Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of K_Ca1.1 channel

Abstract

Pharmacological blockade of cyclic nucleotide phosphodiesterase (PDE) can relax human urinary bladder smooth muscle (UBSM); however, the underlying cellular mechanism is unknown. In this study, we investigated the effects of PDE pharmacological blockade on human UBSM excitability, spontaneous and nerve-evoked contractility, and determined the underlying cellular mechanism mediating these effects. Patch-clamp electrophysiological experiments showed that 3-isobutyl-1-methylxanthine (10 μM), a nonselective PDE inhibitor, caused ∼3.6-fold increase in the transient K_Ca1.1 channel current frequency and ∼2.5-fold increase in the spontaneous transient hyperpolarization frequency in UBSM-isolated cells. PDE blockade also caused ∼5.6-mV hyperpolarization of the UBSM cell membrane potential. Blocking the K_Ca1.1 channels with paxilline abolished the spontaneous transient hyperpolarization and the hyperpolarization effect of PDE blockade on the UBSM cell membrane potential. Live cell Ca²⁺-imaging experiments showed that PDE blockade significantly decreased the global intracellular Ca²⁺ levels. Attenuation of PDE activity significantly reduced spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and muscle tone of human UBSM isolated strips. Blockade of PDE also significantly reduced the contraction amplitude, muscle force integral, and duration of the nerve-evoked contractions induced by 20-Hz electrical field stimulation. Pharmacological inhibition of K_Ca1.1 channels abolished the relaxation effects of PDE blockade on both spontaneous and nerve-evoked contractions in human UBSM-isolated strips. Our data provide strong evidence that in human UBSM PDE is constitutively active, thus maintaining spontaneous UBSM contractility. PDE blockade causes relaxation of human UBSM by increasing transient K_Ca1.1 channel current activity, hyperpolarizing cell membrane potential, and decreasing the global intracellular Ca²⁺.

the large-conductance voltage- and Ca²⁺-activated K⁺ channel, K_Ca1.1, also known as BK channel, is highly expressed in urinary bladder smooth muscle (UBSM) and is a key regulator of human UBSM excitability and contractility (5, 10, 12, 16, 18, 21, 24). Our recent studies demonstrated that the direct pharmacological activation or inhibition of K_Ca1.1 channels with selective openers or inhibitors, respectively, is highly effective in controlling human UBSM cell membrane potential and thereby UBSM contractility (16, 18). Impaired K_Ca1.1 channel activity has been shown to be one of the major contributors to detrusor overactivity (DO) in benign prostatic hyperplasia patients, and it also contributes to neurogenic DO (2, 7, 21). These studies indicate clearly that K_Ca1.1 channels are important regulators of human UBSM function suggesting that K_Ca1.1 channels can be novel therapeutic targets for the treatment of urinary bladder dysfunction related to DO and potentially urinary retention due to UBSM underactivity (18, 21, 24).

K_Ca1.1 channels are activated by Ca²⁺ released from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs), known as “Ca²⁺ sparks” (8, 14, 26). Pharmacological manipulation of intracellular Ca²⁺ can regulate K_Ca1.1 channel activity (26). In freshly isolated guinea pig, mouse, and rat UBSM cells, activation of β-adrenergic receptors that activates the cAMP pathway increases Ca²⁺ sparks which stimulate K_Ca1.1 channel activity (6, 17, 26). K_Ca1.1 channel activation results in UBSM cell membrane potential hyperpolarization and UBSM relaxation via cAMP and cAMP-dependent protein kinase A (PKA) (6, 17, 24, 26).

cAMP is hydrolyzed by phosphodiesterase (PDE); thus, PDE pharmacological blockade can readily elevate cellular cAMP levels and activate PKA signaling pathways (3). There are 11 known PDE isoenzymes of which five (PDE1-PDE5) have been reported in the human urinary bladder (4, 20, 30). Previous studies showed that blockade of PDE1, PDE4, and PDE5 can reduce human UBSM contractility (22, 28, 29). However, the underlying cellular mechanism of PDE regulation of human UBSM function is unknown.

Our recent study, using guinea pig as an animal model, demonstrated that nonselective PDE inhibition can reduce UBSM excitability and contractility (32). However, there is no information on PDE regulation of K_Ca1.1 channels in human UBSM cells, the target species of interest for therapeutic interventions. While animal models are somehow useful for understanding the physiology and pathophysiology of human bladder disorder, such as overactive bladder (OAB), the knowledge gained from animal models cannot be directly applied to humans due to substantial species differences (1, 23). Rodent urination patterns are completely different from humans: male rodents mark their territories of dominance by means of frequent urination (11). Micturition in rodents includes external urethral bursting activity associated with pulsatile urine flow compared with continuous flow in higher mammals, including humans, under normal physiological conditions. Most likely, this distinct rodent behavior is determined by differential regulatory mechanisms, including differences in the regulation of UBSM ion channels. Also, the patterns of action potentials of human and guinea pig UBSM are substantially different (12). Furthermore, studies show that the PDE expression profile in humans is different from guinea pigs (19). PDE1A, PDE1B, PDE3A, PDE3B, PDE4A-C mRNA are expressed in human cardiac myocytes but are not detectable in guinea pig cardiac myocytes (19). The PDE2A, PDE4D, and PDE5A relative mRNA levels in human cardiac myocytes are much lower than in guinea pig myocytes (19). Rolipram, a PDE4-selective inhibitor, inhibits PDE4 in human neutrophil with an IC₅₀ value of 4.52 ± 1.55 μM which is higher than that in guinea pig neutrophil (0.56–1.17 μM) (9). Due to the significant differences between human and guinea pig PDEs, it is important to obtain human data to verify the findings in guinea pig, and to better understand human bladder physiology. The data collected from human UBSM will lay the foundation of further investigation for the therapeutic potential of selective PDE inhibitors in the treatment of bladder dysfunctions.

MATERIALS AND METHODS

Human UBSM tissue collection.

Human UBSM tissue specimens were obtained from routine open bladder surgeries for bladder cancer according to protocol HR 16918, which was reviewed and approved by the Medical University of South Carolina Institutional Review Board. All specimens were obtained from patients without a preoperative history of OAB. We used 13 male and 4 female patients, 12 Caucasian and 5 African American from 46 to 79 years of age (mean 64.9 ± 2.2 yr).

UBSM single cell isolation.

UBSM single cells were freshly isolated as described previously (15, 16, 18). Briefly, 1–2 UBSM strips (5- to 10-mm long, 2- to 4-mm wide) free of mucosa were incubated in 2 ml of dissection solution (DS) at 37°C for 30 min (Solutions and drugs) supplemented with 1 mg/ml bovine serum albumin (BSA), 1 mg/ml papain, and 1 mg/ml dl-dithiothreitol. The UBSM tissues were then transferred to 2 ml of DS supplemented with 1 mg/ml BSA, 0.5 mg/ml collagenase, 0.5 mg/ml trypsin inhibitor, 100 μM CaCl₂ and incubated at 37°C for 20–25 min followed by three washouts with DS supplemented with 1 mg/ml BSA. Individual cells were released from the tissue by passing the enzyme-treated strips through a fire-polished Pasteur pipette. The freshly isolated UBSM cells were used within 8 h following isolation.

Electrophysiological recordings.

The amphotericin-B perforated whole cell patch-clamp technique was used for electrophysiological recordings on freshly isolated single human UBSM cells. Only cells that adhered to the glass bottom, which was not coated with poly-l-lysine, of the recording chamber were used in the experiments. Patch-clamp recordings were conducted using a system equipped with Axopatch 200B amplifier controlled with pCLAMP 10.2 software (Molecular Devices, Union City, CA), Digidata 1440A, and an eight-pole Bessel filter 900CT/9L8L (Frequency Devices, Ottawa, IL). The patch-clamp glass pipettes with fire-polished tips were made from borosilicate glass (Sutter Instruments, Novato, CA) using a Narishige PP-830 puller and a Micro Forge MF-830 fire polisher (Narishige Group, Tokyo, Japan). Pipette resistance was 4 to 6 MΩ. The spontaneous transient outward currents (STOCs), which are mediated by the K_Ca1.1 channels, were recorded at a holding potential of −40 mV and UBSM cell membrane potential was recorded using the current-clamp mode of the patch-clamp technique (16). The average capacitance of 13 cells was 29.4 ± 2.6 pF. All patch-clamp experiments were conducted at room temperature (22–23°C). Voltages were corrected for the junction potential of ∼−10 mV.

Monitoring the change of intracellular Ca²⁺ levels in freshly isolated human UBSM cells.

The intracellular Ca²⁺ levels were monitored using a fluorescent probe, fura 2-AM, as previously described (18). Briefly, UBSM cells were placed on a 35-mm glass bottom dish coated with poly-l-lysine and were incubated with 2 μM fura 2-AM in extracellular solution for 30 min in the dark. Fura 2-AM was washed out three times with fresh extracellular solution. Cells were imaged with an Olympus IX81 motorized inverted microscope equipped with a ×40 oil objective and MetaFluor 7.7.2.0 software. The intracellular Ca²⁺ level was expressed as fura-2 fluorescence emission intensity ratio at 510 nm determined with a 200-ms exposure at 340 and 380 nm (F340/F380) every 3 s. All Ca²⁺ imaging experiments were carried out at room temperature (∼22–23°C).

Isometric UBSM tension recordings.

Isometric contractions of UBSM-isolated strips were measured as previously described (16, 18). Briefly, UBSM strips were secured to isometric force displacement transducers and were suspended in temperature-controlled (37°C) water-jacketed tissue baths containing 10 ml physiological saline solution (PSS) aerated with 95% O₂-5% CO₂, pH 7.4. The UBSM strips were subjected to an initial tension of 1 g, and the strips were washed with fresh PSS every 15 min during an equilibration period of 45–60 min. To minimize the potential effects of neurotransmitters released from the UBSM neurons, the experiments on spontaneous UBSM contractions were performed in the presence of 1 μM tetrodotoxin (TTX), a selective blocker of the neuronal Na⁺ channels.

Nerve-evoked contractions were induced by electrical field stimulation (EFS) using a pair of platinum electrodes mounted in the tissue baths parallel to the UBSM strips. The EFS pulse parameters were as follows: 0.75-ms pulse width, 20-V pulse amplitude, 3-s stimulus duration, and polarity was reversed for alternating pulses. After the equilibration period, the UBSM strips were subjected to continuous repetitive EFS with a frequency of 20 Hz at 1-min intervals. The EFS pulses were generated using a PHM-152I stimulator and the UBSM contractions were recorded using a Myomed myograph system (MED Associates, St. Albans, VT).

Solutions and drugs.

The nominally Ca²⁺-free DS contained (in mM) 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 HEPES, and 2 MgCl₂, pH 7.3, adjusted with NaOH. The extracellular solution for whole cell patch-clamp 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. The Ca²⁺-containing PSS 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. Trypsin inhibitor, BSA, and amphotericin-B were obtained from Thermo Fisher Scientific (Fair Lawn, NJ). Papain was purchased from Worthington Biochemical (Lakewood, NJ). IBMX, paxilline, fura 2-AM, collagenase (type II), and TTX (in citrate buffer) were purchased from Sigma (St. Louis, MO).

Data analysis and statistics.

The membrane potential was analyzed using Clampfit 10.2 (Molecular Devices). The STOCs and spontaneous transient hyperpolarizations (STHs) frequency and amplitude, the UBSM phasic contraction amplitude, muscle force integral, contraction duration, contraction frequency, and muscle tone were analyzed using MiniAnalysis (Synaptosoft, Decatur, GA). Muscle force integral was determined by integrating the area under the phasic contraction force-time baseline curve. The relative change of muscle tone was determined by measuring changes of the phasic contraction baseline curve. The spontaneous and 20-Hz EFS-induced contraction parameters were normalized to the respective control (taken to be 100%). Data were further analyzed with GraphPad Prism 4.03 software (GraphPad Software, La Jolla, CA) and expressed as means ± SE, n = the number of UBSM cells or strips, and N = the number of patients. Statistical significance was tested using paired Student's t-test and P < 0.05 was considered significant.

RESULTS

Blockade of PDE increased the STOCs frequency in human UBSM-isolated cells.

In UBSM cells, localized Ca²⁺ release from SR RyRs, known as “Ca²⁺ sparks,” transiently activates the closely located K_Ca1.1 channels and generates STOCs (16). The STOCs were recorded at a holding potential of −40 mV, which is close to UBSM resting membrane potential (25). In human UBSM-isolated cells, PDE blockade with 3-isobutyl-1-methylxanthine (IBMX; 10 μM) increased the STOCs frequency to 361.1 ± 64.8% of the control activity (n = 4, N = 4; P < 0.05), without a significant effect on the average STOCs amplitude (106.6 ± 5.9%; n = 4, N = 4; P > 0.05; Fig. 1). STOCs cause UBSM cell membrane hyperpolarization. Hence, we next evaluated the effect of PDE blockade on human UBSM cell resting membrane potential.

Fig. 1.

Pharmacological blockade of phosphodiesterase (PDE) with 10 μM 3-isobutyl-1-methylxanthine (IBMX) increased the spontaneous transient outward currents (STOCs) frequency in human urinary bladder smooth muscle (UBSM)-isolated cells. A: original recording illustrating that PDE blockade increased the frequency of STOCs in a single UBSM cell. Portions of the recording are shown on an expanded time scale before and after IBMX application. B: summary data showing that PDE blockade significantly increased the STOCs frequency without a significant effect on the STOCs amplitude [n = 4, N = 4; *P < 0.05; nonsignificant (NS)]. The STOCs frequency and average amplitude under control conditions were taken to be 100%, respectively.

Blockade of PDE increased the STHs frequency and hyperpolarized the membrane potential of isolated human UBSM cells.

The cell membrane potential was recorded using the perforated patch-clamp technique in the current-clamp mode (I_h = 0). In cells exhibiting STHs, blockade of PDE increased STHs frequency to 252.8 ± 27.3% of the control values (n = 4, N = 4; P < 0.05; Fig. 2, A and B), without any effect on the average STHs amplitude (96.6 ± 6.8% of the control values; n = 4, N = 4; P > 0.05; Fig. 2). Blocking K_Ca1.1 channels with paxilline (1 μM), following IBMX application, abolished the STHs and depolarized the cell membrane potential. Under control conditions, the UBSM cell membrane potential was −20.6 ± 3.4 mV, and PDE blockade hyperpolarized the membrane potential to −26.3 ± 3.2 mV (n = 5, N = 4; P < 0.05; Fig. 2C). The subsequent addition of 1 μM paxilline reversed the membrane potential hyperpolarization induced by the blockade of PDE and further depolarized the membrane potential to −17.4 ± 6.5 mV (n = 5, N = 4; P < 0.05 vs. IBMX; Fig. 2, A and C).

Fig. 2.

Pharmacological blockade of PDE with 10 μM IBMX increased the spontaneous transient hyperpolarizations (STHs) frequency and hyperpolarized human UBSM cell membrane potential. A: original current-clamp recording illustrating that PDE blockade increased the STHs frequency and hyperpolarized the UBSM cell membrane potential, and paxilline (1 μM) abolished STHs. Portions of the recording are shown on an expanded time scale. B: summary data showing that PDE blockade significantly increased STHs frequency but had no effect on the STHs amplitude (n = 4, N = 4; *P < 0.05; NS). C: summary data showing that PDE blockade significantly hyperpolarized the UBSM cell membrane potential, and paxilline reversed the hyperpolarization effect of IBMX (n = 5, N = 4; *P < 0.05).

To further confirm that the hyperpolarization effect of PDE blockade was mediated by the K_Ca1.1 channels, K_Ca1.1 channels were inhibited with paxilline before the application of IBMX. Paxilline (1 μM) abolished the STHs and eliminated the hyperpolarization effect of PDE blockade in UBSM cells. The membrane potentials were −19.9 ± 2.9 mV in the presence of paxilline and −19.6 ± 3.1 mV in the presence of both paxilline and IBMX [n = 4, N = 4; nonsignificant (NS); Fig. 3]. These results confirmed that the hyperpolarization effect of PDE blockade on UBSM cell membrane potential is mediated by the K_Ca1.1 channels. Since the human UBSM cell membrane potential directly affects the intracellular Ca²⁺ levels (18), we next investigated whether the hyperpolarization of the membrane potential induced by PDE blockade influences the intracellular Ca²⁺ levels.

Fig. 3.

Pharmacological inhibition of K_Ca1.1 channels with paxilline abolished the hyperpolarizing effect of IBMX in human UBSM-isolated cells. A: original current-clamp recording illustrating that 1 μM paxilline abolished STHs and that the subsequent addition of IBMX (10 μM) did not have any additional effect on the membrane potential. Portions of the recording are shown on an expanded time scale. B: summary data showing that in the presence of 1 μM paxilline, the addition of IBMX did not cause any change in the UBSM cell membrane potential (n = 4, N = 4; NS).

PDE blockade decreased the intracellular Ca²⁺ levels in human UBSM-isolated cells.

The global Ca²⁺ levels decreased significantly from 0.71 ± 0.03 under control condition to 0.55 ± 0.01 following the addition of 10 μM IBMX (n = 22, N = 4; P < 0.05; Fig. 4). We next determined how the decrease of Ca²⁺ levels affects human UBSM spontaneous phasic contractions.

Fig. 4.

Pharmacological PDE blockade with 10 μM IBMX reduced the intracellular Ca²⁺ levels in freshly isolated human UBSM cells. A: original recording illustrating that PDE blockade decreased the cellular Ca²⁺ levels in a single UBSM cell shown as a ratio of fura-2 fluorescence emission intensities at 510 nm with excitation at 340 and 380 nm. B: summary data indicating that PDE blockade significantly decreased the intracellular Ca²⁺ levels (n = 22, N = 4; *P < 0.05).

PDE blockade suppressed the spontaneous phasic (myogenic) contractions of human UBSM-isolated strips.

Human UBSM-isolated strips exhibit spontaneous phasic contractions (Fig. 5). IBMX (10 μM) reduced the UBSM phasic contraction amplitude, muscle force integral, contraction duration, contraction frequency, and muscle tone to 33.5 ± 4.1, 31.7 ± 3.4, 37.7 ± 2.5, 48.8 ± 3.7, and 48.8 ± 3.0% of the control activity, respectively (n = 8, N = 4; P < 0.05; Fig. 5, A and B). To determine the role of K_Ca1.1 channels in PDE-induced inhibition of human UBSM spontaneous contractions, K_Ca1.1 channels were blocked with paxilline. Paxilline (1 μM) alone increased the UBSM phasic contraction amplitude, muscle force integral, duration, frequency, and tone to 243.6 ± 5.7, 211.7 ± 9.6, 142.6 ± 2.3, 158.1 ± 4.3, and 146.1 ± 0.9% of the control activity, respectively (n = 5, N = 5; P < 0.05; Fig. 5, C and D). In the presence of 1 μM paxilline, the subsequent addition of IBMX had no effect on the phasic contraction amplitude, muscle force integral, duration, frequency, and muscle tone (n = 5, N = 5; P > 0.05 vs. paxilline; Fig. 5, C and D). The fact that pharmacological blockade of the K_Ca1.1 channels diminished the UBSM relaxation following PDE blockade suggests that K_Ca1.1 channels are critical to the PDE-mediated suppression of human UBSM spontaneous contractility.

Fig. 5.

PDE blockade with IBMX significantly reduced spontaneous phasic contractions of human UBSM-isolated strips. A: representative recording of a UBSM-isolated strip exhibiting spontaneous phasic contractions and illustrating that IBMX (10 μM) inhibited the contractions. B: summary data showing that PDE blockade reduced the UBSM spontaneous contraction amplitude, muscle force integral, duration, frequency, and tone (n = 8, N = 4; *P < 0.05 vs. control). C: representative recording of a UBSM-isolated strip exhibiting spontaneous phasic contractions showing that the K_Ca1.1 channel blocker paxilline enhanced the spontaneous phasic contractions and that the subsequent addition of IBMX did not have any further effect on the contractions. D: summary data showing that paxilline increased the UBSM spontaneous contraction amplitude, muscle force integral, duration, frequency, and tone and that the subsequent addition of 10 μM IBMX did not have any further effect on UBSM contraction parameters (n = 5, N = 5; *P < 0.05 vs. control; NS vs. paxilline). All these experiments were performed in the presence of 1 μM tetrodotoxin (TTX).

PDE blockade suppressed nerve-evoked contractions of human UBSM-isolated strips.

Nerve-evoked UBSM contractions were induced by 20-Hz EFS. EFS induces acetylcholine and ATP release from bladder nerve terminals and causes an increase in UBSM contractions. PDE blockade significantly reduced the 20-Hz EFS-induced UBSM contraction amplitude, muscle force integral, and duration to 48.2 ± 1.6, 49.7 ± 1.0, and 56.6 ± 4.0% of the control activity, respectively (n = 5, N = 5; P < 0.05; Fig. 6, A and B). Blocking K_Ca1.1 channels with paxilline (1 μM) increased the 20-Hz EFS-induced contraction amplitude, muscle force integral, and duration to 132.6 ± 0.8, 137.2 ± 2.6, and 129.1 ± 2.3% of the control activity, respectively (n = 4, N = 4; P < 0.05; Fig. 6, C and D). In the presence of 1 μM paxilline, PDE blockade had no effect on the EFS-induced contraction amplitude, muscle force integral, and duration (n = 4, N = 4; P > 0.05 vs. paxilline; Fig. 6, C and D). These data indicate that PDE plays a major role in the suppression of nerve-evoked contractions induced by K_Ca1.1 channel blockade.

Fig. 6.

PDE blockade with 10 μM IBMX significantly reduced 20-Hz electrical field stimulation (EFS)-induced phasic contractions of human UBSM-isolated strips. A: representative recording illustrating that PDE blockade suppressed the 20-Hz EFS-induced phasic contractions. B: summary data showing that PDE blockade reduced the 20-Hz EFS-induced UBSM contraction amplitude, muscle force integral, and duration (n = 5, N = 5; *P < 0.05 vs. control). C: representative recording showing that paxilline (1 μM) increased the 20-Hz EFS-induced phasic contractions and that the subsequent addition of 10 μM IBMX did not have any further effect on the contractions. D: summary data showing that 1 μM paxilline increased the 20-Hz EFS-induced contraction amplitude, muscle force integral, and duration and that the subsequent addition of 10 μM IBMX did not have any further effect on UBSM contraction parameters (n = 4, N = 4; *P < 0.05 vs. control; NS vs. paxilline).

DISCUSSION

For the first time, we determined the cellular mechanism of PDE regulation of human UBSM function using pharmacological PDE blockade. Our results indicate that in human UBSM, PDE is constitutively active and that pharmacological PDE blockade significantly 1) increases STOCs frequency, 2) enhances STHs frequency and hyperpolarizes the cell membrane potential, 3) decreases the intracellular Ca²⁺ levels, 4) attenuates human UBSM spontaneous phasic and nerve-evoked contractions, and 5) these effects are mediated by the K_Ca1.1 channels. The results provide compelling evidence that PDE and K_Ca1.1 channels are critical for controlling human UBSM excitability and contractility and that PDE pharmacological manipulation can effectively suppress the contractility of human UBSM via K_Ca1.1 channel activation.

This study, in addition to some earlier studies, demonstrates that blockade of PDEs can induce human UBSM relaxation, and it suggests that PDEs can be potential therapeutic targets for OAB treatment (22, 29, 31). Furthermore, the current study reveals the underlying mechanism of PDE regulation of human UBSM function, which is essential for developing novel strategies to treat OAB by targeting PDE pharmacologically. The data presented here indicate that in human UBSM-isolated cells, PDE blockade increases STOCs and STHs frequency and hyperpolarizes UBSM cell resting membrane potential (Figs. 1 and 2). These results also demonstrate that the hyperpolarization effect of PDE blockade on human UBSM cells is mediated by the K_Ca1.1 channels. The role of K_Ca1.1 channels was confirmed by the fact that pharmacological inhibition of K_Ca1.1 channels with paxilline abolished the hyperpolarization effect of PDE blockade on human UBSM cells (Fig. 3). This study reveals that in human UBSM, PDE is constitutively active and controls the K_Ca1.1 channels, key mediators of human UBSM function.

K_Ca1.1 channels have been shown to be fundamental regulators of human UBSM excitability and contractility by controlling the cell membrane potential and repolarization phase of UBSM action potential (13, 16). Selective K_Ca1.1 channel openers hyperpolarize the cell membrane potential and decrease the global intracellular Ca²⁺ by limiting the Ca²⁺ influx through l-type voltage-dependent Ca²⁺ (Ca_V) channels in human UBSM cells (16). The indirect activation of K_Ca1.1 channels upon PDE blockade results in reduction of intracellular Ca²⁺ (Fig. 4), an effect similar to the one of K_Ca1.1 channel openers, which leads to the suppression of human UBSM contractility.

The transient elevation of intracellular Ca²⁺ concentration due to Ca²⁺ influx through Ca_V channels during the spontaneous action potentials activates UBSM myogenic phasic contractions (5, 12, 18, 24). The Ca²⁺ entry was limited by hyperpolarization of membrane potential via K_Ca1.1 channel activation. For the first time, we demonstrated that PDE blockade significantly reduces the spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and muscle tone in human UBSM-isolated strips and that K_Ca1.1 channels mediated these effects (Fig. 5). When K_Ca1.1 channels were pharmacologically blocked with paxilline, PDE blockade had reduced effect on the human UBSM myogenic contractility, further demonstrating a key role of the interplay between the PDE and K_Ca1.1 channels in the regulation of human UBSM phasic contractions (Fig. 5).

During bladder voiding, the activation of parasympathetic cholinergic nerves in the UBSM evokes forceful contractions. However, an abnormal neuronal activity could excessively increase UBSM contractility and cause DO (1). K_Ca1.1 channels are important regulators of the nerve-evoked UBSM contractions (16, 27). Direct pharmacological K_Ca1.1 channel activation suppresses the nerve-evoked human UBSM contractility (18). The data presented here show that PDE blockade attenuated the nerve-evoked contractions (Fig. 6). Blocking K_Ca1.1 channels with paxilline increased the nerve-evoked human UBSM contractions and completely abolished the relaxation effect of PDE blockade. This suggests a K_Ca1.1 channel-dependent mechanism in the suppression of the nerve-evoked human UBSM contractions by PDE blockade (Fig. 6).

This study fills a gap in our understanding of PDE regulation of human UBSM function. Our data suggest that PDEs are constitutively active and contribute to the maintenance of human UBSM spontaneous contractile activity. The results reported here for the first time suggest that human UBSM response to PDE blockade, including UBSM cell hyperpolarization, reduced intracellular Ca²⁺ levels, and decreased UBSM contractility, results from the activation of the K_Ca1.1 channels. This study contributes a novel insight to human cell physiology revealing that PDE blockade can effectively suppress human UBSM contractility primarily via K_Ca1.1 channel activation. Inhibitors for human UBSM-specific PDEs could be an effective approach to reduce abnormal UBSM contractility without the adverse effects of antimuscarinic drugs, the current main pharmacological treatment for OAB.

GRANTS

This study was supported by National Institutes of Health Grant DK084284 to G. V. Petkov.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: W.X., Q.C., R.P.S., and G.V.P. performed experiments; W.X., Q.C., R.P.S., and G.V.P. analyzed data; W.X., Q.C., R.P.S., E.S.R., and G.V.P. interpreted results of experiments; W.X., Q.C., R.P.S., E.S.R., and G.V.P. prepared figures; W.X., Q.C., R.P.S., E.S.R., and G.V.P. drafted manuscript; W.X., Q.C., R.P.S., E.S.R., and G.V.P. edited and revised manuscript; W.X., Q.C., R.P.S., E.S.R., and G.V.P. approved final version of manuscript; E.S.R. and G.V.P. conception and design of research.