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Characterization of rat intestinal immunoreactive motilin (IR-M)

University of British Columbia

Interdigestive myoelectric activity in rat intestine has been recorded and characterized. The interdigestive pattern of activity can be disrupted by oral glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no effect on the interdigestive myoelectric pattern, nor did high doses of porcine motilin. Attempts were made to develop a hybridoma cell line secreting antibodies that would recognize rat Intestinal immunoreactive motilin (IR-M). The murine myeloma cell line NS1 was fused with murine B-cells primed against porcine motilin. One hundred of the monoclonal cell lines produced secreted monoclonal antibodies that recognized porcine motilin. Attempts to identify a cell line secreting antibodies with the ability to stain rat intestinal tissue, however, produced only negative results. Rat intestinal IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The biological activity of partially purified rat intestinal IR-M has also been evaluated utilizing a rabbit isolated duodenal muscle strip preparation. Five different antisera and one monoclonal antibody directed against natural porcine motilin were utilized in an effort to detect IR-M containing cells in rat intestinal tissues. A variety of techniques were employed including tissue fixation with either Bouins, paraformaldehyde, or benzoquinone. In addition a variety of staining methods including, fluorescein conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques were used. All methods using these antibodies failed to detect IR-H in the rat small intestine. Porcine motilin was able to displace ¹²⁵I-motilin from antisera 13-3, 72X and M03. These antisera were utilized in a motilin RIA to evaluate acid extracts of rat intestinal tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M in gut extracts, and these antisera gave different distributions of IR-M In the proximal small bowel. Rat intestinal tissue was extracted into 2% trifluoroacetic acid and the soluble fraction clarified by centrifugation. This acid extracted material was precipitated with sodium chloride then dissolved in methanol at pH 6.0. Methanol soluble material was precipitated with ether and the ether precipitate then dissolved in water and chromatographed on Sep-Pak C₁₈ cartridges (Waters). Sep-Pak cartridges were eluted with 50% acetonitrile: 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel filtration and reverse phase high pressure liquid chromatography (HPLC). Rat intestinal IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for contractility in a rabbit duodenal muscle strip preparation. Purification after SP-Sephadex-C25 was approximately 20 fold. Desensitization of rabbit duodenum to porcine motilin could be demonstrated by pre-treatment with motilin. Contractile activity of partially purified rat intestinal IR-M was not inhibited by pretreatment with motilin. Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic HPLC, using a linear gradient of water/acetonitrile (10-45% acetonitrile in 30 min), rat intestinal IR-M did not co-elute with natural porcine motilin. In conclusion, the molecular weight of rat intestinal IR-M appeared to be similar to porci ne motilin as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine motilin on HPLC indicates that other molecular characteristics of rat intestinal IR-M, such as hydrophobicity, are not similar to porcine motilin. Furthermore, partially purified rat intestinal IR-M did induce a contractile response in rabbit duodenal muscle strips but porcine motilin did not desensitize this preparation to the contractile activity of rat intestinal extracts. This suggests that the contractile activity of these two compounds is induced via different receptor mechanisms. It is concluded that the immunoreactive motilin found in rat intestinal extracts does not resemble natural porcine motilin in structure or biological activity.

Text

2010-07-19

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http://hdl.handle.net/2429/26658

Physiology

University of British Columbia

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