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Biomedical subjects

A Barnea

Publications and source records attributed to A Barnea.

At least 91 records · Page 5Linked to original sources
PubMed1982

Copper stimulates the release of luteinizing hormone releasing hormone from isolated hypothalamic granules.

Copper, administered to female rabbits, stimulates LHRH release into the hypophyseal portal blood (5) and induces ovulation (1-3). To gain further understanding of the mechanism of action of copper, in this study we addressed the question: Does copper, in the form of CuATP, stimulate LHRH release from isolated hypothalamic granules? Isolated hypothalamic granules, obtained from adult male rats, were incubated under in vitro conditions in the absence (control) or presence of CuATP (0.1 - 2.5 mM). In the presence of CuATP, we noted that LHRH release was stimulated, and the magnitude of stimulation was a saturable function of the concentration of copper, being maximal at 2.5 mM. In addition, we compared the effects of a series of divalent cations (Cu, Zn, Mg, Ca, Fe, Ba, Sr, Mn; 2.5 mM each) on LHRH release and found that copper stimulated release seventeenfold, zinc--sixfold, and the other divalent cations--twofold or less. Thus, copper appears to be a unique releaser of LHRH, and it may act at the level of the LHRH granule.

Adenosine Triphosphate↗
PubMed1982

Evidence that lectin-binding sites are present on the surface of isolated hypothalamic granules containing luteinizing hormone-releasing hormone.

In the present study, we wished to ascertain if lectin-binding sites are present on hypothalamic granules containing luteinizing hormone-releasing hormone (LHRH). LHRH granules (isolated from homogenates of hypothalami of adult male rats by means of hypo-osmotic shock and differential centrifugation) were subjected to affinity chromatography on columns of concanavalin A (Con A)-Sepharose. The amount of LHRH present in the unbound and bound fraction was measured by radioimmunoassay. Of the total LHRH present in the granule suspension chromatographed, 30 to 40% was bound to the columns, and this binding was not related to the binding of the bulk to the proteins. Synthetic LHRH by itself or synthetic LHRH added to the LHRH granules before chromatography did not bind to Con A-Sepharose, indicating that only LHRH contained within granules is retained on the columns. In addition, we found that the binding of LHRH granules to Con A-Sepharose has a requirement for Ca2+ and Mn2+: after equilibration of Con A-Sepharose with Ca2+ and Mn2+ (prior to the chromatography), all of the LHRH granules were bound to the columns, and this binding was prevented by EDTA. To examine the specificity of the binding of LHRH granules to Con A-Sepharose, a competing sugar (alpha-methyl-D-mannoside (alpha-MM)) was added to the LHRH granules, and the columns were equilibrated with alpha-MM prior to chromatography. Under this condition, the Ca2+- and Mn2+-dependent binding of LHRH granules to Con A-Sepharose was inhibited. In addition to Con A-Sepharose, we observed that LHRH granules bind to wheat germ agglutinin-Sepharose but not to Sepharose which does not contain a lectin. These findings are indicative that lectin-binding sites, carbohydrate in nature, are present on the surface of isolated LHRH granules. We propose that such binding sites may play a role in the release of LHRH from hypothalamic neurons.

Animals↗
PubMed1981

Distribution, subcellular localization and identity of immunoreactive alpha-melanotropin in the pituitary gland and brain.

In homogenates of adult human or rat hypothalamic tissue, immunoreactive alpha-MSH (alpha-MSHi) is concentrated in synaptosomes, suggesting that it is localized in neurons. This conclusion is supported by immunohistochemical results on the localization of alpha-MSHi in the brain. The function of alpha-MSHi in the hypothalamus is undefined but the amount of alpha-MSH in this tissue increases in young rats and decreases in old rats. There is little alpha-MSHi in pituitary glands from human abortuses before the 15th gestational week. Thereafter the amount of alpha-MSHi in the anterior and neurointermediate lobes increases appreciably. When acetic acid extracts of each lobe were analysed by high-pressure liquid chromatography the alpha-MSHi in extracts of the anterior lobe had a retention time equal to that of desacetyl alpha-MSH; little if any alpha-MSHi had a retention time identical to that of desacetyl alpha-MSH; alpha-MSH, if present in these extracts, appeared to be a minor component. A similar analysis of extracts of adult human hypothalamic tissue yielded results suggesting that the alpha-MSHi in this tissue is also attributable largely to desacetyl alpha-MSH.

Adult↗
PubMed1981

Characterization of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in human brain tissue.

Adult human hypothalamic tissue was analyzed for the presence of products generated by post-cleavage processing of ACTH 1-13. The displacement curve generated by immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in extracts (5 M acetic acid or acidified ethanol) of adult human hypothalamic tissue was parallel to the alpha-MSH radioimmunoassay standard curve, and alpha-MSHi eluted as a single peak on Sephadex G-10, G-25, and G-50 columns in a manner identical to that of synthetic alpha-MSH. The alpha-MSHi was clearly distinguishable in its mobility on Sephadex G-50 columns from such peptides as beta-MSH, luteinizing hormone releasing hormone, and ACTH. After high performance liquid chromatography of extracts of hypothalamic tissue from young as well as aged men and women, we found that the major peak of alpha-MSHi (75-95% of total alpha-MSHi) coeluted with desacetyl alpha-MSH (ACTH 1-13 amide) rather than alpha-MSH. We suggest that desacetyl alpha-MSH, rather than alpha-MSH, is the predominant alpha-MSHi in adult human brain tissue.

Adrenocorticotropic Hormone↗
PubMed1981

A model system for the study of the release of luteinizing hormone-releasing hormone from isolated storage granules.

We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40-160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KCl resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.

Adenosine Triphosphate↗
PubMed1981

Disproportionate accumulation of immunoreactive corticotropin, melanotropin, and lipotropin in the brain of the maturing rat.

The accumulation of immunoreactive ACTH (ACTHi), alpha MSH (alpha MSHi), and gamma-lipotropin (gamma LPHi) as a function of age (10-120 days) was determined in three regions of the brain of male rats: the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), and the thalamus. In each region of the brain, the concentrations of ACTHi, alpha MSHi, and gamma LPHi increased with age. In the MBH, the increase occurred in such a manner that the molar ratio of alpha MSHi to ACTHi remained constant regardless of the age of the animals. In contrast, in the POA and thalamus, the increase occurred disproportionately in favor of alpha MSHi, and thus the molar ratio of alpha MSHi to ACTHi was 3 times higher in the adult (120 days old) than in the young (10 or 21 days old) animals. Nevertheless, the ratio of (ACTHi plus alpha MSHi) to gamma LPHi was constant at a level of about 2 regardless of the age of the animal or the region of the brain. Extracts of the MBH or POA were fractionated on columns of Sephadex G-75 superfine. The gel filtration profiles of ACTHi were indicative of the presence of five molecular weight forms of ACTH: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. We tentatively identified greater than 40K ACTH as a large form of proopiocortin, 30-40K ACTH as proopiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K as glycosylated ACTH(1-39), and 4.5K ACTH as ACTH-(1-39). Regardless of the age of the animals, the fractional amount of 30-40K ACTH the age of the animals, the fractional amount of 30-40K ACTH was high in the MBH compared to that in the POA. Moreover, the small fractional amount of 30-40K ACTH in the POA was associated with a large fractional amount of small molecular weight forms of ACTHi. However, the predominant form of ACTHi in the POA changed with age: 20-30K ACTH was the major form in the young, whereas 4.5K ACTH was the major form in the adult. These results support the proposal that the production of proopiocortin increases with age, and there is enhanced processing of proopiocortin to ACTH-(1-39) and alpha MSH in the brain of the maturing rat.

Adrenocorticotropic Hormone↗
PubMed1981

The major immunoreactive alpha-melanocyte-stimulating hormone (alpha MSH)-like substance found in human fetal pituitary tissue is not alpha MSH but may be desacetyl alpha MSH (adrenocorticotropin1-13NH2).

Pituitary glands were obtained from human abortuses during the second half of gestation. Acid extracts were made from the anterior and neurointermediate lobes, and alpha MSH immunoreactivity (alpha MSHi) was quantified by RIA. alpha MSHi was found in both lobes of the pituitary gland, with 20-80% of the total pituitary alpha MSHi being present in extracts of the anterior lobe. Anterior and neurointermediate lobe extracts subjected to gel filtration on Sephadex G-50 revealed one peak of alpha MSHi having an elution profile identical to those of alpha MSH and desacetyl alpha MSH (ACTH1-13NH2). To characterize further the alpha MSHi extracts were subjected to high pressure liquid chromatography. No alpha MSH could be identified in extracts of the anterior lobe, and most of the alpha MSHi had an elution profile identical to that of desacetyl alpha MSH. Although small amounts of alpha MSH might be present in the neurointermediate lobe, most of the alpha MSHi in this lobe coeluted with desacetyl alpha MSH. Since alpha MSH was not converted to desacetyl alpha MSH during the extraction and chromatographic procedures, we hypothesize that the predominant form of alpha MSH-like material in the human fetal pituitary gland may be desacetyl alpha MSH.

Adrenocorticotropic Hormone↗
PubMed1979

Release of immunoreactive alpha-MSH by synaptosome-enriched fractions of homogenates of hypothalami.

Immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be concentrated in a synaptosome-enriched fraction prepared by differential centrifugation of rat hypothalamic homogenates. The release of the hormone from this preparation was investigated. After incubation, the synaptosomes were isolated by ultrafiltration and alpha-MSH in the ultrafiltrate was determined by radioimmunoassay. Particle-bound alpha-MSH, recovered by extraction with acid ethanol, and alpha-MSH released from the synaptosome preparation, were immunologically similar to synthetic alpha-MSH and had an accompanying melanotropic activity. Less than 10% of the particle-bound alpha-MSH was released during incubation in 0.32 M sucrose. However, in the presence of 2 mM Ca2+, alpha-MSH release increased with increasing concentrations (30-150 mM) of K+. The stimulatory effect of 60 mM K+ was complete within 2 min and was potentiated by increasing Ca2+ concentrations over the range of 0 to 2 mM. K+-induced release of alpha-MSH was independent of temperature from 1 to 30 degrees C, and neither glucose (10 mM) nor dopamine (10(-10)-10(-2) M) had any effect on the release of the peptide. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains a releasable pool of immunoreactive alpha-MSH that is mobilized by depolarizing concentrations of K+ in a Ca2+-dependent manner.

Animals↗
PubMed1978

Studies on the subsynaptosomal localization of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the rat hypothalamus.

In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal particles which banded at sucrose densities between 0.6-1.0 M. Electron micorscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with dense-cored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles.

Animals↗