Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

(−)-(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): A Novel ATP-Sensitive Potassium Channel Opener Efficacious in Suppressing Urinary Bladder Contractions. I. In Vitro Characterization

Murali Gopalakrishnan, Steven A. Buckner, Kristi L. Whiteaker, Char-Chang Shieh, Eduardo J. Molinari, Ivan Milicic, Anthony V. Daza, Rachel Davis-Taber, Victoria E. Scott, Donna Sellers, Russ Chess-Williams, Christopher R. Chapple, Yi Liu, Dong Liu, Jorge D. Brioni, James P. Sullivan, Michael Williams, William A. Carroll and Michael J. Coghlan
Journal of Pharmacology and Experimental Therapeutics October 2002, 303 (1) 379-386; DOI: http://dx.doi.org/10.1124/jpet.102.034538

Abstract

Alterations in the myogenic activity of the bladder smooth muscle are thought to serve as a basis for the involuntary detrusor contractions associated with the overactive bladder. Activation of ATP-sensitive K+ (KATP) channels has been recognized as a potentially viable mechanism to modulate membrane excitability in bladder smooth muscle. In this study, we describe the preclinical pharmacology of (−)-(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), a novel 1,4-dihydropyridine KATPchannel opener (KCO) that demonstrates enhanced bladder selectivity for the suppression of unstable bladder contractions in vivo relative to other reference KCOs. A-278637 activated KATP channels in bladder smooth muscle cells in a glyburide (glibenclamide)-sensitive manner as assessed by fluorescence membrane potential assays using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (EC50 = 102 nM) and by whole cell patch clamp. Spontaneous (myogenic) phasic activity of pig bladder strips was suppressed (IC50 = 23 nM) in a glyburide-sensitive manner by A-278637. A-278637 also inhibited carbachol- and electrical field-stimulated contractions of bladder strips, although the respective potencies were 8- and 13-fold lower compared with inhibition of spontaneous phasic activity. As shown in the accompanying article [Brune ME, Fey TA, Brioni JD, Sullivan JP, Williams M, Carroll WA, Coghlan MJ, and Gopalakrishnan M (2002)J Pharmacol Exp Ther303:387–394], A-278637 suppressed myogenic contractions in vivo in a model of bladder instability with superior selectivity compared with other KCOs, WAY-133537 [(R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile] and ZD6169 [(S)-N-(4-benzoylphenyl)3,3,3-trifluro-2hydroxy-2-methyl-priopionamide]. A-278637 did not interact with other ion channels, including L-type calcium channels or other neurotransmitter receptor systems. The pharmacological profile of A-278637 represents an attractive basis for further investigations of selective KATP channel openers for the treatment of overactive bladder via myogenic etiology.

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+ (KATP) 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 KATP 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 KATP channels in bladder smooth muscle by low concentrations of KCOs far below that required for substantial KATP 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 KATP 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 KATP 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.

Materials and Methods

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 KATP 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).

View this table:
Table 3

In vitro selectivity of A-278637

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 IC50values. 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

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).

Figure 2
Figure 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 KATP 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 KATP channels in bladder smooth muscle cells.

Figure 3
Figure 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).

Figure 4
Figure 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.

View this table:
Table 1

In vitro functional characterization of A-278637

A major component of the neurogenic stimulus for physiological bladder contractions is acetylcholine-induced stimulation of postjunctional muscarinic receptors on the bladder smooth muscle (Eglen et al., 1999;Hegde and Eglen, 1999). The effect of A-278637 on muscarinic receptor-mediated contractions was assessed in both pig and human bladder strips. Contractions evoked by the muscarinic receptor agonist carbachol were effectively suppressed in a concentration-dependent manner by A-278637 in pig bladder strips with an IC50 value of 164 nM (−log IC50 = 6.78 ± 0.08; n = 6). In human bladder strips, A-278637 relaxed carbachol-evoked contractile response with an IC50 value of 354 nM (−log IC50 = 6.89 ± 0.33; n = 7) comparable with that noted in the pig. These data are summarized in Table 1.

Contractions of pig bladder strips evoked by low-frequency electrical stimulation, thought to reflect presynaptic release principally of both acetylcholine and ATP (Andersson et al., 1999), were also inhibited by A-278637 with an IC50 value of 308 nM (−log IC50 = 6.51 ± 0.09; n = 6). Again, the inhibition of contractions was reversed by addition of glyburide (5 μM). To further define the nature of KATP channel interactions, the effect of glyburide on KCO-evoked relaxation of electrical field-stimulated contractions was quantified by Schild analysis of the concentration-response data (Schild, 1947). Glyburide suppressed racemic A-278637-mediated relaxation of bladder strips in an apparently competitive manner with a pA2 value of 7.40 ± 0.33 and a slope close to unity (1.1 ± 0.10;n = 16).

In Vitro Selectivity.

Vascular KATP . To examine interactions with vascular KATP 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).

View this table:
Table 2

In vitro analysis of tissue selectivity

Selectivity of the interactions of A-278637 was evaluated in assays using the thoracic aorta and portal vein, two defined models of vascular function. A-278637 suppressed contractions in both vascular preparations in a concentration-dependent manner. The IC50 values are summarized in Table 2, along with corresponding values obtained with ZD6169 and WAY-133537. Although A-278637 does not exhibit absolute selectivity, it shows modest bladder selectivity versus thoracic aorta compared with ZD6169 and WAY-133537, which were somewhat more potent in relaxing aortic strips.

Cardiac KATP.

The potential interaction of A-278637 on cardiac KATP 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 KATP channels (Table3).

Pancreatic KATP.

A-278637 was also examined for interaction with sulfonylurea receptor SUR1-containing KATP 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 activate86Rb+ 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 Ki value of 12.49 ± 3.66 μM (n = 3), which is 120-fold higher than the potency to activate bladder KATP 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 BKCa) expressed in various clonal cell lines (n ≥ 3).

A-278637 was evaluated in a binding screen that contained representatives of most G protein-coupled receptors, as well as some ligand- and voltage-gated ion channel binding sites at CEREP (receptor binding and enzyme profile; CEREP, Inc., Redmond, WA). The compound was tested at a single concentration (10 μM) in duplicate. A-278637 did not show significant displacement of binding with the exception of vasopressin V1A receptors where it displaced the binding of [3H]arginine-vasopressin to human recombinant V1A receptors expressed in mammalian cells (82% at 10 μM). Further examination of the concentration response for binding inhibition revealed aKi value 0.92 μM (n= 3). A-278637 was tested for agonist and antagonist activities at V1 receptors in the rat caudal artery. The compound was ineffective when tested as an agonist but did weakly suppress contractions evoked by arginine-vasopressin (3 nM) with a 57% inhibition observed at 10 μM. The potency of A-278637 to inhibit arginine-vasopressin-mediated contractions in the vasculature is at least 100-fold higher than that required to activate bladder KATP channels in the membrane potential studies or to inhibit bladder contractions in vitro.

Discussion

The present study describes the in vitro pharmacological profile of a novel dihydropyridine KATP 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 KATP channels was confirmed in bladder smooth muscle cells where the compound was efficacious in activating glyburide-sensitive currents.

KATP 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 KATP channels compared with ZM244085 (6.1 μM;Gopalakrishnan et al., 1999), a structurally related 1,4-dihydropyridine KATP 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 KATPchannel. 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 KATPchannel. 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 KATP 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).

It is known that activation of the parasympathetic nerves releases acetylcholine from postganglionic nerves, which in turn activates muscarinic receptors present in bladder smooth muscles, resulting in bladder contractions and normal voiding function. Although the muscarinic M2 receptors seem to be the predominant subtype present in most species, it is the postsynaptic muscarinic M3 receptor present in the bladder smooth muscle that is responsible for contractions (Eglen et al., 1999). Although equiefficacious in tissue relaxation assays, A-278637 was 8- to 13-fold less potent in inhibiting contractions evoked by direct muscarinic receptor activation with carbachol or by stimulation of parasympathetic postganglionic nerves that triggers the release of both acetylcholine and ATP compared with the suppression of myogenic phasic activity (Table 1). Previous studies have shown that KATP channel openers, in general, are some 15-fold more potent in suppressing spontaneous activities compared with electrical field-stimulated contractions, primarily via reduction in contraction frequency consistent with the idea that small increases in K+ conductance evoked by KCOs are sufficient to suppress spontaneous myogenic activity (Petkov et al., 2001; Buckner et al., 2002). The suppression of contractions induced by muscarinic receptor activation or nerve stimulation by KCOs, including A-278637, can be attributed to activation of KATP channels that lead to membrane hyperpolarization and attenuation of Ca2+ influx through L-type voltage-gated Ca2+ channels.

In Vitro Selectivity of A-278637.

The cloning and expression of cDNAs for KATP channel subunits indicates a diversity of KATP 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 KATP 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 KATP channels with properties typical of those expressed in pancreas and neurons, whereas the components of plasmalemmal cardiac KATP channel are derived from SUR2A-Kir6.2. A-278637 was found to display selectivity toward activation of KATP channels in bladder smooth muscle (EC50 = 102 nM) relative to KATP channels in cardiomyocytes (EC50 = 40 μM) or pancreatic KATP 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 Ki value that was at least 100-fold higher than the concentrations required to activate bladder smooth muscle KATP channels. Thus, at concentrations effective in suppressing bladder contractility, A-278637 displays substantial selectivity versus other predominant KATP channel subtypes.

Because a key goal in the development of KATPchannel openers is to identify bladder-selective openers with minimal hemodynamic effects, the interactions of A-278637 were assessed in two vascular tissues. Although no absolute in vitro bladder versus vascular selectivity could be demonstrated, our studies show that A-278637 is about 2-fold less potent in relaxing 25 mM K+-stimulated thoracic aorta, whereas both WAY-133537 and ZD6169 are 2- and 6.5-fold more potent in relaxing aortic smooth muscle compared with inhibition of bladder contractions. Similarly, WAY-133537 and ZD6169 are 2- and 6-fold, respectively, more potent in inhibiting portal vein than bladder contractility, whereas A-278637 showed comparable potencies in these two assays. Thus, although no absolute selectivity is apparent, A-278637 seems to be somewhat more selective toward suppression of bladder versus vascular contractility compared with ZD6169 and WAY-133537.

As shown in the accompanying article (Brune et al., 2002), A-278637 showed potent inhibition of bladder contractions in a myogenic model of bladder instability with in vivo selectivity superior to both ZD6169 and WAY-133537. Although A-278637 exhibits a modest degree of relative selectivity at the in vitro level, whether this contributes to the selectivity noted in vivo remains to be established. Considerable diversity exists for the nucleotide-regulated K+channels in smooth muscle tissues with varying conductance and biophysical properties. These have been confirmed by coexpression of the SUR2B subunit with various combinations of Kir6.2 and Kir6.1 subunits in heterologous expression systems (Babenko et al., 1998;Seino, 1999; Yamada et al., 1999). The selectivity of A-278637 could, in principle, arise from distinctions in interactions with KATP channel combinations at the level of SUR-Kir complex or via differential coupling of nucleotide and KCO binding to regulate KATP channel activity in the obstructed bladder. Although SUR2B RNA expression is widespread, splicing of the SUR2 gene has been shown to occur at exon 17 located in the first nucleotide-binding motif with differential expression patterns and nucleotide sensitivities (Chutkow et al., 1999; Davis-Taber et al., 2000). There is also some evidence suggesting that the KATP channel complex functions not only solely as a K+ conductance but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit (Bienengraeber et al., 2000). Whether any of these mechanisms modulate bladder KATP channel function and differentially influence interactions of A-278637 remains to be investigated.

In conclusion, the findings of the present study show that A-278637 is a novel and potent KCO with efficacy in suppressing myogenic activity in vitro in both pig and human detrusor. A-278637 is more selective in suppressing bladder myogenic activity versus inhibition of vascular contractility relative to reference KCOs, and as noted in the accompanying article (Brune et al., 2002), suppresses unstable myogenic contractions in vivo with enhanced bladder selectivity. Such a profile presents an attractive basis for further investigations of selective KATP channel openers with potential for the treatment of overactive bladder.

Footnotes

  • DOI: 10.1124/jpet.102.034538

  • Abbreviations:
    KATP
    ATP-sensitive K+
    KCO
    potassium channel opener
    DIBAC4(3)
    bis-(1,3-dibutylbarbituric acid)trimethine oxonol
    FLIPR
    fluorescent imaging plate reader
    AUC
    area under the curve
    SUR
    sulfonylurea receptor
    WAY-133537
    (R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)cyclobut-1-enylamino]-3-ethyl-benzonitrile
    ZD-6169
    (S)-N-(4-benzoylphenyl)-3,3,3-trifluro-2-hydroxy-2-methyl-priopionamide
    YM934
    2-(3,4-dihydro-2,2-dimethyl-6-nitro-2H-1,4,-benzoxazin-4-yl)pyridine-N-oxide
    ZM 244085
    9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridine dione
    • Received February 11, 2002.
    • Accepted May 30, 2002.

References

  1. ↵
    1. Andersson KE,
    2. Appell R,
    3. Cardozo LD,
    4. Chapple C,
    5. Drutz HP,
    6. Finkbeiner AE,
    7. Haab F,
    8. Navarrete RV
    (1999) The pharmacological treatment of urinary incontinence. BJU Int 84:923–947.
    OpenUrlCrossRefMedline
  2. ↵
    1. Babenko AP,
    2. Aguilar-Bryan L,
    3. Bryan J
    (1998) A view of SUR/KIR6. X, KATP channels. Annu Rev Physiol 60:667–687.
    OpenUrlCrossRefMedline
  3. ↵
    1. Bienengraeber M,
    2. Alekseev AE,
    3. Abraham MR,
    4. Carrasco AJ,
    5. Moreau C,
    6. Vivaudou M,
    7. Dzeja PP,
    8. Terzic A
    (2000) ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex. FASEB J 14:1943–1952.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Bonev AD,
    2. Nelson MT
    (1993) ATP-sensitive potassium channels in smooth muscle cells from guinea pig urinary bladder. Am J Physiol 264:C1190–C1200.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Brading AF
    (1997) A myogenic basis for the overactive bladder. Urology 50:57–67, , 68–73..
    OpenUrlCrossRefMedline
  6. ↵
    1. Brune ME,
    2. Fey TA,
    3. Gauvin DM,
    4. Fabiyi AC,
    5. Brioni JD,
    6. Sullivan JP,
    7. Coghlan MJ,
    8. Gopalakrishnan M
    (2002) A-278637: a novel ATP-sensitive potassium channel opener with potential for the management of overactive bladder. II. In vivo characterization. J Pharmacol Exp Ther 303:387–394.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Buckner S,
    2. Milicic I,
    3. Daza A,
    4. Coghlan M,
    5. Gopalakrishnan M
    (2002) Spontaneous phasic contractions of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators. Br J Pharmacol 135:639–648.
    OpenUrlCrossRefMedline
  8. ↵
    1. Buckner SA,
    2. Milicic I,
    3. Daza A,
    4. Davis-Taber R,
    5. Scott VES,
    6. Sullivan JP,
    7. Brioni JD
    (2000) Pharmacological and molecular analysis of ATP-sensitive K+ channels in the pig and human detrusor. Eur J Pharmacol 400:287–295.
    OpenUrlCrossRefMedline
  9. ↵
    1. Carroll WA,
    2. Drizin I,
    3. Brune ME,
    4. Buckner SA,
    5. Gopalakrishnan M,
    6. Fey TA,
    7. Gauvin DM,
    8. Coghlan MJ,
    9. Scott VE,
    10. Shieh C-C,
    11. et al.
    (2001) In vitro and in vivo profile of A-278637, a novel bladder selective KATP channel opener. Abstracts of Papers, 222nd American Chemical Society National Meeting, Chicago, IL, August 26–30, pp MEDI-195..
  10. ↵
    1. Chapple CR
    (2000) Muscarinic receptor antagonists in the treatment of overactive bladder. Urology 55:33–46.
    OpenUrlCrossRefMedline
  11. ↵
    1. Chutkow WA,
    2. Makielski JC,
    3. Nelson DJ,
    4. Burant CF,
    5. Fan Z
    (1999) Alternative splicing of sur2 exon 17 regulates nucleotide sensitivity of the ATP-sensitive potassium channel. J Biol Chem 274:13656–13665.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Cui Y,
    2. Giblin JP,
    3. Clapp LH,
    4. Tinker A
    (2001) A mechanism for ATP-sensitive potassium channel diversity: functional coassembly of two pore-forming subunits. Proc Natl Acad Sci USA 98:729–734.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Davis-Taber R,
    2. Choi W,
    3. Feng J,
    4. Hoogenboom L,
    5. McNally T,
    6. Kroeger P,
    7. Shieh CC,
    8. Simmer R,
    9. Brioni JD,
    10. Sullivan JP,
    11. Gopalakrishnan M,
    12. Scott VE
    (2000) Molecular characterization of human SUR2-containing KATP channels. Gene 256:261–270.
    OpenUrlCrossRefMedline
  14. ↵
    1. de Groat WC
    (1997) A neurologic basis for the overactive bladder. Urology 50:36–52.
    OpenUrlCrossRefMedline
  15. ↵
    1. Eglen RM,
    2. Choppin A,
    3. Dillon MP,
    4. Hegde S
    (1999) Muscarinic receptor ligands and their therapeutic potential. Curr Opin Chem Biol 3:426–432.
    OpenUrlCrossRefMedline
  16. ↵
    1. Elbadawi A,
    2. Yalla SV,
    3. Resnick NM
    (1993) Structural basis of geriatric voiding dysfunction. III. Detrusor overactivity. J Urol 150:1668–1680.
    OpenUrlMedline
  17. ↵
    1. Elbadawi A,
    2. Diokno AC,
    3. Millard RJ
    (1998) The aging bladder: morphology and urodynamics. World J Urol 16 (Suppl 1) S10–S34.
  18. ↵
    1. Foster CD,
    2. Speakman MJ,
    3. Fujii K,
    4. Brading AF
    (1989) The effects of cromakalim on the detrusor muscle of human and pig urinary bladder. Br J Urol 63:284–294.
    OpenUrlMedline
  19. ↵
    1. Fujii K,
    2. Foster CD,
    3. Brading AF,
    4. Parekh AB
    (1990) Potassium channel blockers and the effects of cromakalim on the smooth muscle of the guinea pig bladder. Br J Pharmacol 99:779–785.
    OpenUrlMedline
  20. ↵
    1. Gopalakrishnan M,
    2. Johnson DE,
    3. Janis RA,
    4. Triggle DJ
    (1991) Characterization of binding of the ATP-sensitive potassium channel ligand, [3H]glyburide, to neuronal and muscle preparations. J Pharmacol Exp Ther 257:1162–1171.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Gopalakrishnan M,
    2. Whiteaker KL,
    3. Molinari EJ,
    4. Davis-Taber R,
    5. Scott VES,
    6. Shieh C-C,
    7. Buckner SA,
    8. Milicic I,
    9. Cain JC,
    10. Postl S,
    11. et al.
    (1999) Characterization of the ATP-sensitive potassium channels (KATP) expressed in guinea pig bladder smooth muscle cells. J Pharmacol Exp Ther 289:551–558.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Grant TL,
    2. Zuzack JS
    (1991) Effects of potassium channel blockers and cromakalim (BRL 34915) on the mechanical activity of guinea pig detrusor smooth muscle. J Pharmacol Exp Ther 259:1158–1164.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Hamill OP,
    2. Marty A,
    3. Neher E,
    4. Sakmann B,
    5. Sigworth FJ
    (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflueger Archiv 391:85–100.
  24. ↵
    1. Hashitani H,
    2. Suzuki H,
    3. Kumazawa J
    (1996) Effects of Y-26763, a novel K-channel opener, on electrical responses of smooth muscles in the guinea pig bladder. J Urol 155:1454–1458.
    OpenUrlCrossRefMedline
  25. ↵
    1. Hegde SS,
    2. Eglen RM
    (1999) Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder. Life Sci 64:419–428.
    OpenUrlCrossRefMedline
  26. ↵
    1. Howe BB,
    2. Halterman TJ,
    3. Yochim CL,
    4. Do ML,
    5. Pettinger SJ,
    6. Stow RB,
    7. Ohnmacht CJ,
    8. Russell K,
    9. Empfield JR,
    10. Trainor DA,
    11. Brown FJ,
    12. Kau ST
    (1995) ZENECA ZD6169: a novel KATP channel opener with in vivo selectivity for urinary bladder. J Pharmacol Exp Ther 274:884–890.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Li JH,
    2. Yasay GD,
    3. Kau ST,
    4. Ohnmacht CJ,
    5. Trainor DA,
    6. Bonev AD,
    7. Heppner TJ,
    8. Nelson MT
    (1996) Studies of the KATP channel opening activity of the new dihydropyridine compound 9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione in bladder detrusor in vitro. Arzneim Forsch 46:525–530.
    OpenUrlMedline
  28. ↵
    1. Malmgren A,
    2. Andersson KE,
    3. Sjogren C,
    4. Andersson PO
    (1989) Effects of pinacidil and cromakalim (BRL 34915) on bladder function in rats with detrusor instability. J Urol 142:1134–1138.
    OpenUrlMedline
  29. ↵
    1. McGhan WF
    (2001) Cost effectiveness and quality of life considerations in the treatment of patients with overactive bladder. Am J Manag Care 7:S62–S75.
    OpenUrlMedline
  30. ↵
    1. Mills IW,
    2. Greenland JE,
    3. McMurray G,
    4. McCoy R,
    5. Ho KM,
    6. Noble J,
    7. Brading AF
    (2000) Studies on the pathophysiology of idiopathic detrusor instability: the physiological properties of detrusor smooth muscle and its pattern of innervation. J Urol 163:646–651.
    OpenUrlCrossRefMedline
  31. ↵
    1. Nurse DE,
    2. Restorick JM,
    3. Mundy AR
    (1991) The effect of cromakalim on the normal and hyperreflexic human detrusor muscle. Br J Urol 68:27–31.
    OpenUrlMedline
  32. ↵
    1. Pandita RK,
    2. Persson K,
    3. Andersson KE
    (1997) Effects of the K+ channel opener, ZD6169, on volume- and PGE2-stimulated bladder activity in conscious rats. J Urol 158:2300–2304.
    OpenUrlCrossRefMedline
  33. ↵
    1. Payne CK
    (1998) Epidemiology, pathophysiology and evaluation of urinary incontinence and overactive bladder. Urology 51:3–10.
  34. ↵
    1. Petkov GV,
    2. Heppner TJ,
    3. Bonev AD,
    4. Herrera GM,
    5. Nelson MT
    (2001) Low levels of KATP channel activation decrease excitability and contractility of urinary bladder. Am J Physiol 280:R1427–R1433.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Quayle JM,
    2. Nelson MT,
    3. Standen NB
    (1997) ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev 77:1165–1232.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Schild HO
    (1947) pA, A new scale for the measurement of drug antagonism. Br J Pharmacol 2:189–206.
    OpenUrlMedline
  37. ↵
    1. Seino S
    (1999) ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61:337–362.
    OpenUrlCrossRefMedline
  38. ↵
    1. Shieh C-C,
    2. Feng J,
    3. Buckner SA,
    4. Brioni JD,
    5. Coghlan MJ,
    6. Sullivan JP,
    7. Gopalakrishnan M
    (2001) Functional implication of spare ATP-sensitive K+ channels in bladder smooth muscle cells. J Pharmacol Exp Ther 296:669–675.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Sullivan J,
    2. Abrams P
    (1999) Pharmacological management of incontinence. Eur Urol 36:89–95.
  40. ↵
    1. Wammack R,
    2. Jahnel U,
    3. Nawrath H,
    4. Hohenfellner R
    (1994) Mechanical and electrophysiological effects of cromakalim on the human urinary bladder. Invest Urol 5:159–164.
  41. ↵
    1. Whiteaker KL,
    2. Davis-Taber R,
    3. Scott VES,
    4. Gopalakrishnan M
    (2002) Fluorescence based assay for ATP sensitive K+ channel activation in cultured neonatal rat ventricular myoctyes. J Pharmacol Toxicol Methods 46:1–6.
  42. ↵
    1. Wojdan A,
    2. Freeden C,
    3. Woods M,
    4. Oshiro G,
    5. Spinelli W,
    6. Colatsky TJ,
    7. Sheldon JH,
    8. Norton NW,
    9. Warga D,
    10. Antane MM,
    11. et al.
    (1999) Comparison of the potassium channel openers, WAY-133537, ZD6169 and celikalim on isolated bladder tissue and in vivo bladder instability. J Pharmacol Exp Ther 289:1410–1418.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Wei XY,
    2. Rutledge A,
    3. Triggle DJ
    (1989) Voltage-dependent binding of 1,4-dihydropyridine Ca2+ channel antagonists and activators in cultured neonatal rat ventricular myocytes. Mol Pharmacol 35:541–552.
    OpenUrlAbstract
  44. ↵
    1. Yamada M,
    2. Satoh E,
    3. Kondo C,
    4. Repunte VP,
    5. Kurachi Y
    (1999) Molecular structure and function of cardiovascular ATP-sensitive potassium channels. Curr Top Membr 46:387–415.
    OpenUrl
  45. ↵
    1. Yu Y,
    2. de Groat WC
    (1999) Effects of ZD6169, a KATP channel opener, on the micturition reflex in the rat. J Pharmacol Exp Ther 290:825–831.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools
Request Permissions
Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

(−)-(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): A Novel ATP-Sensitive Potassium Channel Opener Efficacious in Suppressing Urinary Bladder Contractions. I. In Vitro Characterization

Journal of Pharmacology and Experimental Therapeutics October 2002, 303 (1) 379-386; DOI: http://dx.doi.org/10.1124/jpet.102.034538
Permalink: Copy
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section