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Pharmacological characterization of urinary bladder smooth muscle contractility following partial bladder outlet obstruction in pigs
European Journal of Pharmacology 532 (2006) 107 – 114 www.elsevier.com/locate/ejphar
Pharmacological characterization of urinary bladder smooth muscle contractility following partial bladder outlet obstruction in pigs Ivan Milicic ⁎, Steven A. Buckner, Anthony Daza, Michael Coghlan, Thomas A. Fey, Michael E. Brune, Murali Gopalakrishnan Neuroscience Research, Department R4MN, Building AP9, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA Received 8 September 2005; received in revised form 29 November 2005; accepted 19 December 2005 Available online 17 February 2006
Abstract Partial bladder outlet obstruction of the pig is considered as a valuable preclinical model for evaluating the profile of compounds for the treatment of bladder overactivity. In this study, we characterized the pharmacological properties of isolated bladder smooth muscle from pigs following partial outlet obstruction and its sensitivity to potassium channel openers. Bladder strips from obstructed animals showed significantly lower maximal efficacy (Emax) and sensitivity to stimulation by ATP and carbachol, but not to those evoked by serotonin, compared to agematched controls. Tissue strips from obstructed animals also showed a 2.5-fold increase in the potency and significantly reduced maximum response following K+ depolarization. With respect to spontaneous activity, bladder strips from control strips demonstrated little spontaneous phasic activity at all preloads examined. In contrast, bladder strips from obstructed animals showed large preload-dependent increases in spontaneous phasic activity at preload values of 16–32 g. The potencies of KATP channel openers to relax carbachol-evoked contractions showed a good 1 : 1 correlation (r2 = 0.90) between obstructed and control bladder strips. These studies demonstrate that obstructed pig bladders show enhanced spontaneous phasic activity especially at elevated preloads, which may underlie unstable myogenic bladder contractions reported in cystometrographic measurements in vivo. The impaired responses to electrical field stimulation could be attributed to reduced efficacies and/or lower sensitivities of muscarinic and purinergic signaling pathways. KATP channel sensitivities remain essentially unimpaired in the obstructed bladder and could be effectively modulated by openers with potential for the treatment of overactive bladder secondary to outlet obstruction. © 2006 Elsevier B.V. All rights reserved. Keywords: Potassium channel opener; Detrusor smooth muscle; Smooth muscle relaxation; ATP-sensitive potassium channels; Sulfonylurea receptor; Outlet obstruction; Overactive bladder
1. Introduction Overactive bladder, a condition affecting an estimated 15 million individuals in the United States alone, is characterized by symptoms of urinary frequency and urgency with or without urge incontinence. Of the various etiologies, urinary bladder outlet obstruction is one of the major underlying causes with significant population of men 60 years of age or older having varying degrees of obstruction secondary to benign prostatic hyperplasia (Grayhack et al., 1987). Several characteristics of human outlet obstruction including detrusor instability and increases in micturition pressure can be ⁎ Corresponding author. Tel.: +1 847 938 2222; fax: +1 847 937 9195. E-mail address: [email protected] (I. Milicic). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.076
reproduced in experimental animals including obstructed rats (Malmgren et al., 1987), rabbits (Levin et al., 2000) humans and pigs (Sibley, 1984; Mills et al., 2000). The initial suggestion of abnormal behavior following outlet obstruction was by Sibley (1984) who reported increased sensitivity to agonist simulation of bladder strips from pigs or humans with bladder instability secondary to outflow obstruction. Subsequent studies in pigs demonstrated a change in the ratio of maximal contractile response evoked by muscarinic agonists and intrinsic nerve stimulation in the unstable bladder, the latter often significantly depressed (Speakman et al., 1987). These observations have also been confirmed in patients with obstructive instability consistent with partial denervation of the bladder (Levin et al., 1986). Functional changes in isolated detrusor muscle have also been described in rats where decreases
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in neurogenic contractility after 6 weeks of obstruction (Mattiason et al., 1987) and reduced responsiveness to carbachol, ATP and electrical field stimulation following long term (15 weeks) outlet obstruction (Saito et al., 1996) have been reported. When supersensitivity and partial denervation coexist, the responsiveness to activation of intrinsic nerves may depend on the interaction of the two processes, i.e., attenuated responsiveness because of denervation and increased responsiveness due to the altered properties of the smooth muscle (Brading, 1997; De Groat, 1997). The pig detrusor has been considered a model for investigating the physiology of bladder function, as the pig has comparable urodynamic characteristics to that of human (Sibley, 1984, 1985, 1987; Speakman et al., 1987; Crowe and Burnstock, 1989). There is also evidence from studies in pigs that tissues from the unstable bladder may be more sensitive to exogenous agonists than normal bladder (Sibley, 1987; Foster et al., 1989; Martin et al., 1997). In vitro studies from our laboratory have demonstrated that the pig detrusor is a suitable model for studying the functional pharmacology of K+ channels (Buckner et al., 2000, 2002). More recently, we have characterized in vivo unstable bladder contractions in an anesthetized pig model of partial bladder outlet obstruction by indwelling telemetry and cystometry approaches (Fey et al., 2003). Cystometrographic analysis of isoflurane-anesthetized animals 17–20 weeks post-obstruction showed low amplitude, rhythmic bladder contractions that were sensitive to inhibition by K+ channel openers. In this study, we report the characterization of the in vitro properties of isolated bladder smooth muscle strips from pigs following partial outlet obstruction by assessing spontaneous phasic activity and responsiveness following exogenous agonist- and electrical field-evoked responses. These studies demonstrate that obstructed pig bladder strips show enhanced spontaneous phasic activity, especially at elevated preloads, which may contribute to unstable myogenic bladder contractions observed in vivo. Although responsiveness to electrical field stimulation, muscarinic and purinergic agonists were attenuated in the obstructed bladder, the sensitivities to KATP channel openers were unaffected, consistent with their reported in vivo efficacies in this model (Fey et al., 2003). 2. Materials and methods 2.1. Experimental bladder outlet obstruction Female Landrace/Yorkshire crossbred pigs (Wilson Prairie View Farms, Burlington, WI) 12 weeks of age were obstructed with a 7.5-mm silver omega ring placed around the proximal urethra as described previously by Fey et al. (2003). Studies were carried out in accordance with guidelines outlined by the Animal Welfare Act, the Association for Assessment and Accreditation of Laboratory Animals (AAALAC) and the Institutional Animal Care and Use Committee of Abbott Laboratories. Proper ring placement was confirmed at necropsy in all animals. Seventeen to 20 weeks after placement of the ring, the pigs were instrumented with telemetry transducer/transmitters (Data
Sciences; St. Paul, MN) for the measurement of carotid arterial pressure (unit 1: TA11PA-C40) and intravesical/abdominal pressures (unit 2: TL11M3-D70-PCP). A port catheter (TI-9, Access Technologies) was placed subcutaneously in the side of the abdomen and its distal catheter secured in the bladder lumen. Animals were treated with amoxicillin and buprenorphine for 3 to 5 days post-surgery and allowed to recover for 10 to 14 days before testing. Urine was drained completely via the port catheter from one to five times per week to avoid urinary retention and potential renal failure. 2.2. Cystometry studies Obstructed animals were verified for unstable bladder contractions by in vivo cystometry. Animals were anesthetized with telazol and xylazine, intubated and maintained on an isoflurane : oxygen mixture in the supine position with water blankets to maintain body temperature. Bladder volume was adjusted via the port catheter to establish a regular unstable contraction pattern. Radiotelemetry signals for bladder pressure were obtained using an RMC-1 receiver (Data Sciences, St. Paul, MN). Data acquisition and semi-automated analysis were performed using the cystometry analysis software in the Ponemah Physiology Platform (Gould Instrument Systems, Valley View, OH). Obstructed animals that showed demonstrable unstable contractions, i.e., bladder pressure amplitude ≥ 4 cm H2O, and agematched control animals were utilized for in vitro studies (Fey et al., 2003). 2.3. In vitro studies On the day of the experiment, animals were euthanized with an intraperitoneal injection of pentobarbital (Somlethol®) at a lethal dose of 150–200 mg/kg. The entire urinary bladder was cleared of surrounding adipose tissue, removed and placed in Krebs Ringer bicarbonate solution of the composition, (mM): 120 NaCl, 20 NaHCO3, 11 dextrose, 4.7 KCl, 2.5 CaCl2, 1.5 MgSO4, 1.2 KH2PO4, equilibrated with 5% CO2 : 95% O2 (pH = 7.4 at 37 °C). (±)-Propranolol, (4.0 μM) was included in the study to ensure β-adrenoceptor blockade. The bladder was sectioned into three pieces and the top dome portion and the lower trigonal area were discarded. The remaining tissue (detrusor) was sliced transversely into approximately 4 × 10 mm strips. Tissue strips without urothelium from the area adjacent to the trigone region were used in both control and obstructed pigs throughout the study. Denuded tissue 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 Grass FT03 transducer at a basal preload of 1 g. The stationary rods contained parallel platinum electrodes that fit on each side of the tissue strips. Tissues were rinsed at 10-min intervals and allowed to equilibrate for at least 75 min prior to testing. 2.4. Electrical field stimulation Contractile responses were evoked by a Grass model S88 stimulator at a frequency of 0.05 Hz (except where indicated),
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duration of 0.5 ms, 20 V (supramaximal in terms of the evoked twitch), and current b150 mA. A voltage–current amplifier circuit was placed between the stimulator and the electrodes to maintain constant stimulation parameters. The electrical stimulus was set to deliver square wave pulses for the duration of the protocol. In control experiments, these parameters produced uniform, single shock twitches that returned to baseline levels between each stimulus. These twitches were completely abolished by tetrodotoxin (100 nM). For the generation of frequency response curves, the tissues were stimulated at 0.05, 0.50, 1.0, 2.0, 4.0, 8.0, 16.0 and 32 Hz for 60 s in order to achieve maximum contraction. 2.5. Exogenous agonists Concentration response effects were determined for carbachol, ATP, histamine, and serotonin (1 nM to 30 mM) by a noncumulative addition protocol. The maximum responses were determined and a 20-min rinse cycle was included between successive additions. 2.6. K+ depolarization Concentration response effects were determined for KCl using a protocol identical to that described above for exogenous agonists. KCl was tested over a range of concentrations by noncumulative addition. The maximum responses were determined and a 20-min rinse cycle was included between successive additions. 2.7. Spontaneous phasic activity To examine concentration-dependent effects on spontaneous phasic activity, test compounds were introduced and their effects assessed in terms of area under the contractile curve (AUC) for a 15-min period, normalized to 1-min and expressed as gram seconds per min (AUC g.s./min). After each concentration the tissues were rinsed for 20-min and the cycle repeated. As reported earlier (Buckner et al., 2002), it was found that the area of the detrusor away from the dome region of the bladder provided for the most consistent spontaneous responses. It was also observed that tissues with the urothelium intact demonstrated significantly reduced signal-to-noise ratio compared to adjacent strips where the urothelium was removed. Wherever concentration response curves were generated, each bladder strip was used for only one test compound or condition.
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by generating a cumulative inhibitory response curve to P1075 as previously described (Buckner et al., 2002). The concentration-dependent reduction in the peak amplitude was used for calculating IC50 values. 2.9. Compounds Bay X-9227 [((-)-N-(2-ethoxyphenyl)-N′-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine)], Bay X-9228 (enantiomer of Bay X-9227), (-)-cromakalim and the racemate (±)-cromakalim, nicorandil, P1075 [N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridylguanidine], ZD6169[N-(4-benzoylphenyl)-3,3,3-trifluro-2-hydroxy-2-methylpropion amine], A-278637 [(-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9hexahydrothieno[3,2-b]quinolin and ZM244085 [9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione] were synthesized in house. Diazoxide and pinacidil were purchased from Research Biochemicals International (Natick MA). Stock solutions were prepared fresh daily in distilled water or in methyl sulfoxide (DMSO). All other chemicals including (±)-propranolol HCl, and (DMSO) were purchased from Sigma Chemical Co (St. Louis, MO). 2.10. Data analysis Concentration response curves were analyzed using a fourparameter curve fitting routine, described previously (Zielinski and Buckner, 1998). The maximum peak amplitude response was used for analysis. Results were expressed as gram tension and as percentage of maximum response. Spontaneous phasic activity data were recorded and analyzed using PowerLab (ADI Instruments, Australia). The signal was analyzed as the integrated area under the contractile curve (AUC) for 15 min intervals and corrected to 1 min and expressed as AUC gram seconds per min. Potencies were expressed as the − log 10 (pD2) of the geometric mean of the EC50 or IC50 values ± S.E.M. Data were analyzed for significance using an unpaired, two-tailed t-test, GraphPad Prism version 3.02 (GraphPad software, San
2.8. K+ channel opener pharmacology Carbachol was used as the agonist to assess KATP channel opener sensitivities. The protocol was non-cumulative with rinse cycles between successive additions. Tissues were pretreated with the potassium channel openers for 15 min, then exposed to a fixed concentration (EC100 1 μM) of carbachol until maximum tension developed and rinsed for 15 min and the cycle was repeated. Tissue sensitivities were initially assessed
Fig. 1. (A) Representative cystogram from obstructed, unstable pig bladder in vivo. Contractile responses ≥4.0 cm H20 were considered unstable. (B) Representative cystogram from age-matched control pig bladder. Small oscillations represent breathing artifact. Contractile responses b4.0 cm H20 were considered normal.
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I. Milicic et al. / European Journal of Pharmacology 532 (2006) 107–114 Table 1 Characteristics of agonist-evoked contractility of isolated bladder strips from control and obstructed unstable pigs
Fig. 2. Frequency response relationship of control (open bars) and obstructed (hatched bars) bladder strips. Contractile data shown as the mean maximum response (g) ± S.E.M., n = 8. P b 0.05.
Diego, CA, U.S.A.) and P value b 0.05 was considered significant. 3. Results 3.1. Characterization of unstable contractions Twenty-four hours prior to use, bladders from the obstructed pigs were verified as unstable in vivo by cystometric analysis. Fig. 1 shows representative tracings from obstructed (Fig. 1A) and control (Fig. 1B) unobstructed pigs. As previously reported, obstructed pigs exhibited highly rhythmic, spontaneous nonvoiding contractions starting 17 weeks post-obstruction (Fig. 1A). Approximately 80% of obstructed animals were found to have involuntary bladder contractions as measured by
Agonist
Control
Unstable
pD2
Emax (g)
pD2
Emax (g)
Carbachol ATP Histamine Serotonin KCl
6.54 ± 0.05 (12) 4.85 ± 0.15 (19) 5.58 ± 0.15 (6) 6.26 ± 0.06 (8) 1.08 ± 0.33 (12)
19.38 ± 3.46 4.6 ± 0.85 10.18 ± 1.30 1.91 ± 0.21 27.6 ± 4.02
5.93a ± 0.04 (8) 3.72a ± 0.08 (8) 5.60 ± 0.06 (8) 5.96 ± 0.09 (7) 1.66a ± 0.24 (8)
9.16a ± 0.82 2.61a ± 0.58 2.00a ± 0.20 1.59 ± 0.43 11.20a ± 1.20
Data shown are mean potency (pD2 ± S.E.M.) and mean maximum response (Emax ± S.E.M.) Values in parenthesis are the number of determinations. a represents P b 0.05 vs. obstructed bladders.
cystometry. The remaining 20% of animals showed signs of bladder hypertrophy with no involuntary contractions (Fey et al., 2002) Only bladder strips from obstructed pigs whose bladders were verified as unstable (spontaneous bladder contractions ≥ 4 cm H2O) were used in this study. Obstructed animals used in this study had mean contraction amplitude, frequency and duration of 11.7 ± 0.5 cm H2O, 34.6 ± 4.6 contractions per 30 min and 35.0 ± 2.7 s per contraction (n = 5), respectively. 3.2. Bladder hypertrophy Partial outlet obstruction of the bladder led to a significant increase in bladder weight. The mean values were 77.05 ± 4.99 g (n = 5) for control and 546.00 ± 44.08 g (n = 17) for obstructed pigs (P b 0.01). Consequently, the weights of the isolated
Fig. 3. (A) Contractile responses of bladder strips from control and obstructed pigs showing concentration-dependent increases in contractile responses from control (open circles) and obstructed (filled circles) bladders by carbachol (A), ATP (B), histamine (C) and serotonin (D). In each panel, the inset depicts data expressed as percentage of the maximum response. S.E.M. values are omitted for clarity. Numbers of determinations are summarized in Table 1.
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responses ranging from 3.03 ± 0.76 g at 0.05 Hz to 40.06 ± 7.25 g at 32 Hz (n = 8). Bladder strips from obstructed pigs showed significantly reduced frequency dependent increases in contractions and ranged from 0.98 ± 0.13 at 0.05 Hz to 8.57 ± 1.12 g at 32 Hz (n = 8). However, in both cases, maximal increases were observed at a frequency of 16 Hz (Fig. 2). 3.4. Responses to exogenous agonists
Fig. 4. Contractile response of bladder strips to K+ depolarization. Open circles represent control, closed circles represent strips from obstructed bladders. Data expressed as maximum response (g) mean ± S.E.M. (control n = 12; obstructed n = 8). The inset depicts data expressed as percentage of maximum response.
bladder strips were significantly greater from the obstructed pigs, and therefore, strips were weight adjusted prior to mounting by slicing out the urothelium side to approximate the thickness of control strips. The mean weight of control strips (333.08 ± 29.52 mg; n = 4 strips each from 6 animals) and obstructed bladder strips (333.68 ± 24.67 mg; n = 4 strips each from 8 animals) used in the study were similar. Thus, since tissue strips were weight adjusted, tension responses are compared on a gram-to-gram basis. 3.3. Responses to electrical-field evoked stimulation Strips from control animals contracted in a frequencydependent manner between 0.05 and 32 Hz with mean tension
To examine the basis of decreased electrical field stimulusevoked contractions, responses to carbachol and ATP, agonists of muscarinic and purinergic receptors respectively, were assessed. Carbachol evoked concentration dependent contractions in control strips with a pD2 value of 6.54 ± 0.05 and with a maximum response of 19.38 ± 3.46 g (n = 12). The response to carbachol was tonic with maximal increase in tension responses attained within 5 min. In contrast, carbachol was found to be less potent (pD2 = 5.93 ± 0.04; n = 8) and efficacious (9.16 ± 0.82 g) in obstructed bladder strips (Table 1, Fig. 3A). In contrast to carbachol, the responses to ATP in both control and obstructed bladder strips were phasic returning to basal levels between each pulse. ATP evoked concentration dependent contractions of control bladder strips with a pD2 value of 4.85 ± 0.15 and with a maximum intrinsic activity of 4.6 ± 0.85 g (n = 19). In obstructed bladder strips, ATP evoked contractions with a pD2 value of 3.72 ± 0.08 and significantly lower efficacy (2.61 ± 0.58 g; n = 8, Table 1, Fig. 3B). To determine whether the attenuated responsiveness to muscarinic and purinergic stimulation was not a consequence of a generalized tissue hypertrophy, the effects of other
Fig. 5. (A) Representative responses from control (upper tracing) and obstructed bladder strips (lower tracing) with step increases in preload. Data is shown as spontaneous phasic activity of the baseline and contractile responses triggered by 1 μM carbachol. (B) Spontaneous phasic activity of the baseline preload, control (open bars), obstructed (hatched bars). Data shown as mean area under the curve (AUC g.s./min) ± S.E.M., n = 6. (C) Contractile response of the bladder strips to 1 μM carbachol. Data shown as mean ± S.E.M., in grams, n = 6. P b 0.05.
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Table 2 Effect of preload on contractile activity of isolated bladder strips from control and obstructed unstable pigs Preload (g)
Control
Unstable
Carbachol (g)
Spontaneous phasic activity (AUC g.s./min)
Carbachol (g)
Spontaneous phasic activity (AUC g.s./min)
1 2 4 8 16 32
23.1 ± 3.08 32.0 ± 3.69 42.2 ± 4.55 45.0 ± 4.93 39.9 ± 3.53 28.7 ± 4.30
0 14.5 ± 3.18 40.2 ± 12.82 47.7 ± 16.28 34.2 ± 11.52 52.1 ± 12.17
7.5 ± 1.11a 8.1 ± 3.16a 8.3 ± 1.74a 12.1 ± 1.84a 29.1 ± 1.03a 32.1 ± 3.10
0 0 7.2 ± 3.90 39.2 ± 22.89 115.2 ± 15.47a 342.7 ± 21.50a
Data shown as the mean maximum contractile response ± S.E.M. to carbachol (1 μM) stimulation and basal spontaneous phasic activity expressed as AUC g.s./min±S.E.M. (n=6). Studies were carried out using same tissue strips. a Represents Pb 0.05 vs. obstructed bladders.
neurotransmitters, serotonin and histamine were assessed. Histamine (100 nM–100 μM) evoked concentration-dependent contractions of the isolated pig bladder (pD2 = 5.58 ± 0.15) with a maximum contraction of 10.18 ± 1.30 g (n = 6, Table 1, Fig. 3C). Bladder strips from obstructed animals responded with a potency, pD2 value of 5.60 ± 0.06, similar to that observed in control strips, although the maximal response was significantly reduced (2.00 ± 0.20 g, n = 8, Table 1, Fig. 3C). Serotonin (100 nM–0.1 mM) evoked concentration-dependent contractions of the isolated pig bladder with a pD2 value of 6.26 ± 0.06 with a maximum response (1.91 ± 0.21 g, n = 8, Table 1, Fig. 3D). Obstructed bladder strips also responded with a similar potency (pD2 = 5.96 ± 0.09) and efficacy (1.59 ± 0.43g; n = 7, Table 1, Fig. 3D).
32 g (Fig. 5A, B, Table 2). In this study, we chose to examine both groups at a preload of 1 g. For control strips, the intrinsic activity and maximum response increased with increases in preload peaking at a preload of 4–8 g whereas in obstructed strips the maximal response was shifted to higher preload values (∼16 g). We did not evaluate agonists at the maximum preload since the baseline drifted at preloads N 8 g and since the tension generated by higher preloads were such that the integrity of all strips could not be maintained during the course of the experiment. The differential effect of preload on spontaneous phasic activity in obstructed pigs prompted us to examine its effect on muscarinic receptor activation. Bladder strips taken from control pigs contracted when exposed to carbachol (1 μM) with a slight increase in contractile response as the preload was increased, reaching a peak response of 45.0 ± 4.93 at preload of 8 g (Fig. 5A, C, Table 2). Obstructed bladder strips were less responsive at the lower preloads (1–8 g); however, as the preload was increased (16–32 g) carbachol-evoked responses also increased. At a preload of 32 g, the contractile response was comparable to that of control bladder strips. 3.6. Effects of KATP channel openers K+ channels including ATP-sensitive and calcium activated K channels function as key regulators of resting membrane +
3.5. Myogenic contractility of bladder strips Contractility of bladder strips from obstructed animals was assessed after depolarization with 80 mM K+ and at various preload conditions and compared to the responsiveness of control strips. 3.5.1. Responses to elevated K+ When tissue strips were depolarized by addition of extracellular K+ (3–300 mM), concentration-dependent contractions of obstructed bladder strips were observed. The contractile response was tonic reaching a peak response in about 3 min. Maximal tension responses of 27.6 ± 4.02 g were observed with a pD2 value of 1.08 ± 0.33 (n = 12). Obstructed bladder strips responded with a 2.5-fold higher potency (pD2 = 1.66 ± 0.24), but with reduced maximum response (11.2 ± 1.12 g; n = 8, Table 1, Fig. 4). 3.5.2. Effects of preload on spontaneous phasic activity Control bladder strips demonstrated very little spontaneous phasic activity at preloads of 1 and 2 g but did show modest activity at 4–32 g. In contrast, bladder strips taken from obstructed pigs showed no spontaneous activity at 1 and 2 g preload; however, a substantially larger preload-dependent increases in spontaneous phasic activity were observed at 16 and
Fig. 6. (A) Inhibition of carbachol-evoked contractions in obstructed bladder strips by KATP channel openers. Bladder strips contracted with 1 μM carbachol. (B) Comparison of the potencies of KATP channel openers for the inhibition of carbachol-evoked contractions in control and obstructed bladders. The correlation coefficient was r2 = 0.90 and slope = 0.93 ± 0.11 (n = 4–7).
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potential to modulate excitability of the urinary bladder smooth muscle (Herrera et al., 2000; Buckner et al., 2000, 2002; Gopalakrishnan and Shieh, 2004; Gopalakrishnan et al., 2002). In general, KATP channel openers are 15-fold more potent in suppressing spontaneous phasic activity compared to contractions evoked by electrical field-stimulation (Buckner et al., 2002). To assess interactions with KATP channels, KATP channel openers were examined for their effects on contractions evoked by carbachol (1 μM) from bladder strips taken from control and obstructed pigs (Fig. 6A). Structurally diverse openers examined completely inhibit carbachol-evoked contractions in both obstructed and control tissues. P1075, a pinacidil analog that has been shown to open ATP-sensitive potassium channels (Quast et al., 1993), was the most potent compound, with pD2 values of 7.32 ± 0.02 (n = 4) and 6.78 ± 0.10 (n = 7) for control and obstructed bladder strips, respectively. A-278637, a 1,4dihydropyridine K+ channel opener shown to demonstrate selectivity for the suppression of unstable contractions vs. mean arterial pressure effects (Brune et al., 2002) was found to inhibit carbachol-evoked responses with comparable potencies in both control and obstructed animals. Other agents including ZD6169 and WAY-133537 also showed no substantial differences in sensitivities between control and obstructed pigs. The weakest agent examined was diazoxide with pD2 values of 4.36 ± 0.02 (n = 4) and 4.11 ± 0.10 (n = 4) for control and obstructed bladder strips, respectively. Overall, the potencies of these openers showed good 1:1 correlation between the control and the obstructed bladder strips (Fig. 6B; r2 = 0.90, slope = 0.93 ± 0.12; n = 11). 4. Discussion Neurogenically evoked contractions of the urinary bladder involves participation of both muscarinic and purinergic mechanisms, and contractions evoked by higher frequencies respond better to muscarinic receptor blockade compared to those evoked by lower frequencies (Burnstock et al., 1972; Sjogren et al., 1982). Our studies show that frequency-dependent contractions evoked by electrical field stimulus of the bladder smooth muscle was impaired at all frequencies in bladder strips from obstructed pigs. The reduction in responsiveness to both carbachol and ATP, but not to serotonin, suggests that this decline may relate specifically to these transmitter systems and not attributable to nonspecific changes due to bladder hypertrophy. The large reduction in responses to field stimulation suggests that the smooth muscle from the bladder of obstructed pigs was partially denervated and unable to generate adequate neurotransmission for evoking robust contractile effects. These results are in agreement with the findings of Speakman et al. (1987) who observed a marked deterioration of detrusor muscle contractility to electrical field stimulation in the pig model. In this study, both the potencies and efficacies of carbachol and ATP were reduced. The potency of carbachol was attenuated 4-fold whereas the shift was more robust for ATP with about a 13-fold reduction in potency. On the other hand, the reduction in maximal responses of both carbachol and ATP were comparable (2- and 1.8-fold, respectively). The decreases
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in sensitivity and maximal responses of both purinergic and muscarinic agonists suggest possible alterations in receptor levels and/or affinity although this remains to be directly assessed by biochemical measures. In contrast to our observations, Speakman et al. (1987) showed a slight increase (2-fold) in sensitivity to carbachol in obstructed tissues. These differences may be accounted for by subtle differences in protocols such as duration of obstruction and degree of decompensation. We have utilized smooth muscle strips (4 × 10 mm) in this study as opposed to smooth muscle bundles (1 × 1 × 7 mm) utilized by Speakman et al. (1987). Further, in the present study, the contractile response to carbachol was allowed to reach maximum (60 s) whereas Speakman and colleagues measured tension responses at 10 s. K+ depolarization-evoked contractions were significantly reduced about 60% in the obstructed bladder. However, a 2.5-fold increase in potency of KCl in the contractile response of the obstructed bladder was noted which is consistent with data from Mills et al. (2000) and Speakman et al. (1987). In contrast to studies in pigs, following long-term outlet obstruction (15 weeks) in rats, Lluel et al. (2002) observed a significant reduction in responses to KCl and carbachol-evoked contractility, but interestingly, no difference in response to electrical field stimulus was observed. Bladder strips from control animals contracted in response to carbachol (1 μM) over a preload range of 1–32 g (Fig. 5). The small, spontaneous phasic activity of the baseline was fairly constant over the range of preloads examined. In contrast, tissue strips from obstructed pigs with obstructed bladders showed a preload-dependent increase in the contractile response to carbachol and a similar increase in phasic spontaneous activity of the baseline. At a maximum of 32 g preload, both sets of tissues responded to carbachol in a similar fashion, but the obstructed bladder strips showed greater spontaneous phasic activity at 16 and 32 g preload. These results suggest that obstructed bladders from obstructed pigs could accommodate to the higher outlet pressures necessary for micturition. Several structurally diverse KATP channel openers examined in this study relaxed carbachol-stimulated contractions of the pig bladder strips taken from both control and obstructed pigs. This effect was concentration-dependent; all eleven openers were fully efficacious with potencies spanning a range of about four log units. The potencies of potassium channel openers to inhibit carbachol-evoked contractions showed a good 1 : 1 correlation (r2 = 0.90, slope = 0.93) between obstructed and control bladder strips. This agreement suggests that the interactions of these ligands with KATP channels were not adversely affected in obstructed bladders. In summary, our results support and further extend previous findings of obstruction-induced changes in this animal model. Importantly, our studies provide evidence of enhanced spontaneous phasic activity at elevated preloads post-obstruction. The impaired responses to electrical field stimulation could be attributed to reduced efficacies and lower sensitivities of muscarinic and purinergic signaling pathways. Although the contractile response of the obstructed bladder to electrical fieldstimulation and to muscarinic and purinergic agonists were
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attenuated, the bladder smooth muscle was about 2-fold more sensitive to K+ depolarization. The essentially unimpaired pharmacological profile of K+ channel openers post-obstruction suggests that this class of K+ channels could be effectively modulated by such agents that may have potential for the treatment of overactive bladder secondary to outlet obstruction. Acknowledgements The authors thank Dr Timothy Esbenshade for critical reading and suggestions on this manuscript. References Brading, A.F., 1997. A myogenic basis for the overactive bladder. Urology 50, 57–67. Brune, M.E., Fey, T.A., Brioni, J.D., Sullivan, J.P., Carroll, W.A., Coghlan, M.J., Williams, M., Gopalakrishnan, M., 2002. A-278637 [(-)-(9S)-9-(3-bromo4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno [3,2-b] quinolin-8(4H)-one 1,1-dioxide]: a novel ATP-sensitive potassium channel opener efficacious in suppressing urinary bladder contractions II. In vivo characterization. J. Pharmacol. Exp. Ther. 303, 387–394. Buckner, S.A., Milicic, I., Daza, A., Davis-Taber, R., Scott, V.E., Sullivan, J.P., Brioni, J.D., 2000. Pharmacological and molecular analysis of ATP-sensitive K+ channels in the pig and human detrusor. Eur. J. Pharmacol. 400, 287–295. Buckner, S.A., Milicic, I., Daza, A., Coghlan, M.J., Gopalakrishnan, M., 2002. Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators. Br. J. Pharmacol. 135, 639–648. Burnstock, G., Dumsday, B., Symthe, A., 1972. Atropine resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purine nucleotide. Br. J. Pharmacol. 44, 451–461. Crowe, R., Burnstock, G., 1989. A histochemical and immunohistochemical study of the autonomic innervation of the lower urinary tract of the female pig. Is the pig a good model for the human bladder and urethra? J. Urol. 141, 414–422. De Groat, W.C., 1997. A neurological basis for the overactive bladder. Urology 50, 36–52. Fey, T.A., Gopalakrishnan, M., Strake, J.G., King, L.L., Brioni, J.D., Sullivan, J.P., Coghlan, M.J., Brune, M.E., 2003. Effects of ATP-sensitive K+ channel openers and tolterodine on unstable bladder contractions in a pig model of partial bladder outlet obstruction. Neurourol. Urodyn. 22, 147–155. Foster, C.D., Speakman, MJ., Fujii, K., Brading, A.F., 1989. The effects of cromakalim on the detrusor muscle of human and pig urinary bladder. Br. J. Urol. 63, 284–294. Gopalakrishnan, M., Shieh, C., 2004. Potassium channel subtypes as molecular targets for overactive bladder and other urological disorders. Expert Opin. Ther. Targets 8, 437–458. Gopalakrishnan, M., Buckner, S.A., Whiteaker, K.L., Shieh, C.C., Molinari, E.J., Milicic, I., Daza, A., Davis-Taber, R., Scott, V.E., Sellers, D., ChessWilliams, R., Chapple, C.R., Liu, Y., Liu, D., Brioni, J.D., Sullivan, J.P., Williams, M., Carroll, W.A., Coghlan, M.J., 2002. A-278637 [(-)-(9S)-9-
(3-bromo-4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8 (4H)-one 1,1-dioxide]: a novel ATP-sensitive potassium channel opener efficacious in suppressing urinary bladder contractions I. in vitro characterization. J. Pharmacol. Exp. Ther. 303, 379–386. Grayhack, J.T., Keeler, T.C., Kozlowski, J.M., 1987. Carcinoma of the prostate. Hormonal therapy. Cancer 60, 589–601. Herrera, G.M., Heppner, T.J., Nelson, M.T., 2000. Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am. J. Physiol., Regul. Integr. Comp. Physiol. 279, R60–R68. Levin, R.M., Ruggieri, M.R., Velagapudi, S., Gordon, D., Altman, B., Wein, A.J., 1986. Relevance of spontaneous activity to urinary bladder function: an in vitro and in vivo study. J. Urol. 136, 517–521. Levin, RM., Haugaard, N., O'Connor, L., Buttyan, R., Das, A., Dixon, J.S., Gosling, J.A., 2000. Obstructive response of human bladder to BPH vs rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol. Urodyn. 19, 609–629. Lluel, P., Barras, M., Palea, S., 2002. Cholinergic and purinergic contribution to the micturition reflex in conscious rats with long-term bladder outlet obstruction. Neurourol. Urodyn. 21, 142–153. Malmgren, A., Sjorgren, C., Andersson, K.E., Andersson, P.O., 1987. Effects of atropine on bladder capacity and instability in rats with bladder hypertrophy. Neurourol. Urodyn. 6, 331–339. Martin, S.W., Radley, S.C., Chess-Williams, R., Koortstanje, C., Chapple, C.R., 1997. Relaxant effect of potassium channel openers on normal and hyperreflexic detrusor muscle. Br. J. Urol. 80, 405–413. Mattiason, A., Ekstrom, J., Larsson, B., Uvelius, B., 1987. Changes in the nervous control of the rat urinary bladder induced by outflow obstruction. Neurourol. Urodyn. 6, 37–45. Mills, I.W., Greenland, J.E., McMurray, G., McCoy, R., Ho, K.M., Noble, J.G., Brading, A.F., 2000. Studies of the pathophysiology of idiopathic detrusor instability: the physiological properties of the detrusor smooth muscle and its pattern of inervation. J. Urol. 163, 646–651. Quast, U., Bray, K.M., Andres, H., Manley, P.W., Baumlin, Y., Dosogne, J., 1993. Binding of the K+ channel opener [3H]P1075 in rat isolated aorta: relationship to functional effects of openers and blockers. Mol. Pharmacol. 43, 474–481. Saito, M., Ohmura, M., Kondo, A., 1996. Effects of long-term partial outflow obstruction on bladder function in the rat. Neurourol. Urodyn. 14, 625–633. Sibley, G.N.A., 1984. A comparison of spontaneous and nerve mediated activity in bladder muscle from man, pig and rabbit. J. Physiol. 354, 431–444. Sibley, G.N.A., 1985. An experimental model of detrusor instability in the obstructed pig. Br. J. Urol. 57, 292–298. Sibley, G.N.A., 1987. The physiological response of the detrusor muscle to experimental bladder outflow obstruction in the pig. Br. J. Urol. 60, 332–336. Sjogren, C., Andersson, K.-E., Husted, S., Mattiasson, A., Madsen, B.M., 1982. Atropine resistance of transmurally stimulated isolated human bladder muscle. J. Urol. 128, 1368–1371. Speakman, MJ., Brading, A.F., Gilpin, C.J., Dixon, J.S., Gilpin, S.A., Gosling, J.A., 1987. Bladder outflow obstruction — a cause of denervation supersensitivity. J. Urol. 138, 1461–1466. Zielinski, P.J., Buckner, S.A., 1998. AGANTG: a Microsoft Excel 5.0–visual basic routine for the analysis of dose-response data. Analyst 123, 1661–1668.
Pharmacological characterization of urinary bladder smooth muscle contractility following partial bladder outlet obstruction in pigs Ivan Milicic ⁎, Steven A. Buckner, Anthony Daza, Michael Coghlan, Thomas A. Fey, Michael E. Brune, Murali Gopalakrishnan Neuroscience Research, Department R4MN, Building AP9, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA Received 8 September 2005; received in revised form 29 November 2005; accepted 19 December 2005 Available online 17 February 2006
Abstract Partial bladder outlet obstruction of the pig is considered as a valuable preclinical model for evaluating the profile of compounds for the treatment of bladder overactivity. In this study, we characterized the pharmacological properties of isolated bladder smooth muscle from pigs following partial outlet obstruction and its sensitivity to potassium channel openers. Bladder strips from obstructed animals showed significantly lower maximal efficacy (Emax) and sensitivity to stimulation by ATP and carbachol, but not to those evoked by serotonin, compared to agematched controls. Tissue strips from obstructed animals also showed a 2.5-fold increase in the potency and significantly reduced maximum response following K+ depolarization. With respect to spontaneous activity, bladder strips from control strips demonstrated little spontaneous phasic activity at all preloads examined. In contrast, bladder strips from obstructed animals showed large preload-dependent increases in spontaneous phasic activity at preload values of 16–32 g. The potencies of KATP channel openers to relax carbachol-evoked contractions showed a good 1 : 1 correlation (r2 = 0.90) between obstructed and control bladder strips. These studies demonstrate that obstructed pig bladders show enhanced spontaneous phasic activity especially at elevated preloads, which may underlie unstable myogenic bladder contractions reported in cystometrographic measurements in vivo. The impaired responses to electrical field stimulation could be attributed to reduced efficacies and/or lower sensitivities of muscarinic and purinergic signaling pathways. KATP channel sensitivities remain essentially unimpaired in the obstructed bladder and could be effectively modulated by openers with potential for the treatment of overactive bladder secondary to outlet obstruction. © 2006 Elsevier B.V. All rights reserved. Keywords: Potassium channel opener; Detrusor smooth muscle; Smooth muscle relaxation; ATP-sensitive potassium channels; Sulfonylurea receptor; Outlet obstruction; Overactive bladder
1. Introduction Overactive bladder, a condition affecting an estimated 15 million individuals in the United States alone, is characterized by symptoms of urinary frequency and urgency with or without urge incontinence. Of the various etiologies, urinary bladder outlet obstruction is one of the major underlying causes with significant population of men 60 years of age or older having varying degrees of obstruction secondary to benign prostatic hyperplasia (Grayhack et al., 1987). Several characteristics of human outlet obstruction including detrusor instability and increases in micturition pressure can be ⁎ Corresponding author. Tel.: +1 847 938 2222; fax: +1 847 937 9195. E-mail address: [email protected] (I. Milicic). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.12.076
reproduced in experimental animals including obstructed rats (Malmgren et al., 1987), rabbits (Levin et al., 2000) humans and pigs (Sibley, 1984; Mills et al., 2000). The initial suggestion of abnormal behavior following outlet obstruction was by Sibley (1984) who reported increased sensitivity to agonist simulation of bladder strips from pigs or humans with bladder instability secondary to outflow obstruction. Subsequent studies in pigs demonstrated a change in the ratio of maximal contractile response evoked by muscarinic agonists and intrinsic nerve stimulation in the unstable bladder, the latter often significantly depressed (Speakman et al., 1987). These observations have also been confirmed in patients with obstructive instability consistent with partial denervation of the bladder (Levin et al., 1986). Functional changes in isolated detrusor muscle have also been described in rats where decreases
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in neurogenic contractility after 6 weeks of obstruction (Mattiason et al., 1987) and reduced responsiveness to carbachol, ATP and electrical field stimulation following long term (15 weeks) outlet obstruction (Saito et al., 1996) have been reported. When supersensitivity and partial denervation coexist, the responsiveness to activation of intrinsic nerves may depend on the interaction of the two processes, i.e., attenuated responsiveness because of denervation and increased responsiveness due to the altered properties of the smooth muscle (Brading, 1997; De Groat, 1997). The pig detrusor has been considered a model for investigating the physiology of bladder function, as the pig has comparable urodynamic characteristics to that of human (Sibley, 1984, 1985, 1987; Speakman et al., 1987; Crowe and Burnstock, 1989). There is also evidence from studies in pigs that tissues from the unstable bladder may be more sensitive to exogenous agonists than normal bladder (Sibley, 1987; Foster et al., 1989; Martin et al., 1997). In vitro studies from our laboratory have demonstrated that the pig detrusor is a suitable model for studying the functional pharmacology of K+ channels (Buckner et al., 2000, 2002). More recently, we have characterized in vivo unstable bladder contractions in an anesthetized pig model of partial bladder outlet obstruction by indwelling telemetry and cystometry approaches (Fey et al., 2003). Cystometrographic analysis of isoflurane-anesthetized animals 17–20 weeks post-obstruction showed low amplitude, rhythmic bladder contractions that were sensitive to inhibition by K+ channel openers. In this study, we report the characterization of the in vitro properties of isolated bladder smooth muscle strips from pigs following partial outlet obstruction by assessing spontaneous phasic activity and responsiveness following exogenous agonist- and electrical field-evoked responses. These studies demonstrate that obstructed pig bladder strips show enhanced spontaneous phasic activity, especially at elevated preloads, which may contribute to unstable myogenic bladder contractions observed in vivo. Although responsiveness to electrical field stimulation, muscarinic and purinergic agonists were attenuated in the obstructed bladder, the sensitivities to KATP channel openers were unaffected, consistent with their reported in vivo efficacies in this model (Fey et al., 2003). 2. Materials and methods 2.1. Experimental bladder outlet obstruction Female Landrace/Yorkshire crossbred pigs (Wilson Prairie View Farms, Burlington, WI) 12 weeks of age were obstructed with a 7.5-mm silver omega ring placed around the proximal urethra as described previously by Fey et al. (2003). Studies were carried out in accordance with guidelines outlined by the Animal Welfare Act, the Association for Assessment and Accreditation of Laboratory Animals (AAALAC) and the Institutional Animal Care and Use Committee of Abbott Laboratories. Proper ring placement was confirmed at necropsy in all animals. Seventeen to 20 weeks after placement of the ring, the pigs were instrumented with telemetry transducer/transmitters (Data
Sciences; St. Paul, MN) for the measurement of carotid arterial pressure (unit 1: TA11PA-C40) and intravesical/abdominal pressures (unit 2: TL11M3-D70-PCP). A port catheter (TI-9, Access Technologies) was placed subcutaneously in the side of the abdomen and its distal catheter secured in the bladder lumen. Animals were treated with amoxicillin and buprenorphine for 3 to 5 days post-surgery and allowed to recover for 10 to 14 days before testing. Urine was drained completely via the port catheter from one to five times per week to avoid urinary retention and potential renal failure. 2.2. Cystometry studies Obstructed animals were verified for unstable bladder contractions by in vivo cystometry. Animals were anesthetized with telazol and xylazine, intubated and maintained on an isoflurane : oxygen mixture in the supine position with water blankets to maintain body temperature. Bladder volume was adjusted via the port catheter to establish a regular unstable contraction pattern. Radiotelemetry signals for bladder pressure were obtained using an RMC-1 receiver (Data Sciences, St. Paul, MN). Data acquisition and semi-automated analysis were performed using the cystometry analysis software in the Ponemah Physiology Platform (Gould Instrument Systems, Valley View, OH). Obstructed animals that showed demonstrable unstable contractions, i.e., bladder pressure amplitude ≥ 4 cm H2O, and agematched control animals were utilized for in vitro studies (Fey et al., 2003). 2.3. In vitro studies On the day of the experiment, animals were euthanized with an intraperitoneal injection of pentobarbital (Somlethol®) at a lethal dose of 150–200 mg/kg. The entire urinary bladder was cleared of surrounding adipose tissue, removed and placed in Krebs Ringer bicarbonate solution of the composition, (mM): 120 NaCl, 20 NaHCO3, 11 dextrose, 4.7 KCl, 2.5 CaCl2, 1.5 MgSO4, 1.2 KH2PO4, equilibrated with 5% CO2 : 95% O2 (pH = 7.4 at 37 °C). (±)-Propranolol, (4.0 μM) was included in the study to ensure β-adrenoceptor blockade. The bladder was sectioned into three pieces and the top dome portion and the lower trigonal area were discarded. The remaining tissue (detrusor) was sliced transversely into approximately 4 × 10 mm strips. Tissue strips without urothelium from the area adjacent to the trigone region were used in both control and obstructed pigs throughout the study. Denuded tissue 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 Grass FT03 transducer at a basal preload of 1 g. The stationary rods contained parallel platinum electrodes that fit on each side of the tissue strips. Tissues were rinsed at 10-min intervals and allowed to equilibrate for at least 75 min prior to testing. 2.4. Electrical field stimulation Contractile responses were evoked by a Grass model S88 stimulator at a frequency of 0.05 Hz (except where indicated),
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duration of 0.5 ms, 20 V (supramaximal in terms of the evoked twitch), and current b150 mA. A voltage–current amplifier circuit was placed between the stimulator and the electrodes to maintain constant stimulation parameters. The electrical stimulus was set to deliver square wave pulses for the duration of the protocol. In control experiments, these parameters produced uniform, single shock twitches that returned to baseline levels between each stimulus. These twitches were completely abolished by tetrodotoxin (100 nM). For the generation of frequency response curves, the tissues were stimulated at 0.05, 0.50, 1.0, 2.0, 4.0, 8.0, 16.0 and 32 Hz for 60 s in order to achieve maximum contraction. 2.5. Exogenous agonists Concentration response effects were determined for carbachol, ATP, histamine, and serotonin (1 nM to 30 mM) by a noncumulative addition protocol. The maximum responses were determined and a 20-min rinse cycle was included between successive additions. 2.6. K+ depolarization Concentration response effects were determined for KCl using a protocol identical to that described above for exogenous agonists. KCl was tested over a range of concentrations by noncumulative addition. The maximum responses were determined and a 20-min rinse cycle was included between successive additions. 2.7. Spontaneous phasic activity To examine concentration-dependent effects on spontaneous phasic activity, test compounds were introduced and their effects assessed in terms of area under the contractile curve (AUC) for a 15-min period, normalized to 1-min and expressed as gram seconds per min (AUC g.s./min). After each concentration the tissues were rinsed for 20-min and the cycle repeated. As reported earlier (Buckner et al., 2002), it was found that the area of the detrusor away from the dome region of the bladder provided for the most consistent spontaneous responses. It was also observed that tissues with the urothelium intact demonstrated significantly reduced signal-to-noise ratio compared to adjacent strips where the urothelium was removed. Wherever concentration response curves were generated, each bladder strip was used for only one test compound or condition.
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by generating a cumulative inhibitory response curve to P1075 as previously described (Buckner et al., 2002). The concentration-dependent reduction in the peak amplitude was used for calculating IC50 values. 2.9. Compounds Bay X-9227 [((-)-N-(2-ethoxyphenyl)-N′-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine)], Bay X-9228 (enantiomer of Bay X-9227), (-)-cromakalim and the racemate (±)-cromakalim, nicorandil, P1075 [N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridylguanidine], ZD6169[N-(4-benzoylphenyl)-3,3,3-trifluro-2-hydroxy-2-methylpropion amine], A-278637 [(-)-(9S)-9-(3-bromo-4-fluorophenyl)-2,3,5,6,7,9hexahydrothieno[3,2-b]quinolin and ZM244085 [9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione] were synthesized in house. Diazoxide and pinacidil were purchased from Research Biochemicals International (Natick MA). Stock solutions were prepared fresh daily in distilled water or in methyl sulfoxide (DMSO). All other chemicals including (±)-propranolol HCl, and (DMSO) were purchased from Sigma Chemical Co (St. Louis, MO). 2.10. Data analysis Concentration response curves were analyzed using a fourparameter curve fitting routine, described previously (Zielinski and Buckner, 1998). The maximum peak amplitude response was used for analysis. Results were expressed as gram tension and as percentage of maximum response. Spontaneous phasic activity data were recorded and analyzed using PowerLab (ADI Instruments, Australia). The signal was analyzed as the integrated area under the contractile curve (AUC) for 15 min intervals and corrected to 1 min and expressed as AUC gram seconds per min. Potencies were expressed as the − log 10 (pD2) of the geometric mean of the EC50 or IC50 values ± S.E.M. Data were analyzed for significance using an unpaired, two-tailed t-test, GraphPad Prism version 3.02 (GraphPad software, San
2.8. K+ channel opener pharmacology Carbachol was used as the agonist to assess KATP channel opener sensitivities. The protocol was non-cumulative with rinse cycles between successive additions. Tissues were pretreated with the potassium channel openers for 15 min, then exposed to a fixed concentration (EC100 1 μM) of carbachol until maximum tension developed and rinsed for 15 min and the cycle was repeated. Tissue sensitivities were initially assessed
Fig. 1. (A) Representative cystogram from obstructed, unstable pig bladder in vivo. Contractile responses ≥4.0 cm H20 were considered unstable. (B) Representative cystogram from age-matched control pig bladder. Small oscillations represent breathing artifact. Contractile responses b4.0 cm H20 were considered normal.
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I. Milicic et al. / European Journal of Pharmacology 532 (2006) 107–114 Table 1 Characteristics of agonist-evoked contractility of isolated bladder strips from control and obstructed unstable pigs
Fig. 2. Frequency response relationship of control (open bars) and obstructed (hatched bars) bladder strips. Contractile data shown as the mean maximum response (g) ± S.E.M., n = 8. P b 0.05.
Diego, CA, U.S.A.) and P value b 0.05 was considered significant. 3. Results 3.1. Characterization of unstable contractions Twenty-four hours prior to use, bladders from the obstructed pigs were verified as unstable in vivo by cystometric analysis. Fig. 1 shows representative tracings from obstructed (Fig. 1A) and control (Fig. 1B) unobstructed pigs. As previously reported, obstructed pigs exhibited highly rhythmic, spontaneous nonvoiding contractions starting 17 weeks post-obstruction (Fig. 1A). Approximately 80% of obstructed animals were found to have involuntary bladder contractions as measured by
Agonist
Control
Unstable
pD2
Emax (g)
pD2
Emax (g)
Carbachol ATP Histamine Serotonin KCl
6.54 ± 0.05 (12) 4.85 ± 0.15 (19) 5.58 ± 0.15 (6) 6.26 ± 0.06 (8) 1.08 ± 0.33 (12)
19.38 ± 3.46 4.6 ± 0.85 10.18 ± 1.30 1.91 ± 0.21 27.6 ± 4.02
5.93a ± 0.04 (8) 3.72a ± 0.08 (8) 5.60 ± 0.06 (8) 5.96 ± 0.09 (7) 1.66a ± 0.24 (8)
9.16a ± 0.82 2.61a ± 0.58 2.00a ± 0.20 1.59 ± 0.43 11.20a ± 1.20
Data shown are mean potency (pD2 ± S.E.M.) and mean maximum response (Emax ± S.E.M.) Values in parenthesis are the number of determinations. a represents P b 0.05 vs. obstructed bladders.
cystometry. The remaining 20% of animals showed signs of bladder hypertrophy with no involuntary contractions (Fey et al., 2002) Only bladder strips from obstructed pigs whose bladders were verified as unstable (spontaneous bladder contractions ≥ 4 cm H2O) were used in this study. Obstructed animals used in this study had mean contraction amplitude, frequency and duration of 11.7 ± 0.5 cm H2O, 34.6 ± 4.6 contractions per 30 min and 35.0 ± 2.7 s per contraction (n = 5), respectively. 3.2. Bladder hypertrophy Partial outlet obstruction of the bladder led to a significant increase in bladder weight. The mean values were 77.05 ± 4.99 g (n = 5) for control and 546.00 ± 44.08 g (n = 17) for obstructed pigs (P b 0.01). Consequently, the weights of the isolated
Fig. 3. (A) Contractile responses of bladder strips from control and obstructed pigs showing concentration-dependent increases in contractile responses from control (open circles) and obstructed (filled circles) bladders by carbachol (A), ATP (B), histamine (C) and serotonin (D). In each panel, the inset depicts data expressed as percentage of the maximum response. S.E.M. values are omitted for clarity. Numbers of determinations are summarized in Table 1.
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responses ranging from 3.03 ± 0.76 g at 0.05 Hz to 40.06 ± 7.25 g at 32 Hz (n = 8). Bladder strips from obstructed pigs showed significantly reduced frequency dependent increases in contractions and ranged from 0.98 ± 0.13 at 0.05 Hz to 8.57 ± 1.12 g at 32 Hz (n = 8). However, in both cases, maximal increases were observed at a frequency of 16 Hz (Fig. 2). 3.4. Responses to exogenous agonists
Fig. 4. Contractile response of bladder strips to K+ depolarization. Open circles represent control, closed circles represent strips from obstructed bladders. Data expressed as maximum response (g) mean ± S.E.M. (control n = 12; obstructed n = 8). The inset depicts data expressed as percentage of maximum response.
bladder strips were significantly greater from the obstructed pigs, and therefore, strips were weight adjusted prior to mounting by slicing out the urothelium side to approximate the thickness of control strips. The mean weight of control strips (333.08 ± 29.52 mg; n = 4 strips each from 6 animals) and obstructed bladder strips (333.68 ± 24.67 mg; n = 4 strips each from 8 animals) used in the study were similar. Thus, since tissue strips were weight adjusted, tension responses are compared on a gram-to-gram basis. 3.3. Responses to electrical-field evoked stimulation Strips from control animals contracted in a frequencydependent manner between 0.05 and 32 Hz with mean tension
To examine the basis of decreased electrical field stimulusevoked contractions, responses to carbachol and ATP, agonists of muscarinic and purinergic receptors respectively, were assessed. Carbachol evoked concentration dependent contractions in control strips with a pD2 value of 6.54 ± 0.05 and with a maximum response of 19.38 ± 3.46 g (n = 12). The response to carbachol was tonic with maximal increase in tension responses attained within 5 min. In contrast, carbachol was found to be less potent (pD2 = 5.93 ± 0.04; n = 8) and efficacious (9.16 ± 0.82 g) in obstructed bladder strips (Table 1, Fig. 3A). In contrast to carbachol, the responses to ATP in both control and obstructed bladder strips were phasic returning to basal levels between each pulse. ATP evoked concentration dependent contractions of control bladder strips with a pD2 value of 4.85 ± 0.15 and with a maximum intrinsic activity of 4.6 ± 0.85 g (n = 19). In obstructed bladder strips, ATP evoked contractions with a pD2 value of 3.72 ± 0.08 and significantly lower efficacy (2.61 ± 0.58 g; n = 8, Table 1, Fig. 3B). To determine whether the attenuated responsiveness to muscarinic and purinergic stimulation was not a consequence of a generalized tissue hypertrophy, the effects of other
Fig. 5. (A) Representative responses from control (upper tracing) and obstructed bladder strips (lower tracing) with step increases in preload. Data is shown as spontaneous phasic activity of the baseline and contractile responses triggered by 1 μM carbachol. (B) Spontaneous phasic activity of the baseline preload, control (open bars), obstructed (hatched bars). Data shown as mean area under the curve (AUC g.s./min) ± S.E.M., n = 6. (C) Contractile response of the bladder strips to 1 μM carbachol. Data shown as mean ± S.E.M., in grams, n = 6. P b 0.05.
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Table 2 Effect of preload on contractile activity of isolated bladder strips from control and obstructed unstable pigs Preload (g)
Control
Unstable
Carbachol (g)
Spontaneous phasic activity (AUC g.s./min)
Carbachol (g)
Spontaneous phasic activity (AUC g.s./min)
1 2 4 8 16 32
23.1 ± 3.08 32.0 ± 3.69 42.2 ± 4.55 45.0 ± 4.93 39.9 ± 3.53 28.7 ± 4.30
0 14.5 ± 3.18 40.2 ± 12.82 47.7 ± 16.28 34.2 ± 11.52 52.1 ± 12.17
7.5 ± 1.11a 8.1 ± 3.16a 8.3 ± 1.74a 12.1 ± 1.84a 29.1 ± 1.03a 32.1 ± 3.10
0 0 7.2 ± 3.90 39.2 ± 22.89 115.2 ± 15.47a 342.7 ± 21.50a
Data shown as the mean maximum contractile response ± S.E.M. to carbachol (1 μM) stimulation and basal spontaneous phasic activity expressed as AUC g.s./min±S.E.M. (n=6). Studies were carried out using same tissue strips. a Represents Pb 0.05 vs. obstructed bladders.
neurotransmitters, serotonin and histamine were assessed. Histamine (100 nM–100 μM) evoked concentration-dependent contractions of the isolated pig bladder (pD2 = 5.58 ± 0.15) with a maximum contraction of 10.18 ± 1.30 g (n = 6, Table 1, Fig. 3C). Bladder strips from obstructed animals responded with a potency, pD2 value of 5.60 ± 0.06, similar to that observed in control strips, although the maximal response was significantly reduced (2.00 ± 0.20 g, n = 8, Table 1, Fig. 3C). Serotonin (100 nM–0.1 mM) evoked concentration-dependent contractions of the isolated pig bladder with a pD2 value of 6.26 ± 0.06 with a maximum response (1.91 ± 0.21 g, n = 8, Table 1, Fig. 3D). Obstructed bladder strips also responded with a similar potency (pD2 = 5.96 ± 0.09) and efficacy (1.59 ± 0.43g; n = 7, Table 1, Fig. 3D).
32 g (Fig. 5A, B, Table 2). In this study, we chose to examine both groups at a preload of 1 g. For control strips, the intrinsic activity and maximum response increased with increases in preload peaking at a preload of 4–8 g whereas in obstructed strips the maximal response was shifted to higher preload values (∼16 g). We did not evaluate agonists at the maximum preload since the baseline drifted at preloads N 8 g and since the tension generated by higher preloads were such that the integrity of all strips could not be maintained during the course of the experiment. The differential effect of preload on spontaneous phasic activity in obstructed pigs prompted us to examine its effect on muscarinic receptor activation. Bladder strips taken from control pigs contracted when exposed to carbachol (1 μM) with a slight increase in contractile response as the preload was increased, reaching a peak response of 45.0 ± 4.93 at preload of 8 g (Fig. 5A, C, Table 2). Obstructed bladder strips were less responsive at the lower preloads (1–8 g); however, as the preload was increased (16–32 g) carbachol-evoked responses also increased. At a preload of 32 g, the contractile response was comparable to that of control bladder strips. 3.6. Effects of KATP channel openers K+ channels including ATP-sensitive and calcium activated K channels function as key regulators of resting membrane +
3.5. Myogenic contractility of bladder strips Contractility of bladder strips from obstructed animals was assessed after depolarization with 80 mM K+ and at various preload conditions and compared to the responsiveness of control strips. 3.5.1. Responses to elevated K+ When tissue strips were depolarized by addition of extracellular K+ (3–300 mM), concentration-dependent contractions of obstructed bladder strips were observed. The contractile response was tonic reaching a peak response in about 3 min. Maximal tension responses of 27.6 ± 4.02 g were observed with a pD2 value of 1.08 ± 0.33 (n = 12). Obstructed bladder strips responded with a 2.5-fold higher potency (pD2 = 1.66 ± 0.24), but with reduced maximum response (11.2 ± 1.12 g; n = 8, Table 1, Fig. 4). 3.5.2. Effects of preload on spontaneous phasic activity Control bladder strips demonstrated very little spontaneous phasic activity at preloads of 1 and 2 g but did show modest activity at 4–32 g. In contrast, bladder strips taken from obstructed pigs showed no spontaneous activity at 1 and 2 g preload; however, a substantially larger preload-dependent increases in spontaneous phasic activity were observed at 16 and
Fig. 6. (A) Inhibition of carbachol-evoked contractions in obstructed bladder strips by KATP channel openers. Bladder strips contracted with 1 μM carbachol. (B) Comparison of the potencies of KATP channel openers for the inhibition of carbachol-evoked contractions in control and obstructed bladders. The correlation coefficient was r2 = 0.90 and slope = 0.93 ± 0.11 (n = 4–7).
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potential to modulate excitability of the urinary bladder smooth muscle (Herrera et al., 2000; Buckner et al., 2000, 2002; Gopalakrishnan and Shieh, 2004; Gopalakrishnan et al., 2002). In general, KATP channel openers are 15-fold more potent in suppressing spontaneous phasic activity compared to contractions evoked by electrical field-stimulation (Buckner et al., 2002). To assess interactions with KATP channels, KATP channel openers were examined for their effects on contractions evoked by carbachol (1 μM) from bladder strips taken from control and obstructed pigs (Fig. 6A). Structurally diverse openers examined completely inhibit carbachol-evoked contractions in both obstructed and control tissues. P1075, a pinacidil analog that has been shown to open ATP-sensitive potassium channels (Quast et al., 1993), was the most potent compound, with pD2 values of 7.32 ± 0.02 (n = 4) and 6.78 ± 0.10 (n = 7) for control and obstructed bladder strips, respectively. A-278637, a 1,4dihydropyridine K+ channel opener shown to demonstrate selectivity for the suppression of unstable contractions vs. mean arterial pressure effects (Brune et al., 2002) was found to inhibit carbachol-evoked responses with comparable potencies in both control and obstructed animals. Other agents including ZD6169 and WAY-133537 also showed no substantial differences in sensitivities between control and obstructed pigs. The weakest agent examined was diazoxide with pD2 values of 4.36 ± 0.02 (n = 4) and 4.11 ± 0.10 (n = 4) for control and obstructed bladder strips, respectively. Overall, the potencies of these openers showed good 1:1 correlation between the control and the obstructed bladder strips (Fig. 6B; r2 = 0.90, slope = 0.93 ± 0.12; n = 11). 4. Discussion Neurogenically evoked contractions of the urinary bladder involves participation of both muscarinic and purinergic mechanisms, and contractions evoked by higher frequencies respond better to muscarinic receptor blockade compared to those evoked by lower frequencies (Burnstock et al., 1972; Sjogren et al., 1982). Our studies show that frequency-dependent contractions evoked by electrical field stimulus of the bladder smooth muscle was impaired at all frequencies in bladder strips from obstructed pigs. The reduction in responsiveness to both carbachol and ATP, but not to serotonin, suggests that this decline may relate specifically to these transmitter systems and not attributable to nonspecific changes due to bladder hypertrophy. The large reduction in responses to field stimulation suggests that the smooth muscle from the bladder of obstructed pigs was partially denervated and unable to generate adequate neurotransmission for evoking robust contractile effects. These results are in agreement with the findings of Speakman et al. (1987) who observed a marked deterioration of detrusor muscle contractility to electrical field stimulation in the pig model. In this study, both the potencies and efficacies of carbachol and ATP were reduced. The potency of carbachol was attenuated 4-fold whereas the shift was more robust for ATP with about a 13-fold reduction in potency. On the other hand, the reduction in maximal responses of both carbachol and ATP were comparable (2- and 1.8-fold, respectively). The decreases
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in sensitivity and maximal responses of both purinergic and muscarinic agonists suggest possible alterations in receptor levels and/or affinity although this remains to be directly assessed by biochemical measures. In contrast to our observations, Speakman et al. (1987) showed a slight increase (2-fold) in sensitivity to carbachol in obstructed tissues. These differences may be accounted for by subtle differences in protocols such as duration of obstruction and degree of decompensation. We have utilized smooth muscle strips (4 × 10 mm) in this study as opposed to smooth muscle bundles (1 × 1 × 7 mm) utilized by Speakman et al. (1987). Further, in the present study, the contractile response to carbachol was allowed to reach maximum (60 s) whereas Speakman and colleagues measured tension responses at 10 s. K+ depolarization-evoked contractions were significantly reduced about 60% in the obstructed bladder. However, a 2.5-fold increase in potency of KCl in the contractile response of the obstructed bladder was noted which is consistent with data from Mills et al. (2000) and Speakman et al. (1987). In contrast to studies in pigs, following long-term outlet obstruction (15 weeks) in rats, Lluel et al. (2002) observed a significant reduction in responses to KCl and carbachol-evoked contractility, but interestingly, no difference in response to electrical field stimulus was observed. Bladder strips from control animals contracted in response to carbachol (1 μM) over a preload range of 1–32 g (Fig. 5). The small, spontaneous phasic activity of the baseline was fairly constant over the range of preloads examined. In contrast, tissue strips from obstructed pigs with obstructed bladders showed a preload-dependent increase in the contractile response to carbachol and a similar increase in phasic spontaneous activity of the baseline. At a maximum of 32 g preload, both sets of tissues responded to carbachol in a similar fashion, but the obstructed bladder strips showed greater spontaneous phasic activity at 16 and 32 g preload. These results suggest that obstructed bladders from obstructed pigs could accommodate to the higher outlet pressures necessary for micturition. Several structurally diverse KATP channel openers examined in this study relaxed carbachol-stimulated contractions of the pig bladder strips taken from both control and obstructed pigs. This effect was concentration-dependent; all eleven openers were fully efficacious with potencies spanning a range of about four log units. The potencies of potassium channel openers to inhibit carbachol-evoked contractions showed a good 1 : 1 correlation (r2 = 0.90, slope = 0.93) between obstructed and control bladder strips. This agreement suggests that the interactions of these ligands with KATP channels were not adversely affected in obstructed bladders. In summary, our results support and further extend previous findings of obstruction-induced changes in this animal model. Importantly, our studies provide evidence of enhanced spontaneous phasic activity at elevated preloads post-obstruction. The impaired responses to electrical field stimulation could be attributed to reduced efficacies and lower sensitivities of muscarinic and purinergic signaling pathways. Although the contractile response of the obstructed bladder to electrical fieldstimulation and to muscarinic and purinergic agonists were
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attenuated, the bladder smooth muscle was about 2-fold more sensitive to K+ depolarization. The essentially unimpaired pharmacological profile of K+ channel openers post-obstruction suggests that this class of K+ channels could be effectively modulated by such agents that may have potential for the treatment of overactive bladder secondary to outlet obstruction. Acknowledgements The authors thank Dr Timothy Esbenshade for critical reading and suggestions on this manuscript. References Brading, A.F., 1997. A myogenic basis for the overactive bladder. Urology 50, 57–67. Brune, M.E., Fey, T.A., Brioni, J.D., Sullivan, J.P., Carroll, W.A., Coghlan, M.J., Williams, M., Gopalakrishnan, M., 2002. A-278637 [(-)-(9S)-9-(3-bromo4-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno [3,2-b] quinolin-8(4H)-one 1,1-dioxide]: a novel ATP-sensitive potassium channel opener efficacious in suppressing urinary bladder contractions II. In vivo characterization. J. Pharmacol. Exp. Ther. 303, 387–394. Buckner, S.A., Milicic, I., Daza, A., Davis-Taber, R., Scott, V.E., Sullivan, J.P., Brioni, J.D., 2000. Pharmacological and molecular analysis of ATP-sensitive K+ channels in the pig and human detrusor. Eur. J. Pharmacol. 400, 287–295. Buckner, S.A., Milicic, I., Daza, A., Coghlan, M.J., Gopalakrishnan, M., 2002. Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators. Br. J. Pharmacol. 135, 639–648. Burnstock, G., Dumsday, B., Symthe, A., 1972. Atropine resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purine nucleotide. Br. J. Pharmacol. 44, 451–461. Crowe, R., Burnstock, G., 1989. A histochemical and immunohistochemical study of the autonomic innervation of the lower urinary tract of the female pig. Is the pig a good model for the human bladder and urethra? J. Urol. 141, 414–422. De Groat, W.C., 1997. A neurological basis for the overactive bladder. Urology 50, 36–52. Fey, T.A., Gopalakrishnan, M., Strake, J.G., King, L.L., Brioni, J.D., Sullivan, J.P., Coghlan, M.J., Brune, M.E., 2003. Effects of ATP-sensitive K+ channel openers and tolterodine on unstable bladder contractions in a pig model of partial bladder outlet obstruction. Neurourol. Urodyn. 22, 147–155. Foster, C.D., Speakman, MJ., Fujii, K., Brading, A.F., 1989. The effects of cromakalim on the detrusor muscle of human and pig urinary bladder. Br. J. Urol. 63, 284–294. Gopalakrishnan, M., Shieh, C., 2004. Potassium channel subtypes as molecular targets for overactive bladder and other urological disorders. Expert Opin. Ther. Targets 8, 437–458. Gopalakrishnan, M., Buckner, S.A., Whiteaker, K.L., Shieh, C.C., Molinari, E.J., Milicic, I., Daza, A., Davis-Taber, R., Scott, V.E., Sellers, D., ChessWilliams, R., Chapple, C.R., Liu, Y., Liu, D., Brioni, J.D., Sullivan, J.P., Williams, M., Carroll, W.A., Coghlan, M.J., 2002. A-278637 [(-)-(9S)-9-
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