Introduction

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Overactive bladder, a condition characterized by increased urinary urgency and frequency with or without urge incontinence, continues to be a chronic, highly prevalent condition affecting more than 15 million people in the United States alone (Payne, 1998; McGhan, 2001). Currently available options for the management of this condition, the muscarinic receptor antagonists such as oxybutynin and tolterodine, suffer from limited efficacy, in part, related to poor tolerability as a result of side effects such as dry mouth, blurred vision, and constipation (Andersson et al., 1999; Sullivan and Abrams, 1999; Chapple, 2000). Accordingly, overactive bladder continues to be an unmet medical need with efforts directed to validate mechanisms and identify novel agents with superior efficacy and/or improved side effect profile.

Alterations in the myogenic activity of the bladder smooth muscle have been proposed as a basis for the generation of involuntary detrusor contractions associated with the overactive bladder (Brading, 1997; Elbadawi et al., 1998). Although central nervous system and/or afferent signaling pathways may also participate (de Groat, 1997), overactive bladder may be viewed as a disorder of bladder smooth muscle tone and the underlying changes in spontaneous action potentials and phasic contractions. Tissue reactivity studies have shown that detrusor strips from unstable bladders exhibit spontaneous tetanic activity with enhanced electrical coupling between cells. Studies have shown agonist supersensitivity and altered spontaneous contractile activity in idiopathic detrusor instability, a common cause of lower urinary tract storage symptoms. This is consistent with enhanced electrical coupling of bladder smooth muscle cells (Elbadawi et al., 1993; Mills et al., 2000).

Over the past decade, several openers of ATP-sensitive K⁺ (K_ATP) channels have been evaluated for their effects on bladder function. First generation agents such as ()-cromakalim, YM934, and ZM244085, as well as more recent compounds such as ZD6169 and WAY-133537 have been shown, in vitro, to evoke relaxation of isolated bladder smooth muscle strips from various species precontracted by a variety of stimuli, including electrical field, carbachol, or low external K⁺ (Foster et al., 1989; Fujii et al., 1990; Grant and Zuzack, 1991; Wojdan et al., 1999). These agents selectively open glyburide (glibenclamide)-sensitive K_ATP channels, critical to the control of membrane potential, leading to membrane hyperpolarization, attenuated Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels and decrease in bladder smooth muscle excitability (Bonev and Nelson, 1993; Quayle et al., 1997). In particular, activation of a few K_ATP channels in bladder smooth muscle by low concentrations of KCOs far below that required for substantial K_ATP current activation has been shown to suppress spike action potentials and spontaneous myogenic activity (Petkov et al., 2001; Shieh et al., 2001). In unstable bladders with heightened spontaneous contractility, it is likely that activation by low concentrations of KCOs would serve to inhibit these contractions and dampen smooth muscle (hyper)excitability.

In a preliminary clinical study of patients with detrusor instability or detrusor hyperflexia, 6 of 17 patients responded to cromakalim with modest improvements in symptoms of urinary frequency and increase in mean voided volume (Nurse et al., 1991). Although this observation is consistent with the preclinical efficacy results in animal models (Malmgren et al., 1989), in the absence of placebo-controlled and definitive clinical data, it is thought that cromakalim and subsequent analogs lack sufficient bladder selectivity relative to the vascular effects. Consequently, further clinical proof of principle for KCOs for bladder overactivity mandates a compound with improved selectivity. Improvements in bladder selectivity have been reported for certain chemotypes represented by ZD6169 and WAY-133537 in preclinical models where in vivo efficacies for inhibition of bladder overactivity were demonstrated at doses that do not substantially affect arterial pressure or heart rate (Howe et al., 1995; Pandita et al., 1997; Wojdan et al., 1999; Yu and de Groat, 1999). However, a clear need exists for the identification of agents efficacious in suppressing unstable bladder contractions with superior selectivity versus cardiovascular liabilities.

(9S)-9-(3-Bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno [3,2-b]quinolin-8(4H)-one 1,1-dioxide (A-278637) is a novel K_ATP channel opener (Fig. 1) from a series of 1,4-dihydropyridine analogs (Carroll et al., 2001). In the present and accompanying article (Brune et al., 2002), the initial pharmacological characterization of this compound is described. Herein, we show that A-278637 potently and selectively interacts with K_ATP channels and suppresses contractility of bladder smooth muscle strips, including those from human hyperreflexic bladder. In the accompanying article (Brune et al., 2002), A-278637 is shown to display enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other KCOs, ZD6169 and WAY-133537.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structure of A-278637.

	Materials and Methods

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Materials

All studies were carried out in accordance with guidelines outlined by the Animal Welfare Act, the Association for Assessment and Accreditation of Laboratory Animals, and the Institutional Animal Care and Use Committee of Abbott Laboratories. DiBAC₄(3) was purchased from Molecular Probes (Eugene, OR). Compounds were prepared in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) as a 5 or 10 mM stock, protected from light, and serial dilutions prepared in appropriate assay buffer just before use.

DiBAC₄(3) Fluorescence Studies

Functional activity of K_ATP channels in guinea pig bladder smooth muscle cells was assessed as described previously (Gopalakrishnan et al., 1999) by evaluating changes in membrane potential using the bis-oxonol dye DiBAC₄(3) in a 96-well fluorescent imaging plate reader (FLIPR). Briefly, urinary bladders were removed from anesthetized male guinea pigs (Hartley; Charles River Laboratories, Inc., Wilmington, MA), weighing 250 to 300 g, and cells isolated by enzymatic dissociation using collagenase and pronase. Confluent cells, cultured in black clear-bottomed 96-well plates, were rinsed twice with 200 µl of assay buffer (20 mM HEPES, 120 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 5 mM glucose; pH 7.4 at 25°C) containing 5 µM DiBAC₄(3) and incubated with 180 µl of buffer in a cell incubator for 30 min to ensure dye distribution across the membrane. Assays were carried out at 37°C. After addition of various concentrations of the test compound, changes in DiBAC₄(3) fluorescence were measured at excitation and emission wavelengths of 488 and 520 nm, respectively.

Whole-Cell Patch-Clamp Studies

Whole-cell patch-clamp technique was used to measure changes in ionic currents from guinea pig bladder smooth muscle cells as described previously (Shieh et al., 2001). Urinary bladders were transferred directly into preoxygenated physiological saline solution containing 137 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.42 mM KH₂PO₄, 4.17 mM NaHCO₃, 10 mM HEPES, and 10 mM glucose; pH 7.4 with NaOH. Pieces of bladder smooth muscle were incubated with collagenase, and single smooth muscle cells were obtained by triturating using a fire-polished large-bore Pasteur pipette. The intracellular pipette solution contained 107 mM KCl, 1.2 mM MgCl₂, 1 mM CaCl₂, 10 mM EGTA, 5 mM HEPES, and 0.1 mM ATP; pH 7.2 with KOH; total K⁺ ~140 mM. The bath solution contained 60 mM KCl, 80 mM NaCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, and 5 mM HEPES; pH 7.4 with NaOH. Whole-cell currents were recorded at room temperature and were amplified using Axopatch-200B amplifier (Axon Instruments, Union City, CA) and low pass filtered at 5 kHz (3 dB; four-pole Bessel filter) before digitization (Digidata 1200B; Axon Instruments) at a sampling rate of 10 kHz.

Pig Bladder Relaxation Studies

Bladder strip relaxation studies were performed as described previously (Buckner et al., 2000). Briefly, female Landrace pigs (Wilson's Prairie View Farm, Burlington, WI), weighing 9 to 25 kg, were euthanized with an intraperitoneal injection of pentobarbital (150-200 mg/kg, Somlethol; J.A. Webster Inc., Sterling, MA). The entire urinary bladder was removed and placed in Krebs-Ringer bicarbonate solution (120 mM NaCl, 20 mM NaHCO₃, 11 mM dextrose, 4.7 mM KCl, 2.5 mM CaCl₂, 1.5 mM MgSO₄, and 1.2 mM KH₂PO₄ equilibrated with 5% CO₂, 95% O₂; pH 7.4 at 37°C). The bladder was sectioned after discarding the top dome portion and the lower trigonal area. Approximately 3- to 5- × 20-mm strips were prepared from the remaining tissue adjacent to the trigonal area and cut in a circular manner. The mucosal layer was removed and strips were mounted in 10-ml tissue baths maintained at 37°C with one end fixed to a stationary rod and the other to a FT03 transducer (Grass Instruments, Quincy, MA), at a basal preload of 1.0 g. Tissues were rinsed at 10-min intervals and allowed to equilibrate for at least 70 min.

As previously reported (Buckner et al., 2002), spontaneous phasic activity was observed in many tissues that contracted with transient spikes that varied in frequency, duration, and amplitude. The tissue strips were exposed to varying concentrations of the test agents for 15 min and changes in contractility were assessed. With carbachol-stimulated tissues, the protocol was noncumulative with rinse cycles between each concentration of test compounds because the contractile response tended to wane over time. Tissues were pretreated with test compounds for 15 min, exposed to a fixed concentration of carbachol (300 nM, which corresponds to approximately an EC₇₅ concentration of carbachol), and changes in tension assessed. The tissue was then rinsed for 15 min and the cycle repeated with another concentration of the test compound. For electrical field stimulation studies, two parallel platinum electrodes were included and tissues were stimulated using a frequency of 0.05 Hz, 0.5 ms at 20 V. This low-frequency stimulation produced a stable twitch response of 1 to 5 g (Buckner et al., 2000). Tissues were allowed to equilibrate for at least 70 min and primed with 80 mM KCl before performing cumulative concentration-response curves for test compounds. Glyburide (10 µM) was added at the conclusion of the concentration-response curve to assess antagonist sensitivity. In cases where the potency of glyburide was evaluated by Schild analysis (Schild, 1947), tissues were pretreated with the antagonist for a 30-min period before assessing KCO sensitivities.

Human Bladder Relaxation Studies

Human bladder tissues were obtained from patients undergoing cystectomy for bladder cancer or colposuspension. Fresh tissue was immediately placed in Krebs' solution (118.4 mM NaCl, 4.7 mM KCl, 24.9 mM NaHCO₃, 11.7 mM dextrose, 1.9 mM CaCl₂, 1.15 mM MgSO₄, and 1.15 mM KH₂PO₄ equilibrated with 5% CO₂, 95% O₂; pH 7.4 at 37°C). The mucosa and serosa were removed and strips of detrusor muscle (7 × 2 mm) were dissected for overnight storage. Experiments were performed the following day. Strips were mounted in 5-ml organ baths under 1 g of resting tension. The force developed was recorded via isometric force transducers connected to a Mac/8 data acquisition system. Tissues were equilibrated for 1 h with rinses at 15-min intervals. After this period, carbachol (3 µM) was used to precontract tissue strips before the addition of the test compound. The carbachol-induced contractions were allowed to stabilize before cumulative concentration-response curves (in half-log increments) were obtained in response to increasing concentrations of the test compound. Control experiments were performed to define any vehicle effects.

Vascular Tissue Relaxation Studies

Rat Aorta. The entire thoracic aorta from male Sprague-Dawley rats (200-350 g) was removed and immediately placed into Krebs-Ringer bicarbonate solution as described above for pig bladder strips. The aorta was cleaned of extraneous tissue, endothelium removed, cut into 3- to 4-mm rings, and mounted in 10-ml isolated tissue baths at 37°C. One end was fixed to a stationary glass rod and the other to an FT03 transducer (Grass Instruments) at a basal preload of 1.0 g. Data were recorded on a model 7 polygraph (Grass Instruments). Tissues were rinsed every 10 min for a total of 45 to 60 min. The aorta was primed once with 80 mM KCl rinsed to basal tension and again with phenylephrine (10 µM). Absence of functional endothelium was confirmed by loss of the acetylcholine (10 µM)-induced relaxation. After an additional 60-min equilibration period, tension was established using 25 mM KCl solution, and cumulative concentration-relaxation-response curve was generated for the test compound.

Rat Portal Vein. Male Sprague-Dawley rats (350-400 g) were anesthetized with isoflurane, and the section of the portal vein between the right and left branches and the junction of the splenic vein was isolated. This section (15-20 mm) was removed and placed in Krebs-Ringer bicarbonate solution. The portal vein was cleaned of extraneous tissue and the sides of the vein nicked with a scalpel at 3- to 4-mm intervals and mounted in a 10-ml isolated tissue bath at 37°C. One end was fixed to a stationary glass rod and the other to an FT03 transducer (Grass Instruments) at a basal preload of 1.0 g. Tissues were rinsed every 10 min for a total of 150 to 180 min. The portal vein contracted spontaneously with transient spikes that varied in frequency, duration, and amplitude. Cumulative concentration-response curves (in half-log increments) were obtained for the test compound with a 15-min exposure time after each addition. Data were recorded on a PowerLab/800 data acquisition system and analyzed as the area under the curve (AUC) of the contractile response for 15-min intervals.

Radioligand Binding and Electrophysiology Selectivity Studies

[³H]Glyburide and [³H]isradipine binding to membranes was performed as described previously (Wei et al., 1989; Gopalakrishnan et al., 1991). Whole-cell patch-clamp studies using ion channels expressed in clonal cell lines or native cell types (Table 3) were carried out using standard electrophysiological techniques (Hamill et al., 1981).

Data Analysis

The concentration dependence of maximal steady-state changes in fluorescence or changes in tension responses was fitted by nonlinear regression analysis (GraphPad Prism; GraphPad Software, San Diego, CA) to obtain EC₅₀ or IC₅₀ values. Spontaneous phasic activity was analyzed for changes in frequency, duration, and amplitude and the AUC of the contractile response during a 15-min interval. For carbachol-stimulated tissues, relaxations (measured in grams) were expressed as a percentage of the precontraction produced by carbachol. In electrical field-stimulated tissues, the concentration-dependent reduction in the peak amplitude (measured in grams) was used for calculating the IC₅₀ values. Data are expressed as means ± standard error. Significant differences between group means were assessed by Student's t test, and a p value <0.05 was considered statistically significant.

	Results

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Effects on DiBAC₄(3) Fluorescence Changes. A-278637 evoked concentration-dependent decreases in membrane potential in guinea pig bladder smooth muscle cells as assessed by decreases in DiBAC₄(3) fluorescence with an EC₅₀ value of 102 nM (log EC₅₀ = 7.04 ± 0.11; slope = 1.76 ± 0.12; n = 5; Fig. 2). As shown in Fig. 2A, A-278637-evoked responses were reversed by subsequent addition of glyburide (5 µM). In comparison, A-278637 was 3-fold more potent compared with ZD6169 in this assay (log EC₅₀ = 6.56 ± 0.10; slope = 1.07) but about 5-fold less potent than WAY-133537 (log EC₅₀ = 7.72 ± 0.06; slope = 0.62; n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of A-278637 on membrane potential responses in guinea pig bladder smooth muscle cells assessed by the bis-oxonol dye DiBAC4(3). A, representative experiment showing typical changes in fluorescence responses upon addition (indicated by down arrows) of varying concentrations of A-278637 (3, 10, 30, 100, 300, 1000, and 3000 nM) and its attenuation (indicated by up arrows) by the addition of glyburide (5 µM). B, concentration-response curves for fluorescence changes evoked by A-278637. Depicted are means ± S.E.M. of five separate determinations.

Whole-Cell Patch Clamp. Direct interaction of A-278637 with K_ATP channels in guinea pig bladder smooth muscle cells was studied by whole cell patch clamp. As shown in Fig. 3A, upon addition of 10 µM A-278637, membrane currents were increased by 50.6 ± 5.8 pA (n = 4) under conditions where cells were bathed in solution containing 60 mM K⁺ and voltage clamped at 80 mV with patch pipette containing 140 mM K⁺ and 0.1 mM ATP. The current-voltage relationship of A-278637-evoked responses is presented in Fig. 3B. Experiments performed under current-clamp conditions showed that A-278637 (100 nM) decreased smooth muscle membrane potential by 23.97 ± 8.4 mV (n = 3). A-278637-evoked current and membrane potential effects were both sensitive to inhibition by 5 µM glyburide, suggesting that the compound opens K_ATP channels in bladder smooth muscle cells.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of A-278637 on KATP currents in guinea pig bladder smooth muscle cells. A, application of 10 µM A-278637 evoked an increase in inward whole-cell current in guinea pig bladder smooth muscle cells that was attenuated upon the addition of 5 µM glyburide. Cells were voltage clamped at 80 mV and changes in membrane currents were measured in bath solution containing 60 mM K+ with pipette solution containing 140 mM K+ and 0.1 mM ATP. B, current-voltage relationship of whole-cell currents recorded from smooth muscle cells in presence of A-278637 from cells at test potentials ranging from 100 to +100 mV.

Bladder Strip Relaxation. Isolated pig bladder strips exhibit spontaneous phasic activity that is myogenic in nature (Buckner et al., 2002). Addition of A-278637 resulted in a concentration-dependent suppression of the spontaneous activity that was restored by the addition of 5 µM glyburide (Fig. 4A). The IC₅₀ value of A-278637 measured as changes in AUC of the contractile response was 22.7 nM (log IC₅₀ = 7.64 ± 0.06; n = 6). As shown in Fig. 4B, the reduction in AUC at low concentrations primarily involves a significant reduction in the contractile frequency (37 ± 8.4% at 10 nM), with no significant effect on the duration or the amplitude of the contractile response. A-278637 is about 6- to 10-fold more potent in suppressing phasic activity compared with ZD6169 (log IC₅₀ = 6.68 ± 0.11) and WAY-133537 (log IC₅₀ = 6.99 ± 0.06), respectively (Table 1).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Inhibition of spontaneous phasic activity of isolated pig bladder strips by A-278637. A, representative experiment showing baseline spontaneous phasic activity of the pig bladder strips and its suppression after the addition of increasing concentrations of A-278637. Glyburide (5 µM) reversed these effects. B, concentration-response curves derived from the AUC for the suppression of myogenic contractions by A-278637. C, analysis of frequency, amplitude, and duration effects at varying concentrations of A-278637. Depicted are means ± S.E.M. of six separate determinations, each carried out using duplicate tissue strips.

In Vitro Selectivity. Vascular K_ATP. To examine interactions with vascular K_ATP channels, the effect of A-278637 was examined in 25 mM K⁺-stimulated rat thoracic aorta. A-278637 completely suppressed contractions evoked by 25 mM K⁺ with an IC₅₀ of 45.6 nM (log IC₅₀ = 7.34 ± 0.07; n = 6; Table 2), whereas those evoked by 80 mM K⁺ depolarization were relatively insensitive (IC₅₀ = 140 µM; 47% efficacy; n = 4). On the other hand, under similar conditions, the calcium channel antagonist nifedipine was equipotent under both K⁺ concentrations (data not shown).

Cardiac K_ATP. The potential interaction of A-278637 on cardiac K_ATP channels was examined by assessing decreases in DiBAC₄(3) fluorescence in cultured neonatal rat cardiac myocytes as described previously (Whiteaker et al., 2002). A-278637 evoked responses in cardiac myocytes with an EC₅₀ value of 40.2 µM (log EC₅₀ = 5.52 ± 0.22; n = 4), which is ~400-fold higher than those observed at bladder K_ATP channels (Table 3).

Pancreatic K_ATP. A-278637 was also examined for interaction with sulfonylurea receptor SUR1-containing K_ATP channels that is critical to metabolic regulation in the pancreas (Babenko et al., 1998; Seino, 1999). In rat insulinoma RINm5F cell line, A-278637 did not evoke a decrease in DiBAC₄(3) fluorescence or activate ⁸⁶Rb⁺ efflux up to concentrations of 10 µM. To further examine potential interactions with the SUR1, A-278637 was tested for displacement of [³H]glyburide binding to RINm5F cell membranes. A-278637 weakly displaced [³H]glyburide binding with a K_i value of 12.49 ± 3.66 µM (n = 3), which is 120-fold higher than the potency to activate bladder K_ATP channels as measured by FLIPR DiBAC₄(3) assays.

Ion Channel/Receptor Selectivity. Although A-278637 is structurally related to 1,4-dihydropyridine calcium channel ligands, the compound did not inhibit [³H]isradipine binding to bladder smooth muscle membranes at the highest concentration (30 µM) tested, consistent with its lack of effect in suppressing 80 mM K⁺-depolarized smooth muscle strips. Furthermore, A-278637 had no significant effect on native Ca²⁺ currents expressed in GH3 cells or other representative K⁺ channels (Kv1.5, hERG, Kir2.3, and BK_Ca) expressed in various clonal cell lines (n  3).

	Discussion

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The present study describes the in vitro pharmacological profile of a novel dihydropyridine K_ATP channel opener, A-278637. This KCO evoked concentration-dependent decreases in membrane potential in bladder smooth muscle cells and suppressed spontaneous phasic activity of bladder strips in a glyburide-reversible manner. A-278637 also suppressed contractions evoked by muscarinic receptor activation in pig and human bladder strips with comparable potencies. Direct interaction with native K_ATP channels was confirmed in bladder smooth muscle cells where the compound was efficacious in activating glyburide-sensitive currents.

K_ATP Channel Activity of A-278637. Previous studies from our laboratory have shown a good correlation between KCO potencies to evoke membrane potential changes as measured by evaluating fluorescence changes in bladder smooth muscle cells with relaxation of detrusor strips in vitro (Gopalakrishnan et al., 1999). A-278637 was about 60-fold more potent in evoking membrane potential effects through K_ATP channels compared with ZM244085 (6.1 µM; Gopalakrishnan et al., 1999), a structurally related 1,4-dihydropyridine K_ATP channel opener previously shown to inhibit bladder activity (Li et al., 1996). The reversal by glyburide of A-278637-evoked fluorescence responses in guinea pig bladder smooth muscle cells and its attenuation of spontaneous phasic activity in bladder strips are consistent with the notion that A-278637 interacts with the K_ATP channel. The observation that A-278637 also evoked glyburide-sensitive K⁺ currents in guinea pig bladder smooth muscle cells provides additional direct evidence that membrane hyperpolarization measured in DiBAC₄(3) fluorescence assays and relaxation observed in the bladder smooth muscle are mediated by interactions with the K_ATP channel. Importantly, these effects do not involve interaction with L-type calcium channels because A-278637 did not inhibit [³H]isradipine binding, was ineffective in suppressing aortic strip contractions evoked by 80 mM K⁺, and had no significant effects on native L-type Ca²⁺ currents.

Effects on Bladder Smooth Muscle Function. Urinary bladder smooth muscle, unlike arterial smooth muscle, exhibits action potentials and phasic myogenic activity. Previous studies have shown that KCOs can effectively suppress spontaneous contractions in human urinary bladder (Wammack et al., 1994), guinea pig bladder (Fujii et al., 1990; Hashitani et al., 1996), or hypertrophied rat bladder with instability (Malmgren et al., 1989). In the present study, a comparison of the IC₅₀ values to suppress spontaneous phasic activity showed that A-278637 is 5- to 10-fold more potent compared with both ZD6169 and WAY-133537. It should be noted that WAY-133537 was found to be about 7-fold more potent than A-278637 in the FLIPR assay, but less potent in various tissue reactivity assays; the reason for this discrepancy remains unclear. The suppression of spontaneous phasic activity of bladder strips is attributed to the inhibition of action potentials in smooth muscle cells at relatively low concentrations of KCOs. It has been demonstrated that low concentrations of KCOs could open a fraction of K_ATP channels evoking a small increase in K⁺ conductance sufficient to lower the membrane potential away from the threshold for action potential firing (Petkov et al., 2001; Shieh et al., 2001). In support of this notion, a significant inhibition of the integrated muscle force (AUC) and contraction frequency of isolated bladder strips by A-278637 was noted at concentrations as low as 10 nM. This value is comparable with the estimated plasma concentrations required to inhibit unstable contractions by 50% in the pig model in vivo (~ 8 nM corresponding to 3.3 ng/ml; Brune et al., 2002).

In Vitro Selectivity of A-278637. The cloning and expression of cDNAs for K_ATP channel subunits indicates a diversity of K_ATP channel types arising by heteromeric assembly of SURs and pore-forming inwardly rectifying K⁺ channels. The SUR2B subunit in conjunction with Kir6.2 or Kir6.1 is thought to constitute diverse smooth muscle type K_ATP channels (Babenko et al., 1998; Seino, 1999) although more recently, functional coassembly of both pore-forming subunits, Kir6.1 and Kir6.2, with SUR2B has also been reported (Cui et al., 2001). SUR1-Kir6.2 combination generates K_ATP channels with properties typical of those expressed in pancreas and neurons, whereas the components of plasmalemmal cardiac K_ATP channel are derived from SUR2A-Kir6.2. A-278637 was found to display selectivity toward activation of K_ATP channels in bladder smooth muscle (EC₅₀ = 102 nM) relative to K_ATP channels in cardiomyocytes (EC₅₀ = 40 µM) or pancreatic K_ATP channels expressed in RINm5F cells (EC₅₀ > 10 µM). In radioligand binding studies, A-278637 weakly displaced [³H]glyburide binding to high-affinity sulfonylurea receptors with a K_i value that was at least 100-fold higher than the concentrations required to activate bladder smooth muscle K_ATP channels. Thus, at concentrations effective in suppressing bladder contractility, A-278637 displays substantial selectivity versus other predominant K_ATP channel subtypes.