Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Abstract

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BKbeta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Fig. 1.

Partial bladder outlet obstruction (PBOO)-induced alteration in the voiding pattern and detrusor smooth muscle spontaneous contraction. A: analyzed data for urination frequency and void volume in sham and PBOO groups. B: representative organ bath recording (n = 5) of spontaneous contraction of detrusor muscle strips from sham and PBOO groups. Five small vertical boxes on the chart equal a 2-g force, and 5 small horizontal boxes equal 10 min. The amplitude of spontaneous contractions is ∼4-fold higher in the PBOO group than that for the sham control. C: representative recording of detrusor muscle strips force generation profile in response to 125 mM KCl. D: analyzed contractile data of detrusor muscle strips. Values are means ± SE. *Statistical significance, P < 0.05; n = 5.

Fig. 2.

Downregulation of large-conductance calcium-activated potassium (BK) channel (α- and β-subunits) expression in bladders from rabbits with PBOO. A: representative Western blots for BKα, BKβ, and α-actin (used as an internal control). B: analyzed Western blot data with the quantified expression levels measured by the FUJIFILM Image Reader. Both BK α- and β-subunits are significantly downregulated in bladders from rabbits 2 wk after surgical induction of PBOO. Values are means ± SE. *Statistical significance, P < 0.05; n = 6.

Fig. 3.

Confocal images of BKβ immunostaining in paraffin sections of bladders from rabbits after sham surgery or PBOO. Red fluorescent signal (Cy3) was used to detect BKβ, and the signal is much stronger in the sham sample than in the PBOO sample. Confocal images also show that BKβ is localized to smooth muscle cells. Nuclei are stained with by 4,6-diamidino-2-phenylindole (blue). Representative images are shown; n = 3.

Fig. 4.

Effect of BK opener on PBOO-induced spontaneous contraction and myosin light chain 20 (MLC₂₀) phosphorylation. A: set of representative recordings of detrusor muscle strips after addition of BK opener NS1619 or isoparamic acid. Top tracing is no treatment as a control, middle tracing is after treatment with NS1619, and bottom tracing is after treatment with isoparamic acid. B: set of representative 2-dimensional gels for control and BK opener-treated muscle strips. Top gel is no treatment, middle tracing is after treatment with NS1619, and bottom tracing is after treatment with isoparamic acid. UP-MLC₂₀, unphosphorylated MLC₂₀; P-MLC₂₀, phosphorylated MLC₂₀.

Fig. 5.

BK channel (α- and β-subunits) mRNA expression in bladders from benign prostatic hyperplasia (BPH) patients with and without detrusor overactivity (DO). Quantitative real-time PCR was performed on RNA isolated from bladder tissues from bladder cancer patients and BPH patients with and without DO. BK mRNA levels are expressed as relative quantity units. Values are means ± SE. *Statistical significance, P < 0.05; n = 5.

Fig. 6.

Western blot analysis of BK channel (α- and β-subunits) expression in bladders from BPH patients with and without DO. A: representative Western blots for BKα, BKβ, and α-actin (used as an internal control). B: analyzed Western blot data, with the expression levels measured by the FUJIFILM Image Reader. Values are means ± SE. *Statistical significance, P < 0.05; n = 3.

Fig. 7.

Increased MLC₂₀ phosphorylation in cultured human DSM cells by small interference (si) RNA-mediated downregulation of BKβ. A: Western blot for BKβ, smooth muscle-specific myosin (marker for smooth muscle), and α-actin (internal control) to demonstrate the efficiency of BKβ siRNA. B: representative 2-dimensional gel for control and siRNA-treated cells.