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Biomedical subjects

E Hazum

Publications and source records attributed to E Hazum.

At least 55 records · Page 3Linked to original sources
PubMed1983

Copper and thiol regulation of gonadotropin releasing hormone binding and luteinizing hormone release.

The effects of copper, sulfhydryl and disulfide reagents on gonadotropin, releasing hormone (GnRH) binding to pituitary membrane preparations and on luteinizing hormone (LH) release from pituitaries of immature female rats were studied. Copper ions reduced the specific binding of 125I-labeled [D-Ser(t-Bu)6, des-Gly10-ethylamide]GnRH (Buserelin) to pituitary membranes in a dose responsive manner. Fifty percent inhibition of the specific binding was obtained at 3 x 10(-5)M Cu+2. The decreased binding stems from a 8-fold decrease in the apparent affinity of Buserelin, in the presence of copper ions. Cupric sulfate was examined for its ability to affect basal and GnRH stimulated LH release. Copper stimulated basal LH release 18-fold and 8-fold at 10(-4)M and 10(-5)M, respectively. The LH release in response to Cu+2 was calcium dependent. The effect of Cu+2 on GnRH stimulated LH release was a combined effect of Cu+2 on GnRH binding. GnRH on LH release and Cu+2 on LH release. N-ethylmaleimide, hydrogen peroxide and dithiothreitol did not alter significantly the specific binding of 125I-labeled Buserelin. N-ethylmaleimide and hydrogen peroxide did not affect GnRH stimulated LH release, whereas dithiothreitol (1 mM) significantly inhibited GnRH stimulated LH release. The present findings may explain the induction of ovulation by copper and the reduction in serum LH after injection of reducing agents.

Animals↗
PubMed1983

Gonadotropin releasing-hormone receptors: photoaffinity labeling with an antagonist.

A photoaffinity antagonist of gonadotropin releasing hormone (GnRH), D pGlu-D-Phe-D-Trp-Ser-D-Lys6(N epsilon-azidobenzoyl)-Leu-Arg-Pro-Gly-NH2 (photoaffinity antagonist) was prepared by reacting [D-pGlu1, D-Phe2, D-Trp3, D-Lys6]GnRH with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. The analog appeared homogeneous when analyzed by thin-layer chromatography and its photoreactivity was demonstrated by spectral changes when exposed to light. The photoaffinity antagonist retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in vitro in whole pituitaries. Pituitary membrane preparations were incubated with the radioactive photoaffinity GnRH antagonist and irradiated with light. Sodium dodecyl sulfate gel electrophoresis after solubilization and reduction showed the specific labeling of a single specific protein with an apparent molecular weight of 60,000 daltons. These results indicate that GnRH agonists and antagonists bind to the same receptor.

Affinity Labels↗
PubMed1983

Receptor-mediated internalization of LHRH antagonists by pituitary cells.

A fluorescently labeled antagonist of luteinizing hormone releasing hormone (LHRH), D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Lys6-(tetramethylrhodamine)-Leu-Arg-Pro-Gly-NH2, was prepared. This peptide retained high-affinity binding to the LHRH receptor of pituitary plasma membrane preparations. The analog was able to block LHRH-stimulated LH release from pituitaries incubated in vitro, and exhibited minor agonistic activity. This rhodamine-labeled antagonist was utilized for the microscopic visualization and localization of LHRH receptors in dispersed rat pituitary cells. The fluorescently labeled receptors were initially distributed uniformly on the cell surface. The hormone-receptor complexes were redistributed after incubation at 23 degrees C and formed clusters which subsequently became internalized (at 37 degrees C) into endocytic vesicles. Addition of LHRH (10(-6) M) abolished these processes, indicating specific binding sites for the rhodamine-labeled peptide to the gonadotrope cells. A quantitative comparison of temperature-dependent internalization by iodinated LHRH agonist and antagonist revealed that both analogs were internalized to a similar extent. These findings suggest that LHRH-receptor complex internalization is related to LHRH receptor regulation.

Animals↗
PubMed1983

Covalent linking of photoreactive gonadotropin-releasing hormone to gonadotropes produces a prolonged signal.

A bioactive, photoreactive derivative of gonadotropin-releasing hormone (GnRH; gonadoliberin), [azidobenzoyl-D-Lys6]GnRH, was shown to bind covalently to dispersed pituitary cells after irradiation. Approximately 7% of the total cell-associated radioactivity was covalently bound to the receptors. Photolysis of cultured pituitary cells in the presence of the photoreactive derivative resulted in persistent activation of luteinizing hormone (LH; lutropin) release. This persistent response was time dependent and concentration dependent. No increase in the basal rate of LH release was observed with cells incubated in the presence of photoreactive GnRH analog and maintained in the dark or with hormone derivatives that lack the photoreactive azido group. These results suggest that only the covalently bound cell surface receptors account for the persistent activation of LH release after photolysis.

Affinity Labels↗
PubMed1983

Localization of biotinylated gonadotropin releasing hormone on pituitary monolayer cells with avidin-biotin-peroxidase complexes.

The new avidin-biotin-peroxidase complex (ABC) technique was used to localize the [D-Lys6] analog of gonadotropin releasing hormone (GnRH), labeled with biotin, on pituitary monolayer cultures from female rats. Staining was diffuse, or in patches, on the surface of 10-17% of the cells 30 sec-3 min after the addition of 10(-10)-10(-12) M biotin-labeled GnRH. In parallel studies, double stains for gonadotropins showed label on 16.3 +/- 2% of the monolayers. Capping was evident by 3 min after exposure and the stain appeared in dense patches, vesicles, or granules 10-30 min after exposure. The stain was abolished by the addition of a 10- to 100-fold excess of unlabeled [D-Lys6] GnRH. Biotinylated GnRH released luteinizing hormone (LH) and follicle stimulating hormone (FSH) and was either equipotent or 10 times more potent than the unlabeled analog in multiple dose-response tests. The ED50 of the 4 hr release was 0.075 nM for LH and 0.02 nM for FSH. Competitive binding assays showed that the binding affinity of the biotinylated GnRH was within the range found for the unlabeled analog (0.7 nM-IC50). This report describes the localization of biotinylated GnRH on the surfaces of cells exposed to low concentrations of the analog with a technique that requires minimal manipulation of the cells, and is performed in less than one day.

Animals↗
PubMed1982

GnRH-receptors of rat pituitary is a glycoprotein: differential effect of neuraminidase and lectins on agonists and antagonists binding.

The interaction of 125I-labeled gonadotropin releasing hormone (GnRH) agonist, [D-Ser-(t-Bu)6,des-Gly10-ethylamide]-GnRH, and antagonist [D-pGlu1,D-Phe2,D-Trp3,6]-GnRH with rat pituitary membranes was studied. Their binding was affected differently by pretreatment of membranes with neuraminidase and by wheat-germ agglutinin. Pretreatment of the membranes with neuraminidase abolished the specific binding of both antagonist and agonist, in a dose-response manner, with the former being less affected. Wheat-germ agglutinin affected antagonist binding slightly (22% inhibition at 40 microgram/ml) but inhibited agonist binding more markedly (50% at 40 microgram/ml). This inhibitory effect was specific since it was readily reversed by N-acetylglucosamine. Other lectins, such as concanavalin A and soybean agglutinin affected the binding of both agonist and antagonist to only a small degree. These results suggest that the GnRH-receptor of rat pituitary is a glycoprotein which contains sialic acid residue and that GnRH agonists and antagonists bind differently to the same receptor.

Animals↗
PubMed1982

Photoaffinity-labeling and fluorescence-distribution studies of gonadotropin-releasing hormone receptors in ovarian granulosa cells.

Photoaffinity labeling of rat ovarian granulosa cells and membrane preparations with a bioactive photoaffinity derivative of gonadotropin-releasing hormone resulted in identification of two specific components with apparent molecular weights of 60,000 and 54,000. Fluorescent visualization of gonadotropin-releasing hormone receptors in these cells, by using a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed patches that subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on the granulosa cells. These studies may provide an experimental basis for understanding the molecular events involved in the action of the hormone in the ovary.

Affinity Labels↗
PubMed1982

A novel method for localization of gonadotropin releasing hormone receptors.

Two 125I-labeled analogs of GnRH, [acidobenzoyl-D-Lys6]GnRH (I) and [D-Lys6]GnRH (II) were used for the localization of GnRH receptors in pituitary cells. The analogs exhibited high binding affinity to pituitary membrane preparations and after photoactivation (analog I) or cross-linking with glutaraldehyde (analog II), these analogs are bound covalently to pituitary cells. The distribution of the labeled hormones by light and electron microscopic autoradiography indicated that after exposure of pituitary cells to 125I-labeled hormones at 4 C (90 min), most of the labeled hormones were associated with the cell surface membrane, while at 37 degrees C (30 min) most of the cell-bound labeled hormones were internalized.

Animals↗
PubMed1981

Morphine in cow and human milk: could dietary morphine constitute a ligand for specific morphine (mu) receptors?

Morphine has been found in cow and human milk at concentrations of 200 to 500 nanograms per liter. Multistep purification yields a material that has immunological, biological, pharmacological, and chemical properties identical to those of morphine. Similar morphine-like material, which has been tentatively identified in some common plant sources, may be a ubiquitous dietary constituent and a possible source for the material in milk. Since morphine (mu) receptors have a low affinity for enkephalins, and since morphine-like materials have been described in brain and intestine, it is possible that morphine in food may be the source of this material and a normal ligand specific for mu receptors.

Animals↗