Ali Hassan

Ali Hassan

Department of Zoology, University of Canterbury, Christchurch, New Zealand.

A number of receptors in the G-protein-coupled receptor family are desensitized through phosphorylation of a site in their third intracellular loop by cyclic AMP-dependent protein kinase A (PK-A). Recently the corticotropin-releasing hormone (CRH) receptor expressed in the anterior pituitary, which is known to undergo desensitization, has been cloned and found to contain a potential PK-A phosphorylation site in its third intracellular loop. The aim of this study was to determine whether desensitization of the adrenocorticotrophic hormone (ACTH) response to CRH could be mediated by cyclic AMP. Experiments were carried out using dispersed ovine anterior pituitary cells in a multi-column perifusion system. The response of cells pre-treated with 10 μM forskolin or 0.1 nM CRH for 1 or 3 hours to subsequent stimulation with 10 nM CRH was compared to the response of cells stimulated with 10 nM CRH alone. Cells pre-treated with 10 μM forskolin for 3 hours exhibited a significant (p<0.05) reduction in stimulated ACTH release in response to 10 nM CRH of 36.7 ± 5.6% (n=10) compared to controls, while cells pre-treated with 0.1 nM CRH for 3 hours exhibited a 73.6 ± 6.9% (n=2) reduction (p<0.05) in response. Depletion of intracellular ACTH reserves did not appear to be the cause of the reduction in response as there was a reduction in the percentage of intracellular ACTH released after pre-treatment and the response of cells pre-treated with 10 μM forskolin to 50 mM KCl was not reduced compared to the response of cells treated with 50 mM KCl alone. It is concluded that the ACTH response of perifused ovine anterior pituitary cells to CRH can be mediated by cyclic AMP, most likely through the phosphorylation of the receptor. Phosphorylation of the receptor may functionally uncouple the receptor from adenylyl cyclase or lead to its down-regulation.

B.Sc.(Hons) Thesis, University of Canterbury, Christchurch, New Zealand, 1996.