The establishment of an in vivo model system for studying the control of secretion of human small intestinal mucin

Abstract

In this thesis the output of mucin in the urine of subjects with ileal conduits was studied in order to establish an human model for investigating the control of mucus secretion by the small intestinal tissue. Subjects in this study have had their defective bladders surgically replaced by a segment of ileum (the ileal conduit) with its blood and nerve supplies intact. The 24 h output of urine was collected (containing all the mucus secreted by the ileal conduit) and the mucin component of mucus was measured by ELISA. The daily mucin output of each individual subject fell within a compact range, although there were substantial differences between individuals. The first goal of the research was to prove the model was feasible. The mucin purified from ileal conduit urine was characterised and compared with mucin purified from post-mortem ileal scrapings. Mucins from both sources banded at 1.42 g/ml density in a CsCl density gradient, and their other characteristics such as molecular size, electrophoretic mobility, amino acid composition, monosaccharide composition, and sulphate content were also similar. During the purification of mucin from ileal conduit urine and post-mortem ileal scrapings an unknown component was detected, which has not been previously described. This material co-purified with mucin during chromatography but separated slightly from mucin on CsCl gradients banding at 1.48 g/ml buoyant density. An ELISA was developed to measure this material, and it was purified and partly characterised. Immunohistochemical studies revealed that this material originated from goblet cells. This work has been placed in an appendix as it is not central to the work on mucin. The next goal of the research was to use the established model system to study the effect of pharmacological agents on mucus secretion. Aspirin was the first drug to be tested. During oral aspirin administration (600 mg per day) the mucin outputs increased by an average of 67% in the subjects (n=B). The mucins secreted before and during the aspirin administration were characterised. The mucin from both sources eluted in the excluded volume of the Sepharose CL-2B column and banded at 1.42 g/ml density in the CsCl gradient. A higher threonine content was seen in the mucin secreted during aspirin intake. The model system was then used to study the effect of prostaglandin (Misoprostol, a PGE1 derivative) on mucus secretion. Increased mucin outputs (by an average of 80%) were obtained while subjects (n=4) were taking prostaglandin orally (600 μg per day). The mucins secreted before and during prostaglandin intake were similar (molecular size and buoyant density). Again a higher threonine content was detected in the mucin secreted during the prostaglandin administration. This ileal conduit model has many advantages over other model systems used for studying the rates of mucin secretion. These include the ease of sampling, the same subject can be used repetitively and each acts as his/her own control, mucin is collected by self-flushing of the conduit, no contamination with mucus from other regions occurs, no endogenous enzyme degradation of mucin occurs, the mucin output is reproducible and human subjects can be used. The model can also be used as a reproducible resource for obtaining mucus in vivo.