Purinergic receptor mediated calcium signalling in urothelial cells

Abstract

Non-neuronal ATP released from the urothelium in response to bladder stretch is a key modulator of bladder mechanosensation. Whilst non-neuronal ATP acts on the underlying bladder afferent nerves to facilitate sensation, there is also the potential for ATP to act in an autocrine manner, modulating urothelial cell function. The aim of this study was to systematically characterise the functional response of primary mouse urothelial cells (PMUCs) to ATP. PMUCs isolated from male mice (14–16 weeks) were used for live-cell fluorescent calcium imaging and qRT-PCR to determine the expression profile of P2X and P2Y receptors. The majority of PMUCs (74–92%) responded to ATP (1 μM–1 mM), as indicted by an increase in intracellular calcium (iCa2+). PMUCs exhibited dose-dependent responses to ATP (10 nM–1 mM) in both calcium containing (2 mM, EC50 = 3.49 ± 0.77 μM) or calcium free (0 mM, EC50 = 9.5 ± 1.5 μM) buffers. However, maximum iCa2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X1–6, and P2Y1–2, P2Y4, P2Y6, P2Y11–14, but not P2X7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.

Histamine induces peripheral and central hypersensitivity to bladder distension via the histamine H1receptor and TRPV1

“…Primary urothelial cells were isolated and cultured from mice as previously described (11,28,30). After euthanasia by CO2 inhalation, bladders were removed and dissected in sterile PBS with a single cut from the urethral opening along one side to the dome, pinned and stretched out urothelial side up, and incubated with DMEM containing 2.5 mg/mL Dispase, 10% FCS, and 0.7% HEPES (1 M, pH 7.0, H3375, Sigma) for 3 h at room temperature.…”

Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder

“…P2X-purinoceptors have also been detected in the urothelium of many species including man, however some species differences appears to exist (Birder & Andersson, 2013;Elneil et al, 2001;Shabir et al, 2013). For instance, all seven P2X-purinoceptors have been found in the cat urothelium, while a recent study of primary mouse urothelial cells shows the expression of the P2X1-P2X6purinoceptors but not P2X7 (Birder et al, 2004;Chess-Williams et al, 2019). However, P2X7 has been shown in the urothelium of rats (Lee et al, 2000).…”

BoNT/A in the Urinary Bladder—More to the Story than Silencing of Cholinergic Nerves

“…BoNT/A could be exerting an action on the sensory nerves via an indirect mechanism involving the bladder urothelium. Neuron-like properties of the urothelium were identified after a series of studies discovered the release of neurotransmitters from the urothelium [26,27,30], as well as expression of various receptors for those neurotransmitters [88][89][90][91][92]. Contrary to neuronal ACh release, which occurs through SNAP-25 mediated exocytosis, ACh release from the urothelium is mediated by organic cationic transporters [93].…”