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RECEPTORS AND SIGNAL TRANSDUCTION

Stimulation of β3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca²⁺-activated K⁺ channels

Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina

Submitted 2 January 2008 ; accepted in final form 4 September 2008

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We investigated the role of large-conductance Ca²⁺-activated K⁺ (BK) channels in β3-adrenoceptor (β3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific β3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific β3-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 β3-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 β3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.

BRL 37344; SR59230a; H89; iberiotoxin; detrusor; patch-clamp; spontaneous, transient, outward large-conductance current

Norepinephrine, released from sympathetic nerves, relaxes UBSM via stimulation of β-adrenergic receptors (β-ARs), which is the most plausible major mechanism that sustains bladder relaxation during filling (1, 15, 48). It is generally accepted that activation of β-ARs by agonists stimulates adenylyl cyclase to increase cAMP, which in turn, activates protein kinase A (PKA) to mediate UBSM relaxation. Recent studies show that agonist-induced stimulation of β-ARs causes activation of different K⁺ channels leading to membrane hyperpolarization and relaxation in various smooth muscle tissues (11, 45). In guinea pig UBSM, isoproterenol, a nonselective β-AR agonist, inhibits spontaneous action potentials and hyperpolarizes the membrane through PKA activation (35). In addition, relaxation of guinea pig UBSM in response to isoproterenol is mediated mainly by activation of the BK channels (27, 42). Our previous studies indicate that isoproterenol-induced BK channel activation in UBSM involves increased Ca²⁺ entry through L-type Ca_V channels and Ca²⁺ spark activity (42). The latter effect appears to be mediated by PKA-induced phosphorylation of phospholamban, which, when in a phosphorylated state, activates the SR Ca²⁺-pump, elevates SR Ca²⁺ load and thus RyRs and Ca²⁺ spark activity.

In rat and human UBSM, mRNA that codes for the three known β-ARs subtypes, β1-, β2-, and β3-ARs, has been detected (14, 24, 25, 34, 37, 43, 44). Increasing evidence suggests that the β-AR relaxation of UBSM is mediated mainly by β3-ARs (8, 15, 45, 48). However, the contribution of each of the three separate β-ARs to the BK channel activation in UBSM is unknown. A recent study on human myometrium reports that stimulation of β3-AR with 50–100 µM BRL 37344, a β3-AR-specific agonist, may cause activation of the BK channels and thus smooth muscle relaxation (10). In the urinary bladder, β3-AR stimulation may lead to BK channel activation, suggesting a functional link to facilitate UBSM relaxation.

To test this hypothesis, we employed functional studies on UBSM contractility and patch-clamp electrophysiology using BRL 37344 to stimulate the β3-ARs. We found that β3-AR-induced relaxation of UBSM is mediated by STOCs activation and membrane potential hyperpolarization. To further reveal the cellular mechanism of possible functional coupling between β3-ARs and the BK channels, we applied a variety of patch-clamp protocols and pharmacological tools to elucidate the different regulatory pathways at the level of BK channel Ca²⁺ signaling.

	METHODS

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Animal studies and UBSM tissue harvesting. All animal studies were carried out in accordance with guidelines of the Animal Welfare Act and the Association for Assessment and Accreditation of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of the University of South Carolina (Animal Use Protocol No. 1482). In the present study we used 77 adult Sprague-Dawley rats (54 males and 23 females), 3–5 mo old, with an average weight of 321.9 ± 7.6 g. Rats were euthanized with CO₂, followed by exsanguination. The entire urinary bladder was removed and placed in ice-cold nominally Ca²⁺-free dissection solution (see Solutions and Drugs for composition). The bladder was then pinned to the bottom of a Sylgard-coated petri dish containing dissection solution. After the surrounding adipose and connective tissue were removed, the bladder was cut open with a longitudinal incision beginning from the urethral orifice. The entire mucosal layer of the bladder, including the urothelium, was removed, and the bladder was pinned with the serosal side up for dissection.

Contractility studies. Up to eight small UBSM strips (1–2 mm wide and 5–6 mm long) were excised free from each bladder and transferred to a small petri dish containing dissection solution. Individual strips were placed in thermostatically controlled (37°C) tissue baths (5-ml volume). One end of the strip was attached to a stationary metal hook, whereas the other end was connected to a force-displacement transducer for isometric tension recording. The force generation by the muscle strips was recorded using a MyoMed myograph and MyoViewer data acquisition system (both from MedAssociates, St. Albans, VT). The UBSM strips were suspended under initial 10 mN tension. These procedures were carried out in a nominally Ca²⁺-free dissection solution. Five minutes later, the bath solution was replaced with a Ca²⁺-containing physiological saline solution (see Solutions and Drugs for composition). An equilibration period of at least 45–60 min followed, during which the bath solution was changed every 15 min. Most of the strips exhibited spontaneous phasic contractions during the equilibration period. Only strips that had stable controls and contracted spontaneously for at least 30–45 min were used for the experiments.

UBSM single cell isolation. Small UBSM strips (1–2 mm wide and 5–7 mm long) were excised from the bladder wall. Several muscle strips were placed in a vial containing 2 ml of dissection solution supplemented with 1 mg/ml bovine serum albumin, 1 mg/ml papain (Worthington, Lakewood, NJ), and 1 mg/ml dithioerythritol and incubated for 30–32 min at 37°C. After that, the tissue strips were transferred to 2 ml of dissection solution containing 1 mg/ml BSA, 1 mg/ml collagenase (type II from Sigma), and 100 µM CaCl₂ for 9–14 min at 37°C. After incubation, the digested tissue was washed several times in dissection solution and then was gently triturated to yield single smooth muscle cells. Several drops of the solution containing the dissociated cells were then placed in a recording chamber. Cells were allowed to adhere to the glass bottom of the chamber for about 20 min. Most cells were elongated and had a bright, shiny appearance when examined with a phase-contrast microscope. The average UBSM cell capacitance was 27.7 ± 1.2 pF (n = 71 cells). Cells were used for patch-clamp recordings within 3–4 h after isolation.

Electrophysiological (patch-clamp) recordings. The amphotericin-perforated, whole cell configuration of the patch-clamp technique was employed (16, 22). Whole cell currents were filtered using an eight-pole Bessel filter (model 900CT/9L8L; Frequency Devices) and recorded using an Axopatch 200B amplifier, Digidata 1440A, and pCLAMP version 10.1 software (Molecular Devices, Union City, CA). Patch-clamp pipettes were pulled from borosilicate glass (Sutter Instruments, Novato, CA) using a Narishige PP-830 vertical puller coated with sticky dental wax to reduce capacitance and polished with a Micro Forge MF-830 fire polisher (Narishige) to give a final tip resistance of 4–6 M. STOCs were recorded while the UBSM cells were held at a holding potential (V_h) of –40 mV, a potential similar to the resting membrane potential of intact UBSM preparations (1, 41). To determine the mean amplitude and frequency of the transient BK currents, analysis was performed off-line using Clampfit of the pClamp software version 10.1. The threshold for the STOCs was set at 12 pA, which is more than three times the single-channel amplitude at –40 mV. STOCs were recorded over 10-min periods in the absence (control) and presence of BRL 37344. In a separate series of experiments, voltage-step protocols were used to elicit steady-state K⁺ outward current. UBSM cells were held at –70 mV and then depolarized from –40 mV to +80 mV at 20 mV steps of 200-ms duration. The steady-state K⁺ outward currents were recorded in the presence of ryanodine (30 µM) and thapsigargin (100 nM), which indirectly block STOCs. Nifedipine (1 µM) was also used to block the L-type Ca_V channels. For the voltage-step protocols, 4–6 controls were recorded and the data were averaged. Only cells with stable control currents in response to depolarization steps within at least 15 min were used to study BRL 37344 effects. After BRL 37344 administration, voltage-step protocols were applied every 1–2 min for at least a 15-min period, and steady-state K⁺ outward currents were recorded. The average steady-state K⁺ current value during the last 50 ms of the 200-ms depolarization step was used to plot current-voltage relationships.

In another series of experiments, single BK channel activity was recorded from UBSM cells in whole cell mode as previously described (42). The large amplitude and low open probability (P_o) of the BK channel in UBSM cells permits the measurement of single BK channel currents with the use of the amphotericin-perforated, whole cell configuration of the patch-clamp technique when the cells’ native environment and signal transduction pathways were preserved. To observe single BK channel currents, Ca²⁺ sparks, and hence STOCs were prevented by blocking RyRs and SR Ca²⁺-pump with ryanodine (30 µM) and thapsigargin (100 nM), respectively. The L-type Ca_V channels were inhibited with nifedipine (1 µM), and the cells were clamped at 0 mV, a potential at which L-type Ca_V and voltage-gated K⁺ channels are largely inactivated (46). Under these conditions, single UBSM BK channels were identified by their characteristic large, single-channel conductance, voltage dependence, and sensitivity to IBTX (42). Single BK channel P_o was calculated from continuous recordings of 7- to 10-min intervals in the absence (control) and presence of BRL 37344. Because the total number of BK channels (N) for each individual cell is unknown, the cell NP_o was normalized to the cell capacitance (measured as NP_o/pF). In some cells, at the end of the recordings, IBTX (200 nM) was applied to confirm that the recorded currents were through BK channels. UBSM cell membrane potential was recorded using the current-clamp mode of the patch-clamp technique. All patch-clamp experiments were carried out at room temperature (22–23°C).

Solutions and drugs. The nominally Ca²⁺-free dissection solution had the following composition (in mM): 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 HEPES, and 2 MgCl₂, pH 7.3, adjusted with NaOH. The Ca²⁺-containing physiological salt solution was prepared daily and contained (in mM): 119 NaCl, 4.7 KCl, 24 NaHCO₃, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 MgSO₄, and 11 glucose, and was aerated with 95% O₂-5% CO₂ to obtain pH 7.4. The extracellular (bath) solution used in the electrophysiological experiments contained (in mM): 134 NaCl, 6 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM): 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 HEPES, and 0.05 EGTA, pH adjusted to 7.2 with NaOH and supplemented with freshly dissolved (every 1–2 h) 200 µg/ml amphotericin-B. Atropine, nifedipine, and tetraethylammonium (TEA) were purchased from Acros Organics; BRL 37344, dithioerythritol, H89, IBTX, SR59230A, tetrodotoxin were from Sigma; ryanodine (ryanodine, 9, 21-dehydro-) was from Calbiochem; and bovine serum albumin, thapsigargin, and amphotericin-B were from Fisher Scientific.

Data analysis and statistics. UBSM contraction data were analyzed using MiniAnalysis (Synaptosoft). This software has allowed us to analyze the changes in all four major parameters of the UBSM phasic and tonic contractions (tone)-phasic contraction amplitude, phasic contractions frequency, net muscle force, and muscle tone. Data were further analyzed with GraphPad Prism (version 4) and presented using CorelDraw Graphic Suite X3 (Corel) software. Cumulative concentration response curves for BRL 37344 were obtained by adding increasing concentrations of the drugs directly to the tissue baths every 10 min. A 3-min period from the 7th to the 10th min after exposure to each drug concentration was taken as the analysis period. To compare the phasic contractions parameters, data were normalized to the spontaneous contractions and expressed as percentages. Net muscle force (muscle force integral) was determined by integrating the area under the curve of the phasic contractions component. Tone was determined by measuring changes of the phasic contraction baseline curve. Patch-clamp electrophysiological data were analyzed with Clampfit version 10.1.

Summary data are presented as means ± SE for n, the number of separate UBSM strips or single cells, respectively, and N, the number of individual rats. The data were assessed for statistical significance using the one-tailed paired Student's t-test. A P value < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Stimulation of β3-ARs with BRL 37344 leads to inhibition of UBSM spontaneous contractility: role of the BK channel and PKA. In isolated rat UBSM strips, stimulation of β3-ARs with BRL 37344 inhibits tonic contractions induced by membrane depolarization due to increased external K⁺ (8, 13, 28, 38). High external K⁺ (15–50 mM KCl) restrains the function of the K⁺ channels, including the BK channel, thus masking the native mechanism of β3-AR-induced relaxation of the bladder. In fact, the effects of β3-AR stimulation with BRL 37344 on UBSM spontaneous phasic and tonic contractions in a physiological external K⁺ solution have, to our knowledge, never been studied.

Here, we sought to investigate whether a pharmacological stimulation of β3-ARs with BRL 37344 leads to inhibition of the physiologically relevant spontaneous phasic and tonic contractions that normally decreased during the bladder-filling phase (for a review, see Ref. 1), and to further investigate the underlying mechanism. It is well known that UBSM contractions are modulated by neurotransmitters, released from autonomic nerves located in the bladder wall. To minimize any possible effects caused by neurotransmitter release, all experiments were performed in the presence of 1 µM tetrodotoxin, a neuronal Na⁺ channel blocker. Because BRL 37344 was also reported to have some antimuscarinic effects in rat and guinea pig UBSM (5, 28), we also applied 1 µM atropine, a nonselective muscarinic receptor antagonist.

In isolated rat UBSM strips, a selective stimulation of β3-ARs with BRL 37344 (100 nM-100 µM) caused a concentration-dependent decrease in spontaneous phasic contractions and muscle tone (Fig. 1). Figure 1 also shows the cumulative concentration response curves for BRL 37344 effects on the spontaneous phasic contractions amplitude, frequency, net muscle force, and muscle tone in rat UBSM strips. As illustrated, BRL 37344 (100 nM-100 µM) inhibited the phasic contraction's amplitude and net muscle force, and significantly reduced the muscle tone in isolated UBSM strips. In UBSM, a phasic contraction reflects an elevation of Ca²⁺ entry via L-type Ca_V channels caused by a single action potential or a burst of action potentials (17). The amplitude of a phasic contraction depends on the increase in Ca²⁺ entry caused by membrane depolarization during an action potential, whereas the duration of a phasic contraction depends on the single action potential duration or on a whole burst of action potentials (17, 18). The frequency of phasic contractions reflects mechanisms that temporarily cause action potentials to cease, such as an increase in K⁺ channel conductance (4, 17, 40–42).

View larger version (17K): [in this window] [in a new window]   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.

Because the BRL 37344 concentration response experiments are extended experiments conducted within a 50-min time frame, time control experiments were performed in parallel for the spontaneous and IBTX-induced phasic contractions in which no BRL 37344 was added. The data for the time controls summarized in Table 1 indicate that both spontaneous and IBTX-induced contractions remained stable during the time course for the BRL 37344 concentration response experiments. Therefore, BRL 37344 indeed had inhibitory effects on the contraction parameters as illustrated in Fig. 1, and they were not due to rundown of the UBSM preparations.

View larger version (26K): [in this window] [in a new window]   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.

Stimulation of β3-ARs with BRL 37344 causes activation of STOCs in single, isolated UBSM cells. In UBSM, Ca²⁺ sparks activate the closely located BK channels, thus causing STOCs (20, 42). Therefore, STOCs are indicative for Ca²⁺ sparks. To examine the effect of BRL 37344 on STOCs, STOCs were recorded from single, isolated UBSM cells that were voltage-clamped at –40 mV (see METHODS). BRL 37344 (100 µM) caused a sustained increase in STOC frequency by 46.0 ± 20.1% vs. control (n = 5, N = 5; P < 0.05; Fig. 3), whereas the STOC amplitude was unaffected (92.5 ± 6.4% vs. control, n = 5, N = 5; P > 0.05; Fig. 3). These results support the idea that in UBSM, stimulation of β3-ARs can activate BK channels through STOCs activation. STOCs activation is likely to move the UBSM resting membrane potential away from the threshold of action potential activation and significantly inhibit the action potentials and the associated phasic contractions.

View larger version (17K): [in this window] [in a new window]   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 (Vh) 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).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Stimulation of β3-ARs with BRL 37344 does not significantly change the amplitude and open probability (Po) 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.

View larger version (13K): [in this window] [in a new window]   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 Vh 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.

View larger version (16K): [in this window] [in a new window]   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).

In the last experimental series, illustrated in Fig. 7, the BRL 37344-induced (100 µM) hyperpolarization was not observed in the presence of 30 µM ryanodine (control, –24.9 ± 4.0 mV; BRL 37344, –25.3 ± 4.5 mV; n = 7, N = 5; P < 0.05 vs. control). This final experimental series confirms that pharmacological blockade of the RyR overcomes the hyperpolarizing effect of BRL 37344, indicating that the RyRs are required to facilitate the functional link between β3-ARs and the BK channels.

View larger version (15K): [in this window] [in a new window]   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).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

As key regulators of UBSM membrane excitability, BK channels control the opening and closing of L-type Ca_V channels, thereby affecting UBSM contractility. Our experiments on the spontaneous phasic and tonic contractions showed that stimulation of β3-ARs with BRL 37344 led to concentration-dependent inhibition of phasic contraction amplitude, net muscle force, and muscle tone. BRL 37344 (300 µM) is also effective at inhibiting spontaneous contractions of isolated whole guinea pig bladder (15). In rat UBSM, the potential inhibitory effect on the overall spontaneous contractility upon β3-ARs stimulation with FR165101 and the possible involvement of the BK channel in this process has previously been suggested (47). Here, we have further separated and characterized the β3-AR inhibitory effects on the different contraction parameters. In the presence of IBTX, a rightward shift in the concentration response curves was observed, indicating the importance of the BK channel in the BRL 37344 inhibitory effect on contractility (Fig. 1).

Our current-clamp experiments suggest a prominent role of β3-ARs in controlling the UBSM excitability by utilizing the BK channel (Figs. 6 and 7). The increase in STOCs frequency upon β3-ARs stimulation contributes to membrane hyperpolarization and moves the UBSM resting membrane potential away from the threshold of action potential activation and thus has significant inhibitory effects on action potentials and related phasic contractions. Our experiments on whole cell, K⁺ steady-state currents (Fig. 5) and single BK channel recordings (Fig. 4) do not support a mechanism for a direct activation of the BK channels by BRL 37344 in intact cells with blocked sources of Ca²⁺ necessary for BK channel activation. Rather, they support the concept that β3-AR stimulation activates BK channels by increasing STOCs activity in UBSM cells. Collectively, our data indicate that the physiological coupling between the β3-ARs and BK channels, which is responsible for UBSM hyperpolarization and relaxation, requires active sources of Ca²⁺ for BK channel activation.

Although cAMP/PKA is the main signal transduction pathway that mediates β3-AR effects, both PKA-dependent and -independent mechanisms have been proposed to operate in UBSM (11, 12, 45, 47, 48). Most of the information for a PKA-independent mechanism, however, comes from indirect evidence on UBSM contractility (12, 47, 48). Uchida et al. (47) have further indicated that in "noncontracted" UBSM (spontaneous contractions), relaxation mediated via β3-ARs is achieved solely by a cAMP-dependent mechanism. We have previously found that forskolin increases single BK channel P_o, whereas nonspecific activation of all β-ARs with isoproterenol is not sufficient to increase BK channel P_o (42). It has been shown that physiological coupling between β2-ARs and BK channels occurs by PKA-mediated phosphorylation of the channel pore-forming -subunit (36). It has also been reported that β2-ARs are closely associated with the BK channel -subunit and a PKA-anchoring protein to mediate the β2-AR-agonist responses in UBSM (29). Our data do not provide evidence for a similar colocalization and direct coupling between the β3-ARs and BK channels in rat UBSM. Instead, the functional coupling between the β3-ARs and BK channels in UBSM utilizes BK channel ryanodine-sensitive Ca²⁺ signaling mechanisms at the SR level. Our data indicates that BRL 37344-induced hyperpolarization is eliminated in the presence of ryanodine (Fig. 7). BRL 37344, via PKA activation, can act directly on the RyRs and/or indirectly by phospholamban phosphorylation. Our data show that the BRL 37344 relaxant effect on the UBSM tone is inhibited by H89, consistent with a role for PKA. In skeletal and cardiac muscle, PKA-phosphorylated phospholamban activates the SR Ca²⁺-pump and elevates SR Ca²⁺ load, which increases the P_o of RyRs channels (9). PKA can also increase the P_o of the RyRs channel and thus RyRs activity by direct phosporylation of the receptor (9). Most likely, the observed BRL 37344-induced increase in STOC activity may use both mechanisms.

BK channel activation upon β3-AR stimulation with BRL 37344 (50–100 µM) has been observed in human myometrium (10). Although this study by Doheny et al. (10), was similarly focused, our methodology is more refined for elucidating mechanistic details of the pathway of BK channel activation. The major difference between the two approaches is our utilization of pharmacological tools, such as ryanodine, thapisgargin, and nifedipine, to dissect the different sources of Ca²⁺ for BK channel activation. We have also distinguished between STOCs and steady-state BK currents, which have fundamentally different mechanisms of origin and regulation (20, 42). Additionally, we have improved upon the electrophysiological protocols by completing the experiments in more physiologically relevant conditions. For example, we utilized a V_h = 0 mV, instead of V_h = +40 mV. At V_h = +40 mV, the BK channel is exceedingly activated (40), and further activation upon pharmacological stimulation is unlikely to occur. Also, we used recording intervals of 7–10 min, which provide a more accurate determination of P_o than recording intervals of 10 s. Finally, the unjustifiably high Ca²⁺ concentration in the pipette used by Doheny et al. (10) may be a source of contaminating Ca²⁺ activator for the BK channel, whereas our conditions of 0 Ca²⁺ + 0.05 mM EGTA (to eliminate any residual Ca²⁺) do not provide this interference. These important experimental details allowed us to identify with high accuracy the intrinsic mechanisms of the functional link between β3-ARs and BK channels in rat UBSM. In addition, Doheny et al. (10) did not record any STOCs in human myometrium. Therefore, tissue and/or species differences in the mechanism of BK channel activation upon β3-AR stimulation in rat UBSM and human myometrium cannot be ruled out.

Overactive bladder is a condition characterized by symptoms of urgency, frequency, nocturia, and increased UBSM phasic contractions that often follow bladder outlet obstruction due to prostate enlargement (for review, see Refs. 1 and 2). The etiology of overactive bladder is unknown and the current therapies are limited to antimuscarinics that are largely ineffective with numerous undesirable side effects. Therefore, there is a need to develop alternative therapeutic approaches with novel mechanisms of action.

Because the BK channel is a key modulator of UBSM excitability in experimental animals, mutations of this channel may cause overactive bladder. BK channel inhibition with IBTX leads to increased overall UBSM contractility (19, 40), resembling overactive bladder behavior. Studies from our laboratory have identified the BK channel as one of the most important K⁺ channels that controls membrane excitability in human UBSM (39). BK channel overexpression via gene transfer eliminates UBSM overactivity that is caused by experimental bladder outlet obstruction in rats (6), whereas the opposite phenomenon is observed in mice lacking functional BK channel subunits (32, 40). Plasmid-based BK channel -subunit gene transfer for treatment of overactive bladder is now in clinical trials (30, 31).

Long-term bladder outlet obstruction leads to alteration in the β-AR densities or subtypes changing the bladder response to adrenergic stimuli (33). It may also be speculated that in overactive bladder there is a decrease in the inhibitory response to noradrenaline mediated by β3-ARs. RyRs expression is decreased in rat UBSM with experimental outlet obstruction (26). Data from RyR type 2 (RyR2) heterozygous mice (RyR2^+/–) indicate that RyR2 deficiency changes UBSM membrane potential and excitation-contraction coupling via BK channel modulation (23). Therefore, changes in BK channel functional coupling among β3-ARs, RyR, or PKA may be implicated in overactive bladder etiology.

This study provides evidence that in rat UBSM, β3-ARs and BK channels are functionally coupled at the SR and RyRs level to mediate relaxation. Alterations in this functional coupling may be involved in the pathology of overactive bladder. The results also support the general concept that selective β3-AR agonists might be effective therapeutics to control urinary bladder function. Novel β3-AR selective agonists, such as GW427353, which can suppress spontaneous UBSM contractions in animal and human UBSM tissues are now emerging as potential drugs for treatment of overactive bladder (3, 21).

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-070909 (to G. V. Petkov).

	ACKNOWLEDGMENTS

 
We thank Drs. Adrian Bonev (University of Vermont), Hristo Gagov (University of Sofia), and Mu-Yan Chen (University of South Carolina) for their critical evaluation of this study and Dr. Jennifer G. Schnellmann (Medical University of South Carolina) for improving the quality of the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. V. Petkov, Associate Professor of Pharmacology, Dept. of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, Univ. of South Carolina, Coker Life Sciences Bldg., Rm. 709, 715 Sumter St., Columbia, SC 29208 (e-mail: petkov{at}cop.sc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* K. L. Hristov and X. Cui contributed equally to this paper.

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