Urothelial purine release during filling of murine and primate bladders

Abstract

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD⁺), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD⁺, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

Keywords: adenosine 5′-triphosphate release; nicotinamide adenine dinucleotide release; urothelium.

Fig. 1.

Cystometry recordings in C57BL/6 mice. A: representative cystometrogram recordings using continuous intravesical infusion of saline in conscious C57BL/6 mice. Top, middle, and bottom traces are at 15, 25, and 100 μl/min infusion rates, respectively. B: bar graphs (means ± SE) summarizing bladder pressures at baseline and at 50, 100, and 200 μl infused volumes of saline. Top, middle, and bottom graphs show data from 15, 25, and 100 μl/min infusion rates, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001, significant difference from baseline pressure. Note that there is no change in pressure from baseline with 50 or 100 μl infused volumes of saline, whereas pressure is significantly increased with 200 μl infusion of saline. No. of observations from 3–5 mice are in parentheses.

Fig. 2.

Ex vivo murine bladder filling pressures. A: pressure traces from ex vivo bladder filling experiments in mice at 15 (left) and 100 (right) μl/min infusion rates. Pressure scale (mmHg) applied to all traces. Regions indicated by boxes represent the bladder filling phase before typically micturition occurs (i.e., 0–12 mmHg). Arrows indicate bladder pressures at infused volumes of 50, 100, and 200 μl. B: bar graphs (means ± SE) summarizing bladder pressures at baseline and at 50, 100, and 200 μl infusion of KBS. ***P < 0.001, significant difference from baseline pressure. Note that at 15 and 100 μl/min there is no change in pressure from baseline with 50 or 100 μl infusion of KBS, whereas pressure is significantly increased with 200 μl infusion of KBS. No. of observations in replicates from 3 mice are in parentheses.

Fig. 3.

Increased urothelial release of purines occurs during filling of mouse bladders. A: schematic diagram of the isolated whole bladder model used to collect intraluminal samples for analysis of urothelially released purines. Bladders, placed in 1-ml organ baths and perfused with oxygenated Krebs solution (KBS), were filled with KBS via an infusion pump. The intraluminal solution was collected and processed for detection of purines by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (described in methods). UB, urinary bladder. B: chromatograms of intraluminal fluid collected during low (50 μl) and high (200 μl) infused volumes of isolated C57BL/6 mouse bladders at 15 (left) and 100 (right) μl/min infusion rates. The release of purines from the urothelium is increased during bladder filling. Of note, the samples in B were diluted 10× to obtain chromatography signals in scale. LU, luminescence units. C: averaged data (means ± SE) summarizing the bladder filling-evoked release of purines in pmol/mg tissue from mouse bladder urothelium at 15 and 100 μl/min infusion rates. Total purines (Total PUR) is the sum of ATP, ADP, AMP, adenosine (ADO), and NAD⁺+ADPR+cADPR. Significant difference between groups indicated with horizontal bars (*P < 0.05, **P < 0.01, and ***P < 0.001, data analyzed with 1-way ANOVA or paired t-test). No. of experiments is in parentheses. White bars, 50 μl infused volume; gray bars, 100 μl infused volume; black bars, 200 μl infused volume.

Fig. 4.

Increased urothelial release of purines occurs during filling of monkey bladders. A: chromatograms of intraluminal fluid collected during low (10 ml) and high (50 ml) distention of isolated Cynomolgus monkey bladders at 10 ml/min infusion rate. The release of purines from the urothelium is increased during bladder filling. Of note, the samples in A were diluted 5× to obtain chromatography signals in scale. B: averaged data (means ± SE) summarizing the bladder filling-evoked release of purines in pmol/g tissue from monkey bladder urothelium. **P < 0.01, significant difference from 10 ml infusion. No. of experiments is in parentheses.