Differential regulation of Ca2+-activated K+ channels by {beta}-adrenoceptors in guinea pig urinary bladder smooth muscle

Georgi V. Petkov and Mark T. Nelson

Department of Pharmacology, The University of Vermont, College of Medicine, Burlington, Vermont

Submitted 5 August 2004 ; accepted in final form 19 January 2005


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Stimulation of {beta}-adrenoceptors contributes to the relaxation of urinary bladder smooth muscle (UBSM) through activation of large-conductance Ca2+-activated K+ (BK) channels. We examined the mechanisms by which {beta}-adrenoceptor stimulation leads to an elevation of the activity of BK channels in UBSM. Depolarization from –70 to +10 mV evokes an inward L-type dihydropyridine-sensitive voltage-dependent Ca2+ channel (VDCC) current, followed by outward steady-state and transient BK current. In the presence of ryanodine, which blocks the transient BK currents, isoproterenol, a nonselective {beta}-adrenoceptor agonist, increased the VDCC current by ~25% and the steady-state BK current by ~30%. In the presence of the BK channel inhibitor iberiotoxin, isoproterenol did not cause activation of the remaining steady-state K+ current component. Decreasing Ca2+ influx through VDCC by nifedipine or depolarization to +80 mV suppressed the isoproterenol-induced activation of the steady-state BK current. Unlike forskolin, isoproterenol did not change significantly the open probability of single BK channels in the absence of Ca2+ sparks and with VDCC inhibited by nifedipine. Isoproterenol elevated Ca2+ spark (local intracellular Ca2+ release through ryanodine receptors of the sarcoplasmic reticulum) frequency and associated transient BK currents by ~1.4-fold. The data support the concept that in UBSM {beta}-adrenoceptor stimulation activates BK channels by elevating Ca2+ influx through VDCC and by increasing Ca2+ sparks, but not through a Ca2+-independent mechanism. This study reveals key regulatory molecular and cellular mechanisms of {beta}-adrenergic regulation of BK channels in UBSM that could provide new targets for drugs in the treatment of bladder dysfunction.

Ca2+ sparks; voltage-dependent Ca2+ channel; ryanodine receptor


URINARY BLADDER SMOOTH MUSCLE (UBSM) exhibits spontaneous action potentials, which determine the phasic nature of the contractions in this tissue (6–8, 26, 27). Ca2+ entry through L-type dihydropyridine-sensitive, voltage-dependent Ca2+ channels (VDCC) is responsible for the upstroke of the action potential and gives rise to phasic contractions in UBSM. Blockage of VDCC causes action potentials and spontaneous contractions to be eliminated in UBSM (6–8, 10). The repolarization phase of the UBSM action potential is mediated by the activity of both voltage-dependent K+ (Kv) channels (33) and large-conductance Ca2+-activated K+ (BK) channels (6, 10). Blocking BK channels with iberiotoxin prolongs the action potential, causes membrane potential depolarization (10), and increases the amplitude of phasic contractions of UBSM strips (11, 26). After the repolarization phase, the action potential in UBSM displays a prolonged after-hyperpolarization, which is mediated by apamin-sensitive, small-conductance, Ca2+-activated K+ (SK) channels (4, 13, 14) and Kv channels (33). Thus VDCC, BK, Kv, and SK channels play important roles in regulating UBSM action potentials and related phasic contractions. In addition, recent studies (14, 23, 26) on knockout mouse models of BK channel pore-forming {alpha}- and regulatory {beta}-subunits and SK3 channel isoform suggest that these channels are fundamental in determining UBSM contractility.

During the bladder-filling phase, UBSM relaxes by activation of {beta}-adrenoceptors (1), an effect considered to be mediated by cAMP-dependent protein kinase (PKA). In UBSM, isoproterenol, a nonselective {beta}-adrenoceptor agonist, inhibits spontaneous action potentials and hyperpolarizes the membrane (24). It is generally accepted that in addition to voltage, intracellular Ca2+, and associated regulatory proteins such as {beta}-subunits (26), smooth muscle BK channels are also modulated by protein kinases, including PKA (2, 20, 21, 25, 31, 37). PKA can activate or inhibit BK channel activity, depending on the splice isoform (2, 5, 21, 25, 34). It has been shown that in UBSM, BK channels can be activated by isoproterenol or forskolin, an adenylyl cyclase activator that increases intracellular cAMP levels and PKA activity (18). The latter study, however, did not provide information about the mechanisms of BK channel regulation by {beta}-adrenoceptor and PKA. Direct phosphorylation of the BK channel by PKA has been thought to be the major mechanism by which PKA relaxes smooth muscle (2, 20, 31, 37).

In UBSM, Ca2+ entry through VDCC activates both BK and SK channels, whereas localized sarcoplasmic reticulum (SR) Ca2+ release (Ca2+ sparks) through ryanodine receptors (RyRs) activates only the BK channels (12, 13). These results suggest that Ca2+ signals from VDCC and RyRs are crucial in determining the activation of BK channels in UBSM.

Stimulation of {beta}-adrenoceptors has been shown to activate VDCC in UBSM (32) and other smooth muscle types (17, 19, 36, 41). In some smooth muscle tissues, VDCC can be inhibited by "cross-over" activation of cGMP-dependent protein kinase by cAMP (17, 19). In vascular smooth muscle, PKA activation by forskolin elevates SR Ca2+ release via RyRs, measured as an increase in Ca2+ spark frequency (16, 28). The latter effect appears to be mediated by PKA-induced phosphorylation of phospholamban, which leads to an activation of the SR Ca2+ pump and an elevation in SR Ca2+ load, thus increasing RyRs (Ca2+ sparks) and transient BK channel activity (38).

Stimulation of {beta}-adrenoceptors therefore could activate BK channels by multiple mechanisms, including: 1) increasing Ca2+ influx through VDCC; 2) direct activation of BK channels; and/or 3) elevation of RyR Ca2+ release (Ca2+ sparks). In this study, we examined the contribution of these mechanisms to the activation of BK channels, using isoproterenol to stimulate {beta}-adrenoceptors. Our results support the concept that {beta}-adrenoceptor stimulation does not elevate BK channel directly, but instead acts indirectly via an increase in Ca2+ entry through VDCC and Ca2+ release from SR RyRs (Ca2+ sparks). The latter effect appears to be sustained in intact UBSM cells and is therefore probably more important to maintaining the prolonged bladder relaxation during the filling stage.


    METHODS
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Tissue preparation. Male guinea pigs (250–350 g) were euthanized either by isoflurane or halothane overdose, followed by exsanguination. This procedure was reviewed and approved by the Office of Animal Care Management at the University of Vermont. The entire urinary bladder was removed and placed in ice-cold physiological saline solution (PSS; see below for composition). The bladder was pinned to the bottom of a petri dish containing nominally Ca2+-free dissection/dissociation solution (DS; see below).

UBSM single cell isolation. Small strips (100–300 µm wide and 3–5 mm long) of the detrusor muscle were cut free from the bladder. Several muscle strips were placed in a vial containing 2 ml of DS supplemented with 1 mg/ml BSA, 1 mg/ml papain (Worthington Biochemical, Freehold, NJ), and 1 mg/ml dithioerythritol and incubated for 20 to 25 min at 37°C. After that, the tissue was placed in 2 ml of DS containing 1 mg/ml BSA, 1 mg/ml collagenase (type II from Sigma) and 100 µM CaCl2 for 7–8 min at 37°C. After the incubation, the digested tissue was washed several times in DS and then dispersed with gentle trituration through the tip of a fire-polished Pasteur pipette. Several drops of the solution containing the dissociated cells were then placed in a recording chamber. Most cells were elongated and had a bright, shiny appearance when examined using phase-contrast microscopy.

Electrophysiological recordings. The amphotericin-perforated whole cell configuration of the patch-clamp technique was employed (15). Whole cell currents were recorded using an Axopatch 200 amplifier (Axon Instruments, Union City, CA), and the voltage-clamp protocol was controlled by pCLAMP version 9.2 software (Axon Instruments). To record VDCC and BK currents a brief depolarization protocol was used, similar to the depolarization that occurs during the action potential of UBSM (6, 8, 10). UBSM cells were held at –70 mV and then stepped to +10 mV for 100 ms. This depolarization step evokes an inward VDCC current, followed by an outward current, consisting of steady-state K+ current and transient BK current superimposed (Fig. 1; see also Ref. 13).



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Fig. 1. Original recordings of whole cell current in response to a voltage step depolarization from –70 to +10 mV in isolated urinary bladder smooth muscle (UBSM) cell recorded with the perforated patch-clamp technique. The recorded current consists of an initial L-type dihydropyridine-sensitive voltage-dependent Ca2+ channel (VDCC) current component, followed by a steady-state outward K+ component with transient large-conductance Ca2+-activated K+ channel (BK) current superimposed. The steady-state outward K+ current component consists of BK, small-conductance Ca2+-activated K+ channel (SK), and voltage-dependent K+ channel (Kv) currents. Iberiotoxin (IBTX, 200 nM) inhibits both the transient BK currents and the steady-state BK current. The dotted line indicates a theoretical ryanodine-sensitive current. Ryanodine (10 µM) was used to separate the transient BK currents from the steady-state K+ current, and iberiotoxin (200 nM) was used to separate the steady-state BK current from the SK and Kv currents.

 
The steady-state K+ current was recorded in the presence of ryanodine (10 µM), which blocks the transient BK currents. The average current value during the last 10 ms of the 100-ms depolarization step was taken for the steady-state K+ current. The peak Ca2+ current was measured to plot the data for VDCC current. The steady-state BK current components were determined as iberiotoxin (200 nM)-sensitive currents. For the controls, current traces of up to 30 pulses within 15 min 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 drug effects. To study the time course of the drug effects, 2 or 3 pulses were applied each minute and data were averaged. The leak current was subtracted during the experiments using the Axopatch 200 amplifier.

Transient BK currents were recorded separately while the cells were held at a membrane potential of –40 mV, a potential similar to the resting membrane potential of intact UBSM preparations (6, 8, 27). To determine the mean amplitude and frequency of the transient BK currents, analysis was performed off-line using a custom-designed analysis program, ANN, written by Dr. Adrian Bonev. The threshold of transient BK current was set at three times the single channel amplitude at –40 mV.

Single BK channel activity was recorded as described previously (28). The large amplitude and low open probability (Po) of the BK channel permitted the measurement of single BK channel currents with the use of the amphotericin-perforated whole cell configuration of the patch-clamp technique. To observe single BK channel currents, Ca2+ sparks and hence transient BK currents were prevented by blocking RyRs with ryanodine (10 µM) and SR Ca2+-ATPase with thapsigargin (100 nM). VDCC were inhibited with nifedipine (1 µM), and the cells were clamped at 0 mV. Po was calculated over 8- to 10-min intervals in the absence (control) and presence of 10 µM isoproterenol. Because the total number of BK channels (N) for each individual cell is unknown, the cell NPo was normalized to the cell capacitance (measured as Po/pF). At the end of the recordings, iberiotoxin (200 nM) was applied to confirm that the recorded currents were through BK channels. All experiments were conducted at 22°C.

Confocal fluorescence microscopy and Ca2+ spark detection. Isolated myocytes were loaded with the Ca2+-sensitive fluorophore fluo 4-AM (Molecular Probes) by mixing 1 ml of cell suspension with 4 µM fluo 4-AM, 2 µM Pluronic acid, and adding Ca2+ to achieve a final concentration of 2 mM Ca2+. Five hundred microliters of this fluo 4-AM-containing cell suspension were placed on the bottom surface (glass coverslip) of a recording chamber (1 ml volume). Cells were allowed 20 min to adhere to the bottom of the chamber, at which time dye loading was accomplished. Cells were subsequently washed several times with fresh bath solution to remove extraneous fluo 4-AM from the extracellular fluid. Cells were left for 20–40 min, allowing sufficient time for fluo 4 deesterification. Ca2+ imaging was conducted on a laser-scanning confocal microscope (Oz, Noran Instruments) with a x60 water immersion objective (1.2 numerical aperture; Nikon), and 488-nm line of a krypton-argon laser used for illumination. Images were acquired at 30 Hz and scan durations were 30 s, and the same cells were used for controls (30 s) and after isoproterenol treatment (30 s).

Imaging experiments were analyzed with custom software SparkAn (version 3.3.1; 2004), written by Dr. Adrian Bonev. Ca2+ sparks were defined as local increases in fluorescence of 1.3 F/F0 (where F is the fluorescence at a given time point and F0 is the baseline fluorescence) and automatically detected by SparkAn software. F0 was obtained by averaging 10 images containing no Ca2+ transients. For quantitation of Ca2+ sparks, a square region (2.3 x 2.3 µm or 10 x 10 pixels) was placed over an area of the cell in which Ca2+ sparks were observed, and F/F0 traces were generated for this region of the cell. Event frequency and kinetics were determined from entire experimental recordings lasting 30 s.

Solutions and drugs. PSS was made daily and contained (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, and 11 glucose, aerated with 95% O2 and 5% CO2 to obtain pH 7.4. DS contained (in mM) 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 HEPES, and 2 MgCl2, pH adjusted to 7.3 with NaOH. 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 intracellular (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 200 µg/ml amphotericin B. Ryanodine was from L. C. Laboratories (Woburn, MA). All other drugs and chemicals were from Sigma.

Statistics. Summary data are presented as means ± SE for n, the number of cells isolated from different animals. Statistical analysis of drug effects and the difference between treatment groups were determined using paired Student's t-test. A P value <0.05 was considered significant.


    RESULTS
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 GRANTS
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To determine the effects of {beta}-adrenoceptor stimulation on VDCC and BK channels in their physiological environment, whole cell membrane currents were recorded in freshly isolated UBSM cells using the perforated patch-clamp technique. A brief depolarization protocol was used, similar to the depolarization that occurs during the action potential (6, 8, 10). UBSM cells were held at –70 mV and then stepped to +10 mV for 100 ms. This depolarization step evokes an inward VDCC current, followed by an outward current (Fig. 1). A significant portion of this outward current is conducted by BK channels and consists of a steady-state component, which is dependent on Ca2+ entry through VDCC, and a transient RyR-dependent component (13). Ryanodine (10 µM), which blocks RyRs (Ca2+ spark activity), was used as a tool to separate the steady-state K+ current from the transient BK currents. To isolate the steady-state BK current from the Kv and SK currents, the BK channels were blocked with 200 nM iberiotoxin (Fig. 1).

Isoproterenol activates VDCC current in UBSM cells. The amplitude of the inward VDCC recorded in response to +10 mV depolarization step was transiently increased by 10 µM isoproterenol (Fig. 2). The maximal stimulatory effect of isoproterenol on the VDCC (up to a 50% increase) occurred within the first 1–3 min of drug application (123.4 ± 6.9%; n = 14; P < 0.01; 2 min after isoproterenol application) and was followed by a slight decrease before returning to baseline levels (Fig. 2B; n = 14). Because the early activation of the BK outward current during depolarization could mask the inward VDCC current, we performed experiments under conditions in which BK channels were blocked with iberiotoxin. VDCC activation by isoproterenol was largely unaffected under conditions of blocked BK channel (131.1 ± 14.5%; 2 min after isoproterenol application; n = 4; P < 0.05; Fig. 1E), suggesting that {beta}-adrenoceptor stimulation in UBSM is associated with an increase in VDCC current.



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Fig. 2. Simultaneous increase in VDCC and steady-state K+ currents induced by isoproterenol (ISO; 10 µM). A: original recordings of whole cell currents (averaged traces) in response to a voltage step depolarization (–70 to +10 mV) under control conditions, 2 min after isoproterenol (ISO, 10 µM) application, and after blocking BK current with iberiotoxin (IBTX, 200 nM). BD: summary data showing the time course of isoproterenol effect on the inward VDCC current (n = 14) (B); steady-state outward K+ current (n = 14) (C); and steady-state BK current (n = 11) (D). The steady-state BK currents were determined as iberiotoxin (200 nM) sensitive. Values are means ± SE. *P < 0.05, **P < 0.01. E: original recordings of whole cell currents (averaged traces) in response to a voltage step depolarization (–70 to +10 mV) in the presence of 200 nM iberiotoxin (control) and 2 min after isoproterenol (ISO, 10 µM) application. Isoproterenol increased the VDCC but not the steady-state current. Ryanodine (10 µM) was present throughout the experiments.

 
Isoproterenol modulates the steady-state BK current in UBSM cells. With RyRs blocked (10 µM ryanodine), isoproterenol (10 µM) induces a transient increase in total steady-state K+ current (Fig. 2A). After an initial activation within the first 1–2 min of isoproterenol application, the steady-state outward K+ current returned to its initial baseline levels (Fig. 2C; n = 14), mirroring the pattern of VDCC current activation by isoproterenol.

Because the steady-state component of BK current is dependent on parallel increases in VDCC activity, the prediction is that the observed isoproterenol-mediated increases in the steady-state K+ current reflects an increase in steady-state BK current. To dissect the contribution of the steady-state BK current component to total steady-state K+ current, we used the BK channel inhibitor iberiotoxin (200 nM). We found that the iberiotoxin-sensitive component of the steady-state K+ current was activated by isoproterenol (Fig. 2D; n = 11). In contrast, as illustrated in Fig. 2E, the iberiotoxin-resistant component was not activated by isoproterenol (96 ± 8.5%; 2 min after isoproterenol application; n = 4; P > 0.05). These data suggest that the Ca2+ entry through VDCC activates BK channels and that isoproterenol increases this effect by increasing the steady-state component of BK channel activity.

Isoproterenol-induced activation of the steady-state BK current requires activation of VDCC. To examine further the contribution of VDCC to the steady-state BK current activation, we performed experiments in the presence of nifedipine (1 µM) under conditions in which transient BK currents were blocked (10 µM ryanodine). Under these conditions, isoproterenol (10 µM) did not activate the depolarization-induced (+10 mV steps) steady-state outward K+ current (Fig. 3A; n = 5; P > 0.05). These experiments suggest that the enhancement of steady-state BK current by isoproterenol requires functional VDCC.



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Fig. 3. Blocking Ca2+ influx through VDCC by nifedipine (1 µM) or depolarization to +80 mV suppressed the isoproterenol (10 µM)-induced activation of the steady-state K+ current. Original recordings of whole cell currents (averaged traces) and summary data showing the lack of isoproterenol (10 µM)-induced activation of the steady-state K+ and steady-state BK currents: A, in response to +10 mV depolarization after blocking VDCC with nifedipine (n = 5; P > 0.05); B, in response to +80 mV depolarization (n = 5; P > 0.05); and C, in response to +80 mV depolarization after blocking VDCC with nifedipine (n = 5; P > 0.05). Ryanodine (10 µM) was present throughout the experiments. Values are means ± SE.

 
Isoproterenol does not activate the steady-state BK current in response to +80 mV depolarization steps. To confirm that {beta}-adrenoceptor stimulation acts through VDCC-mediated Ca2+ influx to enhance depolarization-induced BK currents and to rule out a direct effect on BK channel activity, we studied the effect of isoproterenol on the steady-state outward K+ current and the steady-state BK current in response to depolarization steps to +80 mV. Depolarization to +80 mV does not result in any significant VDCC-mediated Ca2+ influx because the driving force for Ca2+ is minimal. Under these conditions, VDCC-dependent mechanisms are inoperative, whereas direct effects on channel activity should be unaffected. The steady-state outward K+ currents and the steady-state BK currents obtained in response to +80 mV depolarization were not significantly different in the absence or presence of isoproterenol (Fig. 3B; n = 5; P > 0.05). Similar results (depolarization to +80 mV) were obtained when the VDCC were blocked with 1 µM nifedipine (Fig. 3C, n = 5; P > 0.05). These experiments suggest that the transient activation of the steady-state BK current is coupled with the VDCC activation and is not due to direct effect on the BK channel.

Isoproterenol does not change significantly Po of single BK channels in the absence of Ca2+ sparks and VDCC activity. A possible explanation for the increase in the steady-state BK current to {beta}-adrenoceptor stimulation is through an elevation of the BK channel Po caused by PKA phosphorylation of the channel, as has been demonstrated by others in excised patches (2, 21, 31, 37) and in whole smooth muscle cells (28). A PKA-independent activation of smooth muscle BK channels by isoproterenol has also been suggested (21). To test the effects of isoproterenol on UBSM BK channels in their native physiological environment, single channel currents were recorded from isolated UBSM cells in the whole cell mode, using the perforated patch-clamp technique. The latter approach allows single channel recordings under physiological conditions when the {beta}-adrenoceptors and PKA are intact. All extra- and intracellular Ca2+ sources were isolated. VDCC were inhibited with nifedipine (1 µM) and the cells were clamped at 0 mV, a potential at which VDCC are largely inactivated. Ca2+ sparks and hence transient BK currents were prevented by blocking RyRs with ryanodine (10 µM) and SR Ca2+-ATPase with thapsigargin (100 nM). Under these conditions, single BK channels were identified by their characteristic large single channel conductance, voltage dependence, and sensitivity to iberiotoxin (10, 26, 28). The advantage of this approach was that single BK channel activity (NPo) was measured across the entire cell membrane.

Isoproterenol (10 µM) did not change statistically significant single BK channel activity: 0.00398 ± 0.00184 NPo/pF (control) 0.00569 ± 0.00291 NPo/pF (isoproterenol) (n = 6; P > 0.05) measured over 8–10 min at 0 mV (Fig. 4). The single BK channel current amplitude was not affected by isoproterenol at 0 mV: 5.0 ± 0.25 pA (control) and 5.1 ± 0.31 pA (isoproterenol) (n = 6; P > 0.05). The observed single channel activity was inhibited by 200 nM iberiotoxin, which confirms that they were BK channel currents (Fig. 4). These results are consistent with the idea that isoproterenol-induced activation in the steady-state BK current is not due to a direct effect on the BK channel.



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Fig. 4. In the absence of Ca2+ sparks and VDCC activity, isoproterenol does not change significantly the open probability (Po) of single BK channels in a UBSM cell. Single BK channel currents were recorded with the whole cell perforated patch-clamp technique from a single smooth muscle cell at 0 mV holding potential. Transient BK currents were inhibited by blocking ryanodine receptors (RyRs) with ryanodine (10 µM) and SR Ca2+ uptake with thapsigargin (100 nM). VDCC were blocked with nifedipine (1 µM). Shown is a series of BK channel openings from a single cell recorded before (top trace) and after application of 10 µM isoproterenol (middle trace). Iberiotoxin (200 nM) blocked the single BK channel activity (bottom trace).

 
To clarify further whether or not PKA can activate BK channels directly in UBSM cells, we examined the effects of forskolin, which activates adenylyl cyclase. We found that forskolin (10 µM) increased statistically significant single BK channel activity: 0.00337 ± 0.00077 NPo/pF (control), 0.00582 ± 0.00142 NPo/pF (forskolin) measured over 8–10 min at 0 mV (n = 8; P < 0.01). The effect of forskolin on BK channels suggests that {beta}-adrenergic activation does not lead to sufficient cAMP increase or PKA activation in the vicinity of the BK channels.

Isoproterenol increases Ca2+ spark activity and transient BK currents. In addition to VDCC, another potential target for PKA after {beta}-adrenoceptor stimulation is phospholamban, a protein that regulates SR Ca2+ pump and thereby modulates the activity of SR Ca2+ release channels, including RyRs. An increase in intracellular Ca2+ sequestration and SR Ca2+ content would be expected to increase Ca2+ spark activity, producing a corresponding increase in transient BK currents.

To test the hypothesis that isoproterenol can increase SR Ca2+ spark frequency in UBSM, Ca2+ sparks were recorded under control conditions and 2 to 5 min after isoproterenol application (10 µM). Figure 5A illustrates images of a single UBSM cell before (control) and after application of isoproterenol. Fluorescence transients are also shown from the same cell under basal conditions and after isoproterenol application (Fig. 5B). Isoproterenol increases Ca2+ spark frequency by 31.7 ± 7.3% (Fig. 5C; n = 10; P < 0.001). These results support the idea that in UBSM, stimulation of {beta}-adrenoceptors activates SR Ca2+ sparks.



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Fig. 5. Isoproterenol-induced {beta}-adrenoceptor activation increases Ca2+ spark activity in single UBSM cells. A: pseudocolor image of a single fluo 4-loaded UBSM cell illustrating Ca2+ sparks under control conditions (top) and in the presence of 10 µM isoproterenol (bottom). Color bar next to the cell images shows the F/F0 scale, where F is the fluorescence at a given time point and F0 is baseline fluorescence. B: F/F0 vs. time plots for a spark site under control conditions (top trace) and in the presence of 10 µM isoproterenol (bottom trace). 1, 2, and 3 indicate the places of the representative images in A. C: summary data showing the effect of isoproterenol (10 µM) on Ca2+ spark frequency (n = 10; ***P < 0.001). Values are means ± SE.

 
In smooth muscle, including UBSM (12), Ca2+ sparks activate nearby BK channels and cause transient BK currents. To examine the effect of isoproterenol on transient BK currents, spontaneous transient outward BK currents were recorded separately in UBSM cells clamped at –40 mV, a potential similar to the resting membrane potential of intact UBSM preparations (6, 8, 27). Isoproterenol caused a sustained increase in the frequency of transient BK currents (40.1 ± 11.9%) that was reversible on washout (Fig. 6; n = 8; P < 0.01). These results support the concept that in UBSM, isoproterenol can activate BK channels through modulation of Ca2+ sparks and the subsequent transient BK currents. Activation of transient BK currents is likely to move the resting membrane potential away from the threshold of action potential activation and thus has significant inhibitory effects on action potentials and related phasic contractions.



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Fig. 6. Isoproterenol-induced {beta}-adrenoceptor activation increases spontaneous transient BK currents in single UBSM cells. A: isoproterenol (10 µM) increases the frequency of the spontaneous transient BK currents in isolated guinea pig UBSM cells. A portion of the experiment is shown on an expanded time scale before and after isoproterenol application. Whole cell currents were recorded with the perforated-patch technique at a holding potential (Vh) of –40 mV. B: differences in spontaneous transient BK current frequency under control conditions, in the presence of isoproterenol (n = 8; **P < 0.01), and after washout of isoproterenol (n = 4). Values are means ± SE.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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The present study proposes new mechanisms of BK channel regulation by {beta}-adrenergic agonists, which have previously been shown to act through cAMP and PKA to promote relaxation in UBSM (Fig. 7). Our data demonstrate that {beta}-adrenergic activation can increase UBSM BK channel activity by two distinct mechanisms. The first mechanism involves activation of VDCC, which provides local Ca2+ to activate BK channels, leading to an increase in the steady-state component of BK current. The second mechanism is characterized by an isoproterenol-induced increase in transient BK channel activity (Fig. 6) and supports the concept that, in UBSM, {beta}-adrenoceptor stimulation can activate BK channels by increasing Ca2+ spark activity (Fig. 5). Our data from whole cell currents and single BK channel recordings do not support a role for direct activation of BK channels by {beta}-adrenergic agonists in intact UBSM cells (Figs. 3 and 4). Similarly, it has been shown that {beta}-adrenergic activation of BK channels is not directly linked to the degree of PKA activation and/or that PKA is not the only determinant of isoproterenol-induced airway smooth muscle relaxation (20).



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Fig. 7. Proposed regulatory mechanisms by which {beta}-adrenergic activation modulates the Ca2+ signals and ion channel activity in UBSM cells to control the resting membrane potential, action potential and contractility. Activity of BK channels depends on Ca2+ entry through VDCC. {beta}-Adrenergic activation enhances this Ca2+ influx, and therefore BK channel activity. In addition, {beta}-adrenergic activation enhances the Ca2+ signaling from RyRs to BK channels (Ca2+ sparks), which leads to transient BK current activation. Pharmacological tools used in this study to inhibit each pathway are indicated. AC, adenylyl cyclase; G, G protein; PHL, phospholamban; SR, sarcoplasmic reticulum.

 
It has been reported that {beta}-adrenoceptor stimulation can cause activation of VDCC in smooth muscle cells that could be either transient (39, 40) or sustained (17, 19, 32, 36). In contrast to our results reported here, Kobayashi et al. (19), who have investigated isoproterenol and PKA effects on VDCC in UBSM, have seen inhibition rather than activation of VDCC.

Unlike our experimental conditions, Kobayashi et al. (19) used conventional whole cell configuration and not the perforated whole cell mode. The latter approach, used in our study, preserves the native environment of the cell, including cAMP and PKA. Under conventional whole cell configuration, the intracellular content is dialyzed by the pipette solution, which may wash out PKA and/or other important components of the {beta}-adrenoceptor/PKA signal transduction pathway. In addition, the study by Kobayashi et al. (19) is also contrary to the study by Smith et al. (32), who, in agreement with our results, found VDCC activation by isoproterenol in guinea pig UBSM cells. Although Smith et al. (32) used the conventional whole cell configuration, these authors also included GTP in the pipette solution, which is different from the Kobayashi et al. (19) experimental conditions. It has been suggested that the stimulatory effect of {beta}-adrenoceptor activation on VDCC is due to a direct modulation of VDCC through a stimulatory Gs protein and/or PKA. In addition, Kobayashi et al. (19) have used high Ba2+ (5 mM) as a charge carrier instead of physiological Ca2+ (2–2.5 mM) used by Smith et al. (32) and in our study. All of these differences in the experimental conditions may account for the discrepancy of the two findings.

It has been suggested that the stimulatory effect of {beta}-adrenoceptor activation on VDCC is due to a direct modulation of VDCC through a stimulatory Gs protein and/or PKA (39). Activation of VDCC by PKA is also believed to involve direct phosphorylation of one or more channel subunits, including the {alpha}1C subunit (17). In vascular myocytes, PKA-induced activation of VDCC after {beta}-adrenoceptor stimulation requires localization of PKA to a specific subcellular environment through association with PKA anchoring proteins (AKAPs) (41). AKAPs are required for the PKA-induced activation of BK channels in tracheal smooth muscle cells (37) and of KATP channels in arterial smooth muscle cells (9). Furthermore, PKA-induced activation of the BK channel requires physiological coupling between {beta}-adrenoceptor and the channel (25). A more recent study (21) reports the requirement of an association of {beta}-adrenoceptor with the pore-forming {alpha}-subunit of BK channel and an AKAP for {beta}-adrenergic regulation in neurons and smooth muscle, including UBSM. Those authors suggested that the {beta}-adrenoceptor can simultaneously interact with both BK and VDCC in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca2+- and phosphorylation-dependent modulation of BK channel (21). The lack of a direct effect of {beta}-adrenergic agonists on the BK channel, observed here, does not preclude that PKA is anchored near BK channel in UBSM.

Although we provide evidence that BK channels are not directly activated by isoproterenol upon {beta}-adrenergic activation, our data do not rule out a mechanism involving direct PKA phosphorylation of the BK channels. We found that forskolin increased single BK channel Po. Unlike isoproterenol, which activates {beta}-adrenergic receptors and adenylyl cyclase that is linked to these receptors, forskolin activates adenylyl cyclase throughout the entire cell, i.e., adenylyl cyclase that is linked to other receptors. We found that {beta}-adrenergic activation increases VDCC currents and calcium sparks, but not BK channel activity. However, the effect of forskolin on BK channels suggests that {beta}-adrenergic activation does not lead to sufficient cAMP increase or PKA activation in the vicinity of the BK channels. This lack of "direct" {beta}-adrenergic activation on BK channels could reflect a number of factors, including local phosphodiesterases buffering on cAMP in the vicinity of the BK channel or distance from the {beta}-adrenergic receptors or AKAPs to the BK channels.

Although Ca2+ entry through VDCC can contribute to the increase in intracellular Ca2+ concentration and promote UBSM contraction, it is also a source of Ca2+ to activate BK channels (13). In smooth muscle, activation of VDCC can contribute to Ca2+ spark activation and thereby increase transient BK currents. We have previously shown that Ca2+ spark frequency in vascular smooth muscle depends on Ca2+ entry through VDCC (16). In guinea pig UBSM cells, transient BK currents are activated by Ca2+ sparks (RyRs) (12), and the frequency of transient BK currents depends on Ca2+ entry through VDCC (3, 13). To study the differential effects of {beta}-adrenoceptor stimulation on UBSM BK channels, we used ryanodine as a tool to separate the two BK current components. Both transient and steady-state BK currents were detected during depolarizing steps to +10 mV (Fig. 1). Thus, by elevating Ca2+ influx through VDCC, {beta}-adrenoceptor stimulation can contribute to both steady-state and transient BK current activation.

Our data do not support a mechanism involving direct activation of the BK channels by {beta}-adrenoceptors. This conclusion is based on the observation that isoproteronol has no effect on the steady-state BK current recorded at +80 mV, a membrane potential that minimizes the driving force for Ca2+ and virtually eliminates the contribution of Ca2+ entry through VDCC (Fig. 3). Similarly, isoproterenol did not activate the steady-state BK current when VDCC were inhibited with nifedipine (Fig. 3). Under conditions when the {beta}-adrenoceptors and PKA are intact (perforated-patch whole cell configuration), isoproterenol did not change single BK channel activity (Fig. 4). The results obtained support the hypothesis that activation of VDCC provides Ca2+ to activate the BK channels. Thus it appears that activation of VDCC leads to compensatory stimulation of BK channels.

{beta}-Adrenoceptor stimulation by isoproterenol increased Ca2+ spark frequency in UBSM. We observed close correlation between the degree of isoproterenol-induced activation of Ca2+ spark frequency and transient BK current frequency (Figs. 5 and 6). Isoproterenol, through PKA activation, could act directly on the RyRs channel and/or indirectly via phospholamban phosphorylation and elevation of SR Ca2+ load, which increases the open state probability of RyR channels in skeletal and cardiac muscle (29, 30, 35). The isoproterenol-induced increase in Ca2+ spark activity and subsequent transient BK current activation should cause membrane potential hyperpolarization and oppose UBSM contraction.

The data obtained reveal multiple mechanisms by which stimulation of {beta}-adrenoceptors can lead to activation of BK channels and a decrease in UBSM excitability and contractility. The increase in steady-state and transient BK currents induced by {beta}-adrenoceptor 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.

The present study represents a step toward understanding the molecular and cellular mechanisms of {beta}-adrenergic regulation of UBSM. The results support the concept that stimulation of {beta}-adrenoceptors activates UBSM BK channels via modulation of localized intracellular Ca2+ signals, increasing VDCC-mediated Ca2+ influx and Ca2+ spark frequency. They do not, however, provide support for a Ca2+-independent mechanism. This study thus reveals key regulatory pathways involved in the control of bladder function that could provide new targets for drugs in the treatment of bladder dysfunction, such as overactive bladder and urinary incontinence.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by a GlaxoSmithKline Young Investigator Grant of The National Kidney Foundation (to G. V. Petkov) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-53832 and DK-065947 (to M. T. Nelson).


    ACKNOWLEDGMENTS
 
We thank Drs. Adrian Bonev and Thomas Heppner for their help with the Ca2+ spark experiments and data analysis, and Drs. A. Bonev, B. Etherton, L. Gonzalez-Bosc, T. Heppner, D. Hill-Eubanks, J. Ledoux, and K. Thorneloe for the critical reading of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. V. Petkov, Dept. of Pharmacology, Univ. of Vermont, Given Bldg., Rm. B-331, 89 Beaumont Ave., Burlington, VT 05405-0068 (e-mail: Georgi.Petkov{at}uvm.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.


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