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Investigations into the regulation of the desensitization of 5-HT4 receptors in the rat gastrointestinal tract

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thesis
posted on 31.01.2017, 05:10 by Megersa, Teshome Nedi
5-HT4 and 5-HT7 receptors contain introns that allow the formation of multiple splice variants which are potential targets for developing tissue selective drugs if they are expressed in specific parts of the gastrointestinal tract. 5-HT induces desensitization of 5-HT4 receptors which is facilitated by G-protein coupled receptor kinases (GRKs). This thesis describes investigations into the expression of 5-HT4, 5-HT7 receptors and GRKs (GRK2, GRK3, GRK5 and GRK6); identification of GRK(s) associated with the regulation of the desensitization of 5-HT4 receptors; and the effect of 5-HT4 receptor activation on the expression level of GRKs in the rat gastrointestinal tract. Reverse transcriptase polymerase chain reaction experiments revealed variations in the distribution of the 5-HT4 receptor splice variants in the rat gastrointestinal tract. Both the 5-HT4(a) and 5-HT4(b) receptor splice variants were consistently expressed in the rat oesophagus, ileum and colon. The 5-HT4(e) receptor splice variant was not detected in any tissue. There was no expression of any of the 5-HT4 receptor splice variants in the jejunum. Three 5-HT7 receptor splice variants (a, b and c) were found in the oesophagus, jejunum, ileum and colon. GRKs were also detected in all parts of the gut. Immunohistochemistry studies showed the expression of 5-HT4 receptors in the tunica muscularis mucosae of the oesophagus. These studies also identified 5-HT4 receptor immunoreactivity in the longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus and muscularis mucosae of both the proximal and distal colon. GRK2 was expressed in longitudinal muscle and occasionally in the myenteric plexus whilst GRK5 showed limited expression in the nerve endings of the myenteric and submucosal plexuses of the colon. GRK3 was expressed in the tunica muscularis mucosae of the oesophagus, circular muscle, submucosal plexus and muscularis mucosae of the colon. GRK6 was expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, circular muscle, and muscularis mucosae of the colon. Stimulation of the tunica muscularis mucosae of the oesophagus and distal colon using the 5-HT4 receptor agonist, tegaserod, followed by analysis of the 5-HT4 receptor antibody immunoprecipitate, revealed the coimmunoprecipitation of GRK6 with 5-HT4 receptors in the tunica muscularis mucosae of oesophagus. In the distal colon, GRK2 and GRK6 were coimmunoprecipitated with 5-HT4 receptors. This study indicates that GRK6 may be involved in the regulation of the desensitization of 5-HT4 receptors in the rat oesophagus whilst GRK2 and GRK6 may be involved in regulation of the desensitization of 5-HT4 receptors in the distal colon. The acute administration of the 5-HT4 receptor agonist tegaserod for 1, 2, 3, 4, 6, and 8 hr did not change significantly the immunodensity of GRK2 in the oesophagus and distal colon when compared with control animals. Also there was no significant change in the immunodensity of GRK6 in the oesophagus. This result shows that acute activation of 5-HT4 receptors using tegaserod did not alter the expression of GRK2 and GRK6. This may indicate that the basal level of GRK2 and GRK6 expression is sufficient to regulate the desensitization of 5-HT4 receptors in acute drug treatment.

History

Campus location

Australia

Principal supervisor

Paul White

Year of Award

2011

Department, School or Centre

Pharmaceutical Biology and Pharmacology

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Pharmacy and Pharmaceutical Sciences