Pannexins: the ‘nexus’ between urothelium ATP production and extracellular release

We take bladder function for granted. We assume that our daily bathroom trips and occasional nighttime visits will continue without difficulty. However, approximately 50 million Americans suffer from bladder control problems that produce physical health problems, poor quality of life and an enormous economic burden (Levy & Muller, 2006). Micturition (urination) is a basic physiological function; however, it may be surprising to learn that the mechanisms underlying micturition continue to be defined. For example, our knowledge of the mechanisms underlying the mechanotransduction of urinary bladder distention to changes in sensory nerve activity and subsequent communication to the central nervous system is limited (Arms, 2009; Birder & Andersson, 2013). How do initial pressure changes stimulate bladder sensory nerves? What mechanoreceptors transduce the change in pressure? What signalling molecules and receptors facilitate mechanotransduction at the level of the urinary bladder? These questions highlight our lack of knowledge concerning basic micturition mechanisms. We continue to document ‘normal’ micturition processes and, in so doing, gain insight into bladder control problems. The lower urinary tract (LUT) is regulated by supraspinal and spinal circuitry and peripheral nervous system input to maintain distinct functions of storage and elimination (Arms, 2009; Birder & Andersson, 2013). The LUT (bladder and urethra) passively stores kidney byproducts until it is appropriate to void. The urinary bladder wall consists of three layers: tunica mucosa, tunica muscularis propria and tunica serosa (Gonzalez et al. 2014). Transitional epithelium or urothelium, and the lamina propria comprise the tunica mucosa. Urothelial cells are arranged in basal, intermediate and apical cell layers. The luminal surface of apical umbrella cells establishes anti-adherence and impermeable properties of the mucosa. The lamina propria may integrate urothelial and smooth muscle function due to its innervations and proximity to the urothelium and tunica muscularis propria, composed of three smooth muscle layers termed the detrusor (Gonzalez et al. 2014). Current and emerging evidence suggests that the urothelium not only acts as a functional barrier against urine solutes, but also exhibits specialized sensory and transduction/signalling properties enabling responses to chemical or mechanical stimuli (Arms, 2009; Birder & Andersson, 2013). The ‘urothelial-associated sensory web’ describes sensory inputs and outputs of the urothelium that result in receptor- and channel-mediated events at the urothelium and subsequent release/secretion of chemical mediators (Birder & Andersson, 2013). The urothelium secretes a variety of signalling molecules (e.g. adenosine triphosphate (ATP), nitric oxide) and is able to communicate, perhaps reciprocally, with underlying cells (Birder & Andersson, 2013). The initial report of ATP release from the urothelium with stretch (Ferguson et al. 1997) demonstrated that the urothelium could respond to stimuli and secrete ATP. We have since learned that urinary bladder distension, under ‘normal’ and pathological conditions, induces urothelially derived ATP release with subsequent participation in autocrine and paracrine LUT signalling (Birder & Andersson, 2013). Urothelial cells and sensory neurons terminating in the suburothelial nerve plexus express purinergic receptors (e.g. P2X and P2Y families; Arms, 2009; Birder & Andersson, 2013). Plasticity in bladder ATP/purinergic signalling mechanisms may contribute to urinary bladder dysfunction including altered sensory processing in bladder pain syndrome (BPS)/interstitial cystitis (IC). Increased ATP expression in urine, enhanced urothelial ATP release with distension, and increased purinergic receptor expression on urothelial cells occur in BPS/IC patients (Birder & Andersson, 2013). Although urothelium releases ATP extracellularly, the mechanisms underlying release were, until recently, undefined (Birder & Andersson, 2013). In this issue of The Journal of Physiology, Beckel and colleagues (2015) report an elegant series of anatomical and functional studies in rat, dramatically advancing understanding of ATP release mechanisms from urinary bladder urothelium. Beckel et al. demonstrate that pannexin channels (i.e. pannexin 1), localized to all three layers of the rat urothelium, are responsible for luminal, but not basolateral, release of ATP following urinary bladder distension or bacterial lipopolysaccharide (LPS)-induced bladder inflammation. Pharmacological and molecular approaches designed to antagonize pannexin channels or to ‘knock down’ urothelial expression of pannexin 1 channels were used in combination with bladder function testing in rats to determine if endogenous ATP contributed to urinary bladder function. Importantly, intravesical (i.e. within the urinary bladder) or systemic instillations with pannexin channel antagonists (e.g. Brilliant Blue-FCF, BB-FCF; carbenoxolone, CBX) were used to distinguish between luminal and basolateral release of ATP. Intravesical instillation of pannexin channel antagonists produced concentration-dependent bladder reflex activity suppression and decreased basal and distension-induced ATP release. In contrast, intravenously delivered pannexin channel antagonists or gap junction blockers did not affect bladder reflex activity or basal or distension-induced ATP expression, providing strong evidence for a role for luminal ATP in micturition reflex modulation. To confirm and extend pharmacological approaches, knock down of urothelial pannexin 1 expression using intravesical instillation of a liposome-based delivery system of anti-Panx1 siRNA reduced voiding frequency and eliminated inhibition of bladder reflex activity by the pannexin channel antagonist BB-FCF. It is important to note that the study by Beckel et al. supports a role(s) for luminal ATP release in bladder reflex modulation in ‘normal’ bladder as well as in bacterial LPS-induced bladder inflammation. Inducing urinary bladder inflammation via intravesical instillation of bacterial LPS, Beckel and colleagues also demonstrated that intravesical infusion of pannexin channel antagonists significantly decreased basal and distension-induced ATP expression. There is much to be learned concerning urothelial ATP release, including mechanisms (non-pannexin mediated) underlying ATP release from the basolateral urothelial surface, identity and location of purinergic receptors that mediate purinergic signalling following pannexin-mediated ATP release, determining if pannexin-mediated ATP release is involved in pain (i.e. referred somatic hypersensitivity) in addition to effects on urinary bladder reflexes and the potential for plasticity in ATP release mechanisms with disease, injury, inflammation or during development (e.g. postnatal maturation, ageing). Beckel et al. have provided much needed insight into basic mechanisms underlying urothelial ATP release in ‘normal’ bladder. Further, the demonstration that pannexin channels play important roles in urothelial ATP release in inflamed urinary bladder makes pannexin channels novel targets for novel treatment development for urinary bladder dysfunction, especially that characterized by bladder overactivity.