Date of Award

2014

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Stephen Keely

Second Supervisor

Professor Frank Murray

Keywords

Ursodeoxycholic Acid, Epithelial Transport, Chloride Secretion, Innate Immunity

Abstract

Background:

Intestinal disorders, including infectious diseases (ID), inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), are common in the European population and on a global scale and are typically associated with inflammation and/or dysregulated intestinal fluid movement, leading to the clinical endpoint of diarrhoea. Although the health and economic burden of diarrhoeal disease is tremendous, there is still a lack of safe and effective drugs for their treatment. Insults to the intestinal epithelium are critical in the pathogenesis of many of the above-mentioned intestinal disorders as these cells govern the transport and barrier functions of the intestine and also participate in initiation of inflammatory responses. As such, these cells represent excellent targets for the development of new drugs to treat intestinal diseases. Ursodeoxycholic acid (UDCA), the therapeutic component of bear bile, used in traditional Chinese medicine, also has a large evidence base and is commonly used in conventional medical practice to treat cholestatic disorders and hepatic inflammation. Other bile acids (BAs) are also known to exert regulatory effects on colonic epithelial transport, cell growth and immunity. Little however is known, to date, about the effects of UDCA and its metabolites on colonic epithelial transport function and immunity.

Methods:

T84 cells, a colonic epithelial cell line used as a reductionist model of chloride (Cl-) secretion, were cultured until they reached confluency and a trans-epithelial resistance of >1000 ohms.cm-2 was achieved. These polarised monolayers of T84 cells, resected colonic mucosa from C57 black mice, Sprague-Dawley rats and surgically resected sections or endoscopic biopsy specimens of human colon were mounted in Ussing chambers and changes in short circuit (Isc) measured. This technique is reflective of changes in Cl- secretion. Confocal imaging was used to visualise localisation of transport proteins. Western blotting and surface biotinylation were used to determine protein expression and abundance. Intracellular calcium (Ca2+) was measured using Fura/ 2AM fluorescence and MetaFluor imaging. ELISA measurements were used to assess cytokine levels.

Results:

The acute effects of UDCA on Cl- secretion in colonic epithelial cell model, in treated mice and on ex vivo rat and human colonic tissues were examined. 18 UDCA, unlike other dihydroxy BAs, did not alter basal Cl- secretion in the T84 colonic epithelial cell line. UDCA in fact exerted potent inhibitory actions in T84 cell monolayers, significantly attenuating responses to both Ca2+ and cyclic-AMP (cAMP) -dependent secretory agonists (Carbachol (CCh) and Forskolin (FSK) respectively, p < 0.001, p < 0.001, n = 18 [UDCA]: 500 M, bilateral addition). UDCA inhibited Cl- secretion across colonic epithelial cells through attenuating activity of basolateral Ca2+-dependent potassium (K+) channels and the sodium/ potassium ATP-ase pump (Na+/ K+ ATPase; p < 0.001, n = 7, p < 0.001, n = 7, respectively). UDCA inhibited channel/ pump activity without altering surface expression or localisation of these proteins. UDCA also stimulated an acute rise in intracellular Ca2+. This was due to influx of extracellular Ca2+ through membrane store operated Ca2+ channels (SOCCs) and L-type Ca2+ channels. In turn, this Ca2+ influx attenuated subsequent CCh-induced release of Ca2+ from intracellular stores, with a consequent inhibition of CCh-induced Cl- secretion. This anti-secretory feedback mechanism involved activation of extracellular regulated kinase/ mitogen activated protein kinase (ERK-MAP kinase) and appeared independent of protein kinase C (PKC). This pathway appeared to be a key mechanism by which UDCA inhibited Ca2+-dependent secretion. UDCA also attenuated cAMP-induced Cl- secretory responses, without altering intracellular levels of this 2nd messenger. Paradoxical to these anti-secretory actions in vitro, secretagogue-induced Cl- secretion was enhanced in the stripped colons of mice treated with UDCA in vivo. This appeared due to its rapid metabolism by colonic bacteria to lithocholic acid (LCA), a pro-secretory BA. Increased LCA levels were noted in the caecal contents of mice treated with UDCA, when compared to control, supporting this hypothesis. 6-methylursodeoxycholic acid (6- MUDCA), a 6-methylated derivative of UDCA, a metabolically stable analogue that is completely resistant to bacterial dehydroxylation, retained the anti-secretory effects of UDCA noted in vitro, inhibiting Cl- secretion across voltage-clamped colonic tissues and T84 cell monolayers. Caecal LCA levels in 6-MUDCA treated mice were similar to controls and significantly less than UDCA-treated animals. UDCA also exerted specific and unique actions on transport function in intact human tissue, stimulating an initial rise in basal Cl- secretion involving activation of muscarinic receptors. 19 Finally, UDCA was also noted to exert both anti-inflammatory and cytoprotective effects on T84 cells and human colonic epithelium. UDCA reduced toll like receptor (TLR)-stimulated cytokine release and was also found to maintain epithelial barrier integrity as demonstrated by trans-epithelial resistance measurement and through inhibition of apoptosis.

Conclusion:

UDCA has predominantly anti-secretory and anti-inflammatory effects in the colonic epithelium. Through direct delivery, modification of its structure or targeting of the molecular targets identified in these studies, these beneficial effects could be utilised to treat inflammatory and secretory colonic diseases.

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2014.