Rabbit blastocysts accumulate [3H]quinuclidinyl benzilate in vitro

Day-6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50% in the presence of 1.5 x 10(-4) M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day-6 blastocysts accumulated 30.3 +/- 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some roll in blastocyst-maternal recognition.     

Inhibitory effects of danazol and medroxyprogesterone acetate on [3H]thymidine incorporation in human endometrial cancer cells

Effects of medroxyprogesterone acetate (MPA) and danazol (1 nM-10 microM) on cultured cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were simultaneously investigated. Of twenty-four endometrial adenocarcinomas examined, five tumors were successfully maintained in primary cell culture. The addition of MPA as well as danazol in culture of cells from the five tumors resulted in a significant inhibition of [3H]thymidine incorporation in cancer cells from three tumors having progesterone receptors (PR). The minimum effective concentrations of MPA and danazol for the inhibition of [3H]thymidine incorporation were found to be 10 and 100 nM, respectively. The difference in effective concentration could be explained by a higher affinity of MPA to PR than that of danazol in cancer cells. On the other hand, neither danazol nor MPA affected [3H]thymidine incorporation in cultured cells from the remaining two tumors, in which PR was absent in one but present in the other. These findings, together with our previous findings that danazol inhibited the growth of a human endometrial cancer cell line with PR, suggest that a growth-inhibitory effect of danazol on human endometrial cancer cells is mediated through PR in the cells.     

Metabolism of glycoconjugates in hypothalamic neurons and glial cells: Comparison of incorporation of [3H]fucose and [3H]N-acetylmannosamine by electron microscopic autoradiography

Summary Incorporation of two glycoconjugate precursors, [³H]fucose and [³H]N-acetylmannosamine, in the arcuate nucleus of the rat hypothalamus is compared 30 min after intraventricular administration. Electron microscopic autoradiographs were analyzed by a sampling technique which provides information about (i) the distribution of radioactivity in the tissue, (ii) the relative volumes, and (iii) the amount of radioactivity per unit volume of the various tissue compartments. With both precursors, the highest incorporation was found in glial cell bodies, being about five to six times that of neurons. This holds true for all three types of glia. The concentration of radioactivity in the neuropil consisting of neuronal and glial processes was exceedingly low, although, due to its large volume, it contained the highest fraction of total tissue radioactivity. The limitations imposed on the interpretation of the data in terms of synthesis of fucosyland sialoglycoconjugates are discussed. It is proposed that the observations support the concept that high rates of synthesis for glycoproteins (and possibly gangliosides) are a characteristic metabolic property of glial cells.Dedicated to Prof. Emil Tonutti on the occasion of his 70th birthday     

The binding of [3H]oestradiol-receptor complex to hypothalamic chromatin of male and female mice

Histones and masking acidic proteins were removed from hypothalamic chromatin in order to evaluate/measure the number of available acceptor sites for the [3H]oestradiol-receptor complex. This number increases after dehistonizing and unmasking and is lower than published values for comparable preparations. No sex-related difference in [3H]oestradiol-receptor binding to hypothalamic chromatin in vitro was observed. Failure to observe such a difference suggests that sexual differentiation and steroid sensitivity cannot be attributed to marked differences in the degree of chromatin masking.     

Medroxyprogesterone acetate, aggression, and sexual behavior in male cynomolgus monkeys (Macaca fascicularis).

Medroxyprogesterone acetate (MPA) is used clinically to treat male sex offenders, but there are conflicting reports about its effects on aggression. To investigate these matters in a nonhuman primate, four intact male cynomolgus monkeys were studied in a testing paradigm that involved the presence of a caged, aggression-arousing stimulus male either immediately before or during a pair-test with an ovariectomized, untreated female partner. After two 4-week periods of pretreatment baseline, males received weekly injections of 40 mg MPA either alone (two 4-week treatment periods) or in combination with testosterone replacement with sc implants (one period) and additional daily injections of 2 mg testosterone propionate (two periods). MPA was then withdrawn while testosterone replacement continued (three periods). The testing paradigm was effective in maintaining aggression, especially male-male aggression, for many months. Male-male aggression increased with MPA treatment, and increased further with testosterone replacement, whereas male-female aggression tended to change in the opposite direction. As in earlier studies, MPA decreased both plasma testosterone and male sexual activity, but restoring plasma testosterone levels in treated males failed to restore their sexual activity. MPA therefore has behavioral effects that are not mediated primarily by its suppression of circulating androgens.     

Evidence for morphine-induced galactorrhea in male cynomolgus monkeys

We evaluated the effect of a daily injection of morphine hydrochloride on galactorrhea in male cynomolgus monkeys. Three groups of three monkeys (nine total) were used. The treatment schedule was separated into three periods: pre-treatment, treatment, and post-treatment. Each group of monkeys was subcutaneously injected daily with 1.5, 3.0, or 6.0 mg/kg (monkey weight) of morphine for 74–130 days, respectively, during the treatment period, and with saline during the pre-treatment and post-treatment periods. We then measured the prolactin (PRL) and testosterone (T) levels from weekly blood samples that were taken 20 hours after injection. No statistically significant differences in either the PRL or T level were detected throughout the treatment period. However, monkeys treated with 3.0 and 6.0 mg/kg/day showed a decrease in T level and an increase in PRL level during the early post-treatment period. Seven of the nine monkeys produced a milk-like secretion from their mammary gland (a symptom of galactorrhea) during the treatment and post-treatment periods. For several months of post-treatment period (average 6.75 months), we monitored the time-course changes in PRL and T levels in all monkeys for 10 hours after a single injection of morphine at the same dose given during the treatment period. Morphine induced a sudden increase in the PRL level (peaked within 30 minutes) and a gradual decrease in the T level (leveled off within 6.5–10 hours), and then returned to basal levels. These results indicate that morphine does not cause a long-term effect on hormonal changes and that a morphine-induced transient rise in PRL levels accompanied by a decrease in T levels can induce spontaneous galactorrhea in male cynomolgus monkeys.     

Axonal transport of [3H] GABA and [3H] glutamate in excitatory and inhibitory neurons innervating lobster exoskeletal musculature

This paper describes the results of intracellular injections of radiolabelled neurotransmitters and transmitter precursor substances, including glutamate, GABA, aspartate, octopamine, tyramine, tryptophan, and choline, into cell bodies of identified excitatory and inhibitory neurons innervating lobster extensor musculature. The distributions and identities of radioactive substances appearing in axons were examined at various times following injection and in vitro incubation. Injected GABA and glutamate were found in appreciable quantities in both excitatory and inhibitory axons and migrated down axons at an estimated rate of between 16 and 22 mm/day at 12 degrees C, whereas the other substances tested were present in substantially smaller quantities and migrated at an estimated rate of less than 7.5 mm/day at 12 degrees C. Injected GABA, D-glutamate and L-glutamate accumulated proximal to ligatures tied around nerves, whereas neither octopamine nor aspartate accumulated proximal to ligatures. Since GABA is the transmitter substance released by inhibitory neurons and L-glutamate is thought to be released from excitatory nerve terminals, these results are consistent with the suggestion that amino acids serving as neurotransmitters are axonally transported. The specificity of axonal transport does not appear to be restricted to the cognate neurotransmitter, as indicated by the movement of L-glutamate in inhibitory axons and GABA in excitatory axons and of D-glutamate in both excitatory and inhibitory axons, but rather may be relaxed to include substances closely related to the neurotransmitter. Some restrictions, however, are apparently placed on axonal transport of small charged molecules in these neurons in that other substances tested migrated down nerves at a considerably slower rate.     

Heat-induced changes in the incorporation of [H3]acetate in membrane lipids

The effects of heat treatments at temperatures from 42 to 47 degrees C on the rate of incorporation of [3H]acetate into different classes of lipids have been studied in V-79 Chinese hamster cells. Thermotolerance induction and subtoxic heat treatments decreased the incorporation of [3H]acetate into phospholipids and caused the ratio [3H]cholesterol/[3H]phospholipids to increase several fold, and a positive correlation between heat dose and the ratio [3H]cholesterol/[3H]phospholipids was obtained for subtoxic hyperthermic treatments. The duration of this hyperthermic effect on the incorporation of [3H]acetate into the different lipid fractions was followed in pulse-label experiments. The highest increase of the ratio [3H]cholesterol/[3H]phospholipids was obtained during the first 24 h, but a significant elevation was also present for the 24-72 h pulse-labelled group. Thermotolerance induction was maximal 24 h after the heat treatment and then declined during the next 24 h. The increased [3H]cholesterol/[3H]phospholipid ratio observed in response to hyperthermia resembles the processes that serve to provide homeoviscous adaptation to sustain thermosensitive membrane-located functional groups, in analogy with the mechanisms responsible for thermal adaptation. However, the lack of a positive correlation between thermotolerance induction and the changes in lipid synthesis, for the whole time interval studied, remains to be further explored before any mechanistic interpretation of the data can be found.     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Effects of social reorganization on cellular immunity in male cynomolgus monkeys

Exposure to acute stressors has been shown to impair cellular immunity in human beings and other animal species. Comparatively little is known, however, about the effects of long-term stressors on immune function and how individual behavioral characteristics may mediate differences in immune function and clinical disease susceptibility. To determine the effects of social stress on cellular immunity and reactivation of a latent herpesvirus, 20 Herpes B virus-positive male cynomolgus monkeys were exposed to four periodic reorganizations of social group memberships over 5 months. Observations were made to categorize individuals as high or low in expression of aggressive, fearful, and affiliative behaviors. Complete blood counts, lymphocyte proliferation tests, and natural killer cell cytotoxicity assays were performed immediately before and 4 days after reorganizations. Herpesvirus-specific immunoglobulin G antibody levels were measured, and oral and conjunctival swabs were cultured for virus. Reorganization was associated with increased lymphocyte counts (P = 0.0009) and decreased lymphocyte proliferation in response to phytohemagglutinin (P < 0.005), particularly among monkeys showing high levels of fear (P = 0.0137). High-aggressive monkeys showed lower baseline natural killer cell activity (P = 0.0013) and higher lymphocyte counts (P = 0.013) than low-aggressive monkeys. Herpesvirus antibody titers decreased over time (P < 0.004) and no positive virus cultures were obtained. Measures of cellular immunity and behavior were unrelated to virus-specific antibody titers. These results suggest that repeated exposure to a social stressor alters several measures of cellular immunity, and that some of these changes may be predicted by individual differences in agonistic behavior. In contrast to human studies, the results suggest that some psychological stressors may not cause reactivation of a common herpesvirus in this species. © 1996 Wiley-Liss, Inc.     

TTX displacement of [H3]-nitrendipine binding in developing spinal cord neurons

[3H] Nitrendipine binding was partially blocked by the presence of tetrodotoxin in developing spinal cord neurons. In young cultures, 1 micron tetrodotoxin displaced 29% and 26% of [3H] nitrendipine binding from the high and low affinity binding sites, respectively. In one month old cultures, tetrodotoxin had no effect on [3H] nitrendipine binding. The interaction between tetrodotoxin and nitrendipine in young cultures suggests ligand binding site similarities during development.     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Distribution of [3H]noradrenaline and [3H]serotonin in photophores of Porichthys notatus

Summary Photophores of Porichthys notatus were examined by electron-microscopic radioautography following incubation in tritiated noradrenaline ([³H]NA) or serotonin ([³H]5-HT). Nerve varicosities surrounding the photocytes were found to accumulate [³H]NA but not [³H]5-HT, providing compelling evidence for the catecholaminergic nature of the monoaminergic innervation of photophores. The photocytes themselves appeared selectively labelled with both tracers, but the intensity of labelling after [³H]5-HT incubation was considerably greater than after [³H]NA. Stereological sampling of organelle content in photocytes showed ultrastructural differences between [³H]NA- and [³H]5-HT-labelled cells, probably related to light emission induced by NA. The main changes noted after incubation with [³H]NA were mitochondrial swelling and disorganization, increased coalescence of photocytic vesicles and extrusion of vesicular material into the extracellular matrix. With respect to the subcellular localization of [³H]NA and [³H]5-HT within the photocytes, statistical analysis of the distribution of silver grains disclosed a preferential affinity of both labels for appositional zones between mitochondria and coalescent vesicles. Moreover, in the case of 5-HT, selective affinity was also exhibited by sites comprising vesicular membrane and adjacent cytoplasm, suggesting binding of this biogenic amine to the entire membrane of photocytic vesicles.Supported by grants from the Natural Sciences and Engineering Research Council (M.A.), and Medical Research Council of Canada (L.D.). Dr. Pierre Legendre kindly provided advice on statistical methods     

Influence of chlormadinone acetate on hypothalamic differentiation in male rats

The effect of chlormadinone acetate on adult male rats during the hypothalamic differentiation phase was studied. Psychic intersexuality with increased male and increased female sexual behavior was observed both before and after postpuberal castration and sex hormone replacement. Organ weights of testes, seminal vesicles, and ventral prostrates were normal, but penis and adrenal gland weights were significantly smaller. Body growth was also significantly reduced compared with control animals. The effects of chlormadinone acetate on androgen-dependent brain differentiation are discussed and compared with analogous effects of cyproterone acetate and orchidectomy.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Biochemical characterization of the uptake and release of [3H]dopamine by dopaminergic hypothalamic neurons: a developmental study using serum-free medium cultures

The development of the biochemical properties of mouse hypothalamic dopaminergic neurons has been analyzed in vivo and in cultures of cell taken on the 16th day of gestation and grown in serum-free medium for up to 3 weeks. In the course of in vivo development, the dopamine (DA) content remains low during fetal life (10% of the adult value), beginning to increase on the 19th fetal day. In contrast, the specific accumulation of [3H]DA increased markedly during the last days of gestation from 20% of the adult value on the 16th fetal day to 70-80% of the adult value on Postnatal Day 3. Hypothalamic DA neurons in culture accumulate endogenous DA although at a lower level than in vivo. They take up [3H]DA by an active transport system which is specific for DA, and which shows time, temperature, and sodium dependency (Km = 1 microM). HPLC analysis showed that the newly taken up [3H]DA was not metabolized in the short run under the conditions used. It was stored in a form that could be released when neurons were depolarized in a high K+ (60 mM) medium. The K+-evoked [3H]DA release was found to be strictly dependent on extracellular Ca2+. Moreover the release of [3H]DA was also stimulated by veratridine in a Ca2+-dependent manner. Similar data have been obtained with the release of endogenous dopamine. No specific uptake and no K+-evoked dopamine release occurred in 2-day-old cultures. The specific [3H]DA uptake and the K+-evoked release appeared in 5-day-old cultures and increased with time in culture at least until Day 15. We examined the effects on [3H]DA release of polyunsaturated fatty acid, triiodothyronine, and corticosterone, all of which have been shown to play an important role in synaptogenesis in culture. These components, either separately or together, did not modify the percentage of the basal or the stimulated [3H]DA release. These results showed that hypothalamic DA neurons grown in serum-free medium progressively acquired the functional properties of adult DA neurons as concerns DA synthesis, DA uptake, and release. From a development point of view, this study suggests that the capacity to specifically take up [3H]DA and to respond to high K+ concentration is not expressed at early stages of neuronal development.(ABSTRACT TRUNCATED AT 400 WORDS)