Neurogastroenterol Motil (2010) 22, e180e191

doi: 10.1111/j.1365-2982.2010.01495.x

Effect of otilonium bromide on contractile patterns in the human sigmoid colon

, , J. ALEU , E. MARTI NEZ , , L. ROFES ,* J. MARTI -RAGUE , M. JIME NEZ * , & P. CLAVE *, , D. GALLEGO ,* M. AULI

ticas y Digestivas (Ciberehd), Instituto de Salud Carlos III, n Biome dica en Red de Enfermedades Hepa *Centro de Investigacio Barcelona, Spain , Mataro , Spain Department of Surgery, Hospital de Mataro de Gastroenterologia Dr. F. Vilardell, Barcelona, Spain Fundacio ` noma de Barcelona, Barcelona, Spain Department of Cell Biology, Physiology and Immunology, Universitat Auto

Abstract Background The mechanism of action of the spasmolytic compound otilonium bromide (OB) on human colonic motility is not understood. The aim of our study was to characterize the pharmacological effects of OB on contractile patterns in the human sigmoid colon. Methods Circular sigmoid strips were studied in organ baths. Isolated smooth muscle cells from human sigmoid colon were examined using the calcium imaging technique. Key Results Otilonium bromide inhibited by 85% spontaneous non-neural rhythmic phasic contractions (RPCs), (IC50 = 49.9 nmol L)1) and stretch-induced tone (IC50 = 10.7 nmol L)1) with maximum effects at micromolar range. OB also inhibited by 50% both on- (IC50 = 38.0 nmol L)1) and off-contractions induced by electrical stimulation of excitatory motor neurons. In contrast, the inhibitory latency period prior to off-contractions was unaffected by OB. OB inhibited acetylcholine-, substance P-, and neurokinin A-induced contractions. The L-type Ca2+ channel agonist BayK8644 reversed the effects of OB on RPCs, on- and off-contractions. Hexamethonium, atropine, the NK2 antagonist, or depletion of intracellular Ca2+ stores by thapsigargin did not prevent the inhibitory effect of OB on RPCs and electrical contractions. KCl-induced calcium
Address for correspondence , MD, PhD, Associate Professor of Surgery, Pere Clave , Universitat Department of Surgery, Hospital de Mataro ` noma de Barcelona, C/Cirera s/n. 08304, Mataro , Spain. Auto Tel: +34 93 741 77 00; fax: +34 93 741 77 33; e-mail: pclave@teleline.es This study was presented in part at the 21st International Symposium on Neurogastroenterology and Motility, Jeju, Korea, August 2007. Received: 15 October 2009 Accepted for publication: 22 February 2010

transients in isolated smooth muscle cells were also inhibited by OB (IC50 = 0.2 lmol L)1). Conclusions & Inferences Otilonium bromide strongly inhibited the main patterns of human sigmoid motility in vitro by blocking calcium inux through L-type calcium channels on smooth muscle cells. This pharmacological prole may mediate the clinically observed effects of the drug in patients with irritable bowel syndrome. Keywords gastrointestinal motility, sigmoid colon, smooth muscle, spasmolytic drugs.

INTRODUCTION
Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder affecting up to 11.5% of the general population1 and is characterized by abdominal pain or discomfort associated with a change in bowel habit. Altered motility in the small bowel and colon may contribute to a change in bowel behavior, and a combination of increased motility and spasm, visceral hypersensitivity, and abnormalities in central pain processing may explain the origin of the abdominal pain.2 Recent in vivo studies showed that pain hypersensitivity and colon hypermotility including increased phasic motility and enhanced smooth muscle tone are independent factors contributing to symptoms in patients with IBS.3 Antispasmodics and muscle relaxants are widely used as rst-line treatment in these patients. However, the biological rationale for the efcacy of antispasmodics is unclear, and their pharmacological effects may vary among compounds. Recent studies found that antispasmodics may act by reducing colonic contraction and increasing transit time and therefore reducing pain and stool frequency in patients with IBS.2 Among antispasmodics, otilonium bromide (OB), a quaternary ammonium

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derivative compound, has shown consistent evidence of efcacy on IBS patients.2,4,5 Clinically, the main effects of OB include a global improvement of IBS symptoms, a reduction in the frequency and severity of abdominal pain, and an increase in the distension pain threshold with the use of a sigmoid balloon.69 However, the effects and the mechanism of action of OB on human colonic motility are not fully understood, and the few studies that have investigated the mechanisms of action of OB in animal models described a heterogeneous pharmacological prole.5,10 Most studies on the pharmacodynamics of OB have been conducted on rodents and suggest several mechanisms of inhibition of OB on spontaneous and stimulated intestinal motility. Animal studies found that OB inhibited contractions induced by acetylcholine (ACh), serotonin, substance P (SP), histamine, barium chloride (BaCl2) and K+ and described how OB exerted its spasmolytic action by a combination of antagonistic actions at muscarinic receptors, by blocking L-type Ca channels, by antagonizing tachykinin NK1 and NK2 receptors and by blocking nicotinic-mediated responses;5,1012 however this has not been fully characterized in humans. We have recently characterized the different types of contractions in human sigmoid colon in vitro. Circular strips of human sigmoid colon in vitro developed spontaneous rhythmic phasic contractions (RPCs) of non-neural origin and responded to electrical stimulation (EFS) of enteric motor neurons (EMNs) with two distinct patterns of strong contractile responses: (i) a phasic contraction during EFS on-contractions or (b) a phasic contraction after EFS off-contraction caused by stimulation of excitatory EMNs following a period of latency caused by stimulation of inhibitory EMNs releasing NO and ATP acting at P2Y1 receptors.13Amplitude of electrical-induced contractions depended on stimulation of excitatory EMNs releasing ACh and tachykinins acting on muscarinic and NK2 receptors located on smooth muscle cells.13 The aim of this study was to assess the pharmacodynamics and effects of the spasmolytic agent OB on the main types of in vitro contractions in human sigmoid colon and to pharmacologically characterize OBs mechanism of action.

approved by the Institutional Review Board of the Hospital de , Barcelona, Spain. Mataro

Preparation of circular muscle strips and isometric tension recording

The colonic segment was cut open longitudinally along the mesenteric border, the mucosal layer was removed and transmural muscle strips (3 mm wide by 10 mm long) were cut in the direction of the circular muscular bers. Weight of the strips was 0.21 0.01 g, N = 31. A silk thread was attached to either end of the strips and they were placed in 10 mL organ baths lled with Krebs solution (37 C, bubbled with a mixture of 5% CO2/95% O2, pH 7.4) as described in previous studies.13,14 Strips were positioned between two parallel platinum wire electrodes 10 mm apart and changes in tension were measured using isometric force transducers, recorded on a chart recorder (model 03 Force Transducer and model 7 Series Polygraph; Grass Instruments Co, Quincy, MA, USA) and digitized (Acqknowledge, MP100; Biopac Systems, Inc, Goleta, CA, USA). In each experiment, up to six strips from the same specimen were simultaneously studied. Strips were initially stretched to 4 g of force and equilibrated for 1 h and then the following patterns of colonic motility were assessed. Spontaneous RPCs After the equilibration period, strips developed spontaneous RPCs. Origin of RPCs was characterized by the ganglionic blocker hexamethonium 100 lmol L)1, the neural blocker tetrodotoxin (TTX) 1 lmol L)1 that interrupts Na+ action potentials along the axon of EMNs, withdrawal of extracellular Ca2+ from the medium (by using a Ca2+-free Krebs solution with EGTA 1 mmol L)1), and depletion of intracellular Ca2+ stores by 20-min incubation with thapsigargin 10 lmol L)1, a blocker of sarco/endoplasmic reticulum Ca2+ ATPase.15 Stretch-induced responses After the period of equilibration, stretch was applied to strips from 0 to 7 g in steps of 1 g each 30 min. Baseline tone and amplitude of RPCs was measured during the last 10 min of each stretch. To avoid neural mediated responses, this study was performed in the presence of the neural blocker TTX (1 lmol L)1). Contractions induced by EFS of EMNs Electrical stimulation was applied by an electrical stimulator (Model S88; Grass Instruments Co) and a power booster (Stimu-Splitter II; Med-Lab Instruments, Loveland, CO, USA).13 Ten-second trains of pulses of 0.4 ms duration at 140 Hz and 26 V were delivered to the electrodes and simultaneously recorded on tension tracings through a synchronized TransistorTransistor logic signal between the electrical stimulator Grass S88 and the computerized Biopac System. In a previous study, we found sigmoid strips responded to EFS with two distinct patterns of contractile responses of neural origin (a) a phasic contraction which began during EFS on-contractions or (b) a phasic contraction which began after EFS off-contractions.13 In the present study, these EFS responses were further characterized by hexamethonium 100 lmol L)1, and depletion of extracellular and intracellular Ca2+ stores. Direct contractions induced by excitatory neurotransmitters In a previous study, we found that sigmoid contractions induced by stimulation of excitatory EMNs were mediated by released ACh and tachykinins mainly acting on muscarinic and NK2 receptors

METHODS Tissue specimens

Tissue specimens of human sigmoid colon were obtained from 70 patients without symptoms of major clinical motility disorders who underwent surgery for rectal cancer (T2/T3 stages, age range 3285 years, 44% women). All strips were obtained from macroscopically non-invaded regions. The experimental protocols were

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located on smooth muscle cells.13 In this study, we assessed the direct effect of ACh (0.01100 lmol L)1), SP (0.00110 lmol L)1), and neurokinin A (NKA) (1 nmol L)1 to 1 mmol L)1) on sigmoid strips.

Calcium image technique

Smooth muscle cells were isolated by a mechanical and enzymatic process. Briey, the circular muscular layer was dissected and cut into small pieces (12 mm). The muscle pieces were placed in Ca2+-free Hanks solution (NaCl 115 mmol L)1, KCl 5.4 mmol L)1, MgCl26H2O 0.5 mmol L)1, NaHCO3 4.2 mmol L)1, Na2HPO4 0.3 mmol L)1, KH2PO4 0.4 mmol L)1, MgSO47H2O 0.4 mmol L)1, glucose 5 mmol L)1). They were incubated with 1.5 mg mL)1 papain (Sigma, St Louis, MO, USA), 1 mg mL)1 bovine serum albumin (BSA) and 2 mg mL)1 soy bean trypsin inhibitor (SBTI) for 3 min at 32 C and stirred at 108 g.16,17 After centrifugation, the muscle pieces were incubated in 1 mg mL)1 BSA, 0.75 mg mL)1 collagenase type II (Worthington, Lakewood, NJ, USA) at 32 C and stirred again at 27 g until isolated cells appeared. Isolated smooth muscle cells were placed in a recording medium (NaCl 145 mmol L)1, KCl 4.8 mmol L)1, MgCl26H2O 1 mmol L)1, CaCl2 1.8 mmol L)1, glucose 10 mmol L)1, HEPES 10 mmol L)1, pH 7.4). Fluo-4/AM (Teabs, Inc., Austin, TX, USA) was used to monitor changes in the calcium level in cytosol. Fluo4 (50 lg) was dissolved in DMSO (25 lL) and 2 lL of this stock solution was added to each culture plate in 1 mL recording medium for 45 min. After washing out the remaining dye, cells were incubated in the recording medium. The cells were imaged with IX-FLA equipment (Olympus Biosystems, Heidelberg, Germany) connected to an Olympus IX70 microscope with an 20 lens. The cells were scanned using CellR software (Olympus Biosystems). Cells were exposed briey (5 s) to high potassium solution (75 mmol L)1 KCl) causing a sudden increase in transient intracellular calcium [Ca2 + ]i in the smooth muscle cells (control), and the effect of 10 min incubation of cumulative concentrations of OB (10 nmol L)1 to 100 lmol L)1) was assessed.

atropine sulphate, from Research Biochemicals International (Natick, MA, USA); NKA, thapsigargin, NK2 receptor antagonist GR 94800 from Tocris, (Bristol, UK); Fluo-4 AM from Teabs Inc. Thapsigargin, n-butyl-hyoscine 20 mg per 1 mL from Boehringer Ingelhemim Espan a SA, (Barcelona, Spain). Stock solutions were made by dissolving drugs in distilled water except for thapsigargin and Fluo-4 AM which were dissolved in DMSO (0.01%) and BayK8644 which was dissolved in ethanol (0.01%).

Data analysis and statistical procedures

Spontaneous RPCs were measured as the area under the curve (AUC), in g min)1 and paired Students t-test was used to assess the effect of antagonists and agonists on the proposed putative neurotransmitters. The effect of antagonists on RPCs was assessed following 20 min incubation, the last 10-min period being analyzed. Data were normalized with respect to a control period (10 min before addition of the antagonist) and concentration response curves analyzed by two-way repeated measure ANOVA analysis. The effect of agonists was measured as the AUC of the 2-min period following their addition to the bath. The concentrationresponse curve for each agonist was computer tted using nonlinear regression, and the IC50 was calculated (GRAPHPAD PRISM, version 4.01; GraphPad Prism Software, San Diego, CA, USA). Latency of EFS contractions was dened as the period of time from the beginning of EFS to the onset of contraction; and maximal amplitude of EFS contractions (in g) was also measured. The effect of OB and the other drugs on latency and amplitude of on- and off-contractions was analyzed by two-way ANOVA for repeated measures. When the two-way ANOVA was signicant, the Bonferroni test was carried out to determine the frequencies or doses of statistically different responses. Data are expressed as mean SEM. Changes in the Fluo4 uorescence were recorded for 30 s at 2.5 Hz with a spatial resolution of 512 480 pixels. At the end of the experiments, the images were analyzed over time by CellR software (Olympus Optical Co., Ltd, Tokyo, Japan) using regions of interest (ROIs). Fluorescence intensity was normalized to the basal uorescence at the onset of the recording for each ROI, and peaks were analyzed as previously described. Paired Students t-test or ANOVA test was used before and after drug addition. A P < 0.05 was considered statistically signicant. N values indicate the number of samples from different patients.

Experimental design
We assessed the effect of OB (10 pmol L)1 to 100 lmol L)1) on the main human sigmoid motility patterns: (i) spontaneous RPCs; (ii) stretch-induced tone; (iii) contractions induced by EFS of EMNs EFS on and off contractions ; (iv) direct contractions induced by ACh, SP and NKA, and (v) calcium transients induced by KCl in isolated muscle cells. We compared the effects of OB with those caused with the specic L-type channel antagonist nifedipine (10 pmol L)1 to 10 lmol L)1), atropine (1 pmol L)1 to 1 lmol L)1), and the NK2 receptor antagonist GR 94800 (1 pmol L)1 to 1 lmol L)1). We also characterized the mechanism of action of OB by pharmacological studies using the specic L-type channel agonist BayK8644 (1 nmol L)1 to 10 lmol L)1), and by assessing the effect of OB on RPCs and EFS contractions following blockade of nAChRs by hexamethonium 100 lmol L)1, blockade of muscarinic receptors by atropine 1 lmol L)1, blockade of NK2 receptors by the NK2 receptor antagonist GR 94800 (1 lmol L)1) and following depletion of intracellular calcium stores by thapsigargin 10 lmol L)1.

RESULTS Effects of OB on spontaneous RPCs

Human circular sigmoid strips developed spontaneous RPCs after 1 h equilibration. The AUC, amplitude, and frequency of RPCs were 72.85 10.87 g min)1, 3.53 0.52 g, and 2.48 0.24 contractions min)1 (N = 20) respectively. The activity of RPCs (AUC) was unaffected by 30-min exposure to the neural blocker TTX 1 lmol L)1 (+6.16 1.5%. ns, N = 11) or hexamethonium (100 lmol L)1) ()3.1 23.57%, ns, N = 5). Blockade of muscarinic receptors by atropine (1 pmol L)1 to 1 lmol L)1) or hyoscine (4.5 pmol L)1 to 4.5 lmol L)1) and blockade of NK2 receptors by the NK2 receptor antagonist GR 94800 (1 pmol L)11 lmol L)1) caused a

Drugs
Otilonium bromide was obtained from Laboratorios Menarini SA, (Badalona, Spain). Acetylcholine chloride (ACh), SP, BayK8644, nifedipine, hexamethonium, BSA, SBTI from Sigma; TTX,

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signicant reduction of RPCs, an effect dependent on their concentration, the design of the experiment (cumulative vs single concentrations) and the time of exposure, but did not block them (Fig. 1). All these results suggest that RPCs have a non-neural origin but are modulated by EMNs. RPCs were greatly reduced ()62.54 18.89%, P < 0.01; N = 6), following 30 s removal of extracellular calcium and in contrast were only slightly reduced by depletion of intracellular calcium stores by 30-min incubation with thapsigargin 10 lmol L)1 ()26.47 16.76%, ns; N = 5) indicating the importance of extracellular calcium in the origin of this pattern of contractions. RPCs were almost abolished by OB ()84.17 16.87% at 100 lmol L)1) in a concentration-dependent manner (IC50 of 49.9 nmol L)1, N = 8). RPCs were also abolished ()96 1.75% at 10 lmol L)1) by the L-type calcium channel blocker, nifedipine, with an IC50 of 1.00 nmol L)1 (N = 7) (Fig. 1, Table 1). The specic L-type channel agonist, BayK8644 (1 nmol L)1 to 10 lmol L)1), increased the amplitude of RPCs in a concentration-dependent manner (1 nmol L)1 to 1 lmol L)1, EC50 = 0.07 lmol L)1); and at 10 lmol L)1 this excitatory effect was reduced (N = 3) (Fig. 2). BayK concentration dependently reversed the inhibitory effects of nifedipine and OB (10 lmol L)1) on RPCs (Fig. 2). In addition, OB 100 lmol L)1 strongly inhibited RPCs following blockade of muscarinic receptors by atropine 1 lmol L)1 ()64.12% 11.62, P < 0.05, N = 5), blockade of NK2 receptors by the antagonist GR 94800 ()70.49 10.32%, P < 0.0001, N = 7), depletion of intracellular calcium by thapsigargin 10 lmol L)1 ()75.16 7.12% P < 0.05, N = 5), and blockade of nicotinic receptors by hexamethonium 100 lmol L)1 ()86.49 9.23% P < 0.05, N = 4) (Fig. 3).

Effect of OB on sigmoid contractions induced by electrical stimulation of EMNs

In a recent study, we found EFS produced two distinct patterns of contractile responses in the human sigmoid colon: (i) contractions which began during EFS, dened as on-contractions caused by selective stimulation of excitatory EMNs and (ii) contractions which began after EFS, dened as off-contractions also caused by stimulation of excitatory EMNs following a period of latency caused by stimulation of inhibitory EMNs.13 In the present study, OB induced a signicant concentrationdependent inhibition in the amplitude of EFS-induced on-contractions (IC50 = 0.38 nmol L)1, )49.39 13.75% at 40 Hz P < 0.05, N = 6) (Table 1). Incubation with the L-type calcium channel blocker nifedipine abolished on-contractions (IC50 = 0.13 nmol L)1, )96.28 3.71% at 40 Hz P < 0.05, N = 6, Fig. 5). EFS on-contractions were fully abolished following incubation with free calcium Krebs solution for 30 s (N = 6), and were reduced by atropine (1 lmol L)1, 40 Hz, )69.58 3.49%, N = 9, P < 0.001), n-butyl-hyoscine 100 lmol L)1 40 Hz, )49.81 8.49%, N = 6, P < 0.05), GR94800 (1 lmol L)1) (40 Hz: )26.07 6.71% N = 6, P < 0.001), and unaffected by hexamethonium (100 lmol L)1) (40 Hz, )9.69 7.82%, N = 5, ns) and thapsigargin (10 lmol L)1, 40 Hz, )10.35 14.56%, N = 5, ns). OB 100 lmol L)1 signicantly inhibited the amplitude of EFS on-contractions following blockade of muscarinic receptors by atropine 1 lmol L)1 ()40.47 9.28%, N = 9, P < 0.01), blockade of NK2 receptors by GR4800 1 lmol L)1 ()58.61 9.08%) blockade of nicotinic receptor by hexamethonium 100 lmol L)1 ()47.33 9.04%) and depletion of intracellular calcium stores by thapsigargin 10 lmol L)1 ()47.19 9.92%). OB also induced a concentration-dependent inhibition in the amplitude of electrical off-contractions at high frequencies of stimulation (2040 Hz) ()58.69 15.61% at 40 Hz, P < 0.05, N = 6). In contrast, latency of offcontractions was unaffected by OB ()5.18 2.26%, at 40 Hz, ns, N = 6). Nifedipine 1 lmol L)1 strongly reduced off-contractions at 140 Hz ()67.86 12.4%, at 40 Hz, P 0.001, N = 5), an effect that was fully reversed by BayK8644 (1 lmol L)1). The inhibition of the amplitude of EFS on- and off-contractions caused by OB 10 lmol L)1 was also fully reversed by the calcium channel activator BayK8644 (1 lmol L)1) (Fig. 6).

Effect of OB on stretch-induced contractions

Stretch of strips did not modify the frequency of spontaneous RPCs. On the contrary, basal tone of strips and amplitude of RPCs were progressively increased by stretch (N = 15, ANOVA P < 0.001). OB was tested at six different concentrations from 1 nmol L)1 to 1 lmol L)1, N = 5. Amplitude of stretch-induced contractions was decreased by OB, signicant differences were observed at the concentration of 1 lmol L)1 ()64.21%). (From 36.08 7.5% in control conditions to 12.9 8.1 after incubation with OB 1 lmol L)1, at 7 g of induced stretch, N = 5 P < 0.001.) Stretch-induced tone was also reduced by OB, with signicant differences observed at 0.1 lmol L)1 ()52.76%) and 1 lmol L)1 ()55.37%; Fig. 4). (From 3.07 0.23 g in control conditions to 1.45 0.18 g with OB 0.1 lmol L)1, N = 5 and to 1.37 0.29 g with OB 1 lmol L)1, N = 5, P < 0.001 each.)

Effect of OB on the responses induced by direct stimulation of sigmoid smooth muscle cells
Otilonium bromide 100 lmol L)1 signicantly inhibited the direct contraction produced by ACh

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Figure 1 Mechanical recordings showing inhibition of spontaneous motility (RPCs) by increasing concentrations of otilonium bromide (A), nifedipine (B), atropine (C), n-butyl-hyoscine (D) and the NK2 antagonist GR94800 (E). Cumulative concentrationresponse curves of (F) L-type calcium channel blockers, otilonium bromide and nifedipine, (G) muscarinic receptor blockers, atropine and n-butyl-hyoscine and (H) the NK2 antagonist GR94800.

(10 lmol L)1, )74.63 8.69%; P < 0.001, N = 10) and SP (10 lmol L)1, )78.8 6.66%; P < 0.05, N = 7) in this preparation. Nifedipine 100 lmol L)1 also inhibited the direct contractions induced by ACh, and SP. Contraction induced by 10 lmol L)1 NKA was also signicantly reduced by 100 lmol L)1 OB )61.61 4.12%; P < 0.001, N = 4) and almost abolished by 100 lmol L)1 nifedipine further suggesting L-type Ca2+ channels contribute to

cholinergic and tachykinergic responses. During experiments with calcium image technique, application of high extracellular potassium (75 mmol L)1) induced an intracellular calcium increase in smooth muscle cells. This increase was concentration dependently reduced by OB (0.01100 lmol L)1). OB inhibited the calcium transients with an IC50 of 0.2 lmol L)1, 15 cells, N = 5) (Fig. 7).

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Table 1 Pharmacodynamics of otilonium bromide and nifedipine on in vitro motor patterns in the human sigmoid colon IC50 + SEM (mol L)1) 4.99 10)8 0.14 1.07 10)8 0.69 7.76 10)6 0.25 3.80 2.52 3.16 1.00 1.32 6.92 5.13 10 0.20 10)2 0.58 10)4 0.45 10)8 0.69 10)10 0.45 10)8 0.31 10)9 0.25
)8

DISCUSSION AND CONCLUSIONS

In the present study, we have assessed the strong inhibitory effect OB has on the main patterns of human sigmoid motility in vitro (RPCs, smooth muscle tone, contractions induced by stimulation of excitatory EMNs, contractions induced by direct effect of excitatory neurotransmitters and calcium transients in isolated colonic smooth muscle cells). In contrast, OB did not affect inhibitory neuromuscular transmission. OB inhibited calcium transients induced by KCl in isolated sigmoid smooth muscle cells in a similar way to the inhibition of inward currents driven by L-type calcium channels in rat colonic smooth muscle cells17 and human small intestine.18 Moreover, binding stud-

Pattern Otilonium bromide RPCs Stretch-induced tone (7 g) Stretch-induced amplitude (7 g) EFS-on ACh contraction SP contraction RPCs EFS-on ACh contraction SP contraction

Emax (% inhibition) 84.17 16.87 54.92 8.21 64.21 4.03 49.39 74.63 78.8 95.35 96.28 73.56 89.59 13.75 8.69 6.66 6.37 3.71 12.51 7.15

Nifedipine

Figure 2 (A) Mechanical recording and histograms showing the increase in the activity of spontaneous rhythmic phasic contractions (RPCs) induced by the L-type Ca2+ channel agonist BayK8644. Incubation of strips with (B) the L-type Ca2+ channel antagonist nifedipine (10 lmol L)1) or (C) otilonium bromide (10 lmol L)1) concentration-dependently antagonized and prevented the effect of BayK8644. AUC, area under the curve of RPCs. Data are expressed as mean SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

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Figure 3 Representative tracings and graph bars showing how single concentrations of OB (100 lmol L)1) strongly inhibit rhythmic phasic contractions following: (A) blockade of muscarinic receptors by atropine 1 lmol L)1, (B) depletion of intracellular Ca by thapsigargin 10 lmol L)1, (C) blockade of nicotinic receptors by hexamethonium 100 lmol L)1, and (D) blockade of NK2 receptors by GR 94800. Data are expressed as mean SEM (*P < 0.05, **P < 0.01).

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Figure 4 (A) Mechanical recording showing the effect of induced tension on spontaneous motility and tone in control conditions (top trace) and in the presence of otilonium bromide (OB) (0.1 lmol L)1) (bottom trace). (B) Plot graph showing (top) the amplitude increase of spontaneous contractions induced by tension and (bottom) the increase of basal tone induced by tension. Data are expressed as mean SEM.

ies also found that OB showed competitive interaction and nmol L)1 afnity for the L-type calcium channel.12 The pharmacological prole of OB in our study suggests that OB might exert its spasmolytic effect mainly by reducing calcium inux through L-type calcium channels on human colonic smooth muscle cells. After oral administration, OB is not absorbed systemically and pharmacokinetic studies in humans have found that OB is mainly eliminated by feces (97.8%) and minimally excreted by urine (0.71%).5,19,20 Animal studies using oral doses similar to those used in humans show maximal and specic accumulation of OB in the colonic circular muscle,21 and peak levels in colonic tissues reaching the micromolar range 8 h after oral drug administration.22 Accordingly, OB might act at the level of the gastrointestinal tract without systemic absorption. The pharmacological prole of the effect of OB in our study shows that OB strongly inhibits spontaneous RPCs (IC50 = 49.9 nmol L)1 for OB and 1.00 nmol L)1 for nifedipine) but with lesser potency than the specic L-type calcium channel blocker nifedipine (Emax = 84.17% and Emax = 95.35% respectively). These results suggest similar afnity but lesser intrinsic activity of OB on L-type channels

compared with nifedipine and agree with our previous studies on the rat colon.17 The effects of OB and nifedipine on RPCs in our study are reversed by the specic L-type Ca2+-channel agonist, BayK8644, conrming the involvement of L-type calcium channels in the effect of OB on RPCs. Stretch is an important stimulus in the gastrointestinal tract and L-type calcium channels are gated by voltage, several chemical mediators and stretch.23 We found that stretch increased the amplitude of spontaneous contractions and baseline tone in the human sigmoid colon through non-neural mechanisms, and stretch-induced contractions and tone were also strongly inhibited by OB. Inhibitory neurotransmission can be quantied with the latency observed before the onset of the off-contraction.13 This latency is caused by stimulation of inhibitory motor neurons releasing NO and a purine acting on P2Y1 receptors.13 The latency is well-correlated with electrophysiological data where the fast and the slow component of the IJP is due to a purine acting on P2Y1 receptors and NO respectively.14 The present study shows that OB does not modify the latency suggesting that inhibitory neurotransmission in the human colon is unaffected by OB. Similar results were reported in the rat colon where the fast component of

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Figure 5 (A) Plot graph showing the effect of otilonium bromide (OB) (top) and nifedipine (bottom) on the amplitude of electrical stimulation (EFS) induced on-contractions. (B) Concentrationresponse curves reecting the inhibitory effect OB and nifedipine on the amplitude of the EFS induced on-contractions at the maximum frequency of stimulation (40 Hz). Data are expressed as mean SEM (*P < 0.05 vs previous concentration).

the IJP was not modied.17 In contrast, we found a strong inhibitory effect of OB on the amplitude of the two main types (on and off) of sigmoid contractions induced by EFS of excitatory EMNs. The mechanisms of action of OB on the inhibition of amplitude of EFS on- and EFS off-contractions both mediated by stimulation of excitatory EMNs co-releasing ACh and tachykinins acting on NK2 receptors13 need careful discussion. Initial in vitro studies in the guinea pig ileum found OB counteracted the spasmogenic effect of ACh with an IC50 in the 20100 nmol L)1 range,

similar to that found for atropine in the same experiments.10 This antimuscarinic activity was conrmed in binding studies that show that OB binds with sub lmol L)1 afnity to many types of muscarinic receptors in different tissues.12 Results from these initial studies described the pharmacological prole of OB as a non-competitive antimuscarinic compound.10 Other in vitro studies demonstrated that OB also behaves as a potent blocker of neuronal nicotinic ACh receptors blocking Ca2+ uptake induced by nicotinic agonists with an IC50 in the lmol L)1 range, and suggested that

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Figure 6 (A) Representative tracings showing electrical stimulation (EFS) off-contractions and (B) EFS on-contractions. Note EFS off responses occur after a period of latency (marked in the tracing). Histograms show the reduction of amplitude of electrical on- and off-contractions by otilonium bromide and the reversion of this effect by the L-type Ca2+ channel agonist, BayK8644 (1 lmol L)1). Data are expressed as mean SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

A
7 6 Fluorescence F/F 5 4 3 2 1
0 5 A B

KCI C OB 0.01 mol L 1 OB 0.1 mol L 1 OB 10 mol L 1 OB 3 mol L 1 OB 10 mol L 1 OB 100 mol L
1

A
OB 0.01 mol L1

10

15 Time (s)

20

25

30

OB 0.1 mol L1

C
Percentage of control 75 50 25 0 8 7 6 5 Log (otilonium) 4

OB 1 mol L1

OB 10 mol L1

Figure 7 (A) Ca2+ transients induced by KCl. (A) This gure illustrates the change in relative Fluo4 uorescence in one cell in response to an application of KCl (75 mmol L)1) (showed with a bar) in the presence of increasing concentrations of otilonium bromide (OB; from 10 nmol L)1 to 100 lmol L)1). (B) Images taken at specic time points and corresponding to the dotted lines in different OB concentrations. (C) Doseresponse data tted to a sigmoid curve of relative Fluo4 uorescence DF/F in response to an application of KCl in the presence of different OB concentrations. Data are expressed as mean SEM.

blockade of nAChRs at the myenteric plexus might contribute to the spasmolytic effect of OB.11 In addition, pharmacological studies on guinea-pig proximal

colon and binding studies on isolated cells transfected with the human tachykinin NK2 receptor demonstrate that OB acts in the lmol L)1 range as muscarinic and

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Neurogastroenterology and Motility

tachykinin NK2 receptor antagonist.24 Taken together, these studies suggest multiple mechanisms of action for the inhibitory properties of OB to reduce stimulated motility of intestinal smooth muscle.5,24 In the present study, we could not distinguish between a putative effect on muscarinic, nicotinic or tachykinergic receptors because the predominant effect of OB in the human sigmoid colon was probably the blockade of L-type calcium channels. In agreement with this hypothesis, nifedipine inhibited both EFS-induced onand off-contractions and the L-type Ca2+ channel activator BayK8644 reversed the inhibitory action of OB on both EFS on- and off-contractions. Altogether, our results suggest that the predominant mechanism of action of OB on these potent contractions induced by stimulation of excitatory EMNs is exerted through blockade of L-type calcium channels, the same mechanisms that we found for the effect of OB on nonneural RPCs. L-type calcium channels have different binding sides to dihydropyridines (nifedipine), benzothiazepines (diltiazem) and phenylalkilamines (verapamil).25 Previous studies have demonstrated that OB binds to the diltiazem binding side of the L-type calcium channel with a minor binding on the dihydropyridine binding side.12 Our results show that BayK, a dihydropiyidine derivative, is able to counteract the effects of OB. This is probably due to the interaction with the fraction of OB bound to the dihydropyridine binding side or to interactions between the different binding sides of the channel.25 The stretch sensor of the L-type calcium channel is located in the alpha(1C)subunit26 where the binding of these different antagonists is located. Contraction of smooth muscle cells from the gastrointestinal tract depends on extra cellular calcium inux mainly through L-type calcium channels and calcium release from intracellular stores. Entry of calcium through L-type voltage-dependent Ca2+ channels provides the major source for the contractile response to ACh in canine circular colonic smooth muscle.27 In contrast, contraction of human sigmoid circular smooth muscle cells in response to NKA requires release of intracellular calcium.28 In our study, contractions induced by ACh, NKA and SP were strongly inhibited by OB as were intracellular calcium transients caused by direct KCl-depolarization of sigmoid smooth muscle cells. It is always difcult to establish a correlation between colonic motility in vivo and in vitro. In vivo human colonic motility is complex and involves three main motor patterns to produce the normal mixing and propulsive motor functions: RPCs, giant migrating contractions (GMCs) and tone, each with specic mechanisms of control.29 Spontaneous RPCs are

caused by direct transmission of electrical slow waves from interstitial cells of cajal to smooth muscle cells30 and contribute to the mixing of colonic contents.29 Colonic tone can modulate the mechanical efcacy of RPCs and GMCs.29 In vitro on-contractions correlate well with in vivo simultaneous contractions, the most common pattern of pressure activity in the human colon, which can slow transit; and in vitro off-contractions might be equivalent to sigmoid GMCs, or high-amplitude propagating contractions causing propulsion of stools and initiation of defecation.31 Latency of in vitro off-contractions determines the velocity of propagation of in vivo GMCs,31 suggesting OB might decrease the amplitude of sigmoid contractions without affecting their propagation. Previous in vivo studies in humans by sigmoid manometry found OB reduced sigmoid motility and enhanced the pain threshold following ination of a endoluminal balloon,69 agreeing with the present study. We believe the evaluation of an antispasmodic drug should include the assessment of its effects on all these specic motor patterns and its effects on direct excitability of sigmoid smooth muscle cells.29 In conclusion, we found a predominant mechanism of action for the strong spasmolytic properties of OB on the normal human sigmoid motility patterns based in its properties as a blocker of L-type calcium channels mediating calcium inux on smooth muscle cells. We believe these pharmacological properties might mediate the clinically proven effects of OB on the spastic motility disturbances described in subsets of patients with IBS.

ACKNOWLEDGMENTS
enz, M. Marti-Gallostra and The authors thank Dr Alex Sa nica Sagrada Familia) and Dr Xavier Sun J. Cases (Cl ol, Dr Oscar Estrada, Dr Fran Espin, Dr Adolfo Heredia, Dr Eva a, and Dr Lu s Antonio Hidalgo (Hospital de Mataro ) for Garc providing human tissue. We also thank Dr S. Evangelista and Mrs Jane Lewis for revising the manuscript. This study was de Gastroenterologia supported by a grant from the Fundacio Salut del Consorci Santari Dr Francisco Vilardell, the Fundacio del Maresme, the Departament dUniversitats, Recerca i Soci (2009-SGR-708), the Fondo de Investigacetat de la Informacio iones Sanitarias del Ministerio de Sanidad y Consumo i Desenvolupa(IF063678-1), the CIDEM (Centre dInnovacio ment Empresarial) (RDITSIND06-1-0174), BFU2006-05055/BFI and by Laboratorios Menarini SA. Ciberehd is funded by the Instituto de Salud Carlos III.

CONFLICT OF INTEREST
has served as a speaker for Menarini International and P. Clave has received research funding from Laboratorios Menarini SAMenarini Group, Badalona, Spain.

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Otilonium bromide inhibits human sigmoid motility

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