal genitourinary sensations, and pain and discomfort with bladder fullness. The diversity of symptoms associated with BPS/IC suggests that the syndrome is heterogeneous and may reflect different subgroups with overlapping symptomology but unique etiology (Mullins et al., 2015).

Etiology

Although the etiology and pathogenesis of BPS/IC are unknown, infection, inflammation, autoimmune disorder, toxic urinary agents, urothelial dysfunction, mast cell involvement and neurogenic causes have been proposed (Driscoll and Teichman, 2001; Sant and Hanno, 2001; Parsons, 2007; Patnaik et al., 2017). Although the primary insult underlying BPS/IC is unknown, it has been suggested that the pathophysiology is a “vicious circle” involving uroepithelial dysfunction, inflammation, afferent nerve hyperexcitability and visceral hyperalgesia and allodynia (Sant et al., 2007) (Figure 1). Visceral inflammation remains a central pathological process in BPS/IC and has been suggested to underlie the development of LUT symptoms. Inflammation within the urinary bladder viscera is characterized by increased vasculature, mucosal irritation that may result in barrier dysfunction and infiltration of inflammatory mediators (Erickson et al., 1997; Grover et al., 2011) (Figure 1). The proliferation and activation of mast cells, in particular, has received considerable attention in the urinary bladder immune response (Sant et al., 2007). Mast cells secrete vasoactive chemicals to promote innate and auto-immunity and their increased activity has been widely demonstrated in BPS/IC (Kastrup et al., 1983; Enerback et al., 1989; Boucher et al., 1995; Theoharides et al., 1995). The subsequent exposure in the bladder interstitium to vasoactive chemicals, inflammatory mediators and neuropeptides from visceral inflammation may lead to afferent nerve hyperexcitability and neurogenic inflammation (Yoshimura and de Groat, 1999; Vizzard, 2001a; Yu and de Groat, 2008) (Figure 1).

Figure 1: Potential etiologic cascade and pathogenesis underlying painful bladder syndrome (BPS)/interstitial cystitis (IC).

It is likely that BPS/IC has a multifactorial etiology that may act predominantly through one or more pathways resulting in the typical symptom-complex. There is a lack of consensus regarding the etiology or pathogenesis of BPS/IC but a number of proposals include a “leaky epithelium,” release of neuroactive compounds at the level of the urinary bladder with mast cell activation, “awakening” of C-fiber bladder afferents, and upregulation of inflammatory mediators including cytokines and chemotactic cytokines (chemokines). Inflammatory mediators can affect CNS and PNS neural circuitry including central “wind-up” and nociceptor sensitization resulting in chronic bladder pain and voiding dysfunction. BPS/IC is associated with diseases affecting other viscera and pelvic floors. See text for additional details. Figure adapted from (Sant et al., 2007; Gonzalez et al., 2014).

Allodynia and hyperalgesia

Pain is the hallmark symptom of BPS/IC and central to the diagnosis of BPS/IC together with the exclusion of other disorders (Fitzgerald et al., 2005). BPS/IC often involves two distinct pain-related symptoms: allodynia and hyperalgesia. Allodynia is characterized by a hypersensitive state in which normally innocuous stimuli (i.e., bladder fullness) causes pain and discomfort occur at lower volumes than normal. Under normal conditions, as the bladder fills, there is no conscious perception of bladder distension until a threshold volume is reached (Nazif et al., 2007). In BPS/IC, changes in bladder afferents lead to awareness of bladder filling much sooner than in normal circumstances, so there is discomfort and often pain with bladder distension that is reduced or eliminated with bladder emptying (Fitzgerald et al., 2005; Nazif et al., 2007). Hyperalgesia can also occur where noxious stimuli are perceived as even more painful (Persu et al., 2010). In BPS/IC, the sensitization of bladder afferents that occurs after insult leads to an increase in excitation that can contribute to an increase in pain sensation (Fitzgerald et al., 2005; Nazif et al., 2007) (Figure 1). Afferent nerve (Aδ and C-fibers) hyperexcitability increases input into the spinal cord that may eventually promote central sensitization or ‘wind-up’ (Sant et al., 2007). Central sensitization can lead to the loss of inhibition of dorsal horn neurons and a decrease in the threshold of nociceptors in the periphery, causing neurogenic inflammation, hyperalgesia, and dysreflexia symptoms that establish a self-perpetuating state that is classic in chronic visceral pain syndromes (Butrick, 2003)(Figure 1). This self-perpetuating state continues with mast cell degranulation, infiltration of mediators from uroepithelial dysfunction, and/or visceral inflammation that sustains peripheral and central sensitization that underlies visceral hyperalgesia/allodynia and symptoms of urinary frequency and urgency (Sant et al., 2007)(Figure 1).

We have hypothesized that pain associated with BPS/IC and changes in urinary bladder function (i.e., increased urinary frequency) involves an alteration of visceral sensation/bladder sensory physiology may be mediated by inflammatory changes in the urinary bladder. For example, neurotrophins (e.g., nerve growth factor, NGF) have been implicated in the peripheral sensitization of nociceptors (Lindsay and Harmar, 1989; Dray, 1995; Dinarello, 1997). Intravenous administration of a humanized monoclonal antibody that specifically inhibits NGF (tanezumab) in patients with BPS/IC demonstrates proof of concept by improving the global response assessment and reducing the urgency episode frequency (Evans et al., 2011).However, clinical trials involving systemic anti-NGF therapies for diverse pain conditions have halted enrollment due to incidence and risk of osteonecrosis (Evans et al., 2011). The need for additional LUT targets beyond NGF is clear. Additional NGF-mediated pleiotropic changes, including changes in neuropeptide expression (e.g., pituitary adenylate cyclase-activating polypeptide, PACAP), may contribute to increases in urinary frequency and pelvic hypersensitivity in BPS/IC.

NGF signaling and nociception

NGF is a peptide that consists of a α, β and γ subunits that bind to TrkA (receptor tyrosine kinase A, tropomyosin-related kinase A) to initiate downstream effects. NGF binds to TrkA at the nerve terminal resulting in receptor dimerization, internalization and the formation of a signaling endosome. The endosome is then trafficked to the cell body via retrograde transport through the axon, where it continues to signal and induce long-term changes including: altering the expression of various receptors and other neuropeptides, synaptic reorganization, mediating neurotransmitter phenotype, increasing synaptic efficiency and controlling function in target organs (Levi-Montalcini et al., 1996; Huang and Reichardt, 2001; Pezet and McMahon, 2006; Mizumura and Murase, 2015).

NGF was the first neurotrophic factor to be identified and was initially found to play a role in the development of sympathetic neurons as well as sensory neurons involved with pain and temperature (Ritter et al., 1991; Levi-Montalcini et al., 1996; Apfel, 2001). Over the last two decades, NGF signaling has been shown to be a major factor in numerous pain conditions: (1) NGF levels are elevated in chronic pain conditions (Aloe et al., 1992; Iannone et al., 2002; Sarchielli et al., 2007); (2) NGF is synthesized and released following repeated tissue injury and subsequent inflammation (Paterson et al., 2009) and (3) NGF blockade decreases pain in acute and chronic pain conditions (Cattaneo, 2010). Notably, it has been determined that NGF plays a role in the development of hyperalgesia in response to urinary bladder inflammation following cystitis. Previous studies have implicated NGF in morphological and functional changes in the sensory and sympathetic neurons that innervate the urinary bladder (Dmitrieva et al., 1997; Clemow et al., 1998; Chuang et al., 2001; Hu et al., 2005; Guerios et al., 2006; Zvara and Vizzard, 2007; Guerios et al., 2008). Moreover, NGF has been detected in the urine and urinary bladder biopsies of women with BPS/IC (Lowe et al., 1997; Okragly et al., 1999) and increased NGF expression has been observed in micturition reflex pathways, spinal cord and peripheral ganglia in animal models of the disease (Dmitrieva et al., 1997; Clemow et al., 1998; Jaggar et al., 1999; Chuang et al., 2001; Hu et al., 2005; Guerios et al., 2006; Zvara and Vizzard, 2007; Guerios et al., 2008).

Neuropeptides in Micturition Reflex Pathways

Bladder afferents contain a variety of neuroactive compounds including neuropeptides: calcitonin-gene related peptide (CGRP), substance P (Sub P), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase- activating polypeptide (PACAP), cholecystokinin and enkephalins (Donovan et al., 1983; De Groat, 1986; Keast and De Groat, 1992; Vizzard et al., 1994; Vizzard, 2000c; 2001a). All of these neuropeptides, except CGRP, are predominantly expressed in small diameter (presumably C-fiber) afferents (Donovan et al., 1983; De Groat, 1986; Keast and De Groat, 1992; Vizzard et al., 1994; Vizzard, 2000c; 2001a). An increase in NGF expression in DRG neurons can induce increased production of neuropeptides (e.g., Sub P, CGRP and PACAP) and alter sensory transduction (Donnerer et al., 1992; Woolf et al., 1997). The following sections will address the expression, distribution and functional role(s) of PACAP/PAC1 in LUT pathways in control conditions, in a transgenic mouse model with chronic urothelial overexpression of NGF, and following urinary bladder inflammation induced by the anti-neoplastic agent, cyclophosphamide (CYP).

PACAP and associated receptors

PACAP is a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family of hormones, originally isolated from the hypothalamus because of its ability to stimulate anterior pituitary adenylate cyclase activity (Ogi et al., 1990; Arimura, 1998). PACAP38 and PACAP27 are the two mature, ∝-amidated forms of PACAP that play roles in cell signaling and neuroendocrine functions in the CNS and PNS (Kimura et al., 1990; Ohkubo et al., 1992; Okazaki et al., 1995; Arimura, 1998; Braas et al., 1998). PACAP and VIP exert a variety of diverse effects by binding to three distinct G-protein-coupled receptors (GPCRs): PAC1, VPAC1, and VPAC2 that exhibit tissue- and cell-type specific expression and function (Ishihara et al., 1992; Hashimoto et al., 1993; Hosoya et al., 1993; Lutz et al., 1993; Spengler et al., 1993; Inagaki et al., 1994; May and Braas, 1995; Braas and May, 1996; Beaudet et al., 1998; Braas et al., 1998; May et al., 1998; Braas and May, 1999; DiCicco-Bloom et al., 2000). When PACAP binds to PAC1, the GPCR is internalized via endocytosis and endosomal signaling leads to diverse downstream effects (May and Parsons, 2017).

PACAP expression and function

PACAP is expressed in peripheral autonomic and sensory neurons and can exert differential downstream effects depending on the receptor subtype expression in the target tissue. Among its diverse functions, PACAP has critical roles in maintaining homeostasis in a multitude of physiological systems, including: endocrine hormone production and secretion, cardiovascular responses, glucose metabolism, gastrointestinal motility, micturition, nociception, and germ cell development (Vaudry et al., 2009). In addition, PACAP regulates a variety of central functions including: feeding/satiety, autonomic responses, nociceptive sensitivity, cognition and learning/memory, and stress-associated behaviors including stress-related psychopathologies (Ressler et al., 2011; Hammack and May, 2015; Pohlack et al., 2015). PACAP or PAC1 receptor deficient mice have given additional insights into the functional significance of PACAP/PAC1 signaling. PACAP or PAC1 receptor deficient mice exhibit blunted anxiety-like behavior, show autonomic and HPA axis dysregulation to stress challenges, and fail to develop nociceptive hypersensitivity to inflammatory pain (Hashimoto et al., 2001; Jongsma Wallin et al., 2001; Otto et al., 2001; Girard et al., 2006; Stroth and Eiden, 2010; Tsukiyama et al., 2011; Hattori et al., 2012; Botz et al., 2013). In human studies, altered PACAP levels and PAC1 receptor polymorphism have been associated with posttraumatic stress disorder (Ressler et al., 2011). Recent studies also suggest an overlap in PACAP expression and involvement in supraspinal neural circuitry underlying pain and anxiety/stress behaviors (Missig et al., 2014; Missig et al., 2017).

Pre-clinical animal models

Cyclophosphamide (CYP)-induced cystitis

CYP-induced bladder inflammation has been extensively used in our laboratory to study BPS/IC using different protocols to model acute (4 hr), intermediate (48 hr) and more chronic (8–10 days) durations of urinary bladder inflammation. Acrolein, the metabolite of CYP, accumulates in the urinary bladder and produces toxic effects, including: edema, ulceration, necrosis, petechial hemorrhage and hemorrhagic cystitis (Batista et al., 2006). CYP treatment in mice and rats is associated with increased referred somatic sensitivity (Guerios et al., 2008; Studeny et al., 2008; Cheppudira et al., 2009). CYP-induced bladder inflammation has also resulted in changes in the inflammatory milieu of the urinary bladder, including the expression and function of cytokines, chemokines (Arms et al., 2010) and NGF (Vizzard, 2000a). CYP-induced bladder inflammation (Cox, 1979; Maggi et al., 1992) produces changes in urinary bladder function that is characterized by increases in voiding frequency, non-voiding bladder contractions during the filling phase, and changes in micturition pressures (Vizzard, 2000b; 2001b; Qiao and Vizzard, 2002; 2004). The inflammatory and functional changes exhibited in rodents following CYP treatment mimic some of the signs and symptoms of the clinical syndrome BPS/IC making it a valuable and reproducible model of clinical disease to evaluate specific hypotheses related to the etiology, pathophysiology and underlying mechanisms of BPS/IC.

Chronic Urothelial Overexpression of NGF (NGF-OE) in Mice

We characterized a transgenic mouse model of urothelium-specific NGF-OE that represents a novel approach to exploring the role of NGF in urinary bladder inflammation and sensory function (Schnegelsberg et al., 2010). NGF-OE mice exhibit an increase in urinary frequency and the presence of non-voiding bladder contractions (NVCs) as well as referred pelvic hypersensitivity (Schnegelsberg et al., 2010). NGF- OE mice may represent a useful animal model of BPS/IC because the increased NGF content and other sensory and local mast cell inflammatory changes observed in the urinary bladders of these mice are consistent with changes observed with the clinical presentation of BPS/ICs. Clinically, increased NGF levels have been detected in the bladder urothelium of patients with BPS/IC (Lowe et al., 1997). Elevated NGF levels were also detected in the urine of patients with BPS/IC or overactive bladder symptoms associated with detrusor overactivity (DO), stress urinary incontinence, or bladder outlet obstruction (BOO) (Andersson, 2002; Kim et al., 2004; Kim et al., 2005; Kim et al., 2006; Liu and Kuo, 2008; 2009). Patients with DO who responded to treatment showed reduced urinary NGF levels (Liu and Kuo, 2007; 2008). Our findings support and extend many previous studies in rodents demonstrating involvement of NGF in altered bladder sensory function and the development of referred hyperalgesia in response to bladder inflammation (Dmitrieva and McMahon, 1996; Dmitrieva et al., 1997; Jaggar et al., 1999; Lamb et al., 2004; Hu et al., 2005; Guerios et al., 2006; Yoshimura et al., 2006; Zvara and Vizzard, 2007; Guerios et al., 2008).

PACAP and Micturition Pathways

PACAP (Adcyap1) and its cognate receptor, PAC1 (Adcyap1r1), have tissue-specific distributions in the LUT. Dense PACAP expression is present in LUT pathways in the CNS and PNS including expression in the urinary bladder. PACAP- immunoreactivity is exhibited throughout the urinary bladder in nerve fibers in the urinary bladder smooth muscle, suburothelial nerve plexus, and surrounding blood vessels (Fahrenkrug and Hannibal, 1998). In neonatal rats treated with the TRPV1-agonist and C-fiber neurotoxin, capsaicin, PACAP expression was significantly reduced in the urinary tract (i.e., ureter, bladder, and urethra), suggesting these PACAP-immunoreactive fibers are derived from small diameter, C-fiber afferents (Fahrenkrug and Hannibal, 1998). In the DRG, PACAP expression was present in small and medium sized DRG neurons including bladder afferent cells. Increases in PACAP expression in lumbosacral DRG are observed after nerve injury, inflammation, spinal cord injury or CYP-induced cystitis (Zhang et al., 1995; Zhang et al., 1996; Larsen et al., 1997; Moller et al., 1997; Vizzard, 2000c).

PACAP knockout (−/−) mice exhibit bladder dysfunction and altered somatic sensitivity

PACAP(−/−) mice exhibited an increase in bladder mass with a significantly thicker lamina propria and detrusor smooth muscle but the urothelium was unchanged (May and Vizzard, 2010). Using functional bladder testing, PACAP(−/−) mice exhibited increased bladder capacity, voided volume and intervals between voiding events (i.e., intercontraction interval) with significantly increased detrusor contraction duration and large residual volume (May and Vizzard, 2010). Although wildtype mice respond to intravesical infusion of acetic acid (0.5%) with a decrease in voided volume and intercontraction interval, PACAP(−/−) mice do not exhibit any changes in bladder function (May and Vizzard, 2010) consistent with a nociceptive response to bladder irritation being mediated, in part, by PACAP-immunoreactive C-fiber bladder afferents. In response to somatic sensitivity testing with calibrated von Frey filaments, PACAP(−/−) mice were less sensitive to hindpaw and pelvic stimulation compared to control mice (May and Vizzard, 2010). These results suggest that PACAP gene disruption contributes to changes in bladder morphology and function, and somatic and visceral sensation.

Functional Expression of PAC1 receptors in rat urothelium

Activation of PAC1 receptors by PACAP27, PACAP38 and VIP to cultured urothelial cells releases ATP, which may activate purinergic receptors expressed on adjacent sensory nerve fibers in the suburothelial plexus as well as on other tissues including detrusor smooth muscle and interstitial cells in the lamina propria (Girard et al., 2008). PAC1 receptor antagonism blocked ATP release (Girard et al., 2008) suggesting that PACAP signaling through PAC1 receptors may regulate micturition reflex function at the level of the urothelium (Girard et al., 2008).

PACAP Expression and Function in Micturition Reflexes following CYP-induced cystitis

PACAP plays a role in urinary bladder dysfunction observed in rodents following CYP-induced cystitis (Vizzard, 2000c; Braas et al., 2006; Herrera et al., 2006; Girard et al., 2008). PACAP and its receptor, PAC1, are expressed throughout the micturition reflex circuitry (Braas et al., 2006), and this expression increased following CYP-induced cystitis. In the spinal cord, PACAP- immunoreactivity dramatically increased in rostral lumbar (L1-L2) spinal cord regions (Vizzard, 2000c), as well as in the lower lumbosacral (L6-S1) spinal cord regions involved in micturition reflexes (Herrera et al., 2006). Intermediate and chronic CYP treatments significantly increased PACAP transcript and protein expression in lumbosacral DRG (Vizzard, 2000c; Girard et al., 2008). CYP-induced cystitis also significantly increased PACAP expression in bladder afferent cells in lumbosacral DRG that were retrogradely labeled from the urinary bladder (Vizzard, 2000c). Intermediate and chronic CYP treatments similarly increased PACAP and/or PAC1 receptor transcript expression in the urothelium and detrusor smooth muscle (Girard et al., 2008). We have recently extended these observations by examining the effects of CYP-induced bladder inflammation (4 h, 48 h, chronic) on LUT pathways in PACAP promoter-dependent BAC transgenic mice (Vizzard et al., unpublished observations). In control animals, low expression of PACAP-EGFP+ fibers was present in the superficial dorsal horn (L1, L2, L4-S1). After CYP-induced cystitis, PACAP-EGFP+ cells increased in spinal segments and dorsal root ganglia (L1, L2, L6, S1) involved in micturition. The density of PACAP-EGFP+ nerve fibers was increased in the superficial laminae (I-II) of the L1, L2, L6, S1 dorsal horn and in the lateral collateral pathway in L6-S1 spinal cord. After cystitis, PACAP-EGFP+ cells and nerve fibers were present in many supraspinal regions, including: locus coeruleus, Barrington’s nucleus or the pontine micturition center, rostral ventrolateral medulla, PAG, raphe, and amygdala. Following cystitis, the number of PACAP-EGFP+ urothelial cells increased with duration of cystitis. PACAP expression in LUT pathways after cystitis may play a role in altered visceral sensation and/or increased voiding frequency.

Functional studies with a PAC1 receptor antagonist have demonstrated a role for PACAP/PAC1 signaling in micturition reflex pathways following urinary bladder inflammation. Intrathecal or intravesical administration of the PAC1 receptor antagonist, PACAP(6–38), increased bladder capacity, reduced voiding frequency and decreased the number and amplitude of non-voiding bladder contractions in CYP-treated rats (Braas et al., 2006)(Figure 2). The expression and upregulation of PACAP/PAC1 in LUT pathways and the functional benefit of PAC1 receptor blockade to the urinary bladder in preclinical animal models suggest that targeting PACAP/PAC1 signaling in micturition pathways may have translational benefit to the clinical syndrome, BPS/IC (Figure 2).

Figure 2: Blockade of the PACAP/receptor system in micturition reflexes is a potential strategy to improve bladder function and reduce pelvic pain following urinary bladder inflammation.

The CYP-induced bladder inflammation model in rodents and the transgenic mouse model of chronic urothelial overexpression of NGF (NGF-OE) are essential to determine underlying mechanisms contributing to urinary bladder dysfunction and pelvic pain in the clinical syndrome BPS/IC. Both models are associated with changes in NGF expression in the urinary bladder that can result in changes in the urinary bladder and be retrogradely transported to lumbosacral dorsal root ganglia (DRG). PACAP and PAC1 receptor exhibit neuroplastic changes in expression and function with CYP-induced bladder inflammation and increased NGF expression in the urinary bladder. Intravesical instillation of the PAC1 receptor antagonist, PACAP (6–38), reduces voiding frequency and somatic sensitivity. Blockade of PAC1 in micturition reflexes may represent a novel therapeutic intervention to improve urinary bladder function and reduce referred, somatic pain. DH, dorsal horn; EUS, external urethral sphincter. Figure adapted from (Girard et al., 2017).

NGF-OE and PACAP signaling

PACAP/PAC1 expression and function has also been evaluated using the NGF-OE mouse that exhibits chronic urothelial overexpression of NGF. PAC1 receptor transcript expression increased in the urothelium of NGF-OE mice whereas PACAP transcript expression decreased in the urothelium of NGF-OE mice (Girard, et al., 2012). Using conscious cystometry, functional studies demonstrated that chronic NGF overexpression in the urothelium increased voiding frequency, non-voiding contractions and referred somatic (i.e., pelvic) sensitivity (Schnegelsberg et al., 2010). Additionally, the intravesical infusion of a PAC1 antagonist, PACAP (6–38), significantly increased the time between voiding events (i.e., intercontraction interval) and the void volume in NGF-OE mice. PACAP (6–38) also significantly reduced pelvic sensitivity in NGF-OE mice (Girard, et al., 2016) (Figure 2). In conclusion, PACAP signaling contributes to the hyperalgesia and increased voiding frequency in NGF-OE mice.

Pleiotropic changes in neuropeptide/receptor systems in LUT pathways in NGF-OE mice

Chronic urothelial overexpression of NGF (NGF-OE) in mice produces changes in PACAP/receptor expression in micturition reflex pathways. PAC1 receptor transcript and PAC1- immunoreactivity were significantly increased in the urothelium of NGF-OE mice whereas PACAP transcript expression and PACAP-immunoreactivity were decreased in the urothelium of NGF-OE mice (Girard et al., 2010). In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice (Girard et al., 2010). VPAC2 receptor transcript was significantly increased in the detrusor smooth muscle in NGF-OE mice (Girard et al., 2010). NGF-OE mice exhibit additional changes in PACAP and associated receptors expression in LUT pathways that may also contribute to altered urinary bladder function in NGF-OE mice.

NGF and PACAP Interactions

Both CYP-induced cystitis in rodents and the NGF-OE mouse model exhibit increased expression of NGF in LUT pathways (e.g., DRG, urinary bladder) with subsequent changes in the expression and function of PACAP/PAC1 signaling in LUT pathways (Figure 2). Reciprocal regulatory interactions between NGF and PACAP have previously been demonstrated in pheochromocytoma (PC)12 cells and sensory ganglia with NGF acting a positive regulator of PACAP expression in DRG cells (Jongsma Wallin et al., 2001). In rat PC12 cells, both NGF and PACAP can induce PC differentiation into a neuronal phenotype (Grumolato et al., 2003). In addition, the neurotrophins, NGF and/or brain-derived neurotrophic factor facilitated expression of the PAC1 receptor in CNS neurons (i.e., cerebellar granule cells) and PC12 cells (Jamen et al., 2002). In complementary studies, PACAP upregulated expression and/or phosphorylation of neurotrophin receptors (e.g., TrkA, TrkB, TrkC) in PC12 cells, CNS neurons (i.e., hippocampal neurons) (Lee et al., 2002) and sympathetic neuroblasts (DiCicco- Bloom et al., 2000). In some contexts, reciprocal regulation of PACAP and NGF signaling pathways may be a feed-forward mechanism to amplify critical, physiological processes (e.g., survival or differentiation) during neuronal development or regeneration. In the context of urinary bladder inflammation, a feed-forward mechanism may be damaging by amplifying painful signals (i.e., allodynia and hyperalgesia) and target organ dysfunction (Figure 2).

Summary and Conclusions

BPS/IC is a chronic, inflammatory pain syndrome that affects both women and men with few effective treatments perhaps due to the heterogeneous nature of the disease. New treatment options, evaluated using preclinical models with signs and symptoms common to the clinical presentation of BPS/IC, are needed. Using the CYP-induced cystitis model in rodents and a transgenic mouse model with chronic, urothelial overexpression of NGF, blockade of the PACAP/PAC1 signaling pathway reduced urinary frequency and referred, somatic sensitivity (Figure 2). In the context of urinary bladder inflammation, blockade of peptide/receptor systems including PACAP/PAC1 and other systems (e.g. CGRP/CGRP receptor) expressed in bladder sensory neurons should be considered as potential targets for pharmacological intervention with the anticipation of pain relief and normalization of urinary bladder function.

Abbreviations

AT

Adenosine triphosphate

BOO

Bladder outlet obstruction

BPS

Bladder pain syndrome

CGRP

Calcitonin-gene related peptide

CNS

Central nervous system

CYP

Cyclophosphamide

DO

Detrusor overactivity

DRG

Dorsal root ganglion

GPCR

G-protein-coupled receptor

HPA

Hypothalamic-pituitary-adrenal

hr

hours

IC

Interstitial cystitis

LUT

Lower urinary tract

NGF

Nerve growth factor

NGF-OE

Nerve growth factor overexpression

NVC

Non-voiding bladder contraction

PAC1

PACAP type I receptor

PACAP

Pituitary adenylate cyclase-activating polypeptide

PAG

Periaqueductal grey

PC

pheochromocytoma

PMC

Pontine micturition center

PNS

Peripheral nervous system

Sub P

Substance P

TrkA

Receptor tyrosine kinase A, tropomyosin-related kinase A

TrkB

Receptor tyrosine kinase B, tropomyosin-related kinase B

TrkC

Receptor tyrosine kinase C, tropomyosin-related kinase C

VIP

Vasoactive intestinal polypeptide

VPAC1

VIP receptor 1

VPAC2

VIP receptor 2