Login to Access Video or Poster Abstract: MP82-14
Sources of Funding: Deutsche Forschungsgemeinschaft (DFG), Chinese Scholarship Council (CSC)

Introduction

Monomeric GTPases (e. g. RhoA, Rac) are critical mediators of smooth muscle contraction in the lower urinary tract, which is involved in pathophysiology and therapy of lower urinary tract symptoms (LUTS). Rac has been recently identified as a novel intracellular mediator of prostate smooth muscle contraction. A similar role in the bladder appears now possible. Here, we addressed Rac function in the human trigone.

Methods

Bladder tissues (trigone, dome, wall, mucosa) were obtained from patients undergoing radical cystectomy. Rac isoforms were detected by RT-PCR, Western blot, and immunofluorescence staining. Active Rac1 was detected by pull down assays using a GST/PBD-PAK (glutathione S-transferase/p21 binding domain of p21-activated kinase) fusion protein, immobilized to glutathione-covered agarose beads. Contractility of trigone tissues was examined in an organ bath.

Results

By RT-PCR, mRNA for Rac1, Rac2 and Rac3 was detectable in trigone tissues (n=5 patients), bladder dome (n=4 patients), and bladder wall (n=2 patients). Western blot analysis for Rac1 revealed bands with expected sizes (ca. 21 kDa), and suggested protein expression in trigone, bladder dome, bladder wall, and mucosa, while Rac2 may lack in the mucosa, and Rac3 may lack in the detrusor. Active Rac1 was detectable in trigone and detrusor tissues by pull down of guanosine triphosphate-loaded Rac1 (GTP-Rac). In the organ bath, the muscarinic agonist carbachol (100 nM-1 mM) induced concentration-dependent contractions of trigone tissues, which were reduced by the Rac inhibitor EHT1864 (30 µM). This inhibition was observed in all included samples (n=5 patients), despite high variations in magnitude of trigone contractions. Reduced contractility after Rac inhibition was reflected by decreased Emax value after curve fitting (111 ±17 % of KCl in the control group; 76 ±16 % of KCl after EHT1864). EHT1864 did not change EC50 values for carbachol, pointing to non-competitive inhibition of carbachol contractions.

Conclusions

Rac GTPases are active in the human trigone and detrusor. Smooth muscle contraction in the human trigone can be inhibited by the Rac inhibitor EHT1864. Rac-mediated trigone contractions may be involved in bladder outlet obstruction, in parallel to prostate-dependent mechanisms. Rac function in the bladder, including detrusor and overactive bladder merits further consideration.

Funding

Deutsche Forschungsgemeinschaft (DFG), Chinese Scholarship Council (CSC)