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K A Eidne

Publications and source records attributed to K A Eidne.

12 recordsLinked to original sources
PubMed1992

Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor.

We have isolated the gonadotropin-releasing hormone receptor (GnRH-R) from a rat anterior pituitary cDNA library, determined its sequence and demonstrated receptor function. The 2.2 kb rat GnRH-R clone encodes a protein of 327 amino acids. A 1.3 kb clone encoding the mouse GnRH-R has previously been described (Tsutsumi et al., 1992). Although both the mouse and rat protein share significant homology with molecules belonging to the family of G protein-coupled receptors, they have certain unusual features, an example being the complete absence of a COOH terminal tail. The 3'-untranslated region reported missing in the mouse is present in the rat cDNA, where an extended 1 kb of 3'-untranslated region extending to the poly-A tail is shown. At the amino acid level, the rat GnRH-R shows considerable homology with that of the mouse. Electrophysiological studies with Xenopus oocytes and transfection of the cDNA into COS-1 cells, have shown that the 2.2 kb cDNA clone encodes a functional receptor.

Action Potentials↗
PubMed1992

Characterization of the gonadotrophin-releasing hormone calcium response in single alpha T3-1 pituitary gonadotroph cells.

Intracellular calcium ([Ca2+]i) was measured in single immortalized gonadotroph alpha T3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. Gonadotrophin-releasing hormone (GnRH, 10(-8) M) produced a biphasic rise in [Ca2+]i which could be abolished by a GnRH antagonist. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. The calcium spike was reduced by nifedipine (10(-6) M), a calcium channel blocker, and thapsigargin (10(-6) M) which inhibits inositol 1,4,5-trisphosphate (IP3) mediated release of [Ca2+]i but abolished by the intracellular calcium antagonist TMB-8 (10(-6) M). The secondary phase was reduced following pretreatment with either nifedipine or the protein kinase C (PKC) antagonist, H-7 (10(-6) M). The PKC agonist PMA (phorbol 12-myristate 13-acetate, 10(-6) M) produced a small rise in basal [Ca2+]i and abolished the GnRH calcium response. The initial calcium response to GnRH therefore involves both an IP3-mediated rise in cytosolic calcium due to the release from intracellular stores and an influx of extracellular calcium through second messenger-operated calcium channels. In contrast the secondary calcium response mainly involves the influx of extracellular calcium through PKC-activated calcium channels.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland.

A new member of the family of G protein-coupled receptors has been isolated from a rat pituitary cDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers. The corresponding protein sequence shows seven transmembrane domains and contains conserved regions of homology characteristic of the G protein-coupled class of receptors. The novel receptor mRNA is expressed in the brain, pituitary gland and testis, and has been localized by in situ hybridization in discrete regions of the brain. Expression of the receptor mRNA in Xenopus oocytes and in transfected mammalian cells has not yet permitted identification of the corresponding ligand for this receptor.

Amino Acid Sequence↗
PubMed1990

Gonadotropin-releasing hormone analogs inhibit primate granulosa cell steroidogenesis via a mechanism distinct from that in the rat.

Gonadotropin-releasing hormone (GnRH) and related peptides are implicated in the local control of rat ovarian function, but evidence to date for direct effects of such peptides on primate ovarian cells is equivocal. In contrast to rat ovaries, where GnRH action is mediated through specific, high-affinity GnRH receptors, no such binding sites have been identified in primate tissue. Using undifferentiated granulosa cells from immature follicles in cyclic (luteal phase) marmoset ovaries, we have observed direct suppression of human (h) FSH-induced steroidogenesis by GnRH analogs in vitro. Granulosa cells from immature (less than 1 mm diameter) follicles were incubated for 4 days in the presence of hFSH and testosterone (aromatase substrate) to stimulate cyclic AMP (cAMP) production and steroidogenesis. The additional presence of GnRH alone (up to 10 microM) had no effect on FSH action. However, the GnRH agonist, [D-Ser(But)6]GnRH 1-9)-ethylamide (Buserelin, 0.1 microM-10 microM), caused time- and dose-dependent inhibition of estradiol (maximum inhibition = 79%; ED50 = 0.55 microM) and progesterone production (maximum inhibition = 93%; ED50 = 0.1 microM). Accumulation of cAMP was also inhibited by up to 54%. Paradoxically, a GnRH antagonist [( N-Ac-D-Nal(2)1,D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10]-GnRH; 10 microM) alone also inhibited hFSH-stimulated cAMP and steroid production by 40% and 70%, respectively. Moreover, the suppressive effects of the GnRH agonist on granulosa cell functions were augmented by the presence of the GnRH antagonist (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA.

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Functional expression of rat pituitary gonadotrophin-releasing hormone receptors in Xenopus oocytes.

Expression of receptors for the hypothalamic regulatory peptide, gonadotrophin-releasing hormone (GnRH), was investigated by intracellular recording from Xenopus oocytes injected with poly(A)+ mRNA isolated from rat anterior pituitary glands. Membrane depolarizations were induced in oocytes in a dose-dependent fashion following the application of GnRH (10nM - 1 microM) or a GnRH superactive agonist, buserelin (1nM - 1 microM). The response was reversibly blocked by the addition of a GnRH antagonist (1 microM). TRH (10nM - 1 microM) had no effect on most of these oocytes. In contrast, some other oocytes which showed no responses to GnRH or to the GnRH agonist, displayed depolarizing responses to TRH (10nM - 1 microM). A relatively small number of oocytes responded to both ligands. Control oocytes did not respond to the GnRH analogues or to TRH. This successful expression of the GnRH receptor could provide a new approach to the study of the receptor, and serve as a means for the isolation and cloning of the encoding genes.

Animals↗
PubMed1987

Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists.

GnRH-binding sites have previously been described in human breast tumors, and a GnRH agonist has been shown to inhibit growth of the MCF-7 human breast cancer cell line. We have investigated the presence of GnRH-binding sites in ZR-75-1, MDA-MB-231, Sk Br 3, MDA-MB-157, and MCF-7 human breast cancer cell lines and the effect of GnRH analogs on the incorporation of [3H]thymidine and 14C-labeled amino acids into DNA and protein. Specific GnRH-binding sites were present in membrane preparations of all five human breast carcinoma cell lines. Studies in three cell lines indicated low affinity (Kd, 1.6-3.0 X 10(-6) M) GnRH binding similar to that reported in human placenta and corpus luteum. In contrast, human pituitary GnRH receptors were of high affinity (Kd, 4.8 X 10(-9) M). Breast carcinoma cell GnRH-binding sites also differed from the pituitary receptor in their inability to discriminate between GnRH and superactive analogs. Binding of a [125I]GnRH analog to ZR-75-1 breast cancer cells and pituitary membranes was affected similarly by various cations. GnRH antagonists rapidly inhibited [3H]thymidine incorporation into DNA (within 3 hr), and this effect was reversible. GnRH antagonists also inhibited cell growth, but only after 6 days. GnRH agonists did not alter either thymidine incorporation or growth. The present observations of low affinity GnRH-binding sites in breast cancer cell lines and inhibitory effects of GnRH antagonists point to the possibility of an autocrine regulatory role of GnRH-like peptides in mammary cells.

Breast Neoplasms↗
PubMed1987

Inhibition of FSH-stimulated granulosa cell function by a synthetic fragment of the porcine inhibin alpha-subunit: evidence for involvement of GnRH receptors.

The bioactivity of a synthetic peptide fragment which mimics the N-terminal sequence of the 134-amino-acid porcine inhibin alpha-subunit (pl-alpha 1-26-Gly27Tyr28-OH) was tested and compared with the bioactivity of GnRH in rat granulosa cell cultures. Granulosa cells from immature female rat ovaries were cultured with hFSH and testosterone to stimulate the production of cyclic AMP, progesterone and oestradiol. Addition of pl-alpha 1-26-Gly27Tyr28-OH to the culture medium caused a dose-dependent suppression of all three parameters (ID50 700-1,000 nmol/l). GnRH caused similar but higher-potency inhibition (ID50 2-4 nmol/l). Suppression of granulosa cell function by both peptides was fully reversible by a synthetic GnRH antagonist. Moreover, specific binding of the porcine inhibin fragment to ovarian GnRH receptors was demonstrated by radioreceptor assay. This is evidence that the porcine inhibin alpha-subunit fragment suppresses FSH-induced rat granulosa cell function via a mechanism of action similar to that of GnRH.

Animals↗
PubMed1985

Gonadotropin-releasing hormone binding sites in human breast carcinoma.

Gonadotropin-releasing hormone analogs can cause regression of hormone-dependent breast carcinomas. These effects are thought to be mediated through the inhibition of gonadotropic and steroid hormones. These analogs may also act directly on the tumor because they are effective in treating breast cancer in some postmenopausal women. The presence of specific binding sites for gonadotropin-releasing hormone was demonstrated in human breast carcinomas by means of a novel approach of ligand immunoblotting. The results indicate a possible mechanism by which the peptide has direct effects on this tissue. These binding proteins were not detectable in non-neoplastic breast tissue.

Adult↗
PubMed1985

Demonstration of a 60K molecular weight luteinizing hormone-releasing hormone receptor in solubilized adrenal membranes by a ligand-immunoblotting technique.

A new technique for the identification of LHRH receptors has been developed and applied to demonstrate an adrenal LHRH binding protein. Solubilized membrane proteins were separated electrophoretically and transferred to nitrocellulose paper. This was followed by sequential incubations with LHRH, anti-LHRH antiserum, peroxidase-conjugated second antibody, and 4-chloro-1-naphthol. Using an antiserum directed towards the middle region of LHRH, a 60K mol wt band was visualized in rat adrenal and pituitary membranes. A band of slightly higher molecular weight was present in membranes of bovine adrenal cortex but was absent in the medulla. The 60K band was not visualized when nonimmune rabbit serum was used. The 60K band was also not visualized when an antiserum requiring the NH2 and COOH termini of LHRH was used, suggesting that these regions of LHRH are not accessible to the antiserum after binding to the receptor. These studies have demonstrated the existence of LHRH binding protein in adrenal cortical tissue with a molecular size similar to that of the pituitary receptor. Adrenal membrane binding sites were less clearly demonstrated by conventional 125I-ligand binding techniques as nonspecific binding was high. The ligand-immunoblotting technique is a sensitive, specific and rapid procedure with potential application in screening normal and tumor tissues for LHRH receptors and studying LHRH interactions with its receptor.

Adrenal Cortex↗
PubMed1985

Gonadotropin-releasing hormone receptors in human pituitary: ligand structural requirements, molecular size, and cationic effects.

A specific, high affinity receptor for GnRH in human pituitaries obtained post mortem is described. The human pituitary GnRH receptor bound GnRH, a GnRH agonist [(D-Ala6,N alpha-MeLeu7,Pro9NEt)-GnRH], and a GnRH antagonist [Ac-D-Nal(2)1,D-alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )-GnRH] with similar affinities (KdS of 4.81 nM, 0.32 nM, and 0.32 nM) to those of the rat pituitary (KdS of 4.71 nM, 0.31 nM, and 0.40 nM). A second GnRH antagonist [(D-pGlu1,D-Phe2,D-Trp3,6)-GnRH], however, was bound with a much lower affinity by the human receptor (Kd of 4.21 nM) than that of the rat (Kd of 0.09 nM). Monovalent and divalent cations affected [125I]GnRH agonist binding to rat and human pituitary receptors differently. In the presence of Mg2+ or Ca2+, binding to the human receptor was significantly lower than in the rat. At near physiological concentrations, Na+ and K+ (100 mM and 10 mM, respectively) inhibited [125I]GnRH agonist binding to the receptors to a similar extent in both species. At unphysiological concentrations (10 mM Na+ and 100 mM K+) these ions decreased binding to the human pituitary receptor to a greater extent than to the rat receptor. Using a ligand-immunoblotting technique, the human receptor or binding component of the receptor complex was found to be of greater molecular size (64,000 daltons) than that of the rat (60,000 daltons). The results show that the human and rat pituitary GnRH receptors have similar binding affinities for GnRH and certain GnRH analogs but differ in their binding of an antagonist, their sensitivity to cationic effects on GnRH agonist binding, and in the molecular size of the receptor GnRH-binding protein.

Adult↗