Abstract
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 peptides,
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 CI" current with superimposed current
fluctuations which reversed around -25mV, compatible with the Eci in
Xenopus oocytes. The current -voltage relation showed a shift agreeable with
the Nernst equation when the external concentration of CI" was changed and the
response was abolished by CT channel blockers. 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 CT channels. A
calcium-ionophore (ionomycin) was used to study the calcium stores
responsible for the activation of a CT 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 GnRH 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 (GRH)
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 -100mV (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
