Purinergic receptor mediated calcium signalling in urothelial cells

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.Russell Chess-Williams, Donna J. Sellers, Stuart M. Brierley, David Grundy, Luke Grund

Phasic contractions in urinary bladder from juvenile versus adult pigs

AIMS: Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs). METHODS: Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib. RESULTS: Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition. CONCLUSIONS: We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals