TRPV1 enhances the afferent response to P2X receptor activation in the mouse urinary bladder

Abstract

Both TRPV1 and P2X receptors present on bladder sensory nerve fibres have been implicated in mechanosensation during bladder filling. The aim of this study was to determine possible interactions between these receptors in modulating afferent nerve activity. In wildtype (TRPV1 +/+ ) and TRPV1 knockout (TRPV1 −/− ) mice, bladder afferent nerve activity, intravesical pressure, and luminal ATP and acetylcholine levels were determined and also intracellular calcium responses of dissociated pelvic DRG neurones and primary mouse urothelial cells (PMUCs). Bladder afferent nerve responses to the purinergic agonist αβMethylene-ATP were depressed in TRPV1 As the bladder fills, the degree of distension is detected by mechanosensitive ion channels located either on sensory nerve terminals or on the specialised epithelial lining of the bladder, the urothelium. This evokes an afferent signal that is conveyed to the CNS via the dorsal root ganglia. Initially this signal lies below the level of consciousness, supplying the autonomic reflexes responsible for controlled bladder filling. As bladder volume increases, however, a sensation of fullness and a desire to void is perceived. In continent individuals this desire can be deferred until it is convenient to empty the bladder, however in patients with lower urinary tract conditions such as overactive bladder syndrome (OAB), bladder pain syndrome (BPS) or interstitial cystitis (IC), this sensation is difficult to defer, occurs when the bladder is not yet full and can even be accompanied by pain (BPS/IC) suggesting that a defect in the ability of the bladder to detect filling may underlie these conditions. Sensory innervation from the bladder is conveyed by myelinated low threshold Aδ fibres and unmyelinated high threshold C fibres carried in the pelvic, hypogastric and pudendal afferent nerves 1 . However, the complex mechanisms which determine how the afferent nerves detect bladder filling remain poorly understood, limiting the development of effective treatments for these bladder sensory defects.There is compelling evidence that purinergic receptors play an active role in mechanosensation of the bladder in rodents. Firstly bladder projecting afferents express a number of P2X puringeric receptors, of which P2X2 and P2X3 are the main subtype. Secondly, ATP is able to activate pelvic nerve afferents arising from the rat urinary bladder and initiate bladder overactivity 2,3 . Thirdly, the P2X1 and P2X3 agonist, αβMethylene-ATP increases firing in both nociceptive and non-nociceptive populations of bladder afferents 4 . Finally, it has been shown that stretch or distension of the urothelium evokes the graded release of non-neuronal ATP 5 which activates purinergic receptors located on the afferent terminal to control afferent firing. Previous studies using P2X3 or P2X2/3 null mice found profound attenuation of the afferent response to bladder filling, increased bladder capacity and decreased voiding frequency, supporting a role for ATP in bladder sensation 5,6 . Purinergic receptors ...

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“…Ex vivo electrophysiology for bladder afferent recordings and contractility studies. Whole pelvic nerve recordings were conducted in response to ramp distension (30 μl/min to 30 mmHg) (37,43,44). Studies also determined the responsiveness of bladder afferents to intravesically applied linaclotide or αβMe-ATP, carbachol, or capsaicin.…”

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“…Mice were maintained under anaesthetic for 10 minutes with either the protamine sulfate or vehicle held within the bladder. After 10 minutes, the bladder was drained via the syringe, the syringe was removed and the mice were allowed to recover, housed individually, and monitored twice daily for two days or until mice were humanely killed (1, 7, or 28 days post procedure) for use in bladder afferent recordings or in the myeloperoxidase assay (Grundy et al, 2018c).…”

“…Bladders isolated from mice immediately following humane euthanasia with CO 2 were sectioned longitudinally, rinsed in ice-cold PBS, and snap frozen in liquid nitrogen (Grundy et al, 2018c). Tissue was defrosted on ice, weighed, and put in 1.5 ml HTAB buffer (0.5% Hexadecyltrimethylammonium bromide, pH6): 2.5 g HTAB (H5882, Sigma, Australia) added to 500 ml of 50 mM PBS, pH6).…”

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“…Transient receptor potential vanilloid subfamily member 1 (TRPV1) and P2X receptors co‐expressed in bladder sensory nerves and implicated in mechanosensation during bladder filling. P2X3 receptor activation evoked bladder afferent activity in the mouse urinary bladder enhanced by TRPV1 (Grundy et al, ).…”