Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures

The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15-30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl-3H]thymidine or [5,6-3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37 degrees C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl-3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr. The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4-10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied. Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures.     

Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor.

A primary culture system of nearly pure neuronal cells from 14-day-old fetal rat spinal cord has been developed by combining a preplating step, the use of a chemically defined serum-free medium, and borated polylysine-coated dishes that prevented the formation of cell aggregates. About 98% of the cells were found to be immunostained with neuron-specific enolase antibodies, confirming their neuronal nature. The cultures are composed essentially of a population of non-motoneurons and contain few motoneurons, characterized by their large size and multipolar aspect, the presence of acetylcholinesterase (AChE), and the intense immunoreaction for growth-associated protein GAP-43. Neuronal precursor cells are also present in these cultures and proliferate during the first 3 days. The addition of bovine brain basic fibroblast growth factor (bFGF) stimulates their proliferation over a period of 2 days, as determined by measurement of [125I]iododeoxyuridine incorporation and by immunocytochemical reaction after bromodeoxyuridine incorporation into nuclei. The proliferating cells were characterized as neurons by immunostaining against neuron-specific enolase. Recombinant human bFGF and bovine brain acidic FGF (aFGF) exerted similar effects. Other growth factors, including epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-beta 1), and thrombin, were without effect on the proliferative activity of these neuronal cells. bFGF has no effect on the survival of motoneurons and on the fiber outgrowth of the whole neuronal population. However, bFGF affects the development of bipolar AChE-positive neurons, probably belonging to the non-motoneuron population. The data indicate that bFGF and aFGF are mitogens for neuroblasts from rat spinal cord in culture and that bFGF influences the development of a subpopulation of spinal neurons that are AChE-positive.     

An interaction between thyroid hormone and nerve growth factor in the regulation of choline acetyltransferase activity in neuronal cultures, derived from the septal-diagonal band region of the embryonic rat brain

Culture conditions have been established for growing neurons from the medial frontal part of the forebrain, containing the septum and the diagonal band of Broca, of 17-day-old rat embryos in a chemically defined medium. At 10 days in vitro, the cultures contained more than 96% nerve cells of which about 18% were cholinergic neurons, while the proportion of astrocytes was less than 1%. The majority of the cells that stained for acetylcholinesterase were bipolar but with different sizes and shapes. During development both the specific activity of choline acetyltransferase (ChAT) and the amount of protein increased markedly in the cholinergic cultures, ChAT activity rising much more than the protein content. Exposure of the cultures to nerve growth factor (NGF) or 3,3',5-triiodo-L-thyronine (T3) enhanced the expression of ChAT activity in a dose-dependent manner. The elevation of ChAT activity was due to an increase in the amount of enzyme per cholinergic cell, since, during the experimental period studied, neither treatment with NGF nor with T3 had significant effects on the total protein content of the cultures or on the number of cells, including the cholinergic neurons. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT activity was much greater than the sum of the individual effects. The observations indicate that subcortical cholinergic neurons, which are affected in Alzheimer's disease and in Down's syndrome, are subject to regulation by an interaction between thyroid hormone and local humoral factors such as NGF.     

Regulation of the system x(C)- cystine/glutamate exchanger by intracellular glutathione levels in rat astrocyte primary cultures

The system x(C)- (Sx(C)-) transporter functions to mediate the exchange of extracellular cystine (L-Cys(2)) and intracellular glutamate (L-Glu). Internalized L-Cys(2) serves as a rate-limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L-Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl-cAMP) and GSH levels on the expression of Sx(C)- were investigated in primary rat astrocyte cultures. Sx(C)- activity in dbcAMP-treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (～threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx(C)- triggered by GSH depletion was only observed in the dbcAMP-treated phenotype and was distinct from the Nrf2-mediated response initiated by exposure to electrophiles. Changes in Sx(C)- activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx(C)- heterodimer, an increase in the V(max) for L-Glu uptake and was linked temporally to GSH levels. This induction of Sx(C)- was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co-administration of the cell-permeant thiols GSH-ethyl ester and N-acetylcysteine. These findings demonstrate that the expression of Sx(C)- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx(C)- activity in CNS injury and disease.     

Arachidonic acid cycloxygenase and lipoxygenase pathways are differently activated by platelet activating factor and the calcium-ionophore A23187 in a primary culture of astroglial cells.

The aim of our study has been to investigate the metabolism of endogenous arachidonic acid or that of radiolabeled arachidonate in astroglial cells, stimulated with platelet activating factor (PAF) and with the calcium-ionphore A23187. Primary cultures of astroglial cells were obtained from brain cortex of one-day-old rats and were characterized by immunofluorescent staining vs glial fibrillary acidic protein. In labeled cells, diacylglycerol was formed after stimulation with platelet activating factor, whereas mainly the release of labeled arachidonic acid from phospholipids was observed after stimulation with calcium-ionophore. Both PAF and the calcium-ionophore A23187 actively stimulated the formation of the cycloxygenase products PGD2, TXB2 and 6-keto-PGF1 alpha, measured by radio- or enzyme-immunoassay. Differences were observed, instead, in the formation of the lipoxygenase metabolites, the hydroxyeicosateraenoic acids, which were measured by high pressure liquid chromatography (HPLC) with on line radiodetection for the labeled products, and Leukotriene C4, measured by radioimmunoassay. The formation of hydroxyacids by stimulated cells was confirmed by gas chromatography-mass spectrometry (GC-MS). In labeled cells, both agonists induced the formation of 12- and 15-hydroxyeicosatetraenoic acids, whereas stimulation of unlabeled cells with calcium ionophore resulted in formation of 12-hydroxyeicosatetraenoic acid and Leukotriene C4. Our results suggest that in astroglial cells, PAF, a compound which is produced in several tissues including brain, mobilizes a selected arachidonic acid pool, possibly associated with diacylglycerol production, from phospholipids, thus activating the conversion of the released fatty acid via the cyclo and the 12-lipoxygenase pathways.     

Effects of the conformationally restricted GABA analogues,Cis‐and Trans‐4‐aminocrotonic acid,on GABA neurotransmission in primary neuronal cultures

The effects of the GABA analogues, cis‐ and trans‐4‐aminocrotonic acid (ACA) on GABA_A receptor function and GABA uptake, together with the presence of ρ‐1 subunit mRNA and putative GABA_C receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl^‐ influx, which was inhibited by bicuculline, t‐butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and γ‐hexachlorocyclohexane (γ‐HCH or lindane). [³H]‐flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the trans‐isomer completely inhibited the [³H]GABA uptake, whereas the cis‐isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABA_C receptors was investigated only in neocortical cultures by using RT‐PCR in order to detect the presence of the mRNA encoding the ρ‐1 subunit which assembles to form homooligomeric Cl^‐ channels. The results presented here show that ρ‐1 subunits, and thus GABA_C receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA_A receptors, although with very different potencies. J. Neurosci. Res. 57:95–105, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, beta-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area

The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or beta-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEB) (5 micrograms) and progesterone (500 micrograms). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or beta-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of beta-endorphin, neither the opiate antagonist nor anti-beta-endorphin-gamma-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of beta-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-beta-endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-gamma-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, β-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area

The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or β-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEP) (5 μg) and progesterone (500 μg). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or β-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of β-endorphin, neither the opiate antagonist nor anti-β-endorphin-γ-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of β-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-β-endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-γ-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA. Conversely, naloxone, anti-β-endorphin-γ-globulin, anti-CRF-γ-globulin and ACTH infused into the MPOA produced high levels of lordosis in female rats normally showing low levels of lordosis by treatment with low doses of OEB (2 μg) and normal doses of progesterone (500 μg). In each case the facilitation could be blocked by pretreatment of the MPOA site with a potent antagonist analogue of LH-RH. The results indicate the key role of LH-RH in the action of endogenous CRF, β-endorphin and ACTH in the regulation of lordosis behaviour. Each of these substances may act directly on the LH-RH neurone. The postulated presynaptic relationship between CRF and β-endorphin neuronal systems that seem to exist in the mediobasal hypothalamus and the central gray may be less predominant in the MPOA. The results, however, provide clear further evidence for the potent effects of CRF and opiocortin peptides in the regulation of LH-RH and reproduction.     

Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP

Introduction

CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation.

Methods

The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study.

Results

CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B₂ (TxB₂) and hydroxyl radical (OH^●) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB₂ formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation.

Conclusion

This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and MAPKs), ultimately inhibiting platelet activation. This novel role of CME-1 indicates that CME-1 exhibits high potential for application in treating and preventing CVDs.     

Ascending components of the medial forebrain bundle from the lower brain stem in the rat, with special reference to raphe and catecholamine cell groups. A study by the HRP method

The afferent connections of the medial forebrain bundle (MFB) arising from the lower brain stem have been investigated by means of horseradish peroxidase (HRP) with sensitive substrate. The injection was made iontophoretically into MFB at various levels.After injection of HRP into MFB, a significant number of HRP-labeled neurons were observed in the following structures of the lower brain stem: (1) raphe nuclear group, (2) locus coeruleus, (3) n. laterodorsalis tegmenti, (4) parabrachial area, (5) A₁, A₂, A₄, A₅ and A₇ areas where noradrenaline-containing neurons were disseminated, (6) A₈, A₉ and A₁₀ areas which contain dopamine neurons, (7) surrounding area of the fasciculus longitudinalis medialis at the level of the n. prepositus hypoglossi, (8) n. prepositus hypoglossi and (9) mesencephalic gray matter. As a rule, the ascending projections are ipsilateral and course in the medial part of MFB.Regarding the raphe nuclei, we have demonstrated that the caudal raphe nuclei, such as n. raphe magnus and obscurus (but not n. raphe pallidus), also send their axons to the hypothalamus. Particularly, the axons of n. raphe magnus ascend in MFB to reach the level of the preoptic or anterior septal area. Furthermore, in accordance with previous reports, HRP-labeled cells were also identified in the n. raphe dorsalis, centralis superior and pontis, respectively. It should be further noted that labeled cells appeared in the n. linearis caudalis.In addition, the present study indicates a number of non-aminergic cell groups as sources of ascending MFB fibers.On the whole, the present study further clarified the organization of the components of the MFB ascending from the lower brain stem, and provided some additional anatomical substrates for the physiology of the control of the forebrain by the lower brain stem neurons.