α-MELANOCYTE-STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN

Abstract— The amount of α-melanocyte-stimulating hormone (α-MSH) in the entire hypothalamus as well as the amount of α-MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1-24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso-osmotic sucrose solution, and free granules and synaptosomes containing α-MSH were fractionated by means of continuous sucrose density gradient centrifugation. α-MSH was quantified by radioimmunoassay. The total amount of α-MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α-MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5-24-month-old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α-MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.     

A Model System for the Study of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Storage Granules

Abstract: 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-KC1 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.     

CHARACTERIZATION OF HYPOTHALAMIC SUBCELLULAR PARTICLES CONTAINING LUTEINIZING HORMONE RELEASING HORMONE AND THYROTROPIN RELEASING HORMONE

Abstract— The 900 g supernatant fluid prepared from male rat hypothalamic homogenates was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone and luteinizing hormone releasing hormone in the gradient fractions were quantified by radioimmunoassays. TRH was associated with two populations of particles separable by means of nonequilibrium density centrifugation (100,000 g for 30min). However, after'equilibrium'centrifugation (100,000 × g for 180 min), a single peak of TRH was observed at 1.07 M-sucrose. Hypo-osmotic shock as well as treatment with 0.1% Triton X-100 or 0.1% deoxycholate (DOC) released TRH from both sets of particles. LRH, as TRH, was associated with two populations of particles which were separable by means of nonequilibrium density gradient centrifugation. After'equilibrium'centrifugation, both sets of LRH-containing particles banded at 1.27M-sucrose as a single symmetrical peak. Although 0.1% Triton X-100 released LRH from both populations of particles, hypo-osmotic shock or 0.1% DOC released LRH only from the large LRH-containing particles. The small LRH-containing particles were resistant to hypo-osmotic shock and to 0.1% DOC. Based on these criteria, it is concluded that in hypothalamic homogenates the TRH-containing particles and the large LRH-containing particles are synaptosomes. The small LRH-containing particles may be of different cellular and/or subcellular origin.     

Apparent Co-Sequestration of Immunoreactive Corticotropin, α-Melanot opin, and γ-Lipotropin in Hypothalamic Granules

Abstract: In the present study, the question of whether immunoreactive α-melanotropin (α-MSH₁), corticotropin (ACTH₁), and β-melanotropin (β-MSH₁) are co-sequestered in hypothalamic granules of adult male rats was addressed. When a 900 ×g supernatant fluid prepared from a hypothalamic homogenate was fractionated on continuous sucrose density gradients under non-equilibrium Conditions, two populations of particles containing α-MSH₁, ACTH₁, or β-MSH₁ Were observed. However, when fractionated under equilibrium conditions, the two populations of particles containing α-MSH₁ ACTH₁, or β-MSH₁ were recovered as a single band. This sedimentation characteristic indicates that the particles containing a given peptide differ in size but are similar in density. In their sedimentation, the small particles containing α-MSH₁, ACTH₁, and β-MSH₁ are indistinguishable from granules containing α-MSH₁, whereas the large particles containing α-MSH₁ (ACTH₁, and β-MSH₁ are indistinguishable from synaptosomes containing α-MSH₁, β-MSH₁ had an apparent molecular weight (M.W.) of about 5,000, which is similar to that of γ-lipotropin. ACTH₁ was comprised of three species of molecules: big (M.W. ≥ 10,000), 5.7K (M.W. ≌ 5,700), and 4.5K (M.W. ≌ 4,500). Big ACTH was the predominant and 5.7K ACTH the minor component of ACTH₁ present in granules as well as in synaptosomes. These results are suggestive that α-MSH, ACTH and its precursors, and γ-lipotropin are co-sequestered in hypothalamic granules.     

MgATP-Stimulated Release of Luteinizing Hormone Releasing Hormone from Isolated Hypothalamic Granules: Evidence for a Selective Requirement for Monovalent Cations

Abstract: The purpose of this study was to characterize the kinetics and monovalent ion requirements of the MgATP-stimulated release of Iuteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. LHRH granules were obtained from homogenates of hypothalami of adult male rats by differential centrifugation and then incubated in buffered media containing KCl. Under these conditions, the pH optimum for the MgATP-stimulated release of LHRH was between 7.5 and 8.0; the rate of release was linear for the first 5 min of incubation; and the magnitude of the release was a saturable function of KCl concentration. When the potassium in the incubation mixture was replaced with various monovalent cations (Li⁺, Na⁺, Cs⁺, choline, or tetraethylammonium), the magnitude of the MgATP-stimulated release of LHRH was inversely related to the size of the cation; release in the presence of Li⁺ was 12%, whereas release in the presence of tetraethylammonium was 0% of the total LHRH. When the chloride in the incubation mixture was replaced with various monovalent anions (Br⁻, I⁻, or isethionate), the magnitude of the MgATP-stimulated release of LHRH was not related to the size of the anion. Moreover, inclusion of anion transport inhibitors in the incubation mixture did not affect LHRH release, suggesting that anion transport is not required for the MgATP-stimulated release of LHRH. In summary, we suggest that the MgATP-stimulated release of LHRH from isolated hypothalamic granules involves an enzymatic event and that this release process does not have a selective requirement for monovalent anions, but has a selective requirement for monovalent cations.     

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism.     

Demonstration of a macromolecule cross-reacting with antibodies to luteinizing hormone releasing hormone and its tissue distribution

Hypothalamic cytosol contains a macromolecule which cross-reacts with antibodies to luteinizing hormone releasing hormone (LRH). This cross-reacting material (macro-CRM) is insoluble in methanol or acid ethanol, and its molecular weight is about 70,000. Macro-CRM is also found in cytosols of extra-hypothalamic regions of the brain, liver, kidney, spleen, and skeletal muscle. Plasma contains only marginal amounts of macro-CRM. This substance inhibits the binding of [¹²⁵I]LRH to LRH antibodies in a reversible, competitive manner.     

A SUBCELLULAR POOL OF HYPO-OSMOTICALLY RESISTANT PARTICLES CONTAINING THYROTROPIN RELEASING HORMONE, α-MELANOCYTE STIMULATING HORMONE, AND LUTEINIZING HORMONE RELEASING HORMONE IN THE RAT HYPOTHALAMUS

Abstract— A 10% homogenate of male rat hypothalami, prepared in 0.32M-sucrose-10μM-CaCl₂, was diluted either with one volume of 0.32M-sucrose-10μM-CaCl₂ (iso-osmotic) or with 10μM-CaCl₂ (hypo-osmotic). A 900 g supernatant fluid fraction (0.9 K-S) was prepared from the diluted homogenates and fractionated on continuous sucrose density gradients under non-equilibrium and equilibrium conditions. In the iso-osmotic 0.9 K-S thyrotropin releasing hormone (TRH), α-melanocyte stimulating hormone (α-MSH), and luteinizing hormone releasing hormone (LHRH) were each found to be sequestered in two populations of particles which were different in size but similar in density. In the hypo-osmotic 0.9 K-S, TRH, α-MSH, and LHRH were each found to be sequestered in a single population of particles. In their sedimentation properties (as judged by differential, non-equilibrium, and equilibrium density centrifugations), the hypo-osmotically resistant particles and the small particles present in the iso-osmotic 0.9 K-S were identical. However, in their peptide content, the two sets of particles differed from each other. If the total quantity of particle-bound peptides recovered after gradient centrifugation of the iso-osmotic 0.9 K-S is taken as 100%, one finds that the amount of TRH, α-MSH. and LHRH recovered in the small particles is 39%, 50%, and 39%. respectively, whereas the amount of TRH, a-MSH, and LHRH recovered in the hypo-osmotically resistant particles is 42%, 68%, and 67%, respectively. This increase in the quantity of peptides sequestered in the small particles occurred concomitantly with the disappearance of peptides from the large synaptosome-like particles. It is estimated that within the large synaptosome-like particles 7% of the TRH, 35% of the a-MSH, and 45% of the LHRH are associated with hypo-osmotically resistant particles. Ultrastructural analysis of purified hypo-osmotically resistant particles containing TRH. α-MSH. or LHRH revealed a predominance of membrane-bounded packets of electron-dense material.