Urinary frequency, urgency and urgency incontinence are common bothersome storage symptoms in men with partial bladder outlet obstruction (BOO) due to BPH.  Bladder function is altered following BOO and often not completely be restored, even after the obstruction is relieved.  Although both sexes have been used for BOO rat models, female rats have been used more commonly because of the simpler anatomy and the absence of accessory sex organs [Ref.1]. 

Adenosine is known to activate four different subtypes of G protein-coupled receptors (A1, A2A, A2B and A3), and it plays an important role in modulating smooth muscle contraction.  Adenosine can be produced and released by the urothelium, and all four adenosine receptor subtypes are expressed in the bladder urothelium.  However, few studies have focused on the subtype specific role of adenosine receptor in BOO-induced bladder dysfunction.  Thus, we examined the expression level of adenosine receptor subtypes in bladder tissues after BOO, and the effects of adenosine receptor A2A & A3 modulation on bladder activity using male BOO rats.

Adult male Sprague-Dawley rats were used, and BOO was induced by partial ligation of the proximal urethra [Ref.1].   At 4 weeks (4w) or 12 weeks (12w) after inducing BOO, conscious rats were assessed by cystometrograms (CMG) with the urethral ligature intact.  The following CMG parameters were evaluated; bladder contraction amplitude (the peak pressure minus the basal pressure during each contraction period); baseline pressure (the pressure immediately after the reflex contraction); threshold pressure (the pressure immediately before the reflex contraction).  Voided volume (VV) was determined by collecting voided saline. After the VV was determined, saline infusion was stopped and the residual volume (RV) was measured by withdrawing intravesical fluid through the catheter by gravity.  The bladder capacity (BC) was calculated as VV + RV, and voiding efficiency (VE) was calculated as (VV/BC) × 100.  Bladder compliance was calculated as bladder capacity divided by the difference of threshold pressure and baseline pressure values, which were obtained after bladder emptying to eliminate the effect of PVR on bladder compliance.  Non-voiding contractions (NVCs) were defined as bladder contractions with amplitudes > 4 cmH20 prior to micturition [Ref.2].  Average values of each rat were calculated by the mean of measured values during two or three voiding cycles.  Saline solution was infused at 0.04 – 0.3 ml/min for approximately 1 h until rhythmic bladder contractions became stable.  In this study, intercontraction intervals were adjusted to around 10 - 15 min intervals to evaluate the same number of voiding cycles because bladder capacity was variable following BOO among animals [Ref.2].  To match the age of rats at analysis to be 20 weeks old, BOO was performed at 16 weeks old in the BOO 4w group, and at 8 weeks old in the BOO 12w group.  In the sham group, 16 weeks-old SD rats were sham-operated without urethral ligation and used as controls at 4 weeks after operation.  Because some of the BOO rats showed urinary retention during CMG, these rats were excluded from the analysis of CMG parameters.

After baseline CMG were obtained with saline infusion, we intravesically applied adenosine receptor A2A antagonist (ZM241385, 15μM), A3 agonist (2CL-IB-MECA, 10-100μM) or A3 antagonist (MRS3777, 10-100μM) in BOO 4w and sham groups. 

Also, changes in mRNA levels of adenosine receptor subtypes such as A1, A2A, A2B and A3 in bladder mucosa and muscle layers were quantified with RT-PCR and normalized by a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase; GAPDH).

All data are represented as means ± SE. Statistical significance was evaluated among groups using one-way ANOVA followed by Dunnett's multiple comparison test.  Thereafter, repeated ANOVA measures followed by Tukey's multiple comparison test were used for the comparison between two groups.  All data were analyzed using the JMP software (ver. 11; SAS Institute, Cary, NC).  P < 0.05 was considered significant.

Bladder weights were significantly higher in BOO 4w rats (362.0 ± 10.9 mg,) and BOO 12w rats (545.8 ± 26.9 mg) compared to sham rats (123.4 ± 5.22 mg, P < 0.001).

Contraction amplitudes were significantly increased in 4w BOO rats compared to 12w BOO rats or sham rats. On the other hand, bladder compliance and capacity were significantly increased in 12w BOO rats compared to 4w BOO rats or sham rats.  VE was decreased in BOO rats vs. sham rats, being more evident in BOO 12w than in BOO 4w.  The number of NVCs was significantly higher in 4w BOO rats (0.950 ± 0.081) compared to 12w BOO (0.502 ± 0.018, P < 0.01) or sham rats (0.309 ± 0.022, P < 0.0001, Fig. 1A, B).  In 12w BOO rats, the number of NVCs were significantly decreased compared to 4w BOO rats, but still higher than sham rats (P < 0.05, Fig. 1A, B).  

Overall differences of mRNA levels were more prominent in the blabber mucosal layer than the detrusor layer (Fig.1C).  In BOO 4w bladder mucosa, A2A and A3 receptors mRNA levels were significantly increased with a reduction in A2B levels vs. sham.  In BOO 12w bladder mucosa, A2A and A3 levels were reduced to the same level as in sham (Fig. 1C).  

In BOO 4w rats, intravesical administration of the A2A antagonist significantly reduced the number of NVCs (P < 0.05, Fig. 2A) while the A3 antagonist had no effects on CMG parameters.  However, the A3 agonist induced a significant NVC increase at a high dose (P < 0.01, Fig. 2B), which was blocked by co-administration of the A3 antagonist (100 µM).  However, the A3 antagonist alone had no significant effects on any CMG parameters in BOO 4w rats at 10 µM or 100 µM.  In sham rats, intravesical application of any drugs had no effects on CMG parameters.

These results indicate that BOO-induced bladder overactivity evident as NVC increases was associated with the increased mucosal expression of adenosine receptors A2A & A3 at 4 weeks after BOO.  Because intravesical application of an A2A antagonist significantly decreased the number of NVCs in male BOO rats, it is likely that up-regulation of A2A receptor expression in the bladder mucosa contributes at least in part to BOO-induced bladder overactivity.

In this study, intravesical application of A3 agonist or antagonist did not influence bladder function in sham rats.  However, in BOO rats, A3 agonist application significantly increased the number of NVCs, and these effects were blocked by an A3 antagonist.  Furthermore, the mRNA level of A3 receptor was significantly increased in the bladder mucosa of BOO rats vs. sham.  Therefore, it is assumed that the up-regulation of A3 receptor expression in the bladder mucosa of BOO rats might increase the susceptibility for the A3 receptor activation.  Thus, exogenous, high-level activation of A3 receptors might exacerbate bladder overactivity in BOO.  These findings were consistent with previous report indicates that activation of A3 receptors can induce contractions of the bladder in some specific conditions [Ref. 3].

The adenosine receptor A2A subtype seems to contribute more significantly to BOO-induced bladder overactivity than the A3 subtype.  Thus, A2A receptors could be a therapeutic target for male patients with OAB due to BPH-induced BOO, but exogenous, high-level activation of A3 receptor might exacerbate bladder overactivity in BOO.  Subtype-selectivity of adenosine receptors should be taken into account when considering the future development of treatment modalities for BOO-related bladder overactivity.

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