Demonstration of Functional Neuronal β3-Adrenoceptors Within the Enteric Nervous System

GASTROENTEROLOGY 2007;133:175–183

Demonstration of Functional Neuronal ␤3-Adrenoceptors Within the Enteric Nervous System SELIM CELLEK,* RAMKUMAR THANGIAH,‡ ANNA K. BASSIL,* COLIN A. CAMPBELL,* KAREN M. GRAY,* JENNIFER L. STRETTON,* OLUTUNDE LALUDE,‡ SHANMUGAM VIVEKANANDAN,‡ ALAN WHEELDON,* WENDY J. WINCHESTER,* GARETH J. SANGER,* MICHAEL SCHEMANN,§ and KEVIN LEE*

Background & Aims: Although the ␤3-adrenoceptor (AR) has been suggested to be involved in regulation of gut motility and visceral algesia, the precise mechanisms have been unknown. ␤3-AR has been postulated to have a nonneuronal expression, being initially characterized in adipocytes and subsequently in the smooth muscle. We aimed to investigate the expression of ␤3-AR in human enteric nervous system and its role in motility and visceral algesia. Methods: The expression of ␤3-AR in human colon myenteric and submucosal plexus was investigated using immunohistochemistry. The effects of a ␤3-AR agonist on nerve-evoked and carbachol-induced contractions as well as somatostatin release were investigated in strips of human colon. The effect of an agonist on diarrhea and visceral pain was investigated in vivo in rat models. Results: ␤3-AR is expressed in cholinergic neurons in the myenteric plexus and submucosal plexus of human colon. Activation of ␤3-AR causes the release of somatostatin from human isolated colon. In a rat model of visceral pain, ␤3-AR agonist elicits somatostatin-dependent visceral analgesia. ␤3-AR agonists inhibit cholinergically mediated muscle contraction of the human colon, as well as chemically induced diarrhea in vivo in a rat model. Conclusions: This is the first demonstration of expression of ␤3-AR in the enteric nervous system. Activation of these receptors results in inhibition of cholinergic contractions and enhanced release of somatostatin, which may lead to visceral analgesia and inhibition of diarrhea. Therefore, ␤3-AR could be a novel therapeutic target for functional gastrointestinal disorders.

␤

(␤3-AR) is a member of the larger family of G-protein-coupled AR. Initially, ␤-ARs were classified into ␤1 and ␤2 subtypes; however, later, as more selective ␤1- and ␤2-AR antagonists became available, an “atypical” ␤-AR has been recognized. This led to cloning of human, mouse, and rat ␤3-AR. ␤3-AR has been shown to be expressed in adipocytes, heart, skeletal muscle, and smooth muscle of the gastrointestinal and urogenital systems.1–5 3-adrenoceptor

Agonists of ␤3-AR have been shown to reduce the elevated tone and inhibit spontaneous contractions in the human isolated colon, but the exact site of action is not clear.6 –9 In addition, activation of ␤-AR via ligands not selective for any of the receptor subtypes is known to elicit the release of somatostatin from the gastrointestinal tract into the systemic circulation.10 Previously, it has been suggested that ␤3-AR might have a role in this release of somatostatin.11 Furthermore, somatostatin analogues are known to inhibit visceral pain.12 Therefore, we hypothesized that activation of ␤3-AR might simultaneously relax human colon smooth muscle and facilitate somatostatin release, which might be a novel target for functional gastrointestinal disorders in which motility disturbance and pain are key features. During our experiments, we discovered that ␤3-AR is expressed not only in the smooth muscle but also in the enteric neurones. Their activation in the submucosal plexus could lead to release of somatostatin, which causes visceral analgesia and inhibits secretomotor nerve activity. Activation of ␤3-AR in the myenteric plexus could inhibit excitatory musculomotor nerve activity, hence cholinergic contractions.

Materials and Methods In Vitro Pharmacology With Human Colon Segments of colon (transverse or sigmoid) were obtained from patients undergoing surgery for colorectal cancer. The study was approved by the local ethics committee, and written informed consent was obtained from patients. The segments were transferred from the hospital to the research laboratories within 2 hours after resection in icecold Kreb’s solution (containing in mmol/L; NaCl 121.5, CaCl2 2.5, KH2PO4 1.2, KCl 4.7, MgSO4 1.2, NaHCO3 25, glucose 5.6) equilibrated with 5% CO2 and 95% O2. Circular muscle strips without mucosa (4 ⫻ 15 mm) obtained from intertaenial segments were mounted in tissue baths (10 mL), and the change in tension was recorded using isometAbbreviations used in this paper: ␤3-AR, ␤3-adrenoceptor; CCh, carbachol; ChAT, choline acetyltransferase. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.05.009

BASIC– ALIMENTARY TRACT

*Neurology and Gastrointestinal Centre of Excellence in Drug Discovery, GlaxoSmithKline, Harlow, United Kingdom; ‡Department of Colorectal Surgery, Princess Alexandra Hospital, Harlow, United Kingdom; and §Human Biology, TU Munchen, Munich, Germany

176

CELLEK ET AL

GASTROENTEROLOGY Vol. 133, No. 1

Table 1. The Details of Primary and Secondary Antibodies Used in the Experiments Primary antibody

Dilution

Secondary antibody

Source

Dilution

Santa Cruz (catalog No: SC1472) Prof. M. Schemann (Munich, Germany) Molecular Probes (catalog No. A21271) Sigma (catalog No. N0142)

1:200

Anti-goat FITC or TRITC Anti-rabbit FITC or TRITC Anti-mouse FITC or TRITC or Cy5

1:100

1:1000

Anti-mouse FITC or TRITC or Cy5

Mouse PGP9.5

Serotec (catalog No. MCA2084)

1:1000

Anti-mouse FITC or TRITC or Cy5

Rabbit somatostatin

Santa Cruz (catalog No. SC13099)

1:500

Anti-rabbit FITC or TRITC

Chemicon (catalog No: AP180F or AP180R) Chemicon (catalog No: AP182F or AP182R) Chemicon (catalog No: AP181F or AP181R or AP 192S) Chemicon (catalog No. AP181F or AP181R or AP192S) Chemicon (catalog No. AP181F or AP181R or AP192S) Chemicon (catalog No. AP182F and AP182R)

Santa Cruz (catalog No. SC1472)

1:500

Anti-goat AMCA or Cy3

Prof. M. Schemann (Munich, Germany) Peninsula Laboratories (catalog No. RIN8001) Molecular Probes (catalog No. A21271)

1:500

Anti-rabbit Cy3

1:1000

Anti-rabbit AMCA

1:50

Anti-mouse Streptavidin Cy2

Frozen sections Goat ␤-3 adrenoceptor Rabbit choline acetyltransferase Mouse HuC/D

Mouse neurofilament-200

Whole mount Goat ␤-3 adrenoceptor

BASIC– ALIMENTARY TRACT

Rabbit choline acetyltransferase Rabbit somatostatin

Mouse HuC/D (biotinylated)

Source

1:1000 1:1000

1:100 1:100

1:100

1:100

1:100

Dianova (catalog No. 705155147 or 05165147) Dianova (catalog No. 711165152) Dianova (catalog No. 711155152)

1:50 for AMCA 1:500 for Cy3

Dianova (catalog No. 016220084)

1:200

1:500 1:50

FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate.

ric force transducers. The stimulation parameters were 50 V (⬃200 mA), 5 Hz, 0.5-ms bipolar pulse duration, for 10 seconds, every 1 minute. Single concentrations of ␤3-AR agonist GW427353 (0.1–10 ␮mol/L)13 were tested on electrical field stimulation-induced cholinergic contractions in the presence of neurokinin receptor-1, ⫺2, and ⫺3 antagonists (L732138, 1 ␮mol/L; MDL-29913, 1 ␮mol/L; and SB-235375, 0.1 ␮mol/L, respectively) and indomethacin (3 ␮mol/L). Some of the strips were pretreated with ␤3-AR antagonist SR-59230A (1 ␮mol/L).

Somatostatin Release From Human Isolated Colon Five ⫻ 5-mm pieces of human colon with intact mucosa were placed into test tubes containing 500 ␮L preoxygenated Kreb’s solution with 10 ␮mol/L bestatin, 1 ␮mol/L phosphoramidon, 10 ␮mol/L amastatin, and 0.025 mg/mL bovine serum albumin and were equilibrated at 37°C for 15 minutes in the presence of either vehicle (dimethyl sulfoxide [DMSO] at a final concentration of 0.01%), SR-59230A (1 ␮mol/L), or tetrodotoxin (1 ␮mol/L). They were then treated with either vehicle (DMSO at a final concentration of 0.05%) or GW427353 (0.1, 1, and 10 ␮mol/L) and further incubated at 37°C for 45 minutes. At the end of the incubation, the tubes were vortex mixed and centrifuged at 3000g for 10 minutes at room temperature,

and the supernatant was recovered and frozen at ⫺20°C for enzyme-linked immunosorbent assay (ELISA). The tissue pieces at the bottom of each tube were blot dried and weighed. Somatostatin in the supernatant was measured using a commercially available ELISA kit according to manufacturer’s instructions (Bachem, St. Helens, United Kingdom). The pH of the buffer was confirmed to be within physiologic levels (pH 7.4 ⫾ 0.1) throughout the experiment using a pH meter.

Immunofluorescence Frozen sections. Sections of human colon with intact mucosa were incubated in 4% paraformaldehyde in 0.1 mol/L phosphate buffer for 72 hours at room temperature and then in 30% sucrose in 0.1 mol/L phosphate buffer for 24 hours at 4°C. Five ⫻ 5 ⫻ 5-mm blocks were cut with a scalpel and frozen in OTC compound on dry ice. Twenty-micrometer sections were cut using a cryostat and dried on coated slides for 2 hours at room temperature. The sections were then incubated with 5% donkey serum and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 2 hours, followed by primary (overnight at 4°C) and secondary antibodies (2 hours at room temperature) as specified in Table 1. The neurons were identified using a combination of neuronal markers: HuC/D, PGP9.5, and neurofilament

␤-3 ADRENOCEPTORS IN ENTERIC NERVOUS SYSTEM 177

BASIC– ALIMENTARY TRACT

July 2007

Figure 1. Expression of ␤3-adrenoceptor in the neurons of the human colonic myenteric plexus. (A) ␤3-adrenoceptor (green) was expressed in 7 neurons of a ganglion in the myenteric plexus (indicated with small arrowheads) and nerve fibers (indicated with large arrowhead). Neuronal phenotype was confirmed using a combination of 3 nerve markers (HuC/D, PGP9.5, and neurofilament 200; red). (B) In some of the ganglia, a ␤3-adrenoceptor-positive neuron (indicated with an arrowhead) was observed next to ␤3-adrenoceptor-negative neurons. Scale bars, 40 ␮m.

200. The images were obtained using a laser-scanning confocal microscope (Leica, Wetzlar, Germany). Whole mount preparations. Human colon was fixed overnight at room temperature in 0.1 mol/L phosphate buffer containing 4% paraformaldehyde and 0.2% picric acid and then washed (3 ⫻ 10 minutes) in phosphate buffer. Whole mount preparations containing the inner submucosal or the myenteric plexus were first incubated in PBS (1 hour, room temperature; buffer changed every 10 minutes) followed by incubation (1 hour, room temperature) in PBS containing 0.3% Triton X-100 and normal goat serum (3%) to block nonspecific binding. The tissues were then incubated with primary (40 hours at room temperature) and secondary antibodies (3.5 hours at room temperature) as specified in Table 1. The fluorescence was detected using an Olympus microscope (BX61 WI; Olympus, Center Valley, PA) equipped with a SIS Fview II CCD camera and analySIS 3.1 software (Soft Imaging System GmbH, Münster, Germany). The quantification of neurones was performed by counting the number of neurones in at least 10 ganglia obtained from each patient. The numbers were then pooled for each patient and expressed as percentage of total cells counted in that patient.

Castor Oil Model Based on previous reports,13 male CD rats (200 – 250 g) were given orally either rat selective ␤3-AR agonist CL-316243 (0.03–1 mg/kg), vehicle, or loperamide (10 mg/kg). One hour later, each rat received an oral dose of castor oil (1 mL/kg body weight). Fecal matter was collected and weighed (wet weight) at 3 hours and 6 hours after castor oil administration.

Mustard Oil Model Based on a previous report,14 male CD rats (150 – 200 g) were given orally either CL-316243 (0.03–1 mg/kg) or vehicle, with or without CYN-154806 (10 mg/kg, subcutaneously [sc]). One hour later, 0.2 mL 3% mustard oil was administered intrarectally. Animals were then returned to the viewing chambers for a period of 25 minutes, and the number of visceral pain-related behaviors (abdominal arches, writhing, stretching, abdominal contractions) was recorded.

Chemicals GW427353 was synthesized in-house according to a recently published report.15 L732138, MDL-29913,

178

CELLEK ET AL

GASTROENTEROLOGY Vol. 133, No. 1

CYN-154806, and tetrodotoxin were from Tocris, Bristol, United Kingdom. All other chemicals were from Sigma, Dorset, United Kingdom.

Statistics The results were analyzed using statistical analysis software (Statistica; StatSoft Inc, Tulsa, OK). The results were expressed as mean ⫾ SEM. In in vivo experiments, the group means were compared using 1-way ANOVA followed by Fisher LSD test. In in vitro experiments, 2-tailed Student t test was used. P ⬍ .05 was considered statistically significant. n denotes the number of human donors or animals. The negative logarithm of EC50, pEC50, values and concentration response curves for somatostatin release experiments were calculated using Statistica Software (V6.1, StatSoft Inc). Median effective concentration (EC50) values in human colon electrical field stimulation experiments were calculated using GraphPad Prism (V4 GraphPad Software Inc, San Diego, CA). BASIC– ALIMENTARY TRACT

Results Immunofluorescence Myenteric plexus. In frozen sections, ␤3-AR immunoreactivity was observed in some of the neurons and nerve fibers in the ganglia of the myenteric plexus (Figure 1A–C). In some ganglia, ␤3-AR-positive neurons could be visualized next to ␤3-AR-negative neurons (Figure 1D–F). ␤3-AR immunoreactivity was observed mostly in choline acetyltransferase (ChAT)-positive neurons (Figure 2A–C). When the primary antibody to ␤3-AR was omitted, no immunofluorescence was observed (Figure 2D–F). The quantitative analysis of ␤3-AR distribution was performed using whole mount preparations (Figure 3A–D): ␤3-AR was observed in 2.6% ⫾ 0.6% of HuC/D (panneuronal marker)-positive neurons in the myenteric plexus (n ⫽ 9 patients, 115 ganglia, 5595 neurons). 40.6% ⫾ 5.7% of HuC/D-positive neurons were ChAT positive. 98.5% ⫾ 1.5% of ␤3-AR-positive neurons were ChAT positive. Only 1.0% ⫾ 0.2% of HuC/D-positive neurons were also positive for somatostatin. None of the somatostatinpositive neurons expressed ␤3-AR (n ⫽ 3 patients, 32 ganglia, 405 neurons). Submucosal plexus. In frozen sections, ␤3-AR immunoreactivity was observed in neurons in close proximity to mucosa. The number of neurons positive for ␤3-AR was generally higher in the submucosal plexus than myenteric plexus. Similar to myenteric plexus, ␤3AR-positive and -negative neurons could be observed in the same ganglion (Figure 4A–C). Most of the ␤3-ARpositive neurons were ChAT positive (Figure 4D–F). Very few neurons containing somatostatin were positive for ␤3-AR (Figure 5). Quantitative analysis of whole mount preparations (Figure 3E–H) revealed that 72.3% ⫾ 3.1% of HuC/D-positive neurons were also positive for ␤3-AR (n

Figure 2. ␤3-adrenoceptor expression was colocalized with choline acetyl transferase (ChAT) in a majority of the neurons in human colonic myenteric plexus. (A) In most of the ganglia, ␤3-adrenoceptor (green) and ChAT (red) were colocalized in the same neurons. (B) When the primary antibody for ␤3-adrenoceptor was omitted, no immunostaining was observed. Neuronal phenotype was confirmed using a combination of 3 nerve markers (Hu C/D, PGP9.5, and neurofilament 200; blue). Scale bars, 40 ␮m.

⫽ 11 patients, 151 ganglia, 1048 neurons). 80.0% ⫾ 11.1% of HuC/D-positive neurons were ChAT positive. 85.3% ⫾ 7.4% of ␤3-AR-positive neurons were ChAT positive (n ⫽ 3 patients, 32 ganglia, 233 neurons). 13.8% ⫾ 5.9% of HuC/D-positive neurons were also positive for somatostatin. Less than 2% of somatostatin-containing neurons were ␤3-AR positive (n ⫽ 3 patients, 35 ganglia, 256 neurons).

Somatostatin Release To investigate the effect of ␤3-AR activation on somatostatin release, somatostatin accumulation in the supernatant of human isolated colon specimens during exposure to a ␤3-AR agonist was measured. ␤3-AR agonist GW427353 (0.1–10 ␮mol/L)15 caused a concentrationdependent increase in the concentrations of somatostatin detected in the supernatant (Figure 6A). ␤3-AR antagonist SR-59230A (1 ␮mol/L) or tetrodotoxin (1 ␮mol/L) reduced basal somatostatin concentrations as well as inhibiting GW427353-induced increase in somatostatin

␤-3 ADRENOCEPTORS IN ENTERIC NERVOUS SYSTEM 179

BASIC– ALIMENTARY TRACT

July 2007

Figure 3. ␤3-adrenoceptor (blue) and ChAT (red) were colocalized in most of the neurons in whole mount preparations of human colonic myenteric (A–D) and submucosal (E–F) plexuses. Three neurons that coexpress ChAT and ␤3-adrenoceptor are indicated with arrowheads (A–D). A neuron which does not express ␤3-adrenoceptor is indicated with an arrow (E–H). Neuronal phenotype was confirmed using an antibody against Hu C/D (green). Scale bars, 40 ␮m.

release. Downward shift of the concentration response curve by SR-59230A was suggestive of a noncompetitive antagonism (Figure 6B).

Visceral Pain Intrarectal administration of mustard oil causes visceral pain in rats that can be measured as the number of abdominal arches the animals display within a set time.14 Systemic administration of a rat selective ␤3-AR agonist, CL-316243 (0.03–1 mg/kg), caused a significant decrease in the number of arches in a dose-dependent manner (Figure 7). This effect was blocked when the animals were pretreated with a somatostatin receptor antagonist, CYN-154806 (10 mg/kg, sc; Figure 7), at a dose that when administered on its own had no algesic activity and reversed the analgesic activity of the somatostatin receptor agonist octreotide (not shown).

In Vitro Motility To determine the functional role of ␤3-AR in the human colon, we investigated the effects of ␤3-AR activation on preparations of human isolated colonic circular muscle. In this preparation, electrical field stimulation (50 V [⬃200 mA], 5 Hz, 0.5-ms bipolar pulse duration, for 10 seconds, every 1 minute) elicited cholinergic con-

tractions in the presence of neurokinin receptor antagonists and indomethacin. These contractions were inhibited by the ␤3-AR agonist GW427353 (0.1–10 ␮mol/L) in a concentration-dependent manner (EC50 ⫽ 300 nmol/L; Figure 8). This inhibition was completely reversed by the ␤3-AR antagonist SR-59230A (1 ␮mol/L; Figure 8). ␤3-AR agonist GW427353 did not alter carbachol-induced contractions (Figure 9).

Diarrhea We investigated the effect of a ␤3-AR agonist on a chemically induced model of diarrhea. Rats were given oral castor oil to induce diarrhea, which was quantified as the weight of fecal matter produced during a set time following administration of the castor oil.13 The rat selective ␤3-AR agonist CL-316243 (0.3–1 mg/kg) given orally 1 hour before castor oil administration significantly reduced fecal weight (Figures 9B and 10).

Discussion We have observed ␤3-AR expression in a majority (⬃85%–90%) of ChAT-positive neurons in the myenteric and submucosal plexuses of the human colon, suggesting that ␤3-AR is exclusive to cholinergic neurons in the

180

CELLEK ET AL

GASTROENTEROLOGY Vol. 133, No. 1

BASIC– ALIMENTARY TRACT

Figure 4. In the human colonic submucosal plexus, ␤3-adrenoceptor is expressed in cholinergic neurons. (A) A ␤3-adrenoceptor-positive (green) neuron is indicated with an arrow in a ganglion among ␤3-adrenoceptor-negative neurons. Neuronal phenotype was confirmed using a combination of 3 nerve markers (Hu C/D, PGP9.5, and neurofilament 200; red). (B) ␤3-adrenoceptor (green) was colocalized with ChAT (red) in most of the neurons. Neuronal phenotype was confirmed using a combination of 3 nerve markers (Hu C/D, PGP9.5, and neurofilament 200; red). Scale bars, 40 ␮m.

human enteric nervous system. None of the somatostatin-containing neurons in the myenteric plexus were stained with ␤3-AR antibody, whereas, in the submucosal plexus, ␤3-AR staining was observed in only a few (⬍2%) somatostatin-containing neurons. Interestingly, the number of ␤3-AR-positive neurons was smaller in the myenteric plexus than in the submucosal plexus (3% vs 72%, respectively). To our knowledge, this is the first demonstration of ␤3-AR in the enteric nervous system. We have also demonstrated in this study that ␤3-AR activation leads to increased somatostatin release. Interestingly, when the mucosa and submucosal plexus were removed, somatostatin release was diminished (unpublished observations). This observation is in accordance with a previous study16 in which the majority of the

somatostatin-containing cells have been located in the human submucosal plexus and mucosa. Our results demonstrating that basal and ␤3-AR agonist-induced somatostatin release is blocked with tetrodotoxin suggest that somatostatin release by ␤3-AR agonism requires neuronal activity. However, ␤3-AR and somatostatin are colocalized only in few neurons in the submucosal plexus, indicating that somatostatin release is very unlikely to be directly from ␤3-AR-positive neurons. A possible scenario therefore can be that somatostatin may be released from ␤3-AR-negative somatostatin-containing neurons or enteroendocrine cells, which may be regulated negatively by ␤3-AR-positive neurons in unstimulated conditions. Activation of ␤3-AR-positive neurons by ␤3-AR agonists may result in dysinhibition of these somatostatin-containing

July 2007

␤-3 ADRENOCEPTORS IN ENTERIC NERVOUS SYSTEM 181

BASIC– ALIMENTARY TRACT

visceral pain-associated behavior. The effect of the ␤3-AR agonist was reversed by treatment with a selective somatostatin receptor-2 antagonist,17 further confirming that ␤3-AR-mediated somatostatin release is the mechanism of action of ␤3-AR-mediated visceral analgesia. Contrary to previous findings in the human isolated colon,6 –9 we did not observe any direct effect of human selective ␤3-AR agonist on smooth muscle tone. This could be due to the use of nonselective ␤3-AR agonists or of not taking the possibility of neuronal involvement into account in the past. We have observed, however, that human selective ␤3-AR agonist inhibited cholinergic contractions in the human isolated colon, an effect fully reversed with a ␤3-AR antagonist. This observation together with the expression of the receptor in the cholinergic nerves suggests that activation of ␤3-AR on the excitatory cholinergic nerves might inhibit their activity and, hence, might result in reduction in colonic motility. This antimotility effect might be partly responsible for the inhibitory effect of ␤3-AR agonist on

Figure 5. In a few neurons in the submucosal plexus, ␤3-adrenoceptor (green) was colocalized with somatostatin (SST; red). Neuronal phenotype was confirmed using a combination of 3 nerve markers (Hu C/D, PGP9.5, and neurofilament 200; blue). Magnification 600⫻.

cells, hence increased somatostatin release. From our study, it is not possible to characterize the mechanism or source of somatostatin release. However, our results clearly demonstrate that activation of ␤3-AR in the human colon leads to somatostatin release. We further investigated by using rat mustard oil model whether somatostatin release by activation of ␤3-AR would result in visceral analgesia. In this model, the rat selective ␤3-AR agonist CL-316243 inhibited mustard oil-induced

Figure 6. Activation of ␤3-adrenoceptor causes release of somatostatin from human isolated colon with intact mucosa. (A) ␤3-adrenoceptor agonist GW427353 (0.1, 1, and 10 ␮mol/L) enhanced somatostatin release from human isolated colon, an effect that was reversed by ␤3-adrenoceptor antagonist SR-59230A (1 ␮mol/L) or tetrodotoxin (TTX; 1 ␮mol/L). (B) Concentration-response curves of GW427353 on somatostatin release in the absence or presence of SR-59230A or TTX (n ⫽ 4). *P⬍.05; significantly different from basal.

182

CELLEK ET AL

GASTROENTEROLOGY Vol. 133, No. 1

BASIC– ALIMENTARY TRACT

Figure 7. ␤3-adrenoceptor agonist inhibits mustard oil-induced visceral pain via somatostatin receptor-2 activation. Rat selective ␤3-adrenoceptor agonist CL316243 (0.03– 0.1 mg/kg, oral) inhibited the number of observed abdominal arches within the first 25 minutes after intrarectal administration of mustard oil to rats; this was reversed by pretreatment with somatostatin receptor antagonist CYN-154806 (10 mg/kg, sc). CYN-154806 did not alter mustard oil-induced pain behavior (n ⫽ 10). *P ⬍ .05; significantly different from vehicle.

Figure 9. ␤3-adrenoceptor agonist does not alter carbachol-induced contractions in human isolated colon. (A) A typical tracing depicting that GW472353 (10 ␮mol/L) did not alter carbachol (CCh, 10 ␮mol/L, EC80)induced contractions in the human isolated colon. (B) GW427353 (0.1–10 ␮mol/L) did not affect carbachol (10 ␮mol/L)-induced contractions (n ⫽ 4).

Figure 8. ␤3-adrenoceptor agonists inhibit cholinergic contractions in human isolated colon. (A) ␤3-adrenoceptor agonist GW427353 inhibited electrical field stimulation-induced cholinergic contractions in the human isolated colon circular muscle (upper panel); this was prevented when the tissue was pretreated with ␤3-adrenoceptor antagonist SR-59230A (1 ␮mol/L; lower panel). The remaining contractions were blocked with scopolamine (10 ␮mol/L), confirming their cholinergic nature. (B) Concentration response curve of GW427353 revealed an EC50 of 300 nmol/L in control conditions (). In the presence of the antagonist SR-59230A, no significant inhibition with GW427353 was observed (□) (n ⫽ 8).

Figure 10. Rat selective ␤3-adrenoceptor agonist inhibited castor oilinduced diarrhea in rats. In a rat model of diarrhea, rat selective ␤3adrenoceptor agonist CL-316243 (0.03–1 mg/kg, oral) decreased fecal matter wet weight measured within the first 3 hours after oral administration of castor oil (n ⫽ 8). Loperamide was used as a positive control. *P ⬍. 05; significantly different from vehicle.

castor oil-induced diarrhea in rats because muscarinic antagonists have been shown to be effective in this model.18,19 Because somatostatin has an antisecretory effect in the gastrointestinal tract,20 ␤3-AR-induced somatostatin release might also be partly responsible for the antidiarrheal effect of the ␤3-AR agonist. Further studies are required to address the relative role for each of these mechanisms in the effects observed. We have used 2 agonists for ␤3-AR: CL-316243 for rat in vivo studies and GW427353 for human in vitro studies. Although GW427353 has similar potency in rat and human ␤3-AR, it is not preferred in the rat for in vivo studies because of its unique pharmacokinetic properties (unpublished observations). CL-316243 was not used in human in vitro studies because it has been shown to be not active in human ␤3-AR.21 Our studies revealed 2 new functions for ␤3-AR: activation of ␤3-AR on the nerves in the colon wall (1) leads to inhibition of cholinergically mediated contractions, probably by suppressing acetylcholine release, and (2) evokes release of somatostatin. The combination of the 2 actions decreases intestinal motility and secretion and induces analgesia. This double activity can be utilized for the treatment of irritable bowel syndrome where pain and disordered intestinal motility are key features of the disease. References 1. Tan S, Curtis-Prior PB. Characterization of the ␤-adrenoceptor of the adipose cell of the rat. Int J Obes 1983;7:409 – 414. 2. Berkowitz DE, Nardone NA, Smiley RM, Price DT, Kreutter DK, Fremeau RT, Schwinn DA. Distribution of ␤-3-adrenoceptor mRNA in human tissues. Eur J Pharmacol 1995;289:223–228. 3. Roberts SJ, Papaioannou M, Evans BA, Summers RJ. Functional and molecular evidence for ␤ 1-, ␤ 2- and ␤ 3- adrenoceptors in human colon. Br J Pharmacol 1997;120:1527–1535. 4. Anthony A, Schepelmann S, Guillaume JL, Strosberg AD, Dhillon AP, Pounder RE, Wakefield AJ. Localization of the ␤(␤)3-adrenoceptor in the human gastrointestinal tract: an immunohistochemical study. Aliment Pharmacol Ther 1998;12:519 –525. 5. Chamberlain PD, Jennings KH, Paul F, Cordell J, Berry A, Holmes SD, Park J, Chambers J, Sennitt MV, Stock MJ, Cawthorne MA, Young PW, Murphy GJ. The tissue distribution of the human ␤3-adrenoceptor studied using a monoclonal antibody: direct evidence of the ␤3-adrenoceptor in human adipose tissue, atrium and skeletal muscle. Int J Obes Relat Metab Disord 1999;23:1057–1065. 6. Bardou M, Dousset B, Deneux-Tharaux C, Smadja C, Naline E, Chaput JC, Naveau S, Manara L, Croci T, Advenier C. In vitro inhibition of human colonic motility with SR 59119A and SR 59104A: evidence of a ␤3-adrenoceptor-mediated effect. Eur J Pharmacol 1998;353:281–287. 7. de Ponti F, Gibelli G, Croci T, Arcidiaco M, Crema F, Manara L. Functional evidence of atypical ␤ 3-adrenoceptors in the human colon using the ␤ 3-selective adrenoceptor antagonist, SR 59230A. Br J Pharmacol 1996;117:1374 –1376. 8. de Ponti F, Modini C, Gibelli G, Crema F, Frigo G. Atypical ␤adrenoceptors mediating relaxation in the human colon: functional evidence for ␤3 rather than ␤4 adrenoceptors. Pharmacol Res 1999;39:345–348.

␤-3 ADRENOCEPTORS IN ENTERIC NERVOUS SYSTEM 183

9. Manara L, Croci T, Aureggi G, Guagnini F, Maffrand JP, Le Fur G, Mukenge S, Ferla G. Functional assessment of ␤ adrenoceptor subtypes in human colonic circular and longitudinal (taenia coli) smooth muscle. Gut 2000;47:337–342. 10. Taborsky GJ Jr, Ensinck JW. Contribution of the pancreas to circulating somatostatin-like immunoreactivity in the normal dog. J Clin Invest 1984;73:216 –223. 11. Levasseur S, Bado A, Laigneau JP, Moizo L, Reyl-Desmars F, Lewin MJ. Characterization of a ␤ 3-adrenoceptor stimulating gastrin and somatostatin secretions in rat antrum. Am J Physiol 1997;272:G1000 –G1006. 12. Hasler WL, Soudah HC, Owyang C. Somatostatin analog inhibits afferent response to rectal distention in diarrhea-predominant irritable bowel patients. J Pharmacol Exp Ther 1994;268:1206–1211. 13. Croci T, Landi M, Emonds-Alt X, Le Fur G, Maffrand JP, Manara L. Role of tachykinins in castor oil diarrhoea in rats. Br J Pharmacol 1997;121:375–380. 14. Boyce S, Combe R, Wheeldon A, Rupniak NM, Hill RG. Antinociceptive activity of the NMDA NR2B receptor subtype selective antagonist CP101606 in a new rat visceral pain assay. P118. International Association for the Study of Pain. 10th World Congress on Pain, 2002. 15. Uehling DE, Shearer BG, Donaldson KH, Chao EY, Deaton DN, Adkison KK, Brown KK, Cariello NF, Faison WL, Lancaster ME, Lin J, Hart R, Milliken TO, Paulik MA, Sherman BW, Sugg EE, Cowan C. Biarylaniline phenethanolamines as potent and selective ␤-3 adrenergic receptor agonists. J Med Chem 2006;49:2758–2771. 16. Keast JR, Furness JB, Costa M. Somatostatin in human enteric nerves. Distribution and characterization. Cell Tissue Res 1984; 237:299 –308. 17. Bass RT, Buckwalter BL, Patel BP, Pausch MH, Price LA, Strnad J, Hadcock JR. Identification and characterization of novel somatostatin antagonists. Mol Pharmacol 1996;50:709 –715. 18. Kobayashi S, Ikeda K, Suzuki M, Yamada T, Miyata K. Effects of YM905, a novel muscarinic M3-receptor antagonist, on experimental models of bowel dysfunction in vivo. Jpn J Pharmacol 2001;86:281–288. 19. Yamamoto O, Niida H, Tajima K, Tanaka M, Makita Y, Ueda F, Yano J. Effect of ␣-2 adrenoceptor antagonists on colonic function in rats. Neurogastroenterol Motil 2000;12:249 –255. 20. McKeen ES, Feniuk W, Humphrey PP. Somatostatin receptors mediating inhibition of basal and stimulated electrogenic ion transport in rat isolated distal colonic mucosa. Naunyn Schmiedebergs Arch Pharmacol 1995;352:402– 411. 21. Cohen ML, Bloomquist W, Kriauciunas A, Shuker A, Calligaro D. Aryl propanolamines: comparison of activity at human ␤3 receptors, rat ␤3 receptors, and rat atrial receptors mediating tachycardia. Br J Pharmacol 1999;126:1018 –1024.

Received July 27, 2006. Accepted March 22, 2007. Address requests for reprints to: Selim Cellek, MD, PhD, Neurology and Gastrointestinal Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park North, Third Ave, Harlow, Essex, CM19 5AW, United Kingdom. e-mail: [email protected]; fax: (44) 0 1279 622470. Supported fully by GlaxoSmithKline and research grants from GlaxoSmithKline (to R.T., O.L., S.V., and M.S.). Financial disclosure: S.C., A.K.B., C.A.C., K.M.G., J.L.S., A.W., W.J.W., G.J.S., and K.L. are employees of GlaxoSmithKline. The authors thank Birgit Kuch for excellent technical assistance in whole mount immunohistochemistry and Simon T. Bate for the statistical analysis.

BASIC– ALIMENTARY TRACT

July 2007