ABSTRACT
Title
Cycloooxygenase isoforms exert differential modulatory effects on human colonic excitatory nerve pathways in diverticular disease
Authors
M. Fornai1, L. Antonioli1, R. Colucci1, R. De Giorgio2, N. Bernardini3, M. Tuccori1, C. Maramai1, P. Buccianti4, M. Chiarugi4, C. Blandizzi1
1Interdepartmental Centre for Research in Clinical Pharmacology and Experimental Therapeutics, University of Pisa, Pisa, Italy, 2Dept. of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy,3Dept. of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy, 4Dept. of Surgery, University of Pisa, Pisa, Italy
1Interdepartmental Centre for Research in Clinical Pharmacology and Experimental Therapeutics, University of Pisa, Pisa, Italy, 2Dept. of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy,3Dept. of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy, 4Dept. of Surgery, University of Pisa, Pisa, Italy
Abstract
Introduction. Prostanoids produced by cyclooxygenase isoforms (COX-1, COX-2) are known to contribute significantly to the modulation of intestinal motor functions. However, the role played by these enzyme pathways in human intestinal motility during inflammation is unknown. The aim of the present study is to evaluate the effects of cyclooxygenase inhibitors on excitatory neuromuscular activity of colonic tissues dissected at surgery from patients with diverticular disease.
Methods. Longitudinal muscle preparations (3 mm width, 20 mm length) were obtained from distal colon of patients undergoing elective surgery for diverticular disease (6 M, 4 F; age range: 35-71) or uncomplicated cancer (sex-age matched controls). Colonic strips were set up in organ baths containing Krebs solution at 37°C bubbled with 95% O2+5% CO2 and connected to isotonic transducers. The effects of indomethacin (COX-1/COX-2 inhibitor, 1 µM), SC-560 (COX-1 inhibitor, 0.1 µM) or DFU (COX-2 inhibitor, 1 µM) were tested on contractions evoked by electrical field stimulation (EFS: 0.5 ms, 30 mA, 10 Hz, 100 pulses), in the presence of guanethidine (10 µM) and Nω-nitro-L-arginine methylester (100 µM). Atropine (1 µM) or L-732,138 (NK1 receptor antagonist, 10 µM) were used to record contractions driven predominantly by tachykinins or acetylcholine, respectively. The distribution pattern and expression of COX-1 and COX-2 in the neuromuscular compartment were assessed by immunofluorescence on paraffin cross-sections of full thickness colonic samples.
Results. In the presence of L-732,138, the application of EFS to normal colonic preparations evoked atropine-sensitive contractions. Under these conditions, indomethacin enhanced EFS-induced cholinergic responses (+101.9%). SC-560 or DFU mimicked the effect of indomethacin, but they were less effective (+64.6% and +52.4%, respectively). When incubated with atropine, normal colonic preparations responded to EFS with phasic contractions which were antagonized by L-732,138. In this setting, the incubation of colonic preparations with indomethacin, DFU or SC-560 blunted EFS-induced tachykininergic responses at different extents (-56.0%, -45.9% and -26.2%, respectively). The application of indomethacin, DFU or SC-560 to colonic tissues from patients with diverticular disease did not modify the cholinergic contractions evoked by EFS in the presence of L-732,138. However, when preparations of diverticular colon were maintained in the presence of atropine, both indomethacin and DFU decreased tachykininergic contractions elicited by EFS (-67.3% and -71.1%, respectively), while SC-560 was without effects. The pattern of COX expression was found to be changed in patients with diverticular disease, mainly for the COX-1 isoform which was decreased in myenteric neurons and abolished in glial cells.
Conclusions. cyclooxygenase isoforms exert different regulatory actions on excitatory myenteric nerves in normal human colon: COX-1 and COX-2 down-regulate the contractile responses driven by cholinergic nerve recruitment, whereas both isoforms enhance the tachykininergic pathways. In the presence of diverticular disease, these regulatory patterns are subjected to remodelling: there is a loss of modulation by both cyclooxygenase isoforms on the cholinergic pathway, while COX-2 ensures an enhanced facilitatory control on tachykininergic motor activity. These functional changes may contribute to an enhanced colonic contractility, and thereby painful stimuli, in patients with diverticular disease.
Methods. Longitudinal muscle preparations (3 mm width, 20 mm length) were obtained from distal colon of patients undergoing elective surgery for diverticular disease (6 M, 4 F; age range: 35-71) or uncomplicated cancer (sex-age matched controls). Colonic strips were set up in organ baths containing Krebs solution at 37°C bubbled with 95% O2+5% CO2 and connected to isotonic transducers. The effects of indomethacin (COX-1/COX-2 inhibitor, 1 µM), SC-560 (COX-1 inhibitor, 0.1 µM) or DFU (COX-2 inhibitor, 1 µM) were tested on contractions evoked by electrical field stimulation (EFS: 0.5 ms, 30 mA, 10 Hz, 100 pulses), in the presence of guanethidine (10 µM) and Nω-nitro-L-arginine methylester (100 µM). Atropine (1 µM) or L-732,138 (NK1 receptor antagonist, 10 µM) were used to record contractions driven predominantly by tachykinins or acetylcholine, respectively. The distribution pattern and expression of COX-1 and COX-2 in the neuromuscular compartment were assessed by immunofluorescence on paraffin cross-sections of full thickness colonic samples.
Results. In the presence of L-732,138, the application of EFS to normal colonic preparations evoked atropine-sensitive contractions. Under these conditions, indomethacin enhanced EFS-induced cholinergic responses (+101.9%). SC-560 or DFU mimicked the effect of indomethacin, but they were less effective (+64.6% and +52.4%, respectively). When incubated with atropine, normal colonic preparations responded to EFS with phasic contractions which were antagonized by L-732,138. In this setting, the incubation of colonic preparations with indomethacin, DFU or SC-560 blunted EFS-induced tachykininergic responses at different extents (-56.0%, -45.9% and -26.2%, respectively). The application of indomethacin, DFU or SC-560 to colonic tissues from patients with diverticular disease did not modify the cholinergic contractions evoked by EFS in the presence of L-732,138. However, when preparations of diverticular colon were maintained in the presence of atropine, both indomethacin and DFU decreased tachykininergic contractions elicited by EFS (-67.3% and -71.1%, respectively), while SC-560 was without effects. The pattern of COX expression was found to be changed in patients with diverticular disease, mainly for the COX-1 isoform which was decreased in myenteric neurons and abolished in glial cells.
Conclusions. cyclooxygenase isoforms exert different regulatory actions on excitatory myenteric nerves in normal human colon: COX-1 and COX-2 down-regulate the contractile responses driven by cholinergic nerve recruitment, whereas both isoforms enhance the tachykininergic pathways. In the presence of diverticular disease, these regulatory patterns are subjected to remodelling: there is a loss of modulation by both cyclooxygenase isoforms on the cholinergic pathway, while COX-2 ensures an enhanced facilitatory control on tachykininergic motor activity. These functional changes may contribute to an enhanced colonic contractility, and thereby painful stimuli, in patients with diverticular disease.