Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: Selective 5-HT4 receptor agonists such as prucalopride are used as human prokinetics, since activation of 5-HT4 receptors on intestinal cholinergic neurons facilitates acetylcholine release. 5-HT4 receptors, linked to adenylyl cyclase, act via generation of cAMP. None of the 4 in vitro studies on 5-HT in horses provided evidence for neuronal 5-HT4 receptors, but none used the protocol as described in human studies [1-4]. OBJECTIVES: To investigate whether functional 5-HT4 receptors are present in the equine small intestine. STUDY DESIGN AND METHODS: In vitro organ bath set up, applying electrical field stimulation (EFS) in longitudinal and circular smooth muscle strips. RESULTS: Results were similar in both muscle layers. In the presence of 0.3 mmol/l NG-Nitro-L-arginine methyl ester and 0.3 µmol/l apamine, excluding effects of the inhibitory transmitters NO and ATP, EFS induced voltage-dependent on-contractions; these were neurogenic as they were abolished by 3 µmol/l tetrodotoxin. At a voltage inducing 50% of the maximal amplitude, the submaximal EFS-induced contractions were cholinergic as atropine (1 µmol/l) abolished them. Prucalopride (0.3 µmol/l) did not increase the amplitude of these submaximal EFS-induced contractions. Even in the presence of the nonselective phosphodiesterase inhibitor IBMX, previously shown to enhance the effect of neuronal 5-HT4 receptors by inhibiting breakdown of their 2nd messenger cAMP [5], prucalopride (3 µmol/l) had no influence. Also 5-HT (10 µmol/l), a full agonist at 5-HT4 receptors, tested in the presence of methysergide and granisetron to exclude interaction with other 5-HT receptor subtypes, did not enhance EFS-induced submaximal contractions. CONCLUSIONS: There is no evidence for presence of 5-HT4 receptors on the cholinergic neurons of the equine small intestine. These results question the application of 5-HT4 prokinetic drugs in horses. Ethical animal research: Research ethics committee oversight not currently required by this conference: the study was performed on material collected at an abattoir. Sources of funding: None. Competing interests: None declared.

Influence of 5-HT4 receptor activation on acetylcholine release in human large intestine with endometriosis.

BACKGROUND: The 5-HT(4) receptor agonist prucalopride enhances large intestinal contractility by facilitating acetylcholine release through activation of 5-HT(4) receptors on cholinergic nerves and is effective in patients with constipation. Patients with intestinal endometriosis can present with constipation. We investigated in vitro whether large intestinal endometriotic infiltration influences contractility and facilitation of acetylcholine release by prucalopride. METHODS: Sigmoid colon or rectum circular muscle strips were obtained at the level of an endometriotic nodule with infiltration of the Auerbach plexus, and at a macroscopically healthy site at least 5 cm cranially from the nodule, in patients undergoing laparoscopic colorectal resection because of symptomatic bowel endometriosis. Responses to muscarinic receptor stimulation and to electrical field stimulation (EFS), and the facilitating effect of prucalopride on acetylcholine release were evaluated. KEY RESULTS: The EC50 and E(max) of the contractile responses to the muscarinic receptor agonist carbachol did not differ between healthy and lesioned strips. EFS-induced on-contractions were not different between the healthy and lesioned strips, while the non-nitrergic relaxant responses induced by EFS were decreased in the lesioned strips. The facilitating effect of prucalopride on acetylcholine release in healthy strips was similar to that reported before in macroscopically healthy colon tissue of patients with colon cancer; in lesioned strips, the effect of prucalopride was fully maintained in 6/8 patients and absent in two. CONCLUSIONS & INFERENCES: Large intestinal endometriosis does not lead to a systematic interference with the cholinergic facilitating effect of prucalopride.

Profilin II is alternatively spliced, resulting in profilin isoforms that are differentially expressed and have distinct biochemical properties.

We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.

Murine adseverin (D5), a novel member of the gelsolin family, and murine adseverin are induced by interleukin-9 in T-helper lymphocytes.

We identified a number of upregulated genes by differential screening of interleukin-9-stimulated T-helper lymphocytes. Interestingly, two of these messengers encode proteins that are similar to proteins of the gelsolin family. The first displays a typical structure of six homologous domains and shows a high level of identity (90%) with bovine adseverin (or scinderin) and may therefore be considered the murine adseverin homolog. The second encodes a protein with only five segments. Sequence comparison shows that most of the fifth segment and a short amino-terminal part of the sixth segment (amino acids 528 to 628 of adseverin) are missing, and thus, this form may represent an alternatively spliced product derived from the same gene. The corresponding protein is called mouse adseverin (D5). We expressed both proteins in Escherichia coli and show that mouse adseverin displays the typical characteristics of all members of the gelsolin family with respect to actin binding (capping, severing, and nucleation) and its regulation by Ca2+. In contrast, mouse adseverin (D5) fails to nucleate actin polymerization, although like mouse adseverin and gelsolin, it severs and caps actin filaments in a Ca2+-dependent manner. Adseverin is present in all of the tissues and most of the cell lines tested, although at low concentrations. Mouse adseverin (D5) was found only in blood cells and in cell lines derived from T-helper lymphocytes and mast cells, where it is weakly expressed. In a gel filtration experiment, we demonstrated that mouse adseverin forms a 1:2 complex with G actin which is stable only in the presence of Ca2+, while no stable complex was observed for mouse adseverin (D5).