The regulation of ATP release from the urothelium by adenosine and transepithelial potential

Authors


Correspondence: Prof Christopher H. Fry, Institute of Biosciences and Medicine, University of Surrey, Guildford GU2 7XH, UK.

e-mail: c.h.fry@surrey.ac.uk

Abstract

What's known on the subject? and What does the study add?

  • Stretch of the urothelium, as occurs during bladder filling, is associated with a release of ATP that is postulated to act as a sensory neurotransmitter. The regulation of ATP release is poorly understood and in particular if there is a feedback mechanism provided by ATP itself.
  • Adenosine, a breakdown product of ATP, is a potent inhibitor of stretch-induced ATP release, acting through and A1 receptor; endogenous levels are about 0.6μM. Data are consistent with ATP release relying on the rise of intracellular Ca2+. Transepithelial potential also controls ATP release, also acting via an A1 receptor-dependent pathway.

Objectives

  • To test the hypothesis that distension-induced ATP release from the bladder urothelium is regulated by adenosine as well as changes to transurothelial potential (TEP).
  • To examine the role of changes to intracellular [Ca2+] in ATP release.

Materials and Methods

  • Rabbit urothelium/suburothelium membranes were used in an Ussing chamber system. Distension was induced by fluid removal from the chamber bathing the serosal (basolateral) membrane face.
  • The TEP and short-circuit current were measured. ATP was measured in samples aspirated from the serosal chamber by a luciferin–luciferase assay.
  • Intracellular [Ca2+] was measured in isolated urothelial cells using the fluorochrome Fura-2. All experiments were performed at 37°C.

Results

  • Distension-induced ATP release was decreased by adenosine (1–10 μm) and enhanced by adenosine deaminase and A1- (but not A2-) receptor antagonists.
  • Distension-induced ATP release was reduced by 2-APB, nifedipine and capsazepine; capsaicin induced ATP release in the absence of distension.
  • ATP and capsaicin, but not adenosine, generated intracellular Ca2+ transients; adenosine did not affect the ATP-generated Ca2+ transient.
  • ATP release was dependent on a finite transepithelial potential. Changes to TEP, in the absence of distension, generated ATP release that was in turn reduced by adenosine.

Conclusion

  • Adenosine exerts a powerful negative feedback control of ATP release from the urothelium via A1 receptor activation.
  • Distension-induced ATP release may be mediated by a rise of the intracellular [Ca2+].
  • Modulation of distension-induced ATP release by adenosine and TEP may have a common pathway.
Abbreviations
TEP

transepithelial potential difference

ENaC

epithelial Na+ channels

SCC

short circuit current

DPCMX

8-cyclopentyl-1,3-dimethylxanthine (A1 antagonist)

DPCPX

8-cyclopentyl-1,3-dipropyl-xanthine (A1 antagonist)

DMPX

3,7-dimethyl-1-propargylxanthine (A2 antagonist)

Introduction

The barrier function of the urothelium, to separate urine from the tissues of the bladder wall, is well understood. In addition, the urothelium is metabolically active and is believed to be crucial in the communication of intraluminal pressure changes upon bladder filling to underlying nervous afferents. Urothelial cells transport Na+ from the apical surface, facing the bladder lumen, to the basolateral (serosal) side [1] and contribute to the generation of a transepithelial potential difference (TEP). One route is through mechanosensitive Na+ channels, identified as amiloride-sensitive epithelial Na+ channels (ENaC). Expression of ENaC is plastic and is increased in human bladders with outlet obstruction [2]. In addition there is active endocytotic and exocytotic traffic at the apical membrane of urothelial cells [3-5] and the serosal face.

A crucial part of the bladder sensory system is the release of transmitters, including acetylcholine and ATP, from the basolateral surface of the urothelium [6, 7]. ATP is proposed ultimately to activate P2X2/3 receptors expressed on sensory nerves that lie in the suburothelium and even penetrate the urothelium [8, 9]. P2X3 receptor knockout mice exhibit decreased voiding frequency and increased bladder capacity with normal filling pressures [10]. Furthermore, several bladder pathologies, such as overactivity and bladder pain syndrome, are associated with increased ATP release [11-13]. After release, ATP is rapidly converted by endonucleotidases to ADP, AMP and adenosine [14], which limits its lifetime, a feature of many chemical transmitters.

A change to the hydrostatic pressure across the urothelium, to mimic urothelial distension on bladder filling, modulates both ENaC-dependent Na+ transport and TEP, as well as ATP release [7]. However, it is not known if these two phenomena are independent, although blockade of ENaC by amiloride, so reducing TEP, does modulate ATP release [7].

Urothelial cells express a range of receptors, including purinergic, adrenergic, muscarinic, transient receptor potential and substance P subtypes, but the functions they mediate are mostly unclear. ATP release has been reported from a number of cells through several possible routes, both Ca2+-dependent and independent [15, 16] and a potential regulatory pathway is via purinergic receptors. Furthermore, ATP has a positive feedback action on ATP release in several tissues, including the urothelium [12, 17, 18]. The role of adenosine and P1 receptors in regulating urothelial function is less well understood. Adenosine release from the urothelium is potentiated by its distension and over many minutes has been proposed to modulate exocytosis by increasing apical membrane surface area [19]. We hypothesize that adenosine also has a more acute effect to modulate distension-induced ATP release from the urothelium. We investigated the cellular pathways whereby adenosine may regulate ATP release and in particular the routes mediated by purinergic receptors.

Methods

New Zealand white rabbits (about 1 kg) of either sex were used. Animals were killed by intravenous injection of sodium pentobarbitone (200 mg/mL) into a superficial ear vein, according to UK Home Office requirements (UK Animals Act, 1986). The urinary bladder was immediately dissected out and washed in Krebs solution. For Ussing chamber experiments, bladders were cut in half from the urethra to the apex and stretched by a silk thread ‘guide rope’ at each corner. The urothelium was separated from the detrusor muscle by injecting Krebs solution between the two tissue planes and removing the muscle using dissecting scissors. For isolated cell experiments urothelium preparations were dissociated with a collagenase-based solution, as described previously [20]; spindle-shaped interstitial or smooth muscle cells were not used.

Superfusing (Krebs) solution contained (mM): NaCl, 123.9; KCl, 5.0; NaHCO3 26.0; CaCl2, 1.1; MgSO4, 1.3; KH2PO4, 1.4; glucose, 10; gassed with 5% CO2/95% O2, pH 7.4, 37°C. Adenosine; adenosine deaminase (calf intestinal mucosa; 1 U deaminates 1.0 μmol adenosine to inosine per minute at pH 7.5); A-317491 sodium salt (P2X2 and P2X2/3 antagonist); 8-cyclopentyl-1,3-dimethylxanthine (A1 antagonist, DPCMX); 8-cyclopentyl-1,3-dipropyl-xanthine (A1 antagonist, DPCPX); 3,7-dimethyl-1-propargylxanthine (A2 antagonist, DMPX); luciferin–luciferase assay mix and Krebs reagents were all supplied by Sigma (Poole, UK).

For the Ussing chamber experiments isolated urothelium halves were mounted in a Perspex Ussing chamber to form a diaphragm (diameter 0.5 mm) between the two halves of the chamber. The orientation of the urothelium as either serosal or apical (bladder lumen) face was noted. Each half chamber (20 mL) was a circulating reservoir of Krebs solution maintained at 37°C by means of a thermostatted water jacket; this permitted the addition of drugs selectively to either side of the urothelium. Distension of the urothelium membrane was achieved by removal of 2 mL from the serosal reservoir, except when specifically described.

Samples of 100 μL bathing fluid, to assay ATP content, were taken from the serosal side of the bladder using a Hamilton syringe via a cannula positioned in the chamber directly adjacent to the urothelium. Samples were taken immediately before distension and at different times afterwards, and assayed using the luciferin–luciferase method (Sigma) with a Turner TD-20e luminometer. Calibration curves were constructed daily using ATP standard (Sigma) dissolved in Krebs solution at constant pH.

To measure TEP and short circuit current (SCC) the potential difference between apical and serosal surfaces of the urothelium, the TEP (apical with reference to the serosal surface), and the current required to clamp the TEP to 0 mV, the SCC, were recorded. TEP and SCC were measured using a DVC-1000 Dual Voltage Clamp (World Precision Instruments, Sarasota, FL, USA). Each side of the preparation was connected to matched calomel electrodes (type SR4, Russell pH Ltd, Auchtermuchty, UK) via 3 m KCl-agar bridges, positioned in the bathing solution close to the preparation. SCC was measured via Ag/AgCl electrodes, in KCl-agar bridges, placed also in the bathing solution in each reservoir. The SCC represents net Na+ transmembrane transport, which flows from the apical to the serosal side [1].

Intracellular [Ca2+], [Ca2+]i was recorded by Fura-2 epifluorescence microscopy, after incubation with the fluorochrome (5 μm) for 20–30 min at room temperature [20]. Cells were excited alternately at 340 and 380 nm at 50 Hz and fluorescence intensity was recorded between 410 and 480 nm. The Fura-2 signal was calibrated using solutions of varying [Ca2+] in the absence of cells, yielding a Kd of 224 nM and β-factor of 13.1 [20].

ATP levels and electrophysiology data are presented as median values [25, 75% interquartiles] of measured values, or percentage changes from baseline values before distension or addition of interventions. Comparison between data sets used paired, non-parametric Wilcoxon signed rank tests and the null hypothesis was rejected when P < 0.05. The inhibitory effect of adenosine on ATP release was fitted to inline image; where maximum release (= 100%) is that in adenosine deaminase; [I] is the inhibitor (adenosine) concentration; and EC50 is the [I] reducing ATP release by 50%.

Results

Baseline [ATP] after equilibration and before experiments was 0.15 [0.10, 0.26] nm (n = 99). A change of the transmural pressure gradient caused bulging of the membrane into the chamber from which fluid was removed; referred to henceforth as distension. An increase of ATP release from the basolateral urothelial surface was measured after distension. When ATP was sampled at 30-s intervals after distension there was a progressive increase between 30 and 60 s, which thereafter stabilized to about twice the pre-distension level (=100%) (Fig. 1A). Subsequent data are reported 3 min after initiation of distension as a percentage of the pre-distension (baseline) value: the percentage increase of [ATP] was independent of the baseline concentration (r = −0.07, P > 0.05). The percentage increase of [ATP] had a non-normal distribution (Fig. 1B) so values are quoted as medians [25, 75% interquartiles], with an increase to 219% [147, 307], n = 99. With interventional experiments ATP release was measured after two distensions at 30-min intervals, one in control and a second in a test solution. The reproducibility of successive releases was tested with two distensions in control solution: respective values were 197% [150, 247] and 190% [160, 245]; n = 7, P > 0.05.

Figure 1.

Distension-induced ATP release. A, Time-course of ATP release after distension (n = 34); *P < 0.05 two-tailed Wilcoxon rank test vs pre-stretch (zero seconds); P < 0.05 vs 60 s and longer times. B, A histogram of the magnitude of ATP release after hydrostatic stretch. The pre-stretch ATP level is indicated as 100% in both panels.

Adenosine was added to the basolateral fluid 20 min before distension. ATP release was significantly attenuated by 1 and 2 μm adenosine and completely abolished at 10 μm (Fig. 2A) (n = 10). In separate experiments adenosine deaminase (n = 6) was added to remove endogenous adenosine and distension-induced ATP release was now significantly greater than in control solution.

Figure 2.

Effect of adenosine on distension-induced ATP release. A, Effect of 1, 2 and 10 μm adenosine and adenosine deaminase. The dotted line in this and subsequent similar plots shows the baseline level of ATP (= 100%). Control represents the increase of ATP release upon distension in Krebs solution; *P < 0.05 vs control. B, The relationship between adenosine concentration and reduction of distension-induced ATP release; the magnitude of ATP release in the presence of adenosine deaminase is scaled as 100%. The double-arrowed datum point is that in control solution with no added adenosine or adenosine deaminase, see text for details.

Increased ATP release with adenosine deaminase implies a significant local adenosine level near the tissue in control solution. Figure 2B plots ATP release as a percentage of that in adenosine deaminase. The line (see Methods section) was fitted to median values in various adenosine concentrations, assuming different background adenosine levels from 0.1 to 1.0 μm in 0.01-μm increments. The background value chosen (0.59 μm) was that which gave a best-fit by maximizing the correlation coefficient; the EC50 value was 0.69 μm. The datum point in control solution (i.e. no added adenosine), with this background amount, is labelled with a double arrowhead.

Both A1- and A2-subtype adenosine receptors are present on the urothelium [19]. The effects of selective adenosine receptor antagonists (all at 1 μm) on distension-induced ATP release were examined. Figure 3A shows that two different A1 receptor antagonists, DPCMX (n = 6, left) and DPCPX (n = 6, middle) significantly increased distension-induced ATP release. By contrast the A2 receptor antagonist DMPX (n = 6, right) had no significant effect.

Figure 3.

The effects of A1 and P2X receptor antagonists on distension-induced ATP release. A, The effect of the A1 receptor antagonists DPCMX and DPCPX (1 μm), and the A2 receptor antagonist DMPX (1 μm). B, The effect of the P2X3/P2X2/3 receptor antagonist A-317491 (10 μm, n = 9). *P < 0.05 vs control. DPCMX, 8-cyclopentyl-1,3-dimethylxanthine; DPCPX, 8-cyclopentyl-1,3-dipropyl-xanthine; DMPX, 3,7-dimethyl-1-propargylxanthine.

Exogenous ATP stimulates further ATP release from urothelial cell cultures [12]. The use of many purinoceptor agonists and antagonists to probe the receptor subtypes that mediate the effect is hampered by their interference with the luminescent ATP assay, and other methods to measure ATP release lacked the required sensitivity. The non-nucleotide P2X3/P2X2/3 receptor antagonist A-317491 [21] offered a means to overcome these problems. Figure 3B shows that A-317491 (10 μm, n = 9) significantly reduced distension-induced ATP release.

Cellular pathways mediating the effect of adenosine were investigated. A1 receptors mediate several signalling pathways and several interventions were used to provide evidence about the important pathways regulating ATP release. Figure 4A shows that 2-APB (75 μm, n = 6), an inhibitor of both store-operated Ca2+ entry and the IP3 receptor attenuated distension-induced ATP release, as did 10 μm nifedipine (n = 6); ethanol (60 μm, n = 4), the vehicle for 2-APB, was without effect. Hence, distension-induced ATP release may be dependent on intracellular Ca2+. The TRPV1 channel activator capsaicin (10 μm), which evokes Ca2+ influx, itself induced ATP release, in turn blocked by the TRPV1-inhibitor capsazepine (10 μm) (Fig. 4B; n = 6). In addition, capsazepine significantly reduced capsaicin-induced ATP release (Fig. 4C; n = 6). Forskolin, an agent expected to increase cAMP, also abolished the distension-induced release of ATP: control 191% [132, 210]; forskolin 127% [114, 177], P < 0.05, n = 6.

Figure 4.

The effect of different modulators on ATP release. A, The action of ethanol (60 μm), 2-APB (75 μm) and nifedipine (10 μm) on distension-induced ATP release; *P < 0.05 vs control. B, The effect of capsaicin (10 μm) in the absence and presence of capsazepine (10 μm) on ATP release in the absence of distension; *P < 0.05 vs capsaicin. C, The effect of capsazepine (10 μm) on distension-induced ATP release; *P < 0.05 vs control.

Intracellular [Ca2+] was measured in the presence of ATP, adenosine and capsaicin (Fig. 5). The resting [Ca2+] was 141 nm [96, 151] (n = 21). ATP (10 μm) caused a rapid and large intracellular Ca2+-transient (part A) with a mean Δ[Ca2+] of 310 nm [246, 391] (n = 16). By contrast, 10 μm adenosine had no consistent effect, or in some instances only a very small effect (part B), Δ[Ca2+] 0 nm [0, 72] (n = 5). Furthermore, adenosine had no significant effect on the magnitude of the ATP Ca2+-transient, 122% [80, 156] (n = 10) control. Finally, 10 μm capsaicin generated a significant Ca2+-transient (part C), Δ[Ca2+] 60 nm [55, 115] (n = 5).

Figure 5.

Intracellular [Ca2+] of isolated urothelial cells. The action of ATP (left), adenosine (middle) and capsaicin (right) in separate urothelial cells.

The relationship between transepithelial electrophysiology and distension-induced ATP release was investigated. The measured ranges of TEP and SCC values, in the absence of distension, were 0.6 to 6.0 mV and 2.0–15.0 μA/cm2 respectively. Distension-induced release of ATP was abolished when TEP was clamped to 0 mV (Fig. 6A, n = 9). Conversely, increasing TEP to –10 mV, in the absence of distension, increased ATP release to 137% [120, 152] (n = 11) of baseline, although the absolute magnitude of this increase was significantly less than distension-induced release. The fact that either distension or an increase of TEP can increase ATP release suggests a close relationship between the two effectors. This potential inter-relationship was further examined by testing the effect of adenosine receptor modulators on: (i) the electrophysiological properties of the urothelium; and (ii) the augmented ATP release induced by increasing TEP.

Figure 6.

ATP release and mucosal electrophysiological properties. A, The influence of transepithelial potential on distension-induced ATP release; *P < 0.05 vs open-circuit transepithelial potential difference (TEP). B, The rate of change of short circuit current (ΔSCC) on distension in control solution, or in the presence of adenosine deaminase or the A1 receptor antagonists DPCPX; *P < 0.5 vs control. C, The increase of ATP release on stepping the TEP to –10 mV, in the absence of distension, in control solution or in the presence of 1 μm adenosine or 1 μm DPCPX; *P < 0.5 vs control. DPCPX, 8-cyclopentyl-1,3-dipropyl-xanthine.

The magnitude of the SCC is an estimate of ion transport across the urothelium and when ATP is released during serosal distension the value is increased [7]. In these experiments the rate of change of SCC (ΔSCC) on distension was used as a more sensitive measure. Figure 6B shows that the ΔSCC induced by distension was augmented both by adenosine deaminase (n = 4) and the A1-receptor antagonist DPCPX (1 μm, n = 16), conditions that also augmented ATP release (Figs 2, 3A). There was no effect of the A2-receptor antagonist DMPX (1 μm), which also had no effect on distension-induced ATP release.

Figure 6C shows how adenosine (1 μm, n = 7) or the A1-receptor antagonist DPCPX (1 μm, n = 6) altered ATP release when TEP was clamped to –10 mV, in the absence of distension. In control solution, ATP release increased to 116% [106, 133] of that before TEP was altered: adenosine reversed this increase while DPCPX further increased it, both effects similar to those on distension-induced ATP release. These data are consistent with the hypotheses that: (i) adenosine, through activation of A1 receptors, reduces ATP release by attenuation of transepithelial ion transport; and (ii) there are sufficient endogenous quantities of adenosine to modulate ATP release.

Discussion

This study addressed several aspects of urothelial ATP release induced by distension: whether adenosine receptor activation modulated ATP release; the role of changes to [Ca2+]i; and the influence of changes to transepithelial potential.

Adenosine was a potent inhibitor of distension-induced ATP release, abolishing it completely at a concentration of 10 μm. Conversely, removal of endogenous adenosine with adenosine deaminase approximately doubled this fraction of ATP release. Assuming that adenosine deaminase completely removed adenosine, this implies an endogenous level of adenosine surrounding the tissue, estimated at about 0.6 μm.

Because ATP exerts a positive feedback effect on urothelial ATP [12], adenosine may enhance endogenous endonucleotidase activity; however, there is no evidence for such an action [22]. Moreover the depressant action of adenosine was receptor-subtype specific, suggesting a direct action on cellular pathways. This study showed that A1, but not A2, receptor antagonism enhanced distension-induced ATP release.

The role of intracellular Ca2+ in distension-induced ATP release was described. A role for a rise of [Ca2+]i in ATP release is consistent with the fact that both ATP and capsaicin increased [Ca2+]i, and capsaicin alone evoked ATP release (see also [23]) – the latter effect blocked by the TRPV1 channel inhibitor capsazepine. In addition, distension-induced ATP release was attenuated by a P2X3/P2X2/3 receptor antagonist, the Ca2+-channel blocker nifedipine or 2-APB, a blocker of store-operated Ca2+ influx and inhibitor of the IP3 receptor [24]. Finally, previous work showed that distension of urothelial cells with hypotonic solutions also increased intracellular [Ca2+] [25].

A1 receptor occupation by adenosine could activate several cellular pathways including: inhibition of adenylate cyclase and hence reduction of cAMP [26]; increased IP3 production and a rise of [Ca2+]i [27, 28]; inhibition of Ca2+ channels [29-31] or activation of K+ channels [32, 33]. There was no consistent evidence in these cells that adenosine raised intracellular [Ca2+]i and the fact that forskolin actually decreased distension-induced ATP release could rule out a role for reduced adenylate cyclase activity. The latter observation might be explained by forskolin depleting cellular ATP for release, through enhanced production of cAMP. We propose that adenosine reduces urothelial ATP release through reducing cellular Ca2+ levels via an action on ion channels, but this requires further electrophysiological experiments.

Urothelial distension increases both ATP release and TEP, but a causal link has not yet been proved, although amiloride modulates both phenomena [7]. This study provided evidence for such a link as ATP release required a finite TEP, and changing TEP in the absence of distension also released ATP. This interdependence between TEP and hydrostatic pressure-induced ATP release may explain the variability of the percentage increase of ATP (see Fig. 1B) as TEP will vary between different preparations. However, this did not affect results reported here because the effect of an intervention on ATP release was always preceded by a control measurement, permitting paired observations in each preparation.

The TEP-dependent ATP release was in turn modulated by adenosine and an A1-receptor antagonist. TEP is determined by ion-transport through an epithelial Na+ channel and the question remains how ion fluxes across the apical surface of the urothelium affect serosal ATP release? One possibility is that the increase of intracellular Na+ generates a rise of intracellular Ca2+ via Na+/Ca2+ exchange, as described in isolated urothelial cells [25]. Hence, we propose that distension-induced ATP release is dependent on ion transport maintaining an adequate level of intracellular Ca2+ that in turn may be regulated by A1-receptors.

Acknowledgements

We thank Pfizer and the EU (FP7 INComb) for financial assistance.

Conflict of Interest

Christopher H. Fry is a Paid Consultant to Eli Lilly.

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