Volume 119, Issue 2, Pages 348-357 (August 2000) Neurotensin stimulates Cl− secretion in human colonic mucosa in vitro: Role of adenosine  Martin Riegler, Ignazio Castagliuolo, Chi Wang, Michael Wlk, Tacettin Sogukoglu, Etienne Wenzl, Jeffrey B. Matthews, Charalabos Pothoulakis  Gastroenterology  Volume 119, Issue 2, Pages 348-357 (August 2000) DOI: 10.1053/gast.2000.9310 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of NT on human colonic Isc. (A) Time course effect of NT on human colonic Isc. (B) Concentration-dependent effect of NT on human colonic Isc (ΔIsc). Colonic mucosae were mounted in Ussing chambers and incubated in the serosal presence (2) or absence (♢, controls) of various NT concentrations. ΔIsc was obtained after serosal administration of NT for 10 minutes. Values represent means ± SEM; n = 6/group; *P < 0.001 vs. controls in A; P values vs. 10−10 mol/L NT in B. Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of NT receptor antagonists. Concentration-dependent effect of the nonpeptide NT receptor antagonists SR 48692 and SR 142948A on human colonic Isc (ΔIsc). Human colonic mucosae were mounted in Ussing chambers and incubated 30 minutes before serosal NT administration (10−6 mol/L) in the serosal presence of 10−10 to 10−6 mol/L SR 48692 (2) and SR 142948A (●). ΔIsc was obtained after serosal administration of NT for 10 minutes. Values represent means ± SEM; n = 6/group.*P < 0.05 vs. SR 142948A. Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of TTX (2) and indomethacin (INDO; ♢). Concentration-dependent effect of the neuronal blocker TTX and the PG synthesis inhibitor indomethacin on NT (10−6 mol/L)-induced Isc increase. Human colonic mucosae were mounted in Ussing chambers and incubated in the serosal presence of 10−9 to 10−5 mol/L TTX or indomethacin, 30 minutes before and during serosal administration of 10−6 mol/L NT. ΔIsc was obtained 10 minutes after administration of NT. Values represent means ± SEM, each with 5 determinations in parallel for each inhibitor (n = 6). P values vs. 10−9 mol/L of respective inhibitor. Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of AdoR antagonists. Concentration-dependent effect of 10−10 to 10−4 mol/L of the nonspecific AdoR antagonist 8-PT (2); the adenosine 1 receptor antagonist 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (AdoR-1 inhibitor) (○); and the adenosine 2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (AdoR-2 inhibitor) (♢) on NT (10−6 mol/L)-induced Isc increase. Human colonic mucosae were mounted in Ussing chambers and incubated in the serosal presence of AdoR antagonists 30 minutes before and during serosal administration of 10−6 mol/L NT. ΔIsc was obtained 10 minutes after administration of NT. Values represent means ± SEM, each with 4–6 determinations in parallel for each inhibitor (n = 6/data point). Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of adenosine uptake inhibitors. (A) Concentration-dependent effect of 10−9 to 10−3 mol/L of the adenosine uptake inhibitors NBTG (2) and NBTI (◊) on NT (10−6 mol/L)-induced Isc increase. (B) Effect of 10−4 mol/L NBTG or NBTI on NT (10−10 to 10−6 mol/L)-induced Isc increase. 2, NT+NBTG; ♢, NT+NBTI; ○, NT alone. Human colonic mucosae were mounted in Ussing chambers and incubated in the serosal presence of adenosine uptake inhibitors 30 minutes before and during serosal administration of NT. ΔIsc was obtained 10 minutes after administration of NT. Values represent means ± SEM, each with 4–6 determinations in parallel for each inhibitor (n = 6/data point). Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of adenosine deaminase inhibitor. (A) Concentration-dependent effect of 10−9 to 10−4 mol/L of the adenosine deaminase inhibitor deoxycoformycin on NT (10−6 mol/L)-induced Isc increase. (B) Effect of 10−4 mol/L deoxycoformycin (◊) on NT (10−10 to 10−6 mol/L)-induced Isc increase. 2, NT alone. Human colonic mucosae were mounted in Ussing chambers and incubated in the serosal presence of the adenosine uptake inhibitor 30 minutes before and during serosal administration of NT. ΔIsc was obtained 10 minutes after administration of NT. Values represent means ± SEM, each with 4–6 determinations in parallel for each inhibitor (n = 6/data point). Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of adenosine on human colonic Isc. (A) Time-course effect of adenosine on human colonic Isc. 2, Adenosine (10−3 mol/L); ♢, controls. (B) Concentration-dependent effect of adenosine on human colonic Isc (ΔIsc). Colonic mucosae were mounted in Ussing chambers and incubated in the serosal presence or absence (controls) of various adenosine concentrations. ΔIsc was obtained after serosal administration of adenosine for 10 minutes. Values represent means ± SEM; n = 6/group.*P < 0.01 vs. controls in A; P values vs. 10−8 mol/L adenosine in B. Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Distribution of NT-1 receptor on nerve cells in human colonic mucosa. Fixed normal human colonic mucosal sections cut in parallel to the crypt surface epithelial axis were stained with rabbit polyclonal antiserum directed against the N-terminal peptides of the NT-1 receptor (FITC conjugated, green fluorescence in A), and a mouse monoclonal IgG1 antibody directed against brain neurofilament (TRITC conjugated, red fluorescence in B). Sections were imaged with a confocal microscope as described in Methods. The merged images (A and B) show colocalization of NT-1 receptor on nerve cells (yellow fluorescence in C). Arrows indicate cells stained with both antibodies (original magnification 400×). Gastroenterology 2000 119, 348-357DOI: (10.1053/gast.2000.9310) Copyright © 2000 American Gastroenterological Association Terms and Conditions