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 (iCa 2+ ). PMUCs exhibited dose-dependent responses to ATP (10 nM–1 mM) in both calcium containing (2 mM, EC 50 = 3.49 ± 0.77 μM) or calcium free (0 mM, EC 50 = 9.5 ± 1.5 μM) buffers. However, maximum iCa 2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X 1–6 , and P2Y 1–2 , P2Y 4 , P2Y 6 , P2Y 11–14 , but not P2X 7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa 2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.