Stimulation of beta3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels

Abstract

We investigated the role of large-conductance Ca(2+)-activated K(+) (BK) channels in beta3-adrenoceptor (beta3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific beta3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific beta3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of beta3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 +/- 20.1%. In whole cell mode at a holding potential of V(h) = 0 mV, the single BK channel amplitude was 5.17 +/- 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 +/- 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from -26.1 +/- 2.1 mV (control) to -29.0 +/- 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of beta3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.

Fig. 1.

Blocking the BK channel with iberiotoxin (IBTX) decreases the inhibitory effect of BRL 37344 on the spontaneous contractile activity in isolated UBSM strips. A: original recordings illustrating the inhibitory effect of BRL 37344 (100 nM-100 μM) on the spontaneous contractions in the absence (top trace) and presence (bottom trace) of 200 nM IBTX. B: concentration response curves for the inhibitory effect of BRL 37344 (100 nM-100 μM) on the spontaneous phasic contraction amplitude, frequency, net muscle force, and muscle tone in the presence and absence of 200 nM IBTX. In the presence of IBTX, the concentration response curves were shifted right. Spontaneous contractions were taken as 100%. Values are means ± SE. (n = 7, N = 5; *P < 0.05, **P < 0.01). TTX (1 μM) and atropine (1 μM) were present throughout the experiments.

Fig. 2.

IBTX overcomes the inhibitory effect of BRL 37344 on the spontaneous contractile activity and further increases contraction amplitude and frequency in isolated UBSM strips. A: original recordings illustrating the inhibitory effect of BRL 37344 (100 μM) on the spontaneous contractions and that IBTX (200 nM) restores and even increases phasic contraction amplitude and frequency. B: summary data for the inhibitory effect of BRL 37344 (100 μM) on the spontaneous phasic contraction amplitude, net muscle force, muscle tone, and frequency; and the effect of IBTX (200 nM) in the presence of BRL 37344 (100 μM). Spontaneous contractions were taken as 100%. Values are means ± SE (n = 9, N = 6; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. BRL 37344). TTX (1 μM) and atropine (1 μM) were present throughout the experiments.

Fig. 3.

Stimulation of β3-ARs with BRL 37344 increases STOCs activity in isolated UBSM cells. A: original recordings illustrating that BRL 37344 (100 μM) increases the frequency of STOCs in single UBSM cells. A portion of the experiment is shown on an expanded time scale before and 8 min after BRL 37344 application. Whole cell currents were recorded with the perforated patch-clamp technique at a holding potential (V_h) of −40 mV. B: summary data illustrating the increase in STOCs frequency in the presence of BRL 37344 (100 μM). STOCs frequency and average STOCs amplitude under control conditions were taken as 100%, respectively, and indicated on the y-axis. Values are means ± SE (n = 5, N = 5; *P < 0.05 vs. control).

Fig. 4.

Stimulation of β3-ARs with BRL 37344 does not significantly change the amplitude and open probability (P_o) of single BK channels under conditions when the Ca²⁺ sources for BK channel activation were pharmacologically inhibited. Single BK channel currents were recorded with the whole cell, perforated, patch-clamp technique from single smooth muscle cells at 0 mV holding potential. A: shown is a series of BK channel openings from a single cell recorded before (control) and after application of 100 μM BRL 37344. B: summary data illustrating lack of BRL 37344 (100 μM) effect on the single BK channel amplitude. Values are means ± SE (n = 5, N = 3; P > 0.05). Ryanodine (30 μM), thapsigargin (100 nM), and nifedipine (1 μM) were present throughout the experiments.

Fig. 5.

Stimulation of β3-ARs with BRL 37344 does not increase the voltage-step-induced steady-state K⁺ currents in single UBSM cells under conditions when the Ca²⁺ sources for BK channel activation were pharmacologically inhibited. A: illustration of the voltage-step protocol that was applied to record whole cell, steady-state K⁺ currents using the perforated-patch technique at a V_h of −70 mV. B: current-voltage relationships for the depolarization-induced steady-state K⁺ currents in the presence and absence of 100 μM BRL 37344 were unchanged. Values are means ± SE (n = 8, N = 6; P > 0.05). Original recordings illustrating the steady-state K⁺ currents in response to voltage-step depolarization (−40 to +80 mV) before (C; control) and after application of 100 μM BRL 37344 (D). Ryanodine (30 μM), thapsigargin (100 nM), and nifedipine (1 μM) were present throughout the experiments.

Fig. 6.

In isolated UBSM cells, stimulation of β3-ARs with BRL 37344 causes membrane potential hyperpolarization, which was prevented by blocking the BK channels with 200 nM IBTX or 1 mM TEA. A: original recordings illustrating the BRL 37344 (100 μM) hyperpolarizing effect on the resting membrane potential under control conditions (top trace), and lack of an hyperpolarizing effect in the presence of 200 nM IBTX (middle trace), or 1 mM TEA (bottom trace). Resting membrane potential was recorded in current-clamp mode of the perforated-patch technique. B: summary data illustrating BRL 37344 (100 μM) hyperpolarizing effect under control conditions. Values are means ± SE (n = 11, N = 6; ****P < 0.0001). C: summary data illustrating lack of BRL 37344 (100 μM) hyperpolarizing effect in the presence of 200 nM IBTX (n = 9, N = 3; P > 0.05). D: summary data illustrating lack of BRL 37344 (100 μM) hyperpolarizing effect in the presence of 1 mM TEA (n = 8, N = 3; P > 0.05).

Fig. 7.

In isolated UBSM cells, stimulation of β3-ARs with BRL 37344 does not cause membrane potential hyperpolarization when the RyRs are inhibited. A: original recordings illustrating the lack of a BRL 37344 (100 μM) hyperpolarizing effect on the resting membrane potential in the presence of 30 μM ryanodine. Resting membrane potential was recorded in current-clamp mode of the perforated-patch technique. B: summary data illustrating lack of a BRL 37344 (100 μM) hyperpolarizing effect in the presence of 30 μM ryanodine (n = 7, N = 5; P > 0.05 vs. control).