Volume 133, Issue 1, Pages 175-183 (July 2007) Demonstration of Functional Neuronal β3-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, Kevin Lee  Gastroenterology  Volume 133, Issue 1, Pages 175-183 (July 2007) DOI: 10.1053/j.gastro.2007.05.009 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Expression of β3-adrenoceptor in the neurons of the human colonic myenteric plexus. (A) β3-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 β3-adrenoceptor-positive neuron (indicated with an arrowhead) was observed next to β3-adrenoceptor-negative neurons. Scale bars, 40 μm. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 β3-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, β3-adrenoceptor (green) and ChAT (red) were colocalized in the same neurons. (B) When the primary antibody for β3-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 μm. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 β3-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 β3-adrenoceptor are indicated with arrowheads (A–D). A neuron which does not express β3-adrenoceptor is indicated with an arrow (E–H). Neuronal phenotype was confirmed using an antibody against Hu C/D (green). Scale bars, 40 μm. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 In the human colonic submucosal plexus, β3-adrenoceptor is expressed in cholinergic neurons. (A) A β3-adrenoceptor-positive (green) neuron is indicated with an arrow in a ganglion among β3-adrenoceptor-negative neurons. Neuronal phenotype was confirmed using a combination of 3 nerve markers (Hu C/D, PGP9.5, and neurofilament 200; red). (B) β3-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 μm. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 In a few neurons in the submucosal plexus, β3-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×. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Activation of β3-adrenoceptor causes release of somatostatin from human isolated colon with intact mucosa. (A) β3-adrenoceptor agonist GW427353 (0.1, 1, and 10 μmol/L) enhanced somatostatin release from human isolated colon, an effect that was reversed by β3-adrenoceptor antagonist SR-59230A (1 μmol/L) or tetrodotoxin (TTX; 1 μmol/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. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 β3-adrenoceptor agonist inhibits mustard oil-induced visceral pain via somatostatin receptor-2 activation. Rat selective β3-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. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 β3-adrenoceptor agonists inhibit cholinergic contractions in human isolated colon. (A) β3-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 β3-adrenoceptor antagonist SR-59230A (1 μmol/L; lower panel). The remaining contractions were blocked with scopolamine (10 μmol/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). Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 9 β3-adrenoceptor agonist does not alter carbachol-induced contractions in human isolated colon. (A) A typical tracing depicting that GW472353 (10 μmol/L) did not alter carbachol (CCh, 10 μmol/L, EC80)-induced contractions in the human isolated colon. (B) GW427353 (0.1–10 μmol/L) did not affect carbachol (10 μmol/L)-induced contractions (n = 4). Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions

Figure 10 Rat selective β3-adrenoceptor agonist inhibited castor oil-induced diarrhea in rats. In a rat model of diarrhea, rat selective β3-adrenoceptor 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. Gastroenterology 2007 133, 175-183DOI: (10.1053/j.gastro.2007.05.009) Copyright © 2007 AGA Institute Terms and Conditions