Neurogastroenterol Motil (2011) 23, 96e10

doi: 10.1111/j.1365-2982.2010.01594.x

Effect of otilonium bromide and ibodutant on the internalization of the NK2 receptor in human colon
G. CIPRIANI ,* P. SANTICIOLI , S. EVANGELISTA , C. A. MAGGI , S. RICCADONNA , M. N. RINGRESSI , P. BECHI , M. S. FAUSSONE-PELLEGRINI *

&

M. G. VANNUCCHI *

*Department of Anatomy, Histology and Forensic Medicine, University of Florence, Firenze, Italy Department of Pharmacology, Menarini Ricerche S.p.A., Firenze, Italy Department of Surgical Critical Care, University of Florence, Firenze, Italy

Abstract Background The present aim was to study the modulation of NK2 receptor internalization by two compounds, the spasmolytic otilonium bromide (OB) endowed with NK2 receptor antagonistic properties and the selective NK2 receptor antagonist ibodutant. Methods Full-thickness human colonic segments were incubated in the presence of OB (0.110 lmol L)1) or ibodutant (0.0010.1 lmol L)1), with or without the NK2 receptor selective agonist [bAla8]NKA(410) and then xed in 4% paraformaldehyde. Cryosections were processed for NK2 receptor immunohistochemical revelation. Quantitative analysis evaluated the number of the smooth muscle cells that had internalized the NK2 receptor. Key Results Immunohistochemistry revealed that in basal condition, the NK2 receptor was internalized in about 23% of total smooth muscle cells. The exposure to the selective NK2 receptor agonist induced internalization of the receptor in more than 77% of the cells. Previous exposure to both OB or ibodutant, either alone or in the presence of the agonist, concentration-dependently reduced the number of the cells with the internalized receptor. Conclusions & Inferences Both OB and ibodutant antagonize the internalization of the NK2 receptor in the human colon. As NK2 receptors are the

predominant receptor mediating spasmogenic activity of tachykinins on enteric smooth muscle, we hypothesize that the antagonistic activity found for both OB and ibodutant should play a specic therapeutic role in gut diseases characterized by hypermotility. Keywords human colon, ibodutant, immunohistochemistry, neurokinin-2 (NK2) receptor, otilonium bromide.

INTRODUCTION
Altered motility in the small bowel and colon may determine increased motility and spasm, visceral hypersensitivity, and abnormalities in the central pain processing which might explain the origin of abdominal pain, a pivotal sign of diseases such as irritable bowel syndrome (IBS). Tachykinins (TKs) have been implied to be involved in these diseases.1 In fact, they are widely distributed in the gastrointestinal tract where they act as excitatory neurotransmitters exerting, via neurokinin-1 (NK1), NK2, and NK3 receptor interaction, several functions.2 In particular, the activation of the NK2 receptor has been shown to affect intestinal motility, as well as secretion and visceral sensitivity.1 In the human intestine, the NK2 receptors have a main role in modulating motility, as demonstrated by in vitro3 and in vivo4 trials and, in the colon, they are localized in the longitudinal and circular smooth muscle, in a population of myenteric neurons and in nerve varicosities of myenteric ganglia as shown by both immunohistochemical5,6 and autoradiographic7 methods. Recent immunohistochemical studies on the human colon have shown that NK2 receptors internalize in smooth muscle cells following exposure to an agonist6,8 as usual for other G-protein coupled receptors.9,10

Address for Correspondence Prof. M. G. Vannucchi, Department of Anatomy, Histology and Forensic Medicine, University of Florence, V.le Pieraccini, 650139 Firenze, Italy. Tel: +39 055 4271389; fax: +39 055 4271385; e-mail: mariagiuliana.vannucchi@uni.it Received: 7 May 2010 Accepted for publication: 29 July 2010

96

2010 Blackwell Publishing Ltd

Volume 23, Number 1, January 2011

NK2 receptor internalization in human colon

Otilonium bromide (OB) is a prototype of a class of 2-aminoethyl-N-benzoylamino-benzoate quaternary salts endowed with spasmolytic activity for the intestinal smooth muscle mainly through its muscarinic and calcium antagonist properties.11 It is used for the treatment of intestinal hypermotility and IBS11 and is shown to be effective in reducing IBS symptoms in controlled clinical trials.12 Binding studies and functional experiments in vitro have shown that OB can bind to the tachykinin NK2 receptor and inhibit its activation.13 In view of the above, we have studied the potential modulation by OB on the NK2 receptor internalization in smooth muscle cells of the human colon induced by the selective NK2 agonist [bAla8]NKA(410)3. As reference compound we have used ibodutant, one of the most potent and selective nonpeptide NK2 receptor antagonists.14

MATERIALS AND METHODS Patients and specimens

Segments of human colon, approximately 10 cm in length, were taken from grossly normal margins of surgical resections from patients (nine males and six females, age range 3980 years) undergoing partial colectomy for adenocarcinoma. Most segments were taken from the descending colon (8) and some from transverse (3), sigmoid (3), and ascending (1) colon. Written informed consent was obtained from all patients. This study has been performed according to the declaration of Helsinki.

potentials in enteric neurons and prevent the potential-dependent release of neurotransmitters.10,15 To investigate the antagonist activity, some segments of gut were preincubated during this period in the presence of OB (0.110 lmol L)1) or the selective NK2 receptor antagonist ibodutant (0.0010.1 lmol L)1). This step was followed by incubation for 1 h at 4 C in Hanks balanced salt solution (HBSS) containing 3 lmol L)1 Nic, 1% bovine serum albumin (BSA), and a mixture of protease inhibitors (4 lg mL)1 chymostatin, 4 lg mL)1 leupeptin, and 40 lg mL)1 bacitracin) in the presence or in the absence (control) of 1 lmol L)1 of the NK2 receptor selective agonist [bAla8]NKA(410). Some segments of gut were incubated in the presence of either OB or the NK2 receptor antagonist ibodutant together with the NK2 receptor agonist. At 4 C, the agonist is able to diffuse into the tissue and binds to its receptor, but the cell membranes are solidied and so receptor endocytosis cannot occur.15,16 Specimens were then rinsed in ice-cold PSS-Nic-TTX and, to induce NK2 receptor internalization, were incubated in PSS-Nic-TTX solution for 10 min at 37 C. When provided, the antagonists (not agonists) were also included in the nal 10 min incubation in PSS-Nic-TTX at 37 C. Lastly, all specimens were rinsed in ice-cold 0.1 N sodium phosphate buffer (PB, pH 7.2) containing 3 lmol L)1 Nic and xed in 4% paraformaldehyde in PB overnight at 4 C. After xation, tissues were rinsed in PB and transferred for 24 h in 30% sucrose in PB at 4 C.

Immunohistochemistry
Transverse sections, 10 lm thick, were cut with a cryostat and collected on polylysine-coated slides. These samples were preincubated in 10% normal donkey serum (NDS) in phosphate buffered saline (PBS, pH 7.4) with 0.3% Triton X-100 for 1 h at room temperature to minimize non-specic binding. The antiserum against the human NK2 receptor (goat polyclonal antiserum, code sc-14121, to the C-terminal 21 amino acids of NK2 receptor of human origin) was used at a 1 : 200 dilution in PBS with 1% NDS and 0.1% Triton X-100, and was incubated overnight at 4 C. At the end of incubation, some sections were processed for light microscope revelation of the antibody. These sections were washed 2 5 min in PBS and then incubated with a biotinylated secondary antiserum (1 : 300 in NDS 1% in PBS + Triton X-100 0.1%) for 2 h at room temperature. The sections were then washed in PBS and incubated with the ABC solution for 30 min and, after being washed with PBS, they were incubated with 3,3diaminobenzidine (DAB). Finally, after washing with PBS, the sections were mounted in an aqueous medium and the immunoreactive products were observed under a Leitz light microscope (Leitz, Mannheim, Germany) and photographed. Other sections were processed for uorescence and confocal microscope examination. After overnight incubation in the primary antiserum, these slides were washed for 3 5 min in PBS, and then incubated for 90 min at room temperature in a donkey secondary antibody directed toward goat IgG-coupled to Alexa 488 (Invitrogen, San Diego, CA, USA) which was diluted at a concentration of 1 : 333. The sections were again washed for 3 2 min in PBS and mounted in an aqueous medium. The immunoreaction products were observed under an epiuorescence Zeiss Axioskop microscope (Zeiss, Mannheim, Germany) and under a Leica TCS SP5 confocal laser scanning microscope (Leica, Mannheim, Germany) equipped with a HeNe/Ar laser source, a Leica Plan Apo X63 oil immersion objective, and differential interference contrast (DIC) optics. The uorescent signal at the confocal microscope was obtained using a 488-nm excitation wavelength. Images of uorescent signal and DIC images 1024 1024 pixels) were taken simultaneously and successively

Tissue preparation
Immediately after resection, colonic segments were placed in icecold Ringer-lactate solution and quickly transported to the laboratory. No patient received radiotherapy or chemotherapy before intervention. All specimens appeared macroscopically normal, with no signs of tumor or inammation. The tissue was transferred into fresh oxygenated (95% O2 and 5% CO2) ice-cold physiological salt solution (PSS) of the following composition (lmol L)1): NaCl 118; NaHCO3 25; NaH2PO4 1.0; MgSO4 1.2; CaCl2 2.5; KCl 4.8; and glucose 11.1, and the serosal fat, mucosal layer, and tenia coli were removed, leaving the smooth muscle bands. Clean colonic segments were then cut in the direction of circular muscular cells into thinner pieces (35 mm wide and about 10 mm long) of muscular tissue. After this preparation, the colonic specimens were incubated in ice-cold (4 C) PSS, equilibrated with carbogen (95% O2 and 5% CO2) for 1 h. Each treatment was performed in three preparations taken from three different patients. Samples treated with the vehicle (control) or the NK2 receptor agonist [bAla8]NKA(410) were always included in each experimental session. Specimens were rinsed in warmed (37 C) PSS and, in order to relocate the NK2 receptor to the cell membrane,6 were incubated in PSS containing 3 lmol L)1 nicardipine (Nic) and 0.3 lmol L)1 tetrodotoxin (PSSNic-TTX), bubbled with carbogen, for 1 h at 37 C. Nicardipine was added to inhibit smooth muscle contraction, which might induce endogenous release of TKs from the intramural nervous system, and TTX was added to block propagation of action

2010 Blackwell Publishing Ltd

97

G. Cipriani et al.

Neurogastroenterology and Motility

merged by using Leica Application Suite software (Leica, Mannheim, Germany). Negative controls were performed omitting the primary antibody and all of them had no labeling.

Quantitative analysis
Quantitative analysis was performed on the sections processed for light microscope by a person blind on the source of the specimens. All the labeled cells present at 63 magnication in three different areas for each specimen for each treatment were considered (total patients for each experimental condition = 3). The number of counted cells for each experiment ranged from 1200 to 1600. These cells were divided into two groups according to the labeling distribution: the cells having the labeling distributed along the cell contour and the cells having deeply intracytoplasmatic labeling. The number and percentage of cells with the intracytoplasmatic labeling were evaluated toward all the labeled cells. The data were tted by sigmoidal non-linear regression (GRAPHPAD PRISM 4.02, Graphpad software, Inc., San Diego, CA, USA) to determine the antagonist concentration producing 50% inhibition (IC50) of the maximal response induced by 1 lmol L)1 [bAla8]NKA(410). Differences between multiple groups of value were examined by analysis of variance (ANOVA) and a Newman Keuls multiple comparison test. Differences between two groups were examined by Students t-test. A value of P < 0.05 was considered statistically signicant. Data were expressed as mean with standard error of the mean (SEM).

Vector, (Burlingame, CA, USA), donkey secondary antibody directed toward goat IgG-coupled to Alexa Fluor 488 (cod. A11055) was from Invitrogen (Milano, Italy). All salts used were of analytical grade and purchased from Merck (Darmstadt, Germany). Stock solutions of [bAla8]NKA(410) (10 mmol L)1) and ibodutant (10 mmol L)1) were prepared in DMSO; OB (100 mmol L)1) was dissolved in distilled water. All these solutions were stored at )30 C and then diluted in PSS or HBSS just before the experiments. The nal maximal concentration of DMSO was 0.2%.

RESULTS Immunohistochemistry
Both under light and uorescence microscopes, all smooth muscle cells of all specimens, either controls or treated ones, were labeled. Labeling was always intense and appeared as small brilliant green granules under the confocal uorescence microscope (Fig. 1A,B) and as brown large granular material under the light microscope (Figs 24). The labeling distribution, however, was different among the cells and this difference was particularly evident in relation to the various treatments (Figs 24). Fundamentally, the cells could be divided into two groups. The cells of one group had non-internalized NK2 receptor immunoreactive granules and the labeling was distributed along the cell contour (Figs 1A and 24). The cells of the second group had the NK2 receptor immunoreactive granules internalized in the cytoplasm (Figs 1B and 24). The number of cells with the internalized receptor varied according to treatment, being highest in those specimens treated with the NK2r agonist (Fig. 2B) and lowest in controls (Fig. 2A) and in those specimens treated with the two antagonists in the absence (Figs 3A,C,E and 4A,C,E; see also quantitative analysis) and in the presence (Figs 3B,D,F and 4B,D,F; see also quantitative analysis) of the agonist.

Chemicals
Otilonium bromide, ibodutant (MEN15596; 6-methylbenzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}ethylcarbamoyl)-cyclopentyl]-amide (batch L3/08) were synthesized at Lusochimica (Menarini Group, Lomagna, Italy), [bAla8]NKA(4 10) was from EspiKem (Firenze, Italy), Triton X-100, DAB, uoremount, nicardipine hydrochloride, leupeptin hemisulfate, chymostatin, bacitracin, BSA, paraformaldehyde and HBSS were from Sigma-Aldrich (Milano, Italy), TTX was from Alomone Labs (Jerusalem, Israel), NDS was from Jackson Immuno Research Europe (Suffolk, UK), goat polyclonal antiserum (NK-2R(C-21)) antibody (cod. sc-14121) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), biotynilated secondary antiserum (cod. BA-5000) and Vectastain Elite ABC kit (cod. pk-6100) were from

Figure 1 Neurokinin-2 (NK2) receptor labeling under confocal microscope. Immunoreactivity appears as small brilliant green granules. (A) Cells with a non-internalized NK2 receptor. The labeling is distributed along the cell contour. (B) Cells with internalized NK2 receptor particles. The immunoreactive granules are deeply located within the cytoplasm. In some cells, the nucleus is completely masked. (A, B) Bar = 10 lm.

98

2010 Blackwell Publishing Ltd

Volume 23, Number 1, January 2011

NK2 receptor internalization in human colon

Figure 2 Neurokinin-2 (NK2) receptor labeling under light microscope. Immunoreactivity appears as large granular material. (A) Control plus vehicle, many cells have peripheral labeling and some have central labeling. (B) Agonist, most of the cells have central labeling. (A, B) Bar = 15 lm.

OB

OB + NKA (1 mol L1)

OB (0.1 mol L1)

OB (1.0 mol L1)

Figure 3 Neurokinin-2 (NK2) receptor labeling under light microscope. Treatment with Otilonium bromide (OB) alone (A, C, E) or with otilonium bromide plus the agonist (B, D, F) at 0.1, 1, and 10 lmol L)1, respectively. Immunoreactivity appears as large granular material; the cells without internalized receptor have peripheral labeling and those with internalized receptors have central labeling. (AF) Bar = 20 lm.

OB (10 mol L1)

Quantitative analysis
In preliminary experiments, the values obtained in unstimulated preparations with the vehicle of OB (0.1% water) and ibodutant (0.1% DMSO) did not signicantly differ among them (25.42 0.54 with H2O 0.1%; 24.69 1.33 with DMSO 0.1%; 21.78 1.97 with DMSO 0.2%). The percentage of cells with internalized receptor relative to all NK2 receptor labeled cells (Fig. 5A,B and Table 1) was signicantly higher in the specimens treated with the agonist (77.06 3.01) as compared with controls (23.59 1.22) and all other treatments (P < 0.001). The exposure of specimens to

lower concentrations of OB (0.11 lmol L)1) or ibodutant (0.0010.01 lmol L)1) did not affect by themselves, the basal values but both compounds at the higher concentrations induced a signicant decrease of the percentage of cells with the internalized receptor as compared with controls (12.32 0.49 with OB at 10 lmol L)1, 17.83 0.66 and 11.54 1.05 with ibodutant at 0.03 and 0.1 lmol L)1, respectively, vs 23.59 1.22 of controls). In the specimens treated with the antagonist plus agonist, the percentage of cells with the internalized receptor was concentration dependently decreased (from 52.28 3.71 to 22.98 0.82 with 0.1 and 10 lmol L)1 of OB and from 55.95 2.84 to 14.90 0.93 with

2010 Blackwell Publishing Ltd

99

G. Cipriani et al.

Neurogastroenterology and Motility

IBO

IBO + NKA (1 mol L1)

IBO (0.001 mol L1)

IBO (0.01 mol L1)

IBO (0.1 mol L1)

Figure 4 Neurokinin-2 (NK2) receptor labeling under light microscope. Treatment with ibodutant (IBO) alone (A, C, E) and with ibodutant plus the agonist (B, D, F) at 0.001, 0.01, and 0.1 lmol L)1, respectively. Immunoreactivity appears as large granular material; the cells without internalized receptor have peripheral labeling and those with internalized receptors have central labeling. (AF) Bar = 20 lm.

A
Percentage

90 80 70

B
Percentage

90 80 70

60 50 40 30 20 10 0

* * * * *

60 50 40 30 20 10 0

* * * *

Figure 5 Quantitative analysis of the percentage of neurokinin-2 (NK2) receptorimmunoreactive smooth muscle cells that have internalized the receptor in control conditions, in the presence of the agonist [bala8]NKA-(410) alone, and in the presence of the two antagonists (A) otilonium bromide (OB), (B) and ibodutant (Ibo), at different concentrations, either alone or in the presence of the agonist. *Signicantly different vs control. Signicantly different vs the agonist alone.

0.001 and 0.1 lmol L)1 of ibodutant), being signicantly lower (P < 0.001) vs the specimens treated with the agonist (Fig. 5A,B and Table 1). The calculated IC50 was 0.59 lmol L)1 (0.40.97, 95% CL) and 8.95 nmol L)1 (6.811.7, 95% CL) for OB and ibodutant, respectively.

DISCUSSION AND CONCLUSIONS

Our study shows that the spasmolytic OB and the selective NK2 receptor antagonist ibodutant are able to 100

C od o Ib uta A ntr od n g ol ut t 0 on Ib an .00 ist od t 0 1 Ib uta .00 m od n 3 t u 0 A go Ib tan .01 m ni od t 0 A st ut .0 m go + a 3 n I nt A ist bo 0.1 m go + 0 .0 A nis Ibo 01 m go t 0. m + n I 0 A ist bo 03 go + 0 m ni Ibo .01 st + 0.0 m Ib 3 o m 0. 1 m

A tro O gon l B is O 0.1 t B 0. m 3 O m B A 1 go O B m n A ist O 3 go + B ni O 10 m A st B 0 m go + O .1 A nis B m go t 0. + 3 n A go ist OB m ni + 1 st O m + B3 O B m 10 m

on

Ib

inhibit, in a concentration-dependent manner, the translocation of the NK2 receptor from the plasma membrane to the cytoplasm of the smooth muscle cells of human colon induced by the NK2 agonist [bAla8]NKA(410). It should be noted that both compounds at the highest concentrations signicantly reduce the NK2 receptor internalization also in the absence of the agonist. Neurokinin-2 receptors belong to the G-coupled receptor family which, when exposed to the agonist, translocate in the cytoplasm as a consequence of their

2010 Blackwell Publishing Ltd

Volume 23, Number 1, January 2011

NK2 receptor internalization in human colon

Table 1 Experimental procedure (see Materials and Methods section for abbreviations) and percentages of neurokinin (NK)2 receptor immunoreactive smooth muscle cells that have internalized the receptor in regards to the several concentrations of ibodutant and otilonium bromide (OB) in presence or absence of NK2 receptor agonist PSS/Nic/TTX (37 C, 60 min) No drug (DMSO) No drug (DMSO) OB 0.1 lmol L)1 OB 0.3 lmol L)1 OB 1 lmol L)1 OB 3 lmol L)1 OB 10 lmol L)1 OB 0.1 lmol L)1 OB 0.3 lmol L)1 OB 1 lmol L)1 OB 3 lmol L)1 OB 10 lmol L)1 Ibodutant 0.001 lmol L)1 Ibodutant 0.003 lmol L)1 Ibodutant 0.01 lmol L)1 Ibodutant 0.03 lmol L)1 Ibodutant 0.1 lmol L)1 Ibodutant 0.001 lmol L)1 Ibodutant 0.003 lmol L)1 Ibodutant 0.01 lmol L)1 Ibodutant 0.03 lmol L)1 Ibodutant 0.1 lmol L)1 PSS/Nic/TTX (37 C, 10 min) No drug (DMSO) No drug (DMSO) OB 0.1 lmol L)1 OB 0.3 lmol L)1 OB 1 lmol L)1 OB 3 lmol L)1 OB 10 lmol L)1 OB 0.1 lmol L)1 OB 0.3 lmol L)1 OB 1 lmol L)1 OB 3 lmol L)1 OB 10 lmol L)1 Ibodutant 0.001 lmol L)1 Ibodutant 0.003 lmol L)1 Ibodutant 0.01 lmol L)1 Ibodutant 0.03 lmol L)1 Ibodutant 0.1 lmol L)1 Ibodutant 0.001 lmol L)1 Ibodutant 0.003 lmol L)1 Ibodutant 0.01 lmol L)1 Ibodutant 0.03 lmol L)1 Ibodutant 0.1 lmol L)1 % intracellular labeling (mean SE) 23.59 77.06 25.59 24.36 24.24 18.02 12.32 52.28 39.28 35.15 28.67 22.98 24.71 24.50 24.87 17.83 11.54 55.95 49.51 43.03 23.78 14.90 1.22 3.01* 1.14 0.70 0.74 0.64 0.49* 3.71 2.29 1.19 0.94 0.82 1.40 1.22 1.16 0.66* 1.05* 2.84 0.97 1.83 1.16 0.93

HBSS/Nic/BSA/PI (4 C, 60 min) No drug (DMSO) [b-Ala8]NKA(410) 1 lmol OB 0.1 lmol L)1 OB 0.3 lmol L)1 OB 1 lmol L)1 OB 3 lmol L)1 OB 10 lmol L)1 [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol Ibodutant 0.001 lmol L)1 Ibodutant 0.003 lmol L)1 Ibodutant 0.01 lmol L)1 Ibodutant 0.03 lmol L)1 Ibodutant 0.1 lmol L)1 [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol [b-Ala8]NKA(410) 1 lmol

L)1

L)1 L)1 L)1 L)1 L)1

plus plus plus plus plus

OB OB OB OB OB

0.1 lmol L)1 0.3 lmol L)1 1 lmol L)1 3 lmol L)1 10 lmol L)1

L)1 L)1 L)1 L)1 L)1

plus Ibodutant 0.001 lmol L)1 plus Ibodutant 0.003 lmol L)1 I plus Ibodutant 0.01 lmol L)1 plus Ibodutant 0.03 lmol L)1 plus Ibodutant 0.1 lmol L)1

*Signicantly different vs control. Signicantly different vs agonist alone.

activation.9,10 In this study, quantitative analysis indicates that, in the absence of the agonist, almost one-fourth of the smooth muscle cells have the NK2 receptor internalized into the cytoplasm. These results could be interpreted as a sign of endogenous receptor stimulation. In other words, in spite of the presence of TTX and the L-type calcium channel blocker Nic, a tonic release of NKA persists. Indeed, the exposure to both the antagonists at their highest doses was able to affect this tonic release reducing signicantly the number of the smooth muscle cells showing the receptor internalized. In this regard, the existence of a tonic release of TKs that contributes to the resting tone maintenance of the rats isolated small intestine has been observed17 as well the TTX and calcium antagonist resistant release of neurotramsmitters.3,18 Finally, it should be considered the possibility that the presence of the NK2 receptor in the cytoplasm might represent a sign of either recycling or new synthesis of the receptor.10 Preincubation of human colon specimens in the presence of NK2 agonist [bAla8]NKA(410) induced a marked increase of cells with NK2 immunoreactivity in the cytoplasm and up to 77% of these cells showed receptor internalization. Addition of OB or ibodutant reduced in a concentration-dependent manner the NK2 receptor internalization down to the basal values or less.

Otilonium bromide is a quaternary ammonium derivative widely used for IBS,11,12 a disease characterized by altered bowel motility and increased visceral sensitivity,19 with a composite mechanism of action, in particular, a mixed antimuscarinic and calcium channel blocker action of OB has been proposed on the basis of the results of functional20,21 and radioligand binding data.22 A distribution study of OB orally administered to rats at a dose close to that used in humans for the IBS treatment, showed that the drug is not systemically absorbed,23 but it is locally taken up by the large intestine wall reaching concentrations in the same range of those known to exert spasmolytic activity in in vitro studies.11,13 The effect on intestinal motility of OB is well established;11 e.g. repeated administration of OB was shown to restore the postprandial motility pattern impaired in IBS patients following a meal with xed values of calories.24 Recently, in in vitro experiments it has been shown that OB is able to inhibit the NK2 receptor-mediated contraction of human colon.25 Furthermore, OB produced a concentration-dependent displacement of [125I]NKA binding in CHO cells transfected to express the human NK2 receptor with a Ki of 7.2 lmol L)1.13 The present ndings conrm that OB acts also as NK2 receptor antagonist in the human colon ex vivo and its maximal efcacy is reached in the same range of concentrations similar to those used in previous

2010 Blackwell Publishing Ltd

101

G. Cipriani et al.

Neurogastroenterology and Motility

in vitro studies.20,21,25 In fact, although OB spasmolytic activity relies on diverse mechanisms of action targeting a variety of ionic channels, such as L- and T-type calcium channels,21,26 and receptors such as muscarinic and NK2 receptors,11,13 in our experimental model, the OB effect is due to the interaction with NK2 receptors as we used the selective NK2 receptor agonist [bAla8]NKA(410)3 and the involvement of Ltype calcium channels can also be excluded due to the use of the blocker Nic. Several NK2 receptor antagonists have been developed such as the cyclic peptide nepadutant and the non-peptide saredutant that were characterized in preclinical and clinical studies.2730 A clinical trial in volunteers4 conrmed that the NK2 receptor antagonist nepadutant was able to inhibit the stimulatory effects of NKA on gut motility and the NKA-induced changes of the fasting migrating motor complex. The NK2 receptor antagonist nepadutant effectively blocked the motility-stimulating effects of NKA and reduced the gastrointestinal IBS-like symptoms (e.g. borborygmi, abdominal pain, nausea, and vomiting) induced by NKA. Among the non-peptide NK2 antagonists, ibodutant14 possesses an afnity for the human NK2 receptor expressed in transfected CHO cells (Ki = 0.014 nmol L)1) which is greater than that of

other previously developed antagonists (nepadutant with a Ki of 2.8 nmol L)1 and the saredutant with a Ki of 0.16 nmol L)1).27,28 Our ndings show that ibodutant concentration-dependently antagonized the internalization of the NK2 receptor in smooth muscle cells of human colon and its efcacy in preventing internalization of the receptor by the agonist ranges between values similar to those reported in vitro.14 These data together with other ibodutant characteristics, such as long duration of action, remarkable oral bioavailability and excellent tolerability in animals and in healthy humans, indicate it as a potential new drug for the oral therapy of IBS.29,30 In conclusion, this study demonstrates that both OB and ibodutant antagonize the internalization of the NK2 receptor in the human colon. As NK2 receptors play a major role in mediating the spasmogenic activity of TKs on intestinal smooth muscle cells, we hypothesize that these two compounds could be useful in the therapy of the intestinal disorders characterized by hypermotility.

ACKNOWLEDGMENT
This study was supported by a grant from the Menarini Group.

REFERENCES
1 Lecci A, Capriati A, Altamura M, Maggi CA. Tachykinins and tachykinin receptors in the gut, with special reference to NK2 receptors in human. Auton Neurosci 2006; 126-127: 232 49. 2 Shimizu Y, Matsuyama H, Shiina T, Takewaki T, Furness JB. Tachykinin and their functions in the gastrointestinal tract. Cell Mol Life Sci 2008; 65: 295311. 3 Santicioli P, Giuliani S, Patacchini R, Tramontana M, Criscuoli M, Maggi CA. MEN 11420, a potent and selective tachykinin NK2 receptor antagonist in the guinea-pig and human colon. Naunyn-Schmied Arch Pharmacol 1997; 356: 67888. 4 Lordal M, Navalesi G, Theodorsson E, Maggi CA, Hellstro m PM. A novel tachykinin NK2 receptor antagonist prevents motility-stimulating effects of neurokinin A in small intestine. Br J Pharmacol 2001; 134: 21523. 5 Renzi D, Pellegrini B, Tonelli F, ` A. Substance P Surrenti C, Calabro

10

(neurokinin-1) and neurokinin A (neurokinin-2) receptor gene and protein expression in the healthy and inamed human intestine. Am J Pathol 2000; 157: 151122. Jaafari N, Khomitch-Baud A, Christen Y. Distribution pattern of MO, Jule tachykinin NK2 receptors in human colon: involvement in the regulation of intestinal motility. J Comp Neurol 2007; 503: 38191. Warner FJ, Liu L, Lubowski DZ, Burcher E. Circular muscle contraction, messenger signalling and localization of binding sites for neurokinin A in human sigmoid colon. Clin Exp Pharmacol Physiol 2000; 27: 92833. Jaafari N, Khomitch-Baud A, Gilhodes Y. Qualitative and JC, Hua G, Jule quantitative analysis of tachykinin NK2 receptors in chemically dened human colonic neuronal pathways. J Comp Neurol 2008; 507: 154258. Koenig JA, Edwardson JM. Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci 1997; 18: 27687. Southwell BR, Seybold VS, Woodman HL, Jenkinson KM, Furness JB.

11

12

13

14

15

Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons. Neuroscience 1998; 87: 92531. Evangelista S. Quaternary ammonium derivatives as spasmolytics for irritable bowel syndrome. Curr Pharm Des 2004; 10: 35618. Lesbros-Pantoickova D, Michetti P, Fried M, Beglinger C, Blum AL. Metaanalysis: the treatment of irritable bowel syndrome. Aliment Pharmacol Ther 2004; 20: 125369. Santicioli P, Zagorodnyuk V, Renzetti AR, Maggi CA. Antimuscarinic, calcium channel blocker and tachykinin NK2 receptor antagonist actions of otilonium bromide in the circular muscle of guinea-pig colon. NaunynSchmied Arch Pharmacol 1999; 359: 4207. Cialdai C, Tramontana M, Patacchini R et al. MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist. Eur J Pharmacol 2006; 549: 1408. Mann PT, Southwell BR, Furness JB. Internalization of the neurokinin 1 rceptor in rat myenteric neurons. Neuroscience 1999; 91: 35362.

102

2010 Blackwell Publishing Ltd

Volume 23, Number 1, January 2011

NK2 receptor internalization in human colon

16 Harrington AM, Hutson JM, Southwell BR. Immunohistochemical localization of substance P NK1 receptor in guinea pig distal colon. Neurogastroenterol Motil 2005; 17: 72737. 17 Maggi CA, Giuliani S. Evidence that tachykinin NK2 rceptors modulate resting tone in the rat isolated small intestine. Br J Pharmacol 1996; 118: 12628. 18 Zagorodnyuk V, Gregory S, Costa M, Tramontana M, Giuliani S, Maggi CA. Spontaneous release of acetylcholine from autonomic nerves in the bladder. Br J Pharmacol 2009; 157: 60719. 19 Kanazawa M, Palsson OS, Thiwan SI et al. Contributions of pain sensitivity and colonic motility to IBS symptom severity and predominant bowel habits. Am J Gastroenterol 2008; 103: 255061. 20 Giachetti A. Pharmacological studies on otilonium bromide. Ital J Gastroenterol 1991; 23(Suppl. 1): 569. 21 Strege PR, Evangelista S, Lyford GL, Sarr MG, Farrugia G. Otilonium bromide inhibits calcium entry through L-type calcium channels in human

22

23

24

25

26

intestinal smooth muscle. Neurogastroenterol Motil 2004; 16: 16774. Evangelista S, Giachetti A, Chapelain B, Neliat G, Maggi CA. Receptor binding prole of otilonium bromide. Pharmacol Res 1998; 38: 1117. Evangelista S, Cochet P, Bromet N, Criscuoli M, Maggi CA. A distribution study with 14C-Otilonium bromide in the rat: evidence for selective tropism for the large intestine after oral administration. Drug Metab Dispos 2000; 28: 6437. Narducci F, Bassotti G, Granata MT, Pelli MA, Palumbo R, Morelli A. Colonic motility and gastric emptying in patients with the irritable colon syndrome. Effect of pretreatment with octylonium bromide. Dig Dis Sci 1986; 31: 2416. M, Aleu J et al. Effect Gallego D, Aul of otilonium bromide on contractile patterns in the human sigmoid colon. Neurogastroenterol Motil 2010; 22: e18091. Strege PR, Sha L, Beyden A et al. T-type Ca2+ channel modulation by otilonium bromide. Am J Physiol Gastrointest Liver Physiol 2010; 298: G70613.

27 Renzetti AR, Catalioto RM, Carloni C et al. Effects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells. Biochem Pharmacol 1999; 57: 899906. 28 Meini S, Bellucci F, Catalani C et al. Multifaceted approach to determine the antagonist molecular mechanism and interaction of ibodutant ([1-(2phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]amide) at the human tachykinin NK2 receptor. J Pharmacol Exp Ther 2009; 329: 48695. 29 Lecci A, Maggi CA. Pheripheral tachykinin receptors as potential therapeutic targets in visceral disease. Expert Opin Ther Targets 2003; 7: 34362. 30 Quartara L, Altamura M, Evangelista S, Maggi CA. Tachykinin receptor antagonists in clinical trials. Expert Opin Investig Drugs 2009; 18: 1843 64.

2010 Blackwell Publishing Ltd

e10