Effects of bladder outlet obstruction on properties of Ca2+-activated K+ channels in rat bladder

Abstract

In this study, we investigated the effects of bladder outlet obstruction (BOO) on the expression and function of large conductance (BK) and small conductance (SK) Ca(2+)-activated K(+) channels in detrusor smooth muscle. The bladder from adult female Sprague-Dawley rats with 6-wk BOO were used. The mRNA expression of the BK channel alpha-subunit, beta1-, beta2-, and beta4-subunits and SK1, SK2, and SK3 channels were investigated using real-time RT-PCR. All subunits except for the BK-beta2, SK2, and SK3 channels were predominantly expressed in the detrusor smooth muscle rather than in the mucosa. The mRNA expression of the BK channel alpha-subunit was not significantly changed in obstructed bladders. However, the expression of the BK channel beta1-subunit and the SK3 channel was remarkably increased in obstructed bladders. On the other hand, the expression of the BK channel beta4-subunit was decreased as the severity of BOO-induced bladder overactivity progressed. In detrusor smooth muscle strips from obstructed bladders, blockade of BK channels by iberiotoxin (IbTx) or charybdotoxin (CTx) and blockade of SK channels by apamin increased the amplitude of spontaneous contractions. These blockers also increased the contractility and affinity of these strips for carbachol during cumulative applications. The facilitatory effects elicited by these K(+) channel blockers were larger in the strips from obstructed bladders compared with control bladders. These results suggest that long-term exposure to BOO for 6 wk enhances the function of both BK and SK types of Ca(2+)-activated K(+) channels in the detrusor smooth muscle to induce an inhibition of bladder contractility, which might be a compensatory mechanism to reduce BOO-induced bladder overactivity.

Fig. 1.

Relative mRNA expression levels of large-conductance (BK) channel α-subunit (BK-α) (A), BK channel β1-subunit (BK-β1) (B), BK channel β2-subunit (BK-β2) (C), BK channel β4-subunit (BK-β4) (D), small-conductance (SK)1 channel (SK1) (E), SK2 channel (SK2) (F), and SK3 channel (SK3) (G) in mucosa-intact whole bladders from control (n = 7), mild bladder outlet obstruction (m-BOO) (n = 6), and severe bladder outlet obstruction (s-BOO) (n = 7) groups. Each value is expressed as a percentage of the expression level of the control group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group. §P < 0.05 vs. m-BOO group.

Fig. 2.

Relative mRNA expression levels of BK-α (A), BK-β1 (B), BK-β2 (C), BK-β4 (D), SK1 (E), SK2 (F), and SK3 (G) in mucosa and detrusor layers separated from bladder tissues in control and BOO groups (n = 5 in each). Each value is expressed as a percentage of the expression level in the mucosa of control group. ***P < 0.001 vs. control group. §P < 0.05, §§§P < 0.001 vs. mucosa of the same group.

Fig. 3.

Representative force tracings produced by detrusor smooth muscle strips. A: effect of cumulative applications of charybdotoxin (CTx; 10, 30, 100 nM) on spontaneous contractions of smooth muscle strips from control and BOO groups. B: effect of 100 nM CTx on contractility in response to cumulative applications of carbachol (10⁻⁸, 3 × 10⁻⁸, 10⁻⁷, 3 × 10⁻⁷, 10⁻⁶, 3 × 10⁻⁶, 10⁻⁵, 3 × 10⁻⁵, 10⁻⁴, and 3 × 10⁻⁴ M). Cumulative application of carbachol before a treatment with 100 nM CTx (1^st) and after a treatment with 100 nM CTx (2^nd).

Fig. 4.

Alterations of amplitudes (A, B, C) and frequency (D, E, F) of spontaneous contractions of detrusor smooth muscle strips elicited by iberiotoxin (IbTx) (A and D: n = 12, N = 4 in control group; n = 14, N = 6 in BOO group, where n represents the number of strips and N represents the number of animals), CTx (B and E: n = 14, N = 6 in control group; n = 15, N = 6 in BOO group), and apamin (C and F: n = 10, N = 5 in control group; n = 11, N = 5 in BOO group). Values are presented as a percentage of the value before an application of each blocker. **P < 0.01, ***P < 0.001 vs. control group.

Fig. 5.

Effect of K⁺ channel blockers on concentration-response curves for carbachol in detrusor smooth muscle strips from the control (A–D) and BOO (E–H) groups. Curves in each panel were obtained from cumulative applications of carbachol in time-matched controls (TMC) without blocking agents (A and E: n = 8, N = 8 in control group; n = 7, N = 4 in BOO group) and in preapplication and postapplication phases of 100 nM IbTx (B and F: n = 11, N = 6 in control group; n = 9, N = 5 in BOO group), 100 nM CTx (C and G: n = 11, N = 6 in control group; n = 11, N = 6 in BOO group) and 100 nM apamin (D and H: n = 10, N = 5 in control group; n = 10, N = 5 in BOO group). 1^st denotes cumulative applications of carbachol before a treatment with each blocker or first carbachol applications in TMC. 2^nd denotes cumulative applications of carbachol after a treatment with each blocker or second carbachol application in TMC. *P < 0.05 vs. TMC (ΔEmax).

Fig. 6.

Comparisons of the effects of K⁺ channel blockers on the carbachol-induced contractile responses in detrusor smooth muscle strip from control and BOO rats. A: percentage changes of Emax between first and second applications of carbachol (ΔEmax). B: differences of −logEC50 between first and second applications of carbachol (Δ−logEC50). Blocking agents were applied before the second application of carbachol. TMC, time-matched controls. *P < 0.05, **P < 0.01, ***P < 0.001 vs. TMC. §P < 0.05 vs. IbTx application (BOO). †P < 0.05, ‡P < 0.001 vs. control group.