Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions

Abstract

The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca²⁺ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M₂-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M₂-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca²⁺ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies.

Keywords: ATP release; Urothelium; muscarinic; neurotransmitters; paracrine effect; purinergic; sensory.

Fig. 1.

Spontaneous surges of ATP release in guinea pig urothelial tissue. A: record of ATP release showing a spontaneous increase in urothelial mucosa. B: averaged data from 20 bladders. Control values were taken as 100%.

Fig. 2.

Effect of purinergic agonists and muscarinic agents on ATP release from guinea pig urothelial mucosa. A: the P2Y agonist UTP (100 μM) increased ATP release. n = 11 bladders. B: the UTP-augmented ATP release persisted in the presence of ARL67156 (100 μM). n = 6 bladders. C: no effect was seen with the P2X agonist α,β-methylene ATP (ABMA; 10 μM). n = 6 bladders. D: carbachol (50 μM) induced ATP release from the guinea pig urothelium. E: oxotremorine (50 μM) generated ATP release. F: effect of muscarinic antagonists on carbachol-induced ATP release [atropine (1 μM), methoctramine (100 nM), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 10 nM)]. The control represents the level of ATP release in the presence of antagonist alone for each intervention and was taken as 100%.

Fig. 3.

Contractile properties of guinea pig urothelial mucosa. A: record of spontaneous contractile activity of a guinea pig bladder mucosal preparation. B: carbachol (50 μM) generated mucosal contraction. Note that carbachol-induced contracture on top of the spontaneous contractions. C: averaged values of mucosal contractions. n, number of bladders. D: relationship between carbachol-induced contraction and ATP release in guinea pig urothelial mucosa. Left, concomitant mucosal contraction and increase of ATP release (●) induced by carbachol (50 μM). ATP was sampled at various time points before and after carbachol application. Right, correlation between the two variables from the experiment.

Fig. 4.

Differential actions of UTP and carbachol on mucosal contraction and intracellular Ca²⁺ concentration ([Ca²⁺]_i). A: changes to urothelial mucosa contraction and ATP release during UTP intervention: UTP (100 μM) generated significant ATP release with only a small effect on contractility, in contrast to carbachol (Fig. 3). B: effect of UTP on intracellular Ca²⁺. i, UTP (100 μM) generated a Ca²⁺ transient in a urothelial cell; ii, averaged data from 8 bladders. C: lack of effect on intracellular Ca²⁺ by carbachol (100 μM). D: comparison of UTP- and carbachol-induced mucosal contractions. Values are averaged data. n, number of bladders. Records are from guinea pig preparations.

Fig. 5.

Mechanical stretch-induced ATP release from guinea pig urothelial mucosa. A: record of ATP release after stretch (20% increase of original length). B: averaged data from 11 bladders. The level of ATP release from the control (without stretch) was taken as 100%. C and D: effect of hypotonic solution on ATP release in guinea pig urothelial mucosa. C: example of urothelial ATP release after challenge with HEPES-buffered Tyrode's solution with tonicity reduced to 70%. D: averaged data from 9 bladders. Control values without hypotonic insult were taken as 100%.

Fig. 6.

Specific pathways for intrinsic and agonist-evoked ATP release in urothelial mucosa. A: conductive and vesicular pathways for intrinsic ATP release. There was a moderate inhibition on intrinsic basal ATP release with the gap junction blocker sodium carbenoxolone (Carbenx; 100 μM), a major suppression of intrinsic ATP release with the vesicular transport inhibitor brefeldin A (Brefld; 10 μM) and an additive effect of both agents. *P < 0.05 and **P < 0.01 vs. control. B: Ca²⁺-dependent pathways for UTP-induced ATP release. i, Removal of extracellular Ca²⁺ did not suppress UTP-augmented ATP release; ii, reducing intracellular Ca²⁺ with BAPTA-AM (10 μM) specifically inhibited UTP-evoked ATP release. C: specific pathways for carbachol-induced ATP release. i, Absence of the excitatory effect of carbachol on ATP release in the presence of the adenylyl cyclase inhibitor SQ-22536 (100 μM); ii, the effect of carbachol was also suppressed by brefeldin A (10 μM).

Fig. 7.

Intrinsic and low concentration carbachol-induced contractile activity in urothelium-intact guinea pig detrusor muscle: involvement of paracrine action. A: urothelium dependence of the intrinsic contractile activity of detrusor muscle. Shown is a record of spontaneous activity in a mucosa-intact detrusor muscle strip; the removal of mucosa diminished the activity. B: record of the experiment showing the attenuating effect of ABMA on urothelium-dependent spontaneous activity. C: there was little effect of AMBA on intracellular Ca²⁺ in a urothelial cell. D: averaged data for ABMA intervention on the contractile activity from 12 bladders. E–G: augmentation of spontaneous contractions in mucosa-attached detrusor smooth muscle by a subthreshold concentration of carbachol and the effect of ABMA. E: record of spontaneous contractions in a urothelium-intact detrusor strip showing an enhanced effect by a low concentration of carbachol and the attenuation of such augmentation after ABMA treatment. F: averaged data for the carbachol effect. n = 7 bladders. G: averaged data for the ABMA effect on carbachol-augmented muscle contractions. n = 6 bladders.

Fig. 8.

Intrinsic ATP release in urothelial mucosa from guinea pig urinary bladders. A: average levels of ATP release in young (2–3 mo) and aging (16–18 mo) bladders. n, number of animals. B: histogram of ATP release (log-transformed values) from both groups showing the greater proportion of aging bladders at the higher level end of the distribution.

Fig. 9.

Characteristics of human urothelium preparations. A: spontaneous rises of ATP release. Data are from 6 subjects. B: neurotransmitter-induced ATP release. i, UTP (100 μM)-induced release; ii, carbachol (50 μM)-induced ATP release. n, number of subjects. C: contractile properties of human bladder mucosa. i, Spontaneous contractions; ii, carbachol (50 μM)-induced response. D: mucosal contraction and ATP release. Shown is the time course of contractile activity and ATP release in response to carbachol (50 μM) intervention in a human bladder mucosa.