Introduction:

Irritative bladder symptoms are the most bothersome symptoms associated with bladder outlet obstruction (BOO) derived from benign prostatic hyperplasia.  Unexpectedly, these irritative symptoms can remain even after effective surgical removal of the obstruction and normalization of urine flow.  Importantly, irritative bladder symptoms are rapidly relieved by a₁ adrenergic receptor (AR) antagonists. One possibility for this relief is that a₁ ARs play an important, de novo role in bladder contractility in obstructed patients. In the current study, we examined expression of a₁ AR subtype mRNA and receptor proteins in detrusor of control and BOO rats.

 

Material and methods:

Female rats were partially obstructed by placing a ligature (1.1 mm opening) around the urethra. After six weeks the voiding behavior was studied with a computer assisted continuous micturition recording system (CMRS). Bladders (12 controls, 12 sham operated, 9 obstructed) were harvested and snap frozen in liquid nitrogen. After tissue RNA extraction with Trizol, a₁ AR mRNA was quantified using competitive RT-PCR. Specific artificial RNA competitors, containing either a_1a, a_1b or a_1d AR specific priming sites, were constructed and added in known amounts to tissue RNA extracts. PCR products were stained with ethidium bromide and quantified with fluorescence image analysis. Cellular membranes were prepared from whole bladders and the membrane protein concentration determined by a bicinchoninic acid assay. The total a₁ AR protein amount was quantified by [¹²⁵I]-HEAT saturation binding and compared to the total a₁ AR mRNA expression.

 

Results:

The surgically obstructed rats showed 6-fold increased bladder mass after six weeks. CMRS results indicated increased micturition frequency (2x) and decreased volume per micturition (30%) in the obstructed rats as compared to sham operated and unoperated control animals. In control and sham operated animals, 71% of the a₁ AR mRNA was of the a_1a subtype, while 24% was a_1d subtype and 5% a_1b. In obstructed animals the opposite relationship was seen with 75% of the a₁ AR mRNA being of the a_1d subtype and 23% of the a_1a subtype; a_1b remained low. This change in relative subtype expression was due to a 60% reduction in a_1a AR expression and a 3-5 fold increase in a_1d mRNA expression (based on pg mRNA/g wet tissue). However, the total a₁ AR mRNA expression per tissue mass increased only slightly (22%). The [¹²⁵I]-HEAT saturation binding shows comparable results for the protein translation of total a₁ ARs.

 

	a1a AR mRNA	a1b AR mRNA	a1d AR mRNA	Total a1 AR mRNA	Total a1 AR protein
Control/Sham	98±82(71%)	7±3(5%)	33±52(24%)	139±138	30±8
Obstruction	38±52(23%)	3±3(2%)	128±112(75%)	170±168	39±4
% Change	-61%	-57%	+388%	+23%	+30%

 

Table

Subtype specific and total a₁ AR mRNA levels (pg mRNA/g wet tissue ± SD (%)) as well as total a₁ AR protein amount (fmol receptor/mg membrane protein ± SD) in normal and obstructed rat detrusor

 

Conclusion:

Our findings indicate a remarkable increase of a_1d AR gene expression in the detrusor of obstructed rats that may contribute to the changes in their micturition behavior. Although the total a₁ AR mRNA expression and protein translation does not change remarkably due to decreased expression of a_1a AR mRNA, the receptor subtype shift itself is causing a 10-fold increase in alpha-adrenergic bladder susceptibility, since both physiological receptor agonists (norepinephrine and epinephrine) show a 10-fold higher affinity towards a_1d AR than towards a_1a or a_1b ARs.  The [¹²⁵I]-HEAT saturation binding supports an overall increase in a₁ ARs in obstructed detrusor.  Competition experiments with subtype-selective antagonists are in progress to determine if the overall increase in a₁ ARs specifically correlates with the increased a_1d ARs mRNA.  If these findings are confirmed in the human, targeting the a_1d AR may provide a new therapeutic approach to controlling bladder hyperactivity associated with BOO.