Date of Award

2023

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmacology

First Advisor

Brigitte Lavoie

Second Advisor

Gary Mawe

Abstract

Serotonin (5-HT) is a neurotransmitter mainly produced in the enteric nervous system (ENS) of the gastrointestinal (GI) system. Serotonin has widespread function throughout the body including regulating mood, cognition, sleep, sex, and appetite. In the context of the gut, the monoamine modulates vasodilation, mucus secretion, pain, and peristaltic reflexes for motility. The 5-HT4 receptor is expressed on enteric epithelial cells and nerve terminals in the intestinal mucosa. The agonists available to act on these receptors are a favorable target to promote epithelial healing, recovery from colitis or constipation, and epithelial cell proliferation. The non-absorbable, luminally restricted, 5-HT4R agonist (5HT4-LA1; Takeda Pharmaceuticals, 10mg/kg) was found to aid mice in recovery from constipation through the ability to assist in the excretion of materials (motility). However, these studies do not directly test if the agonist is acting on epithelial or neuronal receptors. In this experimentation to further investigate the necessary and/or sufficient site of action to elicit prokinetic action, mice are bred to conditionally knock out the epithelial 5-HT4 receptor, with the neuronal receptor still intact. This was done by generating epithelial 5-HT4R CKO mice by the crossing of a cre recombinase mouse (Villin-Cre) with a mouse where the target gene (5-HT4R) is flanked with two loxP sites. The cre recombinase expressed in the epithelium will recognize the loxP sites and excise the 5-HT4R gene at exon 5, rendering it non-functional with all other sites intact, creating the CKO and wildtype littermates. The mice underwent three motility assays at baseline and after agonist treatment. The first was whole gut transit involving the oral gavage of agonist solution and unabsorbable red dye, the mice were monitored until a red fecal pellet was found. The next two assays were run in conjunction, the fecal water content assay involved the collection of pellets for one hour after gavage for wet and dry measurements. The colonic motility assay involved the time to expel a bead inserted into the distal colon to determine the speed of propulsion. The wildtype littermates responded to the agonist in comparison to the baseline with an accelerated time to excrete the red pellet and time to expel the bead as well as an increase in the amount of water present in the pellets – indicating faster transit. The CKO mice did not experience a change in all three assays, demonstrating that the epithelial 5-HT4R is necessary for the agonist to mediate prokinetic action and that the neuronal 5-HT4R does not mediate motility. The second investigation involves the testing of the 5-HT4 receptor agonist (5HT4-LA1) and antagonist (GR113808) on constipation in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) patients. MS is a neurodegenerative disease of the CNS manifesting as an autoimmune disorder resulting in axonal demyelination with constipation as a co-morbidity element. It was hypothesized that the agonist upon EAE induction will rescue symptoms of constipation by stimulation of 5-HT4 receptors and this effect will be blocked by the 5-HT4R antagonist. The whole gut transit and colonic motility assays were able to confirm constipation symptoms at the height of disease. We observed no significant changes for the same three motility assays in response to the agonist or agonist + antagonist which is contrasting to the literature available. As demonstrated, the 5HT4-LA1 agonist is found to act primarily on the epithelial 5-HT4 receptors using the VilCre CKO model and does not act on neuronal receptors. Future studies conducted may conditionally knock out the neuronal receptor to determine any potential differences or effects. The results of the agonist and antagonist administration in EAE mouse model was overall nonconclusive due to the small final cohort and large groups will be needed to determine if 5-HT4R agonists may be an effective treatment for constipation.

Language

en

Number of Pages

67 p.

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