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CELLULAR AND MOLECULAR

BL-1249 [(5,6,7,8-Tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine]: A Putative Potassium Channel Opener with Bladder-Relaxant Properties

Neuroscience Drug Discovery (S.T., M.J.P., G.T., C.G., T.N., L.S., R.A.M., N.J.L.) and Lead Discovery and Profiling (R.J.K., D.G.H., D.W.), Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut

Received October 4, 2004; accepted December 15, 2004.

	Abstract

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BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] produced a concentration-dependent membrane hyperpolarization of cultured human bladder myocytes, assessed as either a reduction in fluorescence of the voltage-sensitive dye bis-(1,2-dibutylbarbituric acid)trimethine oxonol (EC₅₀ = 1.26 ± 0.6 µM) or by direct electrophysiological measurement (EC₅₀ = 1.49 ± 0.08 µM). BL-1249 also produced a membrane hyperpolarization of acutely dissociated rat bladder myocytes. Voltage-clamp studies in human bladder cells revealed that BL-1249 activated an instantaneous, noninactivating current that reversed near E_K. The BL-1249-evoked outward K⁺ current was insensitive to blockade by glyburide, tetraethylammonium, iberiotoxin, 4-aminopyridine, apamin, or Mg²⁺. However, the current was inhibited by extracellular Ba²⁺ (10 mM). In in vitro organ bath experiments, BL-1249 produced a concentration-dependent relaxation of 30 mM KCl-induced contractions in rat bladder strips (EC₅₀ = 1.12 ± 0.37 µM), yet had no effect on aortic strips up to the highest concentration tested (10 µM). The bladder relaxation produced by BL-1249 was partially blocked by Ba²⁺ (1 and 10 mM) but not by apamin, iberiotoxin, 4-aminopyridine, glyburide, or tetraethylammonium. In an anesthetized rat model, BL-1249 (1 mg/kg i.v.) decreased the number of isovolumic contractions, without significantly affecting blood pressure. Thus, BL-1249 behaves as a potassium channel activator that exhibits bladder versus vascular selectivity both in vitro and in vivo. A survey of potassium channels exhibiting sensitivity to extracellular Ba²⁺ at millimolar concentration revealed that the expression of the K_2P2.1 (TREK-1) channel was relatively high in human bladder cells versus human aortic cells, suggesting this channel as a possible candidate target for BL-1249.

A number of K_ATP channel openers, identified over the past several years, have indeed demonstrated that activation of potassium channels can reduce the spontaneous or stimulus-induced contractions of isolated bladder tissues across several species, including humans (Fovaeus et al., 1989; Buckner et al., 2000; for review, see Sellers et al., 2002; Kumar et al., 2003). What is more, several whole-animal studies have shown that spontaneous bladder contractions can be inhibited without reducing the bladder pressure developed during the micturition response (Wojdan et al., 1999; Yu and de Groat, 1999). These data suggested the potential utility of K_ATP channel openers for the treatment of overactive bladder by inhibiting the unwanted spontaneous contractions without affecting normal bladder emptying. Unfortunately, however, reported K_ATP channel openers exhibited only partial or no bladder selectivity either in vitro or in vivo (Edwards et al., 1991; Chess-Williams et al., 1999; Wojdan et al., 1999; Brune et al., 2002; Fabiyi et al., 2003). To date, K_ATP channel openers have not proven to be clinically effective, due in part to cardiovascular side effects (Komersova et al., 1995), casting doubt on whether true bladder selectivity can be achieved by targeting K_ATP channels.

Recently, maxi-K⁺ channels have been proposed as another appealing target for treating bladder overactivity. Several maxi-K⁺ channel openers have been shown to hyperpolarize membrane potential of bladder myocytes (Siemer et al., 2000) and relax precontracted bladder strips (Malysz et al., 2004). However, the reported maxi-K⁺ openers exhibited similar potencies at either bladder or vascular smooth muscle, suggesting that the activation of maxi-K⁺ channels in the smooth muscle membrane is unlikely to provide bladder selectivity (Malysz et al., 2004).

The present paper describes the functional activity and pharmacological properties of BL-1249, a putative KCO, in bladder smooth muscle cells. The study also compares the effect of BL-1249 with the established KCO cromakalim on in vitro and in vivo bladder activity and mean arterial blood pressure (MABP). Finally, the paper surveys the distribution of Ba²⁺-sensitive potassium channels in human bladder and aortic myocytes in search of potential potassium channel targets for the action of BL-1249.

	Materials and Methods

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All protocols involving animal experimentation were approved by the Bristol-Myers Squibb Institutional Animal Care and Use Committee and were in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Materials. BL-1249 and (-)-cromakalim were synthesized at Bristol-Myers Squibb (Wallingford, CT). The following reagents were purchased: DiBAC₄(3) (Molecular Probes, Eugene, OR), papain (Worthington Biochemicals, Freehold, NJ), RNeasy Mini kit 74104 (QIAGEN, Valencia, CA), SYBR Green PCR kit 4304886 (Applied Biosystems, Foster City, CA), RNaseOUT recombinant ribonuclease inhibitor 10777-019 and SuperScript II RT 12371-019 (Invitrogen, Carlsbad, CA), and Hanks' balanced salt solution (no. 14175-095) (Invitrogen). All cell culture products were purchased from Cambrex Bio Science Walkersville (Walkersville, MD). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Cell Culture. Human urinary bladder (part no. CC-2533) and aortic (part no. CC-2571) smooth muscle cells were obtained from Cambrex Bio Science Walkersville and used at passages 3 to 12. To determine the population of smooth muscle-type cells in the culture, the cells were periodically stained with fluorescein isothiocyanate-conjugated antibodies against smooth muscle -actin (part no. F3777; Sigma-Aldrich). Rat bladder cells were acutely isolated as described elsewhere (Thorneloe and Nelson, 2003) and used within 6 h.

Membrane Potential-Sensitive Fluorescence Assay. The functional activity of compounds was initially determined by measuring changes in DiBAC₄(3) fluorescence (Gopalakrishnan et al., 1999), using a 384-well fluorescence plate reader (Molecular Devices, Sunnyvale, CA). Briefly, bladder smooth muscle cells were plated onto clear 384-well plates (part no. 353961; Falcon, Cowley, UK) and allowed to form a 90 to 95% confluent monolayer. Prior to the experiment, the growth media were removed, and cells were incubated in assay buffer (Hanks' Balanced Salt Solution supplemented with 2 mM CaCl₂, 1 mM MgCl₂, 5 mM glucose, and 10 mM HEPES, pH 7.3) containing 5 µM DiBAC₄(3) for 30 to 45 min. Amaranth (2 mM) and Tartrazine (1 mM) were added to the assay buffer to suppress background fluorescence. DiBAC₄(3) was excited with 488-nm wavelength light, and fluorescence was measured using a 535-/25-nm bandpass emission filter. After a baseline readout of 1 min, the assay buffer containing test compounds at various concentrations was added to the cells. Changes in fluorescence were measured for 8 to 10 min after the addition of compounds. The compound-induced fluorescence response was corrected for the background fluorescence, and relative fluorescence intensity change was used as an indication of the change in membrane potential. The assay was performed at room temperature.

Whole-Cell Recording. Whole-cell current and membrane potential measurements were made with pipettes (resistance, 2.5–4 M; tip diameter, 1–2 µm) constructed from 1.5-mm internal diameter borosilicate glass capillaries using a multistage micropipette puller (Sutter Instrument Company, Novato, CA). Extracellular solution contained: 140 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 1.0 mM MgCl₂, 10 mM HEPES, and 5 mM glucose, adjusted to pH 7.3 with NaOH (305–310 mOsM). Intracellular solution contained: 5 mM EGTA, 150 mM KCl, 1.0 mM MgCl₂, 2.5 mM CaCl₂ (free Ca²⁺ concentration estimated to be 113 nM), and 10 mM HEPES, adjusted to pH 7.3 with KOH (290–300 mOsM). Data were acquired and analyzed using HEKA software (Pulse, version 8.6; HEKA Elektronik, Lambrecht, Germany). Currents were measured with an EPC-9 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany), filtered at 1 kHz, and digitized at 5 kHz. Increasing concentrations of test compounds were applied via a local perfusion system (AutoMate Scientific Inc., San Francisco, CA). Based upon measured changes in open pipette potential following solution changes, the estimated solution change time was approximately 20 ms. All the experiments were performed at room temperature.

Bladder and Aorta Relaxation Study. Male rats (Sprague-Dawley, 250–350 g; Harlan, Indianapolis, IN) were sacrificed by decapitation. The bladder was excised and cleaned of connective tissue. Bladder strips cut from the bladder body were mounted in organ baths containing normal Krebs' buffer (118.4 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.3 mM MgSO₄, 1.8 mM CaCl₂, 10.1 mM glucose, and 25 mM NaHCO₃ gassed with 95% O₂/5% CO₂ and maintained at pH 7.4 and 37°C). Changes in muscle tension were measured using isometric force transducers (Grass, FT-03) and recorded with an AcqKnowledge data acquisition system (AcqKnowledge for MP100WS; Biopac Systems Inc., Goleta, CA).

After an equilibration period of at least 45 min at 1 g resting tension, the tissues were stimulated to contract by the addition of KCl (30 mM KCl final). When the contraction became stable, test compounds were added cumulatively with a 30-min incubation period at each concentration. To test the effects of channel blockers on BL-1249-evoked relaxation, tissues were exposed to the blockers for 15 min prior to the application of BL-1249. At the end of each experiment, KCl was added to give a final concentration of 80 mM. Relaxation responses were expressed as a percent relaxation of the 30 mM KCl contraction, unless indicated otherwise. To account for significant spontaneous activity in the bladder strips, average tension measured during a 5-min recording period was used in the data analysis.

For the selectivity studies, the thoracic aorta was removed, cleaned of connective tissues, and cut into 4- to 5-mm-long strips. The vessels segments strips were mounted in the organ baths and treated as described above.

In Vivo Studies. BL-1249 and cromakalim were evaluated in an anesthetized rat model for their ability to alter isovolumic bladder contractions (Guarneri et al., 1993; Testa at al., 2001). Female Wistar rats (Harlan Sprague-Dawley), 200 to 350 g and 6 to 8 weeks old, were used for all studies. BL-1249 and cromakalim were dissolved in 100% polyethylene glycol (PEG400; Sigma-Aldrich), and dosed at a volume of 0.125 ml/kg i.v. Rats were anesthetized with urethane (1.2 g/kg), and the urinary bladder was catheterized via the urethra with polyethylene tubing (PE-50). The catheter, which was tied in placed with a ligature around the external urethral orifice, was connected to a syringe and a pressure transducer. Additional catheters were placed in a femoral vein (for compound or vehicle administration) and in a carotid artery (for MABP and heart rate measurement).

After completion of the surgery, bladder and arterial transducers were connected to an eight-channel Gould TA4000 physiograph and Gould PONEMAH digital acquisition analysis system (Gould Instrument Systems Inc., Cleveland, OH). The bladder was filled with physiological saline (37°C) in incremental volumes until spontaneous bladder contractions occurred, typically at 0.6 to 1.2 ml of infusate. A 15-min baseline was recorded once contractions attained a consistent rhythm. At the completion of the baseline period, BL-1249 (1 mg/kg), cromakalim (0.03–1 mg/kg), or vehicle (100% PEG400, 0.125 ml/kg) was administered intravenously. Bladder contractions were recorded for 30 min post-treatment. The data were separated into two 15-min periods for analysis. The urodynamic parameters measured during the experiment were: the total number of contractions observed, average peak contraction pressure, and average basal pressure; MABP was also measured. Data were recorded from 9 to 11 animals per dose group.

Statistical significance of experimental results was determined using the SAS-JMP statistical package (SAS Institute, Cary, NC). A one-tailed Student's t test was used to determine significance, with p < 0.05 considered significant.

Real-Time PCR. Total RNA was isolated from cultured human bladder and aortic smooth muscle cells using the RNeasy Mini protocol from QIAGEN. Up to 100 µg of total RNA obtained from the cell preparation was treated with DNase I (50 units) and RNaseOUT (90 units). The RNA was further cleaned using the QIAGEN RNA clean-up protocol. Two micrograms of total RNA was reverse transcribed to cDNA using 50 ng of random hexamer primers, 1 mM dNTP, 1x RT buffer, 5 mM MgCl₂, 10 mM dithiothreitol, 40 units of RNaseOUT recombinant ribonuclease inhibitor, and 50 units of SuperScript II RT. For the real-time PCR reaction, the SYBR Green PCR kit protocol (Applied Biosystems) was followed. In brief, 200 ng of RNA, 300 nM forward and reverse primer (see Table 1), 1x SYBR PCR Buffer, 3 mM MgCl₂, 100 nM dNTP blend, and 0.25 units of Amplitaq Gold were used in a 50-µl total reaction. The reactions were incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The real-time data were normalized to the internal control, human -actin, and the relative quantification of samples was calculated using the Ct method, where Ct is a fractional cycle number at which the fluorescence level of the PCR product reaches an arbitrary threshold (set as a level where fluorescence is accumulating exponentially). 2^Ct was used to quantify the fold difference in message level between samples.

	Results

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Effect of BL-1249 on Membrane Potential in Cultured Bladder Smooth Muscle Cells. The activity of BL-1249 was initially evaluated by measuring its effects on the fluorescence of DiBAC₄(3) which had been loaded into human bladder smooth muscle cells. Changes in DiBAC₄(3) fluorescence reflect changes the membrane potential (Gopalakrishnan et al., 1999; Whiteaker et al., 2001a,b). BL-1249 evoked a concentration-dependent decrease in DiBAC₄(3) fluorescence in cultured bladder smooth muscle cells, indicating a hyperpolarization of the membrane potential, with an EC₅₀ of 1.26 ± 0.6 µM (n = 4 experiments; Fig. 1, A and B).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Effect of BL-1249 on membrane potential responses assessed by the membrane potential sensitive dye DiBAC4(3) in cultured human urinary bladder myocytes. A, representative traces showing changes in DiBAC4(3) fluorescence reflective of membrane potential hyperpolarization. B, BL-1249 concentration-response curve, EC50 = 1.26 ± 0.6 µM (n = 4).

Direct measurement of the membrane potential of human bladder cells using electrophysiological methods (Fig. 2, A and B) confirmed the hyperpolarizing properties of BL-1249. BL-1249 produced a concentration-dependent hyperpolarization yielding an EC₅₀ of 1.49 ± 0.08 µM (n = 3–10 cells). In a preliminary study, BL-1249 also hyperpolarized the membrane potential of acutely isolated rat bladder myocytes, but to a lesser degree than in the human bladder cells: -17.2 ± 6.9 mV (n = 3) versus -37.3 ± 5.4 mV (n = 10) at 10 µM, respectively. The smaller hyperpolarization in the rat cells was, most likely, due to a more negative resting potential in the acutely isolated cells; -25.6 ± 2.3 mV (n = 19) and -47.9 ± 10.4 mV (n = 3) in human and rat, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effect of BL-1249 on membrane potential responses assessed by direct measurements of membrane potential in cultured human urinary bladder myocytes. A, representative record of cell membrane potential upon the addition of 0.1, 1, and 10 µM BL-1249. B, BL-1249 concentration-response curve; EC50 1.49 ± 0.08 µM (n = 3–8 cells).

Effects of BL-1249 on Whole-Cell Current. Whole-cell voltage-clamp studies were used to characterize the conductance changes underlying the BL-1249-induced membrane hyperpolarization in human bladder myocytes. Records of typical outward currents produced by a voltage step protocol (holding potential, -60 mV; 250-ms depolarizing voltage steps from -80 to +70 mV) are shown in Fig. 3, A and B. The outwardly rectifying currents measured under control conditions were relatively small up to +40 mV, where maxi-K⁺ currents are likely to be activated (Siemer et al., 2000). Consistent with the postulated contribution of maxi-K⁺ channels, the maxi-K⁺ channel blocker iberiotoxin (300 nM; Galvez et al., 1990) was found to block a large fraction of this current (see Fig. 6, A and B). BL-1249 produced large instantaneously activating, noninactivating outward currents that were readily reversible following drug washout (Fig. 3, A and B). The reversal potential of the BL-1249-induced current was near -80 mV under the physiological K⁺ gradient used in the recording conditions, indicating the current is carried by K⁺ ions (Fig. 3C).

View larger version (22K): [in this window] [in a new window]   Fig. 3. BL-249 activates large whole-cell currents in human bladder smooth muscle cells. A, representative traces of currents before and during application of BL-1249 (2 µM) and after washout. From a holding potential of -60 mV, the cell was voltage-clamped to -80 mV and sequentially depolarized to +70 mV in 10-mV increments for 250 ms. B, current-voltage relationship of the BL-1249-evoked current. The current shown is the calculated difference current obtained by subtracting the control currents at each potential from the currents produced at the corresponding test potentials in the presence of 2 µM BL-1249 (shown are mean ± S.E.M.; n = 4 cells). C, BL-1249-induced current exhibiting a reversal potential near -80 mV, indicating selective K+ conductance (300-ms voltage ramp; -120 to +60 mV from a holding potential of -80 mV; representative trace).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effects of iberiotoxin on control and BL-1249-induced currents in human bladder cells. A, iberiotoxin (300 nM) inhibits whole-cell current in bladder myocytes. B, BL-1249 (2 µM)-induced whole-cell current insensitive to iberiotoxin. Data shown as mean ± S.E.M. for four cells. For clarity, error bars in B are not shown.

Bladder and Aortic Strip Relaxation Studies. To functionally characterize the effects of compounds on bladder and vascular smooth muscle, compounds were tested for their ability to relax rat bladder and aortic strips that had been contracted by exposure to Krebs' solution containing 30 mM KCl. A contraction induced by a depolarizing concentration of KCl has previously been used to evaluate activity of K_ATP channel openers in bladder and aorta smooth muscle (Edwards et al., 1991; Masuda et al., 1991).

BL-1249 relaxed 30 mM KCl precontracted bladder strips in a concentration-dependent manner yielding an EC₅₀ of 1.1 ± 0.37 µM(n = 12; Fig. 4), yet had little inhibitory effect on the rat aorta up to the highest concentration tested (10 µM, n = 3; Fig. 4). Similar to the known KCO, cromakalim, BL-1249 produced little relaxation of 80 mM KCl-induced bladder contractions (27.4 ± 2.3% at 10 µM; n = 5), whereas the calcium channel blocker (nifedipine, 100 nM) fully relaxed both 30 and 80 mM KCl-contracted tissues (data not shown). The partial relaxation of the 80 mM KCl response suggests that another mechanism(s), beyond K⁺ channel activation, plays a minor role in the relaxant response to BL-1249.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Concentration-response curve of BL-1249 for the inhibition of 30 mM KCl-induced contraction of rat bladder and aortic strips (n = 3–12). Data shown as mean ± S.E.M. Bladder EC50 = 1.1 ± 0.37 µM.

Effects of BL-1249 on Isovolumic Bladder Contractions and MABP in the Anesthetized Rats. Isovolumic contractions are elicited by stimulation of the supraspinal micturition reflex which occurs in response to bladder filling (Guarneri et al., 1993; Testa at al., 2001). BL-1249 (1 mg/kg) significantly reduced (p < 0.01) the number of micturition contractions during the 15-min period immediately following dosing; the reduction was less but still significant for the 15- to 30-min period (p < 0.05; Fig. 5, A and B). (Note: initial experiments in rats that were not prepared for isovolumic measurements revealed that 10 mg/kg i.v., but not 1 mg/kg i.v., BL-1249 resulted in hematuria. Thus, the current experiments were restricted to the 1 mg/kg dose). For comparison, the effects of cromakalim (1 mg/kg) are also shown; cromakalim also significantly reduced the number of contractions (p < 0.01) during the 0- to 30-min postadministration period. Vehicle was without effect on the number of contractions (Fig. 5B).

View larger version (23K): [in this window] [in a new window]   Fig. 5. A, representative trace showing the effect of BL-1249 (1 mg/kg) on isovolumic bladder contractions in anesthetized rats. Arrow indicates drug application. Note a quiescence of bladder contractions after drug injection. B, inhibition of isovolumic bladder contractions by BL-1249 and cromakalim. BL-1249 (1 mg/kg), cromakalim (1 mg/kg), and vehicle (PEG400) were administered intravenously in a volume of 0.125 ml/kg. Values are shown as means ± S.E.M. *, p < 0.05; **, p < 0.01 (n = 9–11). C, effect of BL-1249 (1 mg/kg) and cromakalim (0.1 mg/kg) on MABP. BL-1249 and vehicle (PEG400) were administered intravenously at 0.125 ml/kg. Values are shown as means ± S.E.M. (n = 9–11).

Administration of vehicle (PEG400, 0.125 ml/kg) or BL-1249 at 1 mg/kg (n = 9–11) had no effect on MABP during the 0- to 15-min period immediately following administration (Fig. 5C). There was less than 10% increase in MABP during the 15- to 30-min period following BL-1249 administration. In contrast, cromakalim produced a significant decrease in MABP at all doses tested (0.03–1 mg/kg, p < 0.01; 0.1 mg/kg dose shown). For the reference, the lowest effective dose of cromakalim in the isovolumic bladder model was 0.1 mg/kg (no effect at 0.03 mg/kg; data not shown).

Pharmacology of BL-1249-Induced Current. The pharmacology of the potassium conductance activated by BL-1249 in human bladder cells was characterized using a variety of known K⁺ channel inhibitors (inhibition was measured at +70 mV). Iberiotoxin (300 nM; n = 4), a potent inhibitor of maxi-K⁺ channels (Galvez et al., 1990), failed to block the BL-1249-induced current. Figure 6 shows that a large fraction of control current was inhibited by 300 nM iberiotoxin indicating that this current is mediated, at least in part, by maxi-K⁺ channels. In contrast, the amplitude and kinetics of the currents observed during coapplication of BL-1249 and 300 nM iberiotoxin were indistinguishable from the currents evoked by BL-1249 alone, showing that the BL-1249-induced current is unlikely to be mediated by maxi-K⁺ channels. In addition, 300 nM iberiotoxin failed to inhibit BL-1249 (1 and 10 µM)-induced (n = 3) membrane hyperpolarization in bladder myocytes (data not shown).

Several other potassium channel blockers were evaluated for their ability to block the BL-1249-evoked current. Apamin (1 µM), a potent blocker of SK1, SK2, and SK3 currents, failed to inhibit the BL-1249 (2 µM)-induced current (n = 3), suggesting that it is unlikely to be SK current. Glyburide (10 µM; n = 3), a blocker of ATP-sensitive K⁺ channels, 4-aminopyridine (3 mM; n = 6), a nonselective blocker of voltagegated K⁺ channels, and tetraethylammonium (5 mM; n = 4), a nonselective K⁺ channel blocker, produced little or no effect on the properties of the BL-1249-induced current at the concentration tested (data not shown). Glyburide (10 µM; n = 3), 4-aminopyridine (3 mM; n = 6), and tetraethylammonium (5 mM; n = 4) also failed to prevent the BL-1249 (1 and 10 µM)-induced hyperpolarization (data not shown).

The BL-1249-induced current and membrane hyperpolarization were inhibited by Ba²⁺, a nonselective potassium channel blocker. As shown in Fig. 7, A and B, 10 mM Ba²⁺ partially inhibited the membrane hyperpolarization evoked by 2 and 10 µM BL-1249 (93.6 ± 9.5% and 79.4 ± 6.9% inhibition, respectively; n = 4). It should be noted, however, that Ba²⁺ (10 mM) alone produced a membrane depolarization of 4.7 ± 1.6 mV (n = 4). At 0.3 mM, Ba²⁺ inhibited the BL-1249-induced membrane hyperpolarization by 14.0 ± 4.3% (n = 3).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Ba2+ inhibits BL-1249-induced hyperpolarization. A, representative traces of membrane potential measured in human bladder smooth muscle cells. BL-1249 at 2 and 10 µM hyperpolarize membrane potential by 15.7 ± 6 and 30.9 ± 4.7 mV, respectively; Ba2+ (10 mM) alone causes a small depolarization (4.7 ± 1.6 mV) (top trace). When coapplied with BL-1249, Ba2+ inhibits BL-1249-induced hyperpolarization (bottom trace). B, inhibition of BL-1249-induced hyperpolarization; means ± S.E.M. (n = 3).

As shown in Fig. 8, A and B, 10 mM Ba²⁺ significantly inhibited the BL-1249 (2 µM)-induced current. In contrast, the current evoked by BL-1249 was unaffected by the divalent cation Mg²⁺ (10 mM), suggesting that this inhibition is specific for Ba²⁺ rather than a nonspecific effect of divalent cations. Interestingly, the BL-1249-evoked currents measured in the presence of 10 mM Ba²⁺ seem to develop with a slower time course than typically observed. This may be due, at least in part, to the presence of Ba²⁺ inward currents carried by voltage-activated calcium channels. The degree of block measured across a broad range of voltages (-70 to +70 mV) indicates that Ba²⁺ inhibits the BL-1249-induced current at voltages where the Ca²⁺ current is unlikely to have compromised the measurement of the K⁺ current (note also that the effects of Ba²⁺ on Ca²⁺ current cannot explain the inhibition of the BL-1249-induced hyperpolarization by Ba²⁺). Alternatively, Ba²⁺ may truly slow the activation of the BL-1249-induced current.

View larger version (23K): [in this window] [in a new window]   Fig. 8. BL-1249-induced current is inhibited by external Ba2+. A, representative BL-1249-induced current in human bladder smooth muscle cells, recorded from -80 to +70 mV forming a holding potential of -60 mV, with and without external Ba2+ (10 mM). The control current recorded in the absence of BL-1249 was subtracted from both sets of data. B, steady-state I-V relationship for BL-1249-induced current measured in the presence of Ba2+ (10 mM) and Mg2+ (10 mM); mean ± S.E.M. (n = 4–5).

The effects of Ba²⁺ were also evaluated in the bladder strip functional assay. Addition of Ba²⁺ to muscle strips precontracted with 30 mM KCl produced a further concentration-dependent increase in contractile amplitude (46.2 ± 14.3 and 152.1 ± 20.4% increase with 1 and 10 mM Ba²⁺, respectively; n = 4), presumably caused by a further depolarization of the tissue. Subsequent addition of BL-1249 (0.3–10 µM) partially inhibited the tone of the bladder strips, but to a lesser degree than observed in the absence of Ba²⁺. Thus, 1 µM BL-1249 (an approximate EC₅₀ concentration) inhibited force by 39.3 ± 8.8% (n = 4) when given alone and by 10.9 ± 5.6% (n = 4) when administered in the presence of 10 mM Ba²⁺. Tetraethylammonium (1 and 10 mM; n = 3), glyburide (10 µM; n = 6), 4-aminopyridine (1 and 3 mM; n = 3), and iberiotoxin (100 nM; n = 6) had little or no effect on BL-1249-induced bladder strip relaxation (data not shown).

Candidate K⁺ Channel Distribution in Human Bladder versus Aorta. The pharmacological properties, kinetics, and I-V relationship of the BL-1249-activated current rules out a number of channels, including K_ATP, SK1–3, maxi-K⁺, and delayed rectifier K⁺ channels, as candidates channels opened by BL-1249. Interestingly, the currents carried by the members of Two Pore domain family of K⁺ channels K_2P2.1 (other names are KCNK2, TREK-1, and TPKC1), K_2P3.1 (KCNK3, TASK-1, TBAK-1, and OAT-1), K_2P6.1 (KCNK6, TWIK-2, and TOSS), K_2P13.1 (KCNK13 and THIK-1), K_2P16.1 (KCNK16 and TALK-1), K_2P17.1 (KCNK17, TASK-4, and TALK-2), and K_2P5.1 (KCNK-5 and TASK-2) were reported to be relatively sensitive to Ba²⁺ (at 0.1–10 mM concentrations) (the nomenclature and pharmacology referred to in the present paper was adapted from The IUPHAR Compendium of Voltage-Gated Ion Channels, 2002; for review, see O'Connell at al., 2002). Thus, K_2P2.1, K_2P3.1, K_2P5.1, K_2P6.1, K_2P13.1, K_2P16.1, and K_2P17.1 may be candidate targets for the action of BL-1249. Ba²⁺ is also known to block K_ir channels, but at significantly lower concentrations (micromolar).

The mRNA distribution profile of K_2P2.1, K_2P3.1, K_2P5.1, K_2P6.1, K_2P13.1, K_2P16.1, and K_2P17.1 potassium channels in human bladder and aortic smooth muscle cells is presented in Table 2. This profiling revealed a detectable signal for K_2P6.1, K_2P17.1, and K_2P2.1 in both bladder and aortic cells (noted as moderate expression). However, only K_2P2.1 (TREK-1) had a relatively higher expression level in bladder (12-fold increase in signal in bladder cells relative to aorta), whereas K_2P6.1 and K_2P17.1 were expressed in aortic cells at a relatively higher level.

View this table: [in this window] [in a new window]   TABLE 2 Relative distribution of two-pore K+ channels in cultured human bladder and aortic smooth muscle cells Relative expression level (low or moderate) for each target gene was calculated on the basis of cycle number compared with the housekeeping gene (-actin) expression (see Materials and Methods for details). In addition, the expression level of each gene candidate was normalized to the lowest expression either in bladder or aortic cells, and the -fold difference is listed in the table.

Other channels of interest were expressed in both tissues at low levels or levels near the limit of reliable detection (noted as low and very low expression). The expression profiles of the internal control human -Actin and cyclophilin were similar between both tissues in all the experiments.

	Discussion

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Membrane Potential Measurements in Bladder Smooth Muscle Cells. Membrane potential-sensitive oxonol dyes have previously been used to characterize the functional activity of K_ATP channel openers in urinary bladder smooth muscle cells (Gopalakrishnan et al., 1999, 2002; Whiteaker et al., 2001b). Moreover, the changes in fluorescence responses have been shown to correlate with changes in membrane potential produced by activation of K_ATP channels in bladder myocytes (Whiteaker et al., 2001b). In the present study, we employed the oxonol dye DiBAC₄(3) to estimate changes in membrane potential in intact human bladder smooth muscle cells in culture (i.e., under conditions where the normal transmembrane ion gradients were undisturbed). Using this fluorescence technique, BL-1249 produced a concentration-dependent membrane hyperpolarization (EC₅₀ = 1.26 ± 0.6 µM). Similar results were observed using the whole-cell patch-clamp technique (EC₅₀ = 1.49 ± 0.08 µM). Similarly, BL-1249 produced a hyperpolarization of isolated rat bladder myocytes.

Mechanism of Action Studies. The reversal potential for the BL-1249-evoked current was consistent with the activation of a potassium conductance (note: E_Cl would have been close to 0 mV in the voltage-clamp experiments). In addition, the observation that BL-1249 was a less effective relaxant of 80 mM KCl-induced bladder contractions is also consistent with the view that BL-1249's effects are mediated by a K⁺ conductance (i.e., BL-1249 is less effective when the transmembrane K⁺ gradient is reduced). The partial relaxation of the 80 mM KCl response suggests that another mechanism(s), beyond K+ channel activation, play a minor role in the relaxant response to BL-1249.

The possibility that BL-1249 blocks Ca²⁺ current was assessed by measuring depolarization-induced Ca²⁺ influx (measured by changes in Fluo-4 fluorescence) in HEK293 cells endogenously expressing L-type Ca²⁺ channels. This fluorescent response was fully blocked by 1 µM nifedipine, indicating the Ca²⁺ fluorescence change was mediated by L-type Ca²⁺ channels. BL-1249 (up to 30 µM) was without effect on depolarization-induced Ca²⁺ influx, indicating that BL-1249 does not block L-type Ca²⁺ channels (data not shown).

The instantaneous activation and noninactivating properties of the BL-1249-induced current in conjunction with the failure of tetraethylammonium or 4-aminopyridine to inhibit the BL-1249 currents or membrane hyperpolarization makes it unlikely that delayed rectifier channels are the target for BL-1249. Additionally, the BL-1249-induced current was insensitive to iberiotoxin, apamin, or gluburide, further ruling out maxi-K, SK1–3, and K_ATP channels as targets for BL-1249. Furthermore, K_ATP channels display the I-V relationship of an inward rectifier, unlike the BL-1249-induced current described here. Interestingly, the BL-1249-evoked currents and membrane hyperpolarization were inhibited by millimolar concentrations of Ba²⁺, a nonselective potassium channel blocker. Ba²⁺ also seemed to partially inhibit the ability of BL-1249 to relax precontracted bladder strips. However, caution must be exercised when interpreting the relaxation results given the depolarization and further contraction produced by Ba²⁺ itself (i.e., BL-1249's functional potency may be reduced in the face of a stronger contraction).

Recent publications imply a role of the KCNQ2/3 channels in bladder regulation (Argentieri and Sheldon, 2001). However, it has been reported that homomers KCNQ1, KCNQ2, and KCNQ4 and heteromers KCNQ2/3 are blocked by tetraethylammonium with IC₅₀ of 0.3 to 5 mM (Hadley et al., 2000), whereas the BL-1249-induced current was not changed by tetraethylammonium up to 10 mM tested. Using the RT-PCR technique, we investigated the distribution of KCNQ channels in human bladder myocytes and found that they exhibit negligible expression of KCNQ1–4 channels and a low level of expression of KCNQ5 (data not shown). Although it has been reported that KCNQ5 exhibits low sensitivity to tetraethylammonium block (>30 mM), it has also been describe as a slowly activating/slowly deactivating potassium current that displays a marked inward rectification at positive membrane voltages, very unlike the BL-1249-induced current we have observed (Lerche et al., 2000). Thus, based on our own RT-PCR data and reported properties of KCNQ channels, we concluded that it is unlikely that BL-1249 activates a member of KCNQ channel family.

A number of K⁺ channels are known to be inhibited by Ba²⁺, including several members of the two-pore K⁺ channel family, a class of channels that are generally insensitive to traditional K⁺ channel blockers (Catterall et al., 2002; O'Connell et al., 2002). It has been reported that Ba²⁺ at 100 µM blocked 50% of TREK-1 currents expressed in Xenopus oocytes (Fink et al., 1996; O'Connell et al., 2002). We have observed partial inhibition of BL-1249-induced current and membrane hyperpolarization by 300 µM Ba²⁺ (data not shown) and full inhibition at 10 mM, indicating that BL-1249-induced current is at least moderately sensitive to the inhibition by Ba²⁺.

The two-pore K⁺ channels are an emerging family of background (leak) K⁺ channels that are thought to contribute to setting the resting membrane potential in various tissues. To explore the possibility that BL-1249 may activate a member of the two-pore family of K⁺ channels, we used an RT-PCR technique to survey the mRNA distribution of two-pore channels in bladder and aortic cell cultures. The mRNA distribution profile revealed a detectable signal for K_2P6.1 (TWIK-2), K_2P17.1 (TASK-4), and K_2P2.1 (TREK-1) in bladder cells. However, only K_2P2.1 (TREK-1) had a higher expression level relative to the aorta (12-fold increase in signal in bladder cells relative to aorta). In addition, TREK-1 current has been described as instantaneously activating, noninactivating current with outwardly rectifying characteristics (Fink et al., 1996), not unlike the observed BL-1249-induced current. In contrast, TWIK-2, which is also present in bladder smooth muscle cells, although structurally similar to TREK-1 (Fink et al., 1996), has been reported to have inward-rectifying properties (Lesage et al., 1996).

Clearly, the observation of higher abundance of a given channel in the bladder relative to the aorta does not necessarily reflect an increased functional importance of this channel in the bladder. Nevertheless, the presence of this channel in the target tissue, as well as the kinetics and pharmacology of the BL-1249-evoked current, are consistent with the hypothesis that activation of a TREK-1-like K⁺ channel may account for BL-1249's actions and tissue selectivity.

The human TREK-1 channel is highly expressed in central nervous system tissues, ovary, and small intestine and was implicated in neural response to temperature, arachidonic acid, mechanical stretch, and volatile anesthetics (Goldstein at al., 1998, 2001). Although is not possible to unambiguously define the specific type of a K⁺ channel activated by BL-1249, one may speculate that stretch-activated leak potassium channel in bladder myocytes could deliver an important function of maintaining bladder compliance during bladder filling. Finally, our data do not exclude a possibility that BL-1249 activates a novel type of a K⁺ channel.

In Vitro and in Vivo Selectivity of BL-1249. Preliminary results obtained by measuring changes in DiBAC₄(3) fluorescence in human aortic smooth muscle cells showed that BL-1249 produced a concentration-dependent hyperpolarization, with an EC₅₀ of 21.0 ± 0.9 µM (n = 3, data not shown), a value substantially weaker than the BL-1249 EC₅₀ obtained using cultured human bladder myocytes (1.26 ± 0.6 µM; n = 4). However, given the observation that the expression of ion channels in general, and K⁺ channels in particular, may be altered in cultured smooth muscle cells (Tang and Wang, 2001), we wanted to assess whether this apparent bladder versus aortic selectivity would be seen in acutely isolated bladder and aortic tissues. Thus, the relaxant effect of BL-1249 was assessed using precontracted rat bladder and aortic tissue strips. BL-1249 was found to be a considerably more potent inhibitor of 30 mM KCl-induced contractions in bladder strips (EC₅₀ = 1.1 ± 0.37 µM) than aortic strips (no significant relaxation observed at 10 µM). Consistent with the inhibitory activity of BL-1249 against the bladder strips in vitro, BL-1249 (1 mg/kg) was found to inhibit isovolumic bladder contractions in vivo. The short duration of the effect of BL-1249 on bladder contraction (<30 min) was likely due to a fast elimination half-life of the compound after i.v. administration (0.69 ± 0.07 h; L. Pajor, unpublished data). BL-1249 (1 mg/kg) was also shown to have little effect on MABP, an observation again consistent with the in vitro bladder to vascular relaxant selectivity.

The K_ATP channel opener cromakalim has been shown to exhibit selectivity for vascular tissue relative to bladder (Howe et al., 1995; Fabiyi et al., 2003). In contrast, the K_ATP channel opener ZD-6169 has been reported to exhibit some degree of bladder/vascular selectivity (Howe at al., 1995). However, in a recent publication, the bladder selectivity of ZD-6169, as well as its analog WAY-133537, was not confirmed in two in vivo bladder models: a partial outlet obstruction model and an isovolumic model (Fabiyi et al., 2003). Thus, BL-1249 seems to exhibit greater bladder versus vascular selectivity than either cromakalim or ZD-6169.

In summary, the present paper describes the activity of BL-1249, a putative potassium channel opener that seems to be relatively selective for bladder versus vascular tissue. The specific K⁺ channel mediating the action of BL-1249 has not been identified. Nevertheless, the kinetics and pharmacology of the BL-1249-evoked currents as well as the higher abundance of TREK-1 in bladder cells relative to aortic cells raises the intriguing possibility that a two-pore leak-type channel may underlie BL-1249's actions.

	Acknowledgements

 
We thank Beata Braginski for technical support.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.078592.

ABBREVIATIONS: KCO, potassium channel opener; MABP, mean arterial blood pressure; BL-1249, (5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine; DiBAC₄(3), bis-(1,2-dibutylbarbituric acid)trimethine oxonol; PCR, polymerase chain reaction; RT, reverse transcriptase; PEG400, polyethylene glycol; ZD-6169, (S)-N-(4-benzoylphenyl)-3,3,3-trifluro-2-hydroxy-2-methyl-priopionamide; WAY-133537, (R)-4-[3,4-dioxo-2-(1,2,2,-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile.

¹ Current address: Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL.

² Current address: Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical School, Nashville, TN.

Address correspondence to: Dr. Svetlana Tertyshnikova, Bristol-Myers Squibb Pharmaceutical Research Institute, Neuroscience Drug Discovery, 5 Research Parkway, Wallingford, CT 06492-7660. E-mail: svetlana.tertyshnikova{at}bms.com

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