UBC Theses and Dissertations
Immunological techniques in the investigation of the physiological functions of gastric inhibitory polypeptide and motilin Dryburgh, Jill Robertson
A radioimmunoassay was developed, specific for the gastrointestinal polypeptide, motilin. Antisera were raised in guinea pigs and rabbits. The immunogen was porcine motilin, conjugated to bovine serum albumin by the carbodiimide condensation reaction. The routine antiserum behaved identically towards endogenously- released motilin and the pure standard preparation. A radioactive tracer of high 125 specific activity was obtained after incorporation of - iodine into the motilin molecule by the chloramine-T method. The optimum conditions for all other assay variables were established to produce the most sensitive displacement Cstandard) curve. Motilin antiserum, coupled directly to an agarose matrix, retained full antibody activity and sensitivity. It is a feasible technique for use in both the radioimmunoassay and in the extraction of motilin from both serum and tissue extracts. The fasting serum levels of IR- motilin was 190 - 131 pg/ml in men and 294 -'.44 pg/ml in dogs (mean - SD) . The increase in motor activity in the extrinsically denervated fundic pouch of the dog after duodenal alkalinization was associated with a concomitant elevation in serum IR- motilin levels. This increase in serum IR- motilin was in the same range as that achieved by the exogenous administration of the porcine polypeptide which produced the same motor response. Duodenal acidification produced an apparent increase in serum IR-motilin with no associated increase in gastric motor activity. Only one peak of motilin immunoreactivity was detected when serum containing alkali-stimulated motilin or a partially purified duodenal extract were subjected to gel filtration on Sephadex G-50. The distribution of motilin throughout the hog gastrointestinal tract, determined by radioimmunoassay on partially purified extracts, agreed with the immunocytochemical findings that motilin was predominantly located in the duodeninn and jejunum, with traces.in the upper ileum. Virtually the intact molecule was required for the expression of full biological potency. The individual amino acids were important inasmuch as they contributed to the charge distribution and conformation of the molecule. The physiological release and function of motilin have yet to be determined. Elevated levels of circulating IR- motilin have not been associated with any gastro-intestinal function, although they appear to be depressed by feeding. Motilin has been implicated in the control of the interdigestive phase of gastric motor activity. It may be acting in a local or paracrine manner. Motilin has not been implicated in any .•cU'in±cal.rst"ait"eC&s sjffetfce i The hormonal status of gastric inhibitory polypeptide (GIP) has been studied with the existing radioimmunoassay, modified to improve the label specific activity (by ion exchange chromatography). Direct coupling of GIP antisera to agarose beads was unsatisfactory, antibody activity and sensitivity being greatly reduced by the close proximity of the solid matrix. The postulated role of GIP as the enterogastrone1 of Kosaka and Lim, suggested by studies with exogenously-administered polypeptide, was confirmed by experiments in the dog. Pentagastrin-stimulated gastric acid secretion was inhibited by intra-duodenal infusion with glucose or fat; this inhibition being associated with a significant elevation in the circulating serum IR- GIP levels, within the range produced by ingestion of a mixed meal. GIP does not appear to be involved in the inhibition of gastric acid secretion produced by duodenal acidification. Endogenous;GIP.stimulated by either fat or glucose exhibited at least 3 Immunoreactive components after column chromatography. The IR- GIP eluting in the void volume appeared to represent a non-specific complex between GIP and a serum protein and is possibly biologically inactive. A second IR-GIP component with a molecular weight of 7500-8000 (ProGIP), eluted ahead of the established form of GIP (molecular weight = 5105). ProGIP has been found to be relatively unstable. ProGIP and GIP^QQQ have also been detected in extracts of hog duodenal mucosa. The established insulinotropic effect of GIP correlates best with that percentage of the total IR- GIP composed of ProGIP and GIP500(). The relative proportions of IR- GIP500Q and IR- ProGIP in serum samples taken at different times after ingestion of either fat or glucose, suggest that ProGIP is either a precursor of GIP or that the ProGIP-producing cells occupy a more distal region of the duodenal and jejunal mucosa than the GIP- producing cells. Exogenous administration of synthetic somatostatin in dogs and man will inhibit both.GIP release by either fat or glucose and the insulino-tropic action of GIP at the level of the 8/-cell. Naturally-occurring intestinal or pancreatic somatostatin may contribute to the control of GIP release and serve to modulate the GIP- mediated response of the gastric parietal or pancreatic β-cell.
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