Modulation of bladder function by luminal adenosine turnover and A1 receptor activation

Abstract

The bladder uroepithelium transmits information to the underlying nervous and musculature systems, is under constant cyclical strain, expresses all four adenosine receptors (A(1), A(2A), A(2B), and A(3)), and is a site of adenosine production. Although adenosine has a well-described protective effect in several organs, there is a lack of information about adenosine turnover in the uroepithelium or whether altering luminal adenosine concentrations impacts bladder function or overactivity. We observed that the concentration of extracellular adenosine at the mucosal surface of the uroepithelium was regulated by ecto-adenosine deaminase and by equilibrative nucleoside transporters, whereas adenosine kinase and equilibrative nucleoside transporters modulated serosal levels. We further observed that enriching endogenous adenosine by blocking its routes of metabolism or direct activation of mucosal A(1) receptors with 2-chloro-N(6)-cyclopentyladenosine (CCPA), a selective agonist, stimulated bladder activity by lowering the threshold pressure for voiding. Finally, CCPA did not quell bladder hyperactivity in animals with acute cyclophosphamide-induced cystitis but instead exacerbated their irritated bladder phenotype. In conclusion, we find that adenosine levels at both surfaces of the uroepithelium are modulated by turnover, that blocking these pathways or stimulating A(1) receptors directly at the luminal surface promotes bladder contractions, and that adenosine further stimulates voiding in animals with cyclophosphamide-induced cystitis.

Fig. 1.

Adenosine turnover at the mucosal surface of the uroepithelium. A: schematic of adenosine biosynthesis and turnover in the intracellular and extracellular spaces. Intracellular adenosine is formed either by the conversion of S-adenosylmethionine (SAM) to S-adenosylhomocystine (SAH) to adenosine, or by the hydrolysis of ATP. The latter is expelled from the cells via nucleotide channels (NC). The extracellular ATP is converted to adenosine by the ecto-nucleotide triphosphate diphosphohydrolases (NTDPases), ecto-NTDPase (CD39), and ecto-5′ nucleotidases (CD73). Extracellular adenosine can have multiple fates: 1) transport back into the cell by nucleoside transporters (NT), 2) binding to and activation of adenosine receptors (AR), 3) metabolism to inosine by adenosine deaminase, or 4) conversion to AMP by ecto-adenosine kinase. EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride; IDT, 5-iodotubericidin; NBTI, S-(4-nitrobenzyl)-6-thioinosine. B-D: rabbit uroepithelium was mounted in Ussing stretch chambers and pretreated with the indicated drug for 60 min before the start of the experiment. The tissue was left in its quiescent state for 60 min and then stretched by increasing the fluid in the mucosal chamber for 30 min. The tissue was then left in this stretched state for an additional 60 min. Samples were drawn at the indicated times and then analyzed by mass spectrometry to determine the concentration of adenosine, inosine, and AMP. The experiment was performed on 3 separate occasions and the data are expressed as means ± SE (control n = 7, EHNA n = 7, IDT n = 7, NBTI n = 4). *Statistically significant differences (P < 0.05) between control samples and treated ones.

Fig. 2.

Adenosine turnover at the serosal surface of the uroepithelium. A–C: tissue was treated as in Fig. 1, but samples were taken and analyzed from the serosal surfaces of the tissue. The data are expressed as means ± SE (control n = 7, EHNA n = 7, IDT n = 7, NBTI n = 4). *Statistically significant differences (P < 0.05) between control samples and treated ones.

Fig. 3.

Effect of EHNA, NBTI, or IDT on adenosine release in the rat bladder. A–C: rat bladders were filled with PBS containing DMSO (control), EHNA, NBTI, or IDT at a rate of 6 μl/min, and the intravesicle fluid was collected for analysis. Subsequently, the bladder was slow filled for 60 min and then maintained in its filled state for an additional 60 min before collecting samples. The concentration of adenosine (A), inosine (B), or AMP (C) is indicated. Data are means ± SE (n ≥ 4). *Significant differences (P < 0.05).

Fig. 4.

Effect of adenosine on bladder function. A: sample cystometrogram. B–F: continuous cystometry was performed using the indicated concentration of adenosine or adenosine deaminase dissolved in buffered urine substitute (BUS). The parameters measured included intercontraction interval (ICI; B), basal pressure (C), threshold pressure (D), peak pressure (E), and fluid recovered (F). Data are expressed as means ± SE (control n = 7, 100 μM adenosine n = 8, 10 μM adenosine n = 8, 1 μM adenosine n = 8, 100 nM adenosine n = 9, 10 nM adenosine n = 6, 0.3 U adenosine deaminase n = 8, 0.15 U adenosine deaminase n = 6). No parameters were significantly different from those of controls.

Fig. 5.

Activation of apical A₁ receptors with 2-chloro-N⁶-cyclopentyladenosine (CCPA) lowers the threshold pressure for voiding. Continuous cystometry was performed using the indicated concentration of drug dissolved in BUS. A–E: cystometry parameters were measured as described in Fig. 4. Data are expressed as means ± SE (DMSO control n = 10, 1 mM CCPA n = 7, 100 nM CCPA n = 13, 10 nM CCPA n = 12, 1 nM CCPA n = 9). *Statistically significant differences (P < 0.05) between DMSO-treated bladders and CCPA-treated ones. F: specificity of the effect of CCPA was tested by including 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in the cystometry buffer. Data are expressed means ± SE (CCPA n = 7, DPCPX + CCPA n = 8). DPCPX significantly antagonized the effects of CCPA alone.

Fig. 6.

Inhibitors of adenosine turnover lower the threshold pressure for voiding. Continuous cystometry was performed using the indicated concentration of drug dissolved in BUS. A–E: comparison of cystometrogram (CMG) parameters for bladders treated with EHNA, NBTI, IDT, or a cocktail containing all 3 inhibitors. The DMSO values in Fig. 5 are reproduced in A–E to aid in making comparisons (DMSO control n = 10, 10 μM EHNA n = 7, 10 μM NBTI n = 8, 100 nM IDT n = 8, drug cocktail n = 7). *Statistically significant differences (P < 0.05) between DMSO-treated bladders and those treated with drugs.

Fig. 7.

Cyclophosphamide treatment induces urinary bladder cystitis. Rats were either injected with saline (sham treatment) or with 150 mg/kg cyclophosphamide. Four hours later, BUS was intravesically infused at a constant rate of 50 μl/min for a period of 90 min. A: representative images of CMGs obtained from sham- or cyclophosphamide-treated bladders. B–F: CMG parameters for sham- or cyclophosphamide-treated bladders. Data are expressed as means ± SE (sham n = 8, cyclophosphamide n = 9). *Statistically significant differences (P < 0.05).

Fig. 8.

CCPA exacerbates the hyperactive bladder phenotype. A–E: rats were treated with cyclophosphamide for 4 h to induce an acute cystitis and then cystometry was performed. Data are expressed as means ± SE (Cyp-control n = 9, 10 μM adenosine n = 8, DMSO control n = 7, 1 nM CCPA n = 7). *Statistically significant differences (P < 0.05). F–G, I: cross sections of frozen uroepithelial tissues obtained from sham-treated animals or cyclophosphamide-treated ones. Images were captured with a confocal microscope and the z-series was projected. The A₁ receptor is shown in green, actin staining is shown in red, and nuclei are shown in blue. The location of umbrella cells (UCs) is indicated. The * in I shows a region of cyclophosphamide-treated bladders where the submucosal tissue is expanded, most likely a result of edema. H: Western blot showing A₁ receptor expression in sham (control) or cyclophosphamide-treated animals. Epithelial lysates from at least 2 rats were pooled for this analysis.