Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

Abstract

Background and purpose: Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood.

Experimental approach: We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [(3) H]-ACh overflow experiments.

Key results: Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [(3) H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid.

Conclusions and implications: Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors.

Keywords: ATP release; P2Y6 receptor; UDP; acetylcholine release; anaesthetized rat; micturition reflex; urinary bladder; urothelium.

Figure 1

(A) Bladder cystometry recordings during normal saline (0.9%·w/v of NaCl) infusion into the urinary bladder of urethane-anaesthetized rats: comparison of the effects of UDP (100 μM) and PSB0474 (100 nM) in the absence and in the presence of the selective P2Y₆ receptor antagonist, MRS2578 (50 nM). Large-amplitude rhythmic bladder contractions correspond to voiding contractions when they were accompanied by urine draining through the urethra. The inset shows the urodynamic parameters evaluated: ICI (min), PTh (cm of H₂O), amplitude (A, cm of H₂O) and duration (Δt, s) of the voiding contractions. Stable urodynamic responses to UDP and PSB0474 were reached in 10–15 min. Co-application of MRS2578 with the agonists preceded urodynamic measurements by at least 20 min. (B) Scatter plots representing the percentage change of the ICI and of the duration (Δt) of voiding contractions as compared with control values (Ctr, 0%). The vertical bars represent SEM of a n number of animals (shown in parenthesis). P values as shown; significantly different from control samples (saline infusion) or from the effects of UDP or PSB0474, applied alone; unpaired Student's t-test with Welch's correction.

Figure 2

Concentration-dependent inhibition of the voiding frequency by the P2Y₆ receptor antagonist, MRS2578, during normal saline (0.9%·w/v of NaCl) infusion into the urinary bladder of urethane-anaesthetized rats. MRS2578 (50–300 nM) was applied in a cumulative manner into the bladder lumen by changing the content of the syringe connected to the automated perfusion system. Stable urodynamic responses to MRS2578 were reached in 10–15 min.

Figure 3

Modification of UDP (100 μM)-induced increase in the voiding frequency by i.v. application of A317491 (100 nM, a selective P2X3 receptor antagonist) and MRS2179 (0.3 μM, a P2Y₁ receptor antagonist) in the anaesthetized rat. (A) Bladder cystometry recordings during normal saline (0.9%·w/v of NaCl) infusion into the urinary bladder in the absence and in the presence of UDP (100 μM). A317491 (100 nM) and MRS2179 (0.3 μM) were continuously perfused through the catheter inserted into the left jugular vein starting at least 20 min before UDP application. (B) Scatter plots representing the percent change in the ICI and of the duration (Δt) of voiding contractions, compared with control values (Ctr, 0%). The vertical bars represent SEM of a n number of animals (shown in parenthesis). P values as shown; significantly different from UDP alone; unpaired Student's t-test with Welch's correction.

Figure 4

Immunolocalization of P2Y₆ receptors in the uroepithelium and sub-urothelial layers of transverse sections of the rat urinary bladder by confocal microscopy. Two distinct P2Y₆ receptor antibodies, APR-011 and ABIN1386282, were used as indicated. Terminally differentiated urothelial cells (umbrella cells) and sub-urothelial myofibroblasts are labelled with cytokeratin 20 (CK20, red, in panel A) and vimentin (Vim, red, in panel B) respectively. Nuclei are stained with DAPI (blue). Yellow staining denotes co-localization of P2Y₆ receptor (green) with CK20 (red) or Vim (red). Pre-adsorption with the peptide corresponding to the amino acid sequence 311–328 of the rat P2Y₆ receptor abolished staining with the APR-011 antibody. Differential interference contrast (DIC) image is shown for comparison in the latter condition. Scale bar = 50 μm.

Figure 5

Immunolocalization of P2X3 and P2Y₁ purinoceptors in the uroepithelium and sub-urothelial layer of transverse sections of the rat urinary bladder by confocal microscopy. Both, P2X3 and P2Y₁, receptor subtypes are expressed throughout the bladder epithelium. P2X3, but not P2Y₁, receptors (green) co-localize (yellow staining) with NF160 (red) labelling in small sub-urothelial nerve fibres. Nuclei are stained with DAPI (blue). Scale bars = 20 or 50 μm (as indicated).

Figure 6

(A) Setup for myographic recordings of the whole urinary bladder of the rat in vitro. (B) Spontaneous contractile activity of the rat urinary bladder in response to bladder filling with Tyrode's solution, up to 150 μL, infused at a constant flow rate (40 μL·min⁻¹) to mimic in vivo cystometry experiments. UDP (300 μM) was superfused either into the bladder lumen (by changing the syringe connected to the automated perfusion system) or directly to the bathing solution outside the bladder. (C) Quantification of the frequency and magnitude of spontaneous contractions of the whole bladder in vitro in the absence and in the presence of UDP (300 μM) applied inside and outside the bladder. The vertical bars represent SEM of four isolated bladders. *P < 0.05; significantly different from control (saline superfusion); one-way anova followed by Dunnett's modified t-test. (D) Confocal micrographs of transverse sections of rat urinary bladder detrusor muscle immunostained for P2Y₆ (APR-011) and P2X1 (APR-001) receptors. Scale bars = 50 μm.

Figure 7

(A) Comparison of bladder cystometry recordings obtained during infusion of ARL 67156 (100 μM, an E-NTPDase inhibitor) and PSB0474 (100 nM, a selective P2Y₆ receptor agonist) into the urinary bladder lumen of anaesthetized rats. (B) Intravesical infusion of PSB0474 (100 nM) increases ATP levels in the urinary fluid collected during cystometry recordings. The ATP content of the samples was quantified by the luciferin-luciferase bioluminescence assay. The vertical bars represent SEM of five animals. *P < 0.05; significantly different from control (saline infusion); one-way anova followed by Dunnett's modified t-test.

Figure 8

(A) Effect of UDP (100 μM) on electrically-evoked [³H]-ACh release from intact urinary bladder strips and in preparations without the urothelium UDP (100 μM) was applied 8 min before S₂. MRS2578 (50 nM) and MRS2179 (300 nM) were added to the incubation media at the beginning of the release period (time zero) and were present throughout the assay, including S₁ and S₂. The ordinates represent evoked tritium outflow expressed by S₂/S₁ ratios, i.e. the ratio between the evoked [³H]-ACh release during the second period of stimulation (in the presence of UDP) and the evoked [³H]-ACh release during the first stimulation period (without UDP). The vertical bars represent SEM. *P < 0.05; significantly different from control; #P < 0.05: significantly different from UDP alone; unpaired Student's t-test with Welch's correction. (B) Representative microscopic images of rat urinary bladder strips stained with haematoxylin-eosin to confirm the presence or the absence of the urothelium. Magnification, 40×.

Figure 9

Confocal micrographs showing P2Y₆, P2Y₁ and NTPDase2 immunoreactivity in transverse sections of the detrusor smooth layer of rat urinary bladder. To facilitate visualization of small cholinergic nerve terminals staining for VAChT (red) images correspond to the intensity projections over Z axis of five to six confocal microscopy stacks taken at the smooth muscle layer. No co-localization was found between P2Y₆ receptor (green) and VAChT (red) immunoreactivity. Conversely, VAChT-positive cholinergic nerve terminals (red) stained positively with antibodies against the P2Y₁ receptor and E-NTPDase2 (green); yellow staining denotes co-localization. Nucleic DNA is stained with DAPI (blue). Scale bars = 50 μm.

Figure 10

Histochemical E-NTPDase activity in the rat urinary bladder. Phosphate deposition resulting from extracellular catabolism of ATP (30 μM, A) was found predominantly in the urothelium (arrows), but was also present in the sub-urothelial (square) and smooth muscle (star) layers: Extracellular ADP (30 μM, B) was dephosphorylated predominantly in apical urothelial cells (arrows) and in the smooth muscle (stars), but only at very low levels in the sub-urothelial layer (square). These details are better appreciated in the higher magnification (1.5×) images in the lower panels. Scale bars = 150 μm.

Figure 11

Schematic representation of the putative mechanisms underlying the control of the voiding frequency by urothelial UDP-sensitive P2Y₆ receptors in the anaesthetized rat. Activation of P2Y₆ receptors on distended umbrella cells during bladder filling increased, by threefold, the release of ATP from the urothelium. Released ATP, acting via multiple urothelial P2 purinoceptors, triggers a self-regenerating purinergic wave propagating to sensory nerve afferents endowed with P2X3 receptors to initiate the voiding reflex. Bladder activity may be partly reversed by the hydrolysis of ATP into ADP by E-NTPDases, namely E-NTPDase2 located in the lamina propria (probably on interstitial cells) and on cholinergic nerve efferents. ADP accumulation at the neuromuscular synapse decreases ACh release and smooth muscle contraction through the activation of prejunctional inhibitory P2Y₁ receptors. The diagram also shows the locus of action of the main drugs used in this study.