Beta-adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca2+-activated K+ channel

Abstract

In urinary bladder smooth muscle (UBSM), stimulation of beta-adrenergic receptors (beta-ARs) leads to activation of the large-conductance Ca2+-activated K+ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255-C1263, 2005). In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to beta-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K+ or 1 microM carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with approximately 12 mV. Isoproterenol, a nonspecific beta-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentration-dependent manner. In the presence of IBTX, the concentration-response curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol- and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the beta-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in beta-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.

Fig. 1.

Original recordings illustrating the inhibitory effect of isoproterenol on the contractility of urinary bladder smooth muscle (UBSM) strips isolated from wild-type (WT) and large-conductance Ca²⁺-activated K⁺ (BK) channel α-subunit knockout (KO) mice. Isoproterenol (0.1 nM–1 μM) inhibited in a concentration-dependent manner both phasic and tonic contractions of WT UBSM strips (top trace), WT UBSM strips pretreated with the BK channel inhibitor iberiotoxin (IBTX; 200 nM; middle trace), and KO UBSM strips (bottom trace). The overall contractility was increased in the presence of IBTX and in the absence of functional BK channel (BK-KO mice). Note that isoproterenol was less effective when the BK channel was inhibited with IBTX but had similar potency in the BK-KO compared with WT mice. Phasic and tonic contractions were induced by addition of 20 mM K⁺ in the bath solution. Dotted lines indicate the initial baseline level, which corresponds to the muscle tone.

Fig. 2.

Concentration-response curves for the effects of isoproterenol (0.1 nM–100 μM) on the phasic contraction amplitude (A), phasic contraction frequency (B), muscle force integral (C), and muscle tone (D) in UBSM strips isolated from WT mice under control conditions and after the BK channel was blocked with IBTX (200 nM). In the presence of IBTX, the concentration-response curves were shifted to the right. Data are normalized to the 20 mM K⁺-induced contractions. Values are means ± SE (n = 4). *P < 0.05; **P < 0.01; ***P < 0.005 (paired test).

Fig. 3.

Concentration-response curves for the effects of isoproterenol (0.1 nM–100 μM) on the phasic contraction amplitude (A), phasic contraction frequency (B), muscle force integral (C), and muscle tone (D) in UBSM strips isolated from WT and BK-KO mice. Surprisingly, there was no rightward shift in the concentration-response curves for the contraction amplitude (A), frequency (B), and muscle force (C) such as that observed in the WT animals in the presence of IBTX (see Fig. 2), and even a leftward shift was noted on the effects on the phasic contraction amplitude (A). Data are normalized to the 20 mM K⁺-induced contractions. Values are means ± SE (n = 14–21). *P < 0.05 (unpaired test).

Fig. 4.

Original recordings illustrating the inhibitory effect of forskolin on the contractility of UBSM strips isolated from WT and BK-KO mice. Forskolin (30 nM–10 μM) inhibited in a concentration-dependent manner both phasic and tonic contractions of WT UBSM strips (top trace), WT UBSM strips pretreated with the BK channel inhibitor IBTX (200 nM; middle trace), and KO UBSM strips (bottom trace). The overall contractility was increased in the presence of IBTX and in the absence of functional BK channel (KO mice). Note that forskolin was less effective when the BK channel was inhibited with IBTX but had similar potency in the KO compared with WT mice. Phasic and tonic contractions were induced by addition of 1 μM carbachol in the bath solution. Dotted lines indicate the initial baseline level, which corresponds to the muscle tone.

Fig. 5.

Concentration-response curves for the effects of forskolin (30 nM–10 μM) on the phasic contraction amplitude (A), muscle force integral (B), phasic contraction frequency (C), and muscle tone (D) in UBSM strips isolated from WT mice under control conditions and after the BK channel was blocked with IBTX (200 nM). In the presence of IBTX, the concentration-response curves were shifted to the right. Data are normalized to the 1 μM carbachol-induced contractions. Values are means ± SE (n = 5). *P < 0.05; **P < 0.01 (paired test).

Fig. 6.

Concentration-response curves for the effects of forskolin (10 nM–30 μM) on the phasic contraction amplitude (A), muscle force integral (B), phasic contraction frequency (C), and muscle tone (D) in UBSM strips isolated from WT and BK-KO mice. Surprisingly, with the exception of the forskolin effect on contraction frequency (C), there was no rightward shift in the concentration-response curves such as that observed in the WT animals in the presence of IBTX (see Fig. 5). Instead, a leftward shift was noted on the effects on the phasic contraction amplitude (A), muscle force (B), and muscle tone (D). Data are normalized to the 1 μM carbachol-induced contractions. Values are means ± SE (n = 8–10). *P < 0.05; **P < 0.01 (unpaired test).

Fig. 7.

Concentration-response curves for the effects of isoproterenol (0.1 nM–1 μM) on the phasic contraction amplitude (A), phasic contraction frequency (B), muscle force integral (C), and muscle tone (D) in UBSM strips isolated from WT and BK-KO mice in the presence of 3 μM H-89, a PKA inhibitor. When the PKA was inhibited with H-89 (3 μM), a rightward shift in the concentration-response curves for the contraction amplitude (A), frequency (B), and muscle force (C) was observed in the BK-KO mice. Data are normalized to the 20 mM K⁺-induced contractions. Values are means ± SE (n = 11–16). *P < 0.05; **P < 0.01 (unpaired test).

Fig. 8.

IBTX has no effect on the contractility of UBSM strips isolated from the BK-KO mice. A: original recordings illustrating the lack of effect of IBTX (200 nM) on the contractility of a UBSM strip isolated from the BK-KO mouse. Phasic and tonic contractions were induced by addition of 20 mM K⁺ in the bath solution. B: summary data illustrating the effect of IBTX (200 nM) on contraction amplitude (CA), contraction frequency (CF), muscle force (MF), and muscle tone (MT) in UBSM strips isolated from BK-KO mice. Data are normalized to the 20 mM K⁺-induced contractions (control). Values are means ± SE (n = 5). P > 0.05 (paired test).

Fig. 9.

Proposed mechanisms by which BK channels mediate β-adrenergic relaxation in UBSM cells with illustrations of the differential outcomes following channel pharmacological inhibition or genetic ablation in the WT (top) and the BK-KO mouse (bottom), respectively. In UBSM cells from WT mice, functionally active BK channels regulate Ca²⁺ entry via L-type voltage-gated Ca²⁺ (Ca_V) channels, and thus contractility (top). In addition, BK channels are under the local control of the so-called “Ca²⁺ sparks” caused by Ca²⁺ release from the ryanodine receptors of the sarcoplasmic reticulum, adjacent to the cell membrane (9, 20). After permanent BK channel gene deletion in the BK-KO mouse, an adaptive compensatory upregulation of the β-adrenergic receptor (β-AR)-cAMP-PKA signaling pathway develops. The enhanced β-AR/PKA activity compensates for the increased Ca²⁺ entry via L-type Ca_V channels that occurs due to sustained membrane depolarization in the absence on the BK channels. AC, adenylyl cyclase; G_s, stimulatory G protein; PKA, cAMP-dependent protein kinase; PLB, phospholamban; RyR, ryanodine receptor; SR, sarcoplasmic reticulum.