Delesalle, Catherine

Deprez, Piet

Lefebvre, Romain

SCHUURKES, J

Ghent University Academic Bibliography

In vitro effects of 5-hydroxytryptamine on the Jejunum of Healthy and Colic Horses: Characterization of a Putative 5-HT1A Receptor in the Equine Gut.   C. Delesalle, , P. Deprez, J.A.J. Schuurkes, R.A. Lefebvre  Department of Large Animal Internal Medicine, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium (Delesalle, Deprez); Heymans Institute of Pharmacology, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium (Lefebvre); Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutical N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium (Schuurkes).    Prokinetic use for intestinal stasis in horses is mainly extrapolated from human medicine, with limited scientific proof that the targeted enteric receptor populations are similar in the equine and human gastrointestinal tract. As the prokinetic cisapride acts at serotonin (5-HT) receptors, we aimed to characterize the 5-HT receptors mediating the contractile response to 5-HT in equine jejunum longitudinal muscle. We compared the results of healthy horses with those from colic patients, who developed non-responsive post-operative ileus after surgery for small intestinal strangulation.   Strips of the mid-jejunum of healthy horses were collected at the slaughterhouse. Colic patient material was obtained after euthanasia with T61a. Longitudinal muscle strips, deprived of mucosa were prepared for isotonic measurement. All strips showed basal phasic activity. After stabilisation the tissue was challenged twice with 1x10-6M carbachol. The second carbachol-induced contraction was used as internal standard and all contractions to 5-HT are expressed as % of this second response. Results are given as mean ± SEM; the number of horses used is denoted by n.  5-HT induced a tonic contraction with an increase in the amplitude of the phasic activity. Cumulative administration of 5-HT in the same strip led to rapid fading of the contractile response at the higher concentrations of 5-HT, in contrast to what has been reported in former studies on equine ileum and pelvic flexure1,2. The isolated concentration-response curve for 5-HT (10-10M – 3x10-5M) has the features of a monophasic sigmoidal concentration-response curve, consistent with a single-site interaction. The curve parameters are given in Table 1.  Table 1: Curve fit parameters of the concentration-response curves to 5-HT and 5-CT in jejunum from healthy and colic horses    Emax pEC50 Hill slope 5-HT Healthy (n=24) 79.04 ± 2.47 7.88 ± 0.07 1.07 ± 0.08  Colic (n=7) 70.72 ± 11.21 7.41 ± 0.23** 0.79 ± 0.08 5-CT Healthy (n=6) 85.86 ± 3.78 6.02 ± 0.18 0.76 ± 0.09  Colic (n=7) 66.95 ± 9.31** 6.44 ± 0.11* 1.17 ± 0.11** *P<0.05, **P<0.01: Significantly different from the response in healthy horses   Tetrodotoxin (3x10-6M), atropine (3x10-6M) and the combination of both, did not affect the responses to 5-HT, suggesting a non-neurogenic, non-cholinergic contractile effect mediated by 5-HT receptors located directly on smooth muscle cells. The specific 5-HT receptor antagonists ketanserin (5-HT2A; 3x10-7M), granisetron (5-HT3; 3x10-7M); GR 113808 (5-HT4; 1x10-7M); SB 269970 (5-HT7; 3x10-7M), GR 127935 (5-HT1B,D; 1x10-7M), SB 204741 (5-HT2B; 3x10-7M), RS 102221 (5-HT2C, 3x10-7M) had no effect on the responses to 5-HT, excluding interaction at 5-HT1B,1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4 and 5-HT7 receptors.  The 5-HT1, 5-HT2, 5-HT5, 5-HT6 and 5-HT7-receptor antagonist methysergide (1x10-9M, 1x10-8M, 1x10-7M) antagonised non-surmountably the concentration-response curve of 5-HT.  NAN 190 (5-HT1A; 1x10-7M, 3x10-7M, 1x10-6M ) behaved as a competitive antagonist. WAY 100635 (3x10-9M) shifted the concentration-response curve to 5-HT to the right in a parallel way without a change in Emax, but the higher concentrations of WAY 100635 (3x10-8M and 3x10-7M) significantly depressed the Emax of 5-HT. The pKb values of NAN 190 and WAY 100635 were estimated from the antagonist inhibition curve in the presence of a fixed concentration of 5-HT. The pKb values (NAN 190, 8.13 ± 0.06; WAY 100635, 8.69 ± 0.07) are in accordance with the pKb values of these antagonists for the 5-HT1A receptor reported in the literature.  The specific 5-HT1, 5-HT7 agonist 5-carboxamidotryptamine (5-CT) elicited responses similar to those of 5-HT. The concentration-response curve parameters are given in Table 1.  The responses to 5-CT were not influenced by application of tetrodotoxin plus atropine (both 3x10-7M), and the 5-HT7-receptor antagonist SB 269970 (5-HT7; 3x10-7M). The specific 5-HT1A receptor antagonist WAY 100635 (3x10-9M) antagonised the contractions to 5-CT. When WAY 100635 was applied at a concentration of 3x10-8M and 3x10-7M, the concentration-response curve to 5-CT was further shifted to the right but there was a clear concomitant suppression of the maximum effect elicited by 5-CT.    Responses to carbachol were not different in healthy horses (1.70 ± 0.08 cm) and colic horses (1.53 ± 0.21cm). 5-HT was less potent in colic horses than in healthy horses (Table 1).   The parameters of the concentration-response curves of 5-HT and 5-CT in colic horses are given in Table 1. The pEC50 of 5-HT in colic horses was decreased. The Emax of 5-CT was significantly decreased in colic horses, however pEC50 and Hill slope were increased.  Our data clearly suggest that muscular 5-HT1A receptors mediate the contractions to 5-HT in equine jejunal longitudinal muscle. The receptor shows rapid desensitization, which could have important implications in horses with colic or ileus, where a status of hypercoagulability or necrotic intestinal segments could serve as a source of 5-HT overload.   References and Footnotes  1. Nieto JE, Snyder JR, Kollias-Baker C, et al. In vitro effects of 5-hydroxytryptamine and cisapride on the circular smooth muscle of the jejunum of horses. AJVR 2000;61:1561-1565. 2. Weiss R, Abel D, Scholtysik G, et al. 5-Hydroxytryptamine mediated contractions in isolated preparations of equine ileum and pelvic flexure: pharmacological characterization of a new 5-HT4 agonist. J Vet Pharmacol Ther 2002;25:49-58. 3. T61® ad us. Vet. Embutramide, mebenzoniumiodide, tetracaïn hydrochloride, dimethylformamide, Intervet Belgium, Ragheno Parc, Mechelen, Belgium      

In vitro effects of 5-hydroxytryptamine on the jejunum of Healthy and colic Horses: Characterization of a putative 5-HT1A receptor in the Equine Gut

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In vitro effects of 5-hydroxytryptamine on the jejunum of Healthy and colic Horses: Characterization of a putative 5-HT1A receptor in the Equine Gut

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