Gonadotropin-releasing hormone in milk.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) has been found in milk of man, cow, and rat. Radioimmunoassays of acidified milk indicate concentrations of GnRH ranging between 0.1 and 3 nanograms per milliliter. Multistep extractions, followed by electrophoresis, reveal gonadotropin-releasing activity in the fraction that comigrates with the GnRH-marker. A second hypothalamic hormone, thyrotropin-releasing hormone, is present in milk at a much lower concentration. "Milk -GnRH" may influence the secretion of the gonadotropic hormones in neonates.

Specific nonopiate receptors for beta-endorphin.

Iodinated beta H-[2-D-alanine]endorphin exhibits specific binding to cultured human lymphocytes. The binding is inhibited by low concentrations of beta-endorphin and its D-alanine derivative, but is not affected by opiate agonists and antagonists, or by enkephalin analogs, beta-lipotropin, adrenocorticotrophic hormone, or alpha-melanocyte-stimulating hormone; this suggests the existence of a specific, non-opiate binding site (receptor) for beta-endorphin. The carboxy-terminal region of beta-endorphin is essential for this binding activity, since alpha-endorphin is not active. beta-Endorphin may be a circulating hormone with peripheral physiological effects that are not primarily mediated through interactions with opiate or enkephalin receptors.

Opiate (Enkephalin) receptors of neuroblastoma cells: occurrence in clusters on the cell surface.

A bioactive, fluorescent derivative of enkephalin, Tyr-D-Ala-Gly-Phe-Leu-Lys-rhodamine, was used to determine the distribution of opiate receptors in living neuroblastoma cells. The receptors appeared in clusters on the cell surface, and no internalization was detected. No specific fluorescence or clusters were observed in the presence of [D-Ala2, Leu5]enkephalin or at 4 degrees C, and the clusters were much reduced under ionic conditions (that is, with 100 millimolars sodium) that specifically decrease the binding of opiate agonists.

Morphiceptin (NH4-tyr-pro-phe-pro-COHN2): a potent and specific agonist for morphine (mu) receptors.

The synthetic peptide NH2-Tyr-Pro-Phe-Pro-CONH2 (morphiceptin), which is the amide of a fragment of the milk protein beta-casein, has morphinelike activities and is highly specific for morphine (mu) receptors but not for enkephalin (delta) receptors. It is as active as morphine in the guinea pig ileum but much less active in the mouse and rat vas deferens. The discovery of this specific morphine receptor ligand substantiates the hypothesis of multiple opiate receptors. The ligand, which may be of physiological significance since a very similar, or identical, activity can be detected in enzymatic digests of beta-casein, may prove useful for further investigation of the functions of opiate receptor subtypes.

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.

Structure-activity relationships of luliberin substituted at position 8.

Substitution of arginine at position 8 of luliberin by the basic amino acids homoarginine, lysine and diaminobutyric acid resulted in analogues in which the luteinizing hormone-releasing activity is markedly reduced, whereas the cross reactivity with specific antibodies to luliberin is preserved. Fluorimetric titrations of these analogues, carried out as with luliberin, revealed pK values of 6.00 +/- 0.05 and of 9.75 +/- 0.15 for His 2 and Try 5 respectively which are essentially the same as in luliberin. However, the rate of collisions between the side chains of His 2 and Trp 3 in these analogues was found to decrease by 36-39%. Substitution at position 8 with the non-basic amino acid omega-nitro arginine yielded an analogue possessing a very low hormonal activity as well as poor recognition of antibodies specific to luliberin. The fluorescence properties of this peptide are markedly different from those of luliberin and its three basic analogues. These results indicate that the functional integrity of the active unit His 2 . . . Tyr 5 . . . Arg 8 in luliberin depends both on size and basicity of the amino acid side chain at position 8.

Neuroendocrine peptides in milk.

Milk, which is a mammal-specific biologic fluid, contains several neuroendocrine peptides at concentrations higher than those found in plasma. These neuroendocrine peptides can be synthesized or processed in the mammary gland or excreted into milk through various pathways. In addition, certain milk proteins, notably casein, can be enzymatically processed to release "exorphins," peptides with opioid activities. In suckling mammals, hormones and neuropeptides are absorbed through the gastrointestinal tract and appear intact in the plasma. This absorption is age dependent and could have physiologic significance in neonatal development.

Photoaffinity labeling of luteinizing hormone releasing hormone receptor of rat pituitary membrane preparations.

Specific LHRH receptor proteins of plasma membrane preparation from pituitary glands of the rat were identified using an 125I-labeled photoreactive LHRH derivative, [D-Lys6-N epsilon-azidobenzoyl]LHRH. This analog retained high binding affinity (apparent Kd value of 0.1 nM) to a single class of receptors. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated the specific photolabeling of two proteins, a major band with an apparent molecular weight of 60,000 daltons and a minor band of 48,000 daltons. The latter is probably a degradation product of the receptor.

Tunicamycin and neuraminidase effects on luteinizing hormone (LH)-releasing hormone binding and LH release from rat pituitary cells in culture.

We have studied the effects of tunicamycin (TM) and neuraminidase on the binding of 125I-labeled Buserelin, a GnRH agonist, and on GnRH-stimulated LH release in cultured rat pituitary cells. Treatment with TM, an antibiotic which inhibits protein glycosylation, abolished the development of elongated cell processes without any effect on cell viability. Concomitantly, TM caused a time- and dose-dependent inhibition of specific binding of Buserelin and of GnRH-stimulated LH release. The inhibition of binding was due to a decrease in the number of GnRH receptors without any significant effect on binding affinity. Protein synthesis was not affected under these experimental conditions, suggesting that the aglycosylated GnRH receptors are probably intracellularly accumulated and are not expressed on the cell surface. Treatment with neuraminidase inhibited only 50% of GnRH agonist binding and did not affect GnRH-stimulated LH release. These results indicate that the oligosaccharide portion is essential for the functional properties of the GnRH receptor.

Bovine cerebellum endothelin receptor: solubilization and identification.

Endothelin receptors were solubilized from bovine cerebellum membrane preparations in an active form by using the zwitterionic detergent CHAPS, [3-(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid. The solubilized receptors displayed high affinity, saturability, and specificity. The dissociation constant (Kd) for endothelin was 7 +/- 2 nM, and the number of binding sites was 600 +/- 200 fmol/mg protein. These results are similar to those obtained for the membrane-bound receptor and suggest that during solubilization the binding characteristics of the receptor are preserved. Attempts to purify the solubilized receptors in an active form using affinity chromatography techniques, i.e. Affi-gel 15 column coupled to endothelin, were not successful. Nevertheless, identification of the receptors was achieved by affinity chromatography of the solubilized proteins and subsequent iodination. Autoradiographic analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major protein with an apparent mol wt of 50 kD. Taken together with our previous findings, this result suggests that the 50-kD band represents the endothelin receptor.

Identification of endothelin receptors by chemical cross-linking.

Specific binding sites for human/porcine endothelin have been found in rat brain membrane preparations using an [125I]endothelin. The apparent Kd value was 3 +/- 2 nM with a Bmax value of 2,200 +/- 200 fmol/mg protein. Chemical cross-linking of [125I]endothelin to rat brain membranes by using the cross-linking reagent disuccinimidyl suberate (DSS) revealed two specific proteins of Mr = 52,000 and Mr = 30,000. The same results were obtained by photoaffinity labeling of [125I]azidoendothelin to rat brain membranes. The labeling of the two proteins was inhibited in a dose-dependent manner by unlabeled endothelin but not by unrelated peptides. Peptide map comparisons of the Mr = 52,000 and Mr = 30,000 protein bands using Staphylococcus aureus V8 protease and papain revealed that the Mr = 30,000 band is a proteolytic degradation product of the Mr = 52,000. The apparent mol wt of the endothelin receptor is, therefore, 50,000, subtracting the mol wt of the iodinated endothelin.

Identification of a single binding protein for endothelin-1 and endothelin-3 in bovine cerebellum membranes.

Competition binding experiments of [125I]endothelin-3 ([125I]ET-3) to bovine cerebellum membrane preparations in the presence of either ET-3 or ET-1 have indicated the presence of a single class of high affinity binding sites for these two peptides in the brain. Cross-linking of [125I]ET-3 to cerebellum membrane preparations with dissuccinimidyl suberate (DSS) resulted in the labeling of two bands with apparent mol wt of 52 and 30 kDa. Under these conditions the labeling intensities of these two bands were similar. However, addition of 5 mM EDTA to the protease inhibitor mixture during membrane preparations as well as the binding and cross-linking reaction increased the labeling of the 52-kDa protein while reducing the labeling of the 30-kDa protein. Peptide map comparisons of the 52- and 30-kDa protein bands using Staphylococcus aureus V8 protease and papain revealed that the 30-kDa band is a proteolytic degradation product of the 52-kDa protein. These results suggest that the 52-kDa protein represents the specific binding protein of ET-3, and thus, the apparent mol wt of the ET receptor is 50 kDa, subtracting the mol wt of the iodinated ET. Since cross-linking of [125I]ET-1 to cerebellum membrane preparations revealed the same two bands of 52 and 30 kDa, peptide mapping of the 52-kDa proteins, cross-linked with either [125I]ET-1 or ET-3, was conducted. Under these experimental conditions, identical peptide fragments were generated by both Staphylococcus aureus V8 protease and papain. These results suggest that ET-1 and ET-3 bind to a common brain binding protein with an apparent mol wt of 50 kDa.

Luteinizing hormone release and gonadotropin-releasing hormone (GnRH) receptor internalization: independent actions of GnRH.

Three different approaches are described which provide independent and new evidence that gonadotropin-releasing hormone (GnRH) internalization and GnRH-stimulated LH release are distinct actions of the releasing hormone. 1) Removal of GnRH from medium bathing the pituitary cell cultures resulted in the prompt return of LH release to basal levels. This finding indicated that a continuous supply of externally applied GnRH is required for the stimulation of LH release. 2) Covalent immobilization of D-Lys6-des-Gly10-Pro9-ethylamide GnRH (a GnRH agonist) on agarose beads resulted in a derivative which stimulated LH release with full efficacy. At concentrations of immobilized releasing hormone analog sufficient to evoke gonadotropin release, the quantity of LH release was restricted by the number of beads added. This finding was interpreted as evidence that the attachment of immobilized agonist was stable during the bioassay and indicated that LH release could be stimulated with full efficacy without the requirement for GnRH internalization. 3) Comparative studies using image-intensified microscopy and the cell culture bioassay showed that 100 microM vinblastin markedly inhibited large scale patching and capping of the GnRH receptor (viewed by image-intensified microscopy), but did not alter the EC50 or efficacy of LH release stimulated by GnRH or the agonist described above. These observations indicated that internalization as well as large scale patching and capping of the GnRH receptor are not required for LH release.

Receptors for gonadotropin releasing hormone are present in rat oocytes.

Specific receptors for gonadotropin releasing hormone (GnRH) in the rat oocyte have been identified by using two independent methods. Light microscopic autoradiography, utilizing an iodinated biologically active photoaffinity derivative of GnRH, revealed specific binding of the neurohormone to rat oocytes. Furthermore, the presence of GnRH-receptor is also evident from indirect fluorescent immunocytochemistry that shows binding of GnRH-receptor antibodies to rat oocytes which is neither detected with non immune serum nor with antiserum depleted of GnRH-receptor antibodies. These antibodies to the GnRH-receptor, also bind to both cumulus and granulosa cells but not to rat basophilic leukemia cells. The presence of specific GnRH receptors on rat oocytes provides an experimental basis for understanding the molecular events involved in GnRH-induced oocyte maturation.

Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling.

In this study, two melanotropin binding proteins from M2R melanoma cells have been identified based on the photochemical cross-linking of [125I]iodinated porcine beta-MSH ([ 125I]iodo-beta-MSH) to melanoma cell membranes, using N-hydroxysuccinimidyl-azidobenzoate. Autoradiography of photoaffinity-labeled M2R membrane protein, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the specific labeling of two separate bands with an apparent molecular mass of 43 and 46 kilodaltons, respectively. Photoaffinity labeling of both bands was of near-equal intensity and could be inhibited, in a dose-dependent manner, by the addition of unlabeled beta-MSH before photolysis. In addition, agents known to inhibit the binding of beta-MSH to its cellular receptor, such as EGTA, GTP, guanosine 5'-O-(3-thio)triphosphate, and a synthetic analog of the calmodulin-binding domain of myosin light chain kinase-M5, were all found to specifically inhibit the labeling of these two protein bands by the azido derivative of [125I]iodo-beta-MSH. In contrast, addition of a nonrelated peptide, vasoactive intestinal peptide, had no effect upon the labeling of these melanotropin-binding proteins. On the basis of these results we suggest that the two proteins may function as the binding domain(s) of the cellular receptor for melanotropins, or may represent entire receptor moieties themselves.

Cytochemical evidence for different routes of gonadotropin-releasing hormone processing by large gonadotropes and granulosa cells.

The route and rate of internalization of GnRH were compared in studies of dispersed ovarian granulosa cells and large pituitary gonadotropes from fractions enriched by centrifugal elutriation. GnRH receptors were localized with the use of a biotinylated [D-Lys6]GnRH analog, followed by avidin gold or avidin-biotin-peroxidase complex stains. Both target cell types bound the [biotinyl-D-Lys6]GnRH (Bio-GnRH) in 1 min, and there were multiple patches of label on microvilli and coated or uncoated pits by 3 min. Quantification of the avidin-gold stains showed a significant increase in the number of labeled sites per cell profile at 3 min, followed by a decrease 15 min after exposure. No staining was seen in cells treated with medium only or with Bio-GnRH competing with a 100-fold excess of unlabeled [D-Lys6]GnRH. Internalization of the Bio-GnRH occurred during the first 3 min in both target cell types. However, the initial sites of processing appeared to be different. In granulosa cells, label was in vesicles and receptosomes (endosomes) and a few small multivesicular bodies. No stain was seen in the Golgi region for at least 15 min, at which time the stain was of low intensity. At later times (15-30 min), most of the label appeared in large multivesicular bodies. In contrast, gonadotropes exhibited labeling in Golgi complex cisternae, condensing vesicles, and immature granules as early as 3 min after exposure. Label was also seen on a subpopulation of granules in the cytoplasm and in a few multivesicular bodies. These comparative studies of two different target cells suggest that whereas the rates of internalization of GnRH are similar, the initial sites of processing may be different. Granulosa cells may degrade or separate the ligand from its receptor in multivesicular bodies. Large pituitary gonadotropes appear to use the Golgi complex route, and the processing may be associated with the formation of granules. The staining pattern correlates with early immunocytochemical studies that showed staining for GnRH on gonadotrope granules. We hypothesize that the granules may be sites for degradation of the ligand, separation of the ligand from its receptor, recycling of the receptor to the plasma membrane, or all three.

Endothelin-1 as a luteinization inhibitor: inhibition of rat granulosa cell progesterone accumulation via selective modulation of key steroidogenic steps affecting both progesterone formation and degradation.

Endothelin (ET)-1, is a 21 amino acid vasoactive peptide subject to regulation by cellular oxygen tension. However, an increasing body of information now suggests that ET-1 is a multifunctional peptidergic regulator the actions of which are not limited to the vascular system. Although ET-1 has been shown to inhibit the gonadotropin-supported accumulation of progesterone by cultured granulosa cells, the precise cellular mechanism(s) involved remain unknown. It was therefore the objective of this study to examine in greater detail the effects of ET-1 on progestin economy in cultured granulosa cells from immature rats. Treatment with ET-1 was inhibitory to the FSH-supported accumulation of progesterone in a dose-dependent manner, an action characterized by a median inhibitory dose of 2 x 10(-11) M and a maximal inhibitory effect of 90%. This inhibitory action of ET-1 was reversible following extensive washing and could not be accounted for by a decrease in the viable cell mass. Evaluation of the activities of progesterone-forming enzymes revealed ET-1 to be a potent (P < 0.01) inhibitor of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase (HSD)/isomerase (76.1 +/- 1.2% and 47.3 +/- 8.6% inhibition, respectively). Cellular radiolabeling with [3H]pregnenolone confirmed an ET-1-induced inhibition of the FSH-supported accumulation of radiolabeled progesterone. However, this effect was concomitant with enhancement of the accumulation of more distal metabolites, i.e. 20 alpha-dihydroprogesterone, 5 alpha-pregnane-3 alpha, 20 alpha-diol, and 5 alpha-pregnane-3 alpha-ol-20-one. Analysis of the FSH-supported activities of the progesterone-degrading enzymes revealed ET-1 as a potent (P < 0.05) stimulator of 20 alpha-HSD and 5 alpha-reductase (3.6 +/- 1.0 and 1.7 +/- 0.3-fold, stimulation respectively). In contrast, no significant changes were observed in 3 alpha-HSD activity. Taken together, our findings demonstrate that the ET-1 induced inhibition of gonadotropin-supported progesterone accumulation constitutes a complex phenomenon wherein ET-1 inhibits the activities of steroidogenic enzymes concerned with progesterone formation while enhancing the activities of enzymes concerned with progesterone degradation. We speculate that ET-1, possibly of intraovarian origin, acts as a luteinization-inhibitor to suppress premature luteinization at a time when continued preovulatory expression of ET-1 (in the intact but not ruptured follicle) may be contingent upon relative intrafollicular hypoxia.

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.

Isolation and identification of neuroendocrine peptides from milk.

Hormone, hormone-like substances, and neuroendocrine peptides are natural constituents of milk, and they may have an important physiological and pharmacological role in neonate development. The concentration of these peptides in milk greatly exceeds those in serum and implies an active concentration mechanism in the mammary gland. The large quantities in which milk can be supplied and the development of highly efficient procedures for the purification of peptides suggest that milk may prove to be an excellent source for identifying as yet "unknown" hormones and neuroendocrine peptides.

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.