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Title: Electrophysiology of rat anterior pituitary receptors expressed in Xenopus laevis oocytes
Author: Plant, S.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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Messenger RNA was isolated from rat anterior pituitary glands and was injected into Xenopus laevis oocytes. The anterior pituitary receptors which the mRNA encoded were then expressed into the oocyte membrane. Expression of anterior pituitary receptors was investigated by intracellular recording and two-electrode voltage-clamp methods. With intracellular recording, membrane depolarisations were induced in oocytes following activation of the expressed receptors for the hypothalamic regulatory pep-tides, gonadotrophin releasing hormone (GnRH) and thyrotrophin-releasing hormone (TRH). The response to GnRH or a GnRH superactive agonist, buserelin, was reversibly blocked by the addition of a GnRH antagonist and that to TRH by the TRH blocker chlordiazepoxide. Control oocytes did not respond to the GnRH analogue or to TRH. Under voltage clamp activation of exogenous GnRH and TRH receptors was shown to cause a slow, transient inward Cl~ current with superimposed current fluctuations which reversed around -25mV, compatible with the EC| in Xenopus oocytes. The current-voltage relation showed a shift agreeable with the Nemst equation when the external concentration of Cl~ was changed and the response was abolished by Cl~ channel block-ers. Incubation of the oocytes with the G-protein inhibitor pertussis toxin abolished the response to GnRH and TRH. The addition of the calmodulin inhibitor chlordiazepoxide and the blocker of intracellular Ca2+ release TMB-8 inhibited the response to GnRH and TRH. Thus receptor activation appears to involve a G-protein and the release of intracellular calcium from stores to open Ca2+-dependent Cl~ channels. A calcium-ionophore (ionomycin) was used to study the calcium stores responsible for the activation of a Cl~ current. The response to ionomycin consisted of two phases, which had a differing sensitivity to metabolic inhibitors and to intracellular but not extracellular calcium chelation. The response to ionomycin is shown to mimic the response to receptor activation and appears to be due to the release of two different intracellular Ca2+ stores. The successful expression of anterior pituitary gland receptors, as measured using two-electrode voltage clamp method, was used to commence the search for the genes encoding the GnTH and TRH receptors with a view to the eventual isolation and cloning of these genes. Studies of oocytes which had intact follicular layers revealed that the Xenopus oocyte has endogenous growth hormone-releasing hormone receptors in the follicular layer. Activation of these receptors caused the production of a transient outward K+ current in a dose-dependent manner, which reversed around -lOOmV (compatible with EK in Xenopus oocytes) and was inhibited by the K+ channel blocker TEA+ . The activation of a K+ current by GRH is shown to be dependent on a rise in cAMP. The possible function of this receptor is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available