Poster, Podium & Video Sessions
Presentation Authors: Ricardo Pineda*, Balachandar Nedumaran, Joseph Hypolite, Shandra Wilson, Randall B Meachan, Anna P Malykhina, Aurora, CO
Introduction: Detrusor Overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. Although the mechanisms underlying this response are poorly understood, increasing evidence suggest that TREK-1 channel, a mechanosensitive member of the two-pore potassium channel family (K2P, KCNK), is a key regulator of detrusor response to stretch. We hypothesized that changes in detrusor TREK-1 protein expression underlie excitability and force transduction alterations in DO. We aimed to: 1) study the changes in expression and function of TREK-1 channels and, 2) explore the basis of TREK-1 force transduction under pathological conditions associated with detrusor overactivity (DO) in the human detrusor.
Methods: We performed immunocytochemistry labeling, qRT-PCR, whole cell patch-clamp electrophysiological recordings and detrusor muscle strip contractility studies on freshly dissociated bladder smooth cells isolated from normal (AUA SS <8; n= 15) and DO (AUA SS >22; n= 33) human bladders.
Results: A two-fold depletion in TREK-1 channel protein expression was observed in specimens obtained from DO bladders in comparison to controls (4.6±2.1 vs 9.1±2.7. ≤ 0.05). This change was associated with a significant reduction of whole cell patch-clamp recorded TREK-1 currents (Control, 1087.0±99.3 pApF vs DO, 98.6±66.3 pApF at 60 mV, n=6 each. ≤0.001). Further, detrusor strips obtained from DO patients failed to relax when exposed to 10 µM of arachidonic acid, a TREK-1 channel opener. To study how the mechanical force is transduced, we performed confocal analysis of immunocytochemically labeled freshly dissociated detrusor smooth muscle cells against cytoskeletal elements. Our results revealed colocalization of TREK-1 channels with members of the caveolin protein family and cytoskeletal proteins. Interestingly, caveolae microdomains were severely disrupted in DO specimens.
Conclusions: Our data demonstrate a significant reduction of TREK-1 channel expression and function in human smooth muscle cells obtained from DO bladders. Further, we provided evidence of close association between TREK-1 channels, different elements of the cell cytoskeleton and caveolins suggesting the presence of TREK-1 enriched caveolae microdomains.
Source Of Funding: NIH/NIDDK RO1-DK095817