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.

Calcium-sensitive accumulation of norepinephrine in rat cerebral cortex.

The accumulation of high and low concentrations of [3H]-norepinephrine has been examined in a crude synaptosomal preparation of rat cerebral cortex in the presence and absence of uptake1 inhibitors. When uptake1 was blocked, [3H]-norepinephrine accumulation exhibited very rapid initial rates. It was not inhibited by 10 mM normetanephrine, a potent inhibitor of peripheral uptake2, but it was inhibited by 10 mM metaraminol. This accumulation was markedly reduced when calcium ions were omitted from the incubation medium, and is named here 'calcium-sensitive accumulation' (CSA) to distinguish it functionally from the sodium-dependent, high affinity, uptake1 process. CSA may be localized in nerve endings since it was found predominantly i the synaptosomal fraction of homogenates subjected to density gradient centrifugation in sucrose or in Ficoll-in-sucrose. At high concentrations of [3H]l-norepinephrine (1.0 microM) and short incubation times, CSA accounted for most of the total accumulation of [3H]l-norepinephrine whereas uptake1 contributed only a small portion. Since extracellular concentrations of brain norepinephrine are thought to reach levels in excess of 1.0 microM, CSA may be a significant factor in noradrenergic neuronal transmission.

Two distinct events underlie the MgATP-stimulated release of luteinizing hormone releasing hormone from isolated hypothalamic granules.

We have previously demonstrated that MgATP stimulates the release of luteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules and that this stimulation has a requirement for monovalent ions. To assess the role of the monovalent ions in the release process, granules (isolated from hypothalami of adult male rats) were incubated in buffered medium containing MgATP in the presence or absence of KCl. When granules were incubated in the absence of KCl, MgATP did not lead to LHRH release. However, when KCl was added to the MgATP-treated granules, a rapid (1 min) release of LHRH occurred. Based on these results we propose that the process of MgATP-stimulated release of LHRH from isolated hypothalamic granules is comprised of two distinct events: a priming event and a release event. During the priming event, MgATP interacts with the LHRH granule which leads to a change in the granule such that the release event, initiated by monovalent ions, can occur.

Acute stress and the brain norepinephrine uptake mechanism in the rat.

The kinetic constants for norepinephrine uptake in cerebral cortical homogenates were determined in vitro immediately following an acute stress consisting of either forced immobilization, cold-wet exposure, combined cold-plus-restraint, swim stress, or electric footshock in the rat. The kinetic constants, apparent Km and Vmax, for uptake of 3H-l-norepinephrine were significantly increased only following 10 min swim at 22 degrees or following 5 min electric footshock. When severe hypothermia accompanied the stress, the findings suggested that a profound reduction in body temperature was associated with depressed responsiveness of brain noradrenergic mechanisms to stress including decreased uptake kinetic constants. In a series in which the duration of electric footshock was varied from 2 to 30 min, it was noted that the NE uptake kinetic constants were increased at 5 min, but were similar to paired controls at 2, 10 and 30 min following the onset of footshock. It was concluded that various acute stresses did not elicit a generalized response of the cortical NE uptake mechanism to stress in the rat. Furthermore, when uptake kinetic constants did change with stress, the values were often within the range of normal values seen in the rat.

Cortical glucose utilization patterns in primary degenerative dementias of the anterior and posterior type.

Regional cortical function was evaluated in 13 patients with primary degenerative dementia and six age-matched, neurologically normal control individuals. Five patients were selected on the basis of preponderant signs of posterior dementia, five had mainly signs of anterior dementia, and the remaining three evidenced mixed anterior-posterior cognitive deficits. Positron emission tomographic scans following the intravenous administration of fluorodeoxyglucose revealed a characteristic pattern of abnormalities in each group: those with posterior dementia, as primarily manifested by verbal and visuospatial deficits, had a predominant focus of hypofunction in the posterior parietal and temporal regions; those with the less common anterior dementia, evidenced by prominent personality and social behavioral changes, had their most conspicuous abnormality in the anterior frontal cortex; those with approximately equal signs of anterior and posterior dementia had abnormalities in both anterior frontal and posterior parietal-temporal areas. The results indicate that the primary degenerative dementias may reflect a selective abnormality of the frontal and parietal association corticies. The continuous distribution of cortical involvement in patients with these disorders suggests that diagnostic entities as neuropathologically distinct as Alzheimer's disease (mainly posterior involvement) and Pick's disease (mainly anterior involvement) could share a common pathophysiology. A focusing of histological and biochemical efforts in areas of major functional abnormality could hasten the elucidation of such mechanisms and the development of effective therapies.

Structural and functional studies of Gilles de la Tourette syndrome.

Preliminary studies with PET-scanner and nuclear magnetic resonnance have shown no abnormalities in Gilles de la Tourette syndrome. Estimation of homovanillic acid, the main metabolic of dopamine, in the cerebrospinal fluid of patients with Gilles de la Tourette disease suggests that brain dopaminergic receptors are hypersensitive. Such an hypersensitivity of dopamine receptors is in agreement with the clinical improvement of patients treated with neuroleptic drugs. The study with PET-scanner presented here suggests the existence of a neuronal hypofunction in some striatal and corticolimbic area in patients.

Comparison of the effects of ATP, Mg2+, and MgATP on the release of luteinizing hormone-releasing hormone from isolated hypothalamic granules.

The effect of ATP, Mg2+, or MgATP on the release of luteinizing hormone-releasing hormone (LH-RH) from hypothalamic granules was examined under in vitro conditions. Granules, isolated from adult make hypothalami, were incubated at 37 degrees C in a buffered (pH 7.8) medium containing 0.15 M-KCl. The addition of ATP to the incubation mixture did not stimulate the release of LH-RH. In contrast, the addition of MgATP stimulated the release of LH-RH, the release being 62% greater than control. The addition of Mg2+ to the incubated granules also stimulated the release of LH-RH. However, the magnitude of this Mg2+-stimulated release of LH-RH was significantly (P less than 0.01) lower than that of the MgATP-stimulated release, indicating that ATP stimulates LH-RH release in a Mg2+-dependent manner. As both MgATP and Mg2+ alone stimulated LH-RH release, we characterized further these two release processes by incubating the granules under one of the following conditions: incubation at 4 degrees C in a buffered medium containing 0.15 M-KCl or incubation at 37 degrees C in a medium that does not contain KCl. Under these two incubation conditions, the MgATP-stimulated release of LH-RH was not manifested, whereas the Mg2+-stimulated release of LH-RH was manifested. On the basis of these differences, we propose that two different processes can lead to the release of LH-RH from isolated hypothalamic granules: one process involves ATP and Mg2+ (MgATP) and another process involves Mg2+ alone.

MgATP-stimulated release of luteinizing hormone releasing hormone from isolated hypothalamic granules: evidence for a selective requirement for monovalent cations.

The purpose of this study was to characterize the kinetics and monovalent ion requirements of the MgATP-stimulated release of luteinizing 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 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.

A new clonal strain of rat pituitary tumour cells: a model for non-regulated secretion of prolactin.

A new clonal strain of Prl-secreting cells derived from the transplantable rat pituitary tumour, 7315a, has been established in culture. The cells of this strain, designed 235-1, have a highly developed Golgi complex, an extensive rough endoplasmic reticulum, and a few small but no large dense-core granules. When inoculated into athymic mice and rats of the Buffalo strain, the 235-1 cells produce tumours, and the host animals have hypertrophied mammary glands that produce milk, indicating that Prl secreted by these cells has mammotrophic activity. In monolayer culture, the doubling time of 235-1 cells is 31 +/- 1 h (mean +/- SE). The cells secrete Prl, a trace quantity of GH, but no LH, FSH, TSH, ACTH, or alpha-MSH. Prl is released at a rate of 257 +/- 12 fg per h per cell. The cellular content of Prl is 424 +/- 23 fg per cell. Prl secretion by 235-1 cells is not affected by dopaminergic agonists and antagonists, TRH, or oestradiol-17 beta but is inhibited in the presence of EGTA or monensin, an ionophore that is believed to act at the level of the Golgi complex. The subcellular distribution of Prl in 235-1 cells is different from that in rat pituitary cells. In 235-1 cells, Prl is associated not with a single set of dense particles as it is in pituitary cells but with 2 sets of subcellular particles, of which 1 set cosedimented with particles having lysosomal enzyme activity. These findings suggest that Prl secretion by 235-1 cells involves secretory pathways that are different from those seen in normal lactotrophs.

Neuroendocrine control of gonadotropin secretion.

Luteinizing hormone releasing hormone (LHRH), a hypothalmic peptide that is concentrated in granules of neurons, has the capacity to release gonadotropins (luteinizing hormone (LH) and follicle stimulating hormone) from the pituitary gland. LHRH has been found in hypophysial portal blood of rats, monkeys, and rabbits. Antibodies to LHRH depress plasma LH concentrations in castrated animals and evoke testicular atrophy, but passive immunization against LHRH does not block the LH surge induced by estrogen in monkeys. Estrogens, progestin, prolactin, and dopamine have marked effects on LH secretion, yet an association between these effects and altered hypophysial portal blood concentrations of LHRH is not established. In view of the paucity of evidence demonstrating such a cause and effect relationship, two alternative proposals have become tenable. One, hormones and neurotransmitters may not alter the levels of portal blood LHRH, but rather alter the frequency of pulsatile LHRH secretion. Two, hormones, such as estrogens, progesterone, and prolactin, may alter the responsiveness of the gonadotropin-secreting cells to LHRH by affecting the secretion of dopamine.