Thyroid hormone stimulates gamma-glutamyl transpeptidase in the developing rat cerebra and in astroglial cultures

Hypothyroidism in the developing rat brain is associated with enhanced oxidative stress, one of the earliest manifestations of which is a decline in the level of glutathione (GSH). To investigate the role of thyroid hormone (TH) on GSH homeostasis, the effect of TH on gamma-glutamyl transpeptidase (gammaGT), the key enzyme involved in the catalysis of GSH, was studied. Hypothyroidism declined the specific activity of cerebral gammaGT at all postnatal ages examined (postnatal day 1-20) with a maximum inhibition of 42% at postnatal day 10. Intraperitoneal injection of TH to 15-day-old rat pups increased the specific activity of gammaGT by 25-30% within 4-6 hr. Treatment of primary cultures of astrocytes by TH also enhanced the specific activity of gammaGT by 30-40% within 4-6 hr. The induction of gammaGT by TH was blocked by actinomycin D or cycloheximide. gammaGT is an ectoenzyme that is normally involved in the catabolism of GSH released by astrocytes. In the presence of the gammaGT-inhibitor, acivicin, GSH released in the culture medium of astrocytes increased linearly for at least 6 hr and TH had no effect on this accumulation pattern. In the absence of acivicin, GSH content of the medium from TH-treated cells was significantly lower than that of untreated controls due to activation of gammaGT by TH and a faster processing of GSH. Because the products of gammaGT reaction are putative precursors for neuronal GSH, the activation of gammaGT by TH may be conducive to GSH synthesis in neurons and their protection from oxidative stress.     

Thyroid hormone action on astroglial cells fromdistinct brain regions during development

Astrocytes are target to triiodothyronine (T3) hormone action during rat braindevelopment. In this work, we show that astrocytes from distinct developing brain regions aredifferently responsive to thyroid hormone. Distinctly from embryonic or newborn cerebralhemisphere and mesencephalic astrocytes, newborn cerebellar and embryonic hippocampalastrocytes do not change their morphology in response to hormone treatment. We also analysedprotein synthesis and secretion from these T3-treated astrocytes. The results showed a significantincrease in protein synthesis in astrocytes from older brain regions. Maximum effect, however,was observed in cerebral hemisphere astrocytes from newborn rats. The protein secretion effectwas also more evident in the cerebral hemisphere as well as in cerebellar astrocytes fromnewborn rats. In addition, we examined T3 effects on GFAP/vimentin expression by culturing6-day old cerebellar astrocytes. In this case T3 seems to induce GFAP expression which might beoccurring as a first step to astrocyte differentiation.     

Nitric oxide synthase induction in astroglial cell cultures: effect on heat shock protein 70 synthesis and oxidant/antioxidant balance

Glial cells in the nervous system can produce nitric oxide in response to cytokines. This production is mediated by the inducible isoform of nitric oxide synthase. Radical oxygen species (ROS) and nitric oxide (NO) derivatives have been claimed to play a crucial role in many different processes, both physiological such as neuromodulation, synaptic plasticity, response to glutamate, and pathological such as ischemia and various neurodegenerative disorders. In the present study we investigated the effects of NO synthase (iNOS) induction in astrocyte cultures on the synthesis of heat shock proteins, the activity of respiratory chain complexes and the oxidant/antioxidant balance. Treatment of astrocyte cultures for 18 hr with LPS and INFgamma produced a dose dependent increase of iNOS associated with an increased synthesis of hsp70 stress proteins. This effect was abolished by the NO synthase inhibitor L-NMMA and significantly decreased by addition of SOD/CAT in the medium. Time course experiments showed that iNOS induced protein expression increased significantly by 2 hr after treatment with LPS and INFgamma and reached a plateau at 18 hr; hsp70 protein synthesis peaked around 18 and 36 hr after the same treatment. Addition to astrocytes of the NO donor sodium nitroprusside resulted in a dose dependent increase in hsp70 protein that was comparable to that found after a mild heat shock. Additionally, a decrease in cytochrome oxidase activity, a marked decrease in ATP and protein sulfhydryl contents, an increase in the activity of the antioxidant enzymes mt-SOD and catalase were found which were abolished by L-NMMA. These findings suggest the importance of mitochondrial energy impairment as a critical determinant of the susceptibility of astrocytes to neurotoxic processes and point to a possible pivotal role of hsp70 in the signalling pathways of stress tolerance.     

甲状腺激素与脑发育中神经细胞迁移

甲状腺激素在脑正常发育中具有重要作用，对脑发育中细胞迁移、分化、神经元的生长、突触形成、髓鞘形成等具有调节作用。现就甲状腺激素与脑发育中神经细胞迁移的研究现状及相关理论作一综述。     

Thyroid hormone and conditioned medium effects on astroglial cells from hypothyroid and normal rat brain: Factor secretion,cell differentiation,and proliferation

The effects of triiodothyronine (T₃)on cell morphology were examined in cerebral hemisphere and cerebellar astrocyte cultures obtained from normal and hypothyroid neonatal rats. T₃-treatment induced morphological changes in astrocytes from cerebral hemispheres. This morphological effect was produced earlier if astrocytes were treated with conditioned medium obtained from cerebral hemisphere astrocyte cultures previously exposed to 50 nM T₃. T₃ or conditioned medium-treatment produced faster morphological changes in hypothyroid rat cerebral hemisphere astrocyte monolayers. Cerebellar astrocytes from normal brain did not respond to thyroid hormone with morphological changes, but proliferated after T₃-treatment. However, hypothyroid cerebellar astrocyte cultures exhibited morphological changes, differently than normal cells. We verified that T₃ may induce astrocyte secretion of factor(s) that promotes morphological differentiation in cerebral hemisphere astroglial cultures and stimulates the proliferation of cerebellar astrocytes. Astrocytes obtained from hypothyroid animals were more sensitive to secreted factors than normal cells. These results emphasize the heterogeneity and the importance of glial cells to normal brain development and open new questions about thyroid hormone therapy in hypothyroidism. © 1995 Wiley-Liss, Inc.     

Amyotrophic lateral sclerosis: Thyroid and prolactin hormone changes in thyrotropin-releasing hormone therapy

13 patients with amyotrophic lateral sclerosis (ALS) were treated with intravenous infusion of thyrotropin-releasing hormone (TRH). In 6 patients 2 mg/day of TRH was i. v. given over 2 hours for 10 days. In 7 others 2 mg/day of TRH was continuously infused by means of a pump. An increase of thyroid hormones related to the duration of the treatment was observed. A surprising finding was the onset of prolactin (PRL) response to growth hormone releasing hormone (GHRH), previously absent.

---

Sommario 13 pazienti con sclerosi laterale amiotrofica furono trattati con TRH endovena. In 6 pazienti TRH, alla dose di 2 mg al dì, fu somministrato in 2 ore per 10 giorni. In 7 altri pazienti 2 mg di TRH furono continuativamente infusi, per mezzo di una pompa, per 15 giorni. Un aumento degli ormoni tiroidei T3 e T4 fu osservato. Fu anche osservata la comparsa di una risposta, normalmente assente, della prolattina alla stimolazione con GHRH.

     

Antioxidant treatment inhibited glutamate-evoked NF-kappaB activation in primary astroglial cell cultures

In glial cells, glutamate exposure causes alterations in cell redox status, mainly mediated by glutathione depletion and reactive oxygen species generation. These effects finally lead to astrocyte dysfunction which contributes to the pathogenesis of several neurological disorders. This study was aimed to investigate the involvement of the NF-kappaB pathway in oxidative stress induced by glutamate exposure in primary cultures of astrocytes. Further, we evaluated the power of the antioxidants genistein (0.1-10 microM) and IRFI 016 (20-80 microM), a synthetic tocopherol analogue, compared with glutathione ethyl ester (10-50 microM) and cysteamine-HCl (100-500 microM), to antagonize the effects elicited by glutamate (500 microM). Alterations of cell redox status were reduced, in a dose-dependent way, by antioxidants; in particular, 80 microM IRFI 016 and 10 microM genistein almost completely restored glutathione basal levels and significantly diminished ROS production, as well as 100 microM glutathione ethyl ester. These antioxidant effects were stronger than those caused by 500 microM cysteamine-HCl. Further, glutamate promoted the up-regulation of p50 and p65 NF-kappaB subunits and their nuclear translocation, as revealed by Western blot analysis and electrophoretic mobility shift assay of both subunits. The activation of p50 and p65 NF-kappaB subunits induced by glutamate exposure was significantly reduced by IRFI 016, acting in a dose-dependent manner. Altogether, these data confirm that the NF-kappaB pathway is involved in cell response to oxidative stress induced by glutamate injury in primary astrocyte cultures, and suggest that the use of antioxidants, such as IRFI 016, may be a helpful pharmacological strategy for neuroprotection.     

Thyroid hormone metabolism in primary cultures of fetal rat brain cells

The metabolism of thyroxine 3,5,3',5'-tetraiodothyronine, (T4) and 3,5,3'-triiodothyronine (T3) was studied in primary cultures of dispersed fetal rat brain cells. Cultured brain cells actively metabolized both T4 and T3 by enzyme catalyzed deiodination reactions which increase (type II 5'-deiodinase) or decrease (type I 5'-deiodinase and type III 5-deiodinase) the bioactivity of thyroid hormone. Homogenates of cultured brain cells showed both type I and type II 5'-deiodinating activities and these two enzymes tended to differ in their time course of appearance. Cultures exposed to 10 microM cytosine arabinoside for 16 h showed up to a 70% reduction in type I activity without decreasing the type II enzyme suggesting that the type II enzyme is associated with non-dividing neuronal cells. The predominant pathway for T4 and T3 metabolism in situ was tyrosyl-ring or type III 5'-deiodination which followed first order kinetics with a t1/2 of 70 min. T4 to T3 conversion by the type II enzyme was consistently observed after correcting for the degradation of newly formed T3 by the type III enzyme. In situ, both type II and type II enzymes were thiol-dependent and both activities were inhibited by iopanoic acid. Type III 5-deiodination of T4 produced 34 fmol 3,3,5'-triiodothyronine (rT3)/h per 10(6) cells at 10 mM dithiothreitol (DTT) and 97 fmol of rT3/h per 10(6) cells at 50 mM DTT. T3 production by the type II enzyme was 1.2 and 4.4 fmol of T3/h per 10(6) cells at 10 and 50 mM DTT, respectively. Thyroid hormone deficient culture conditions increased type II enzyme activity by 4-5-fold within 48 h and this was prevented in a dose-dependent fashion by supplementing the media with increasing amounts of T3. These data indicate that primary cultures of dispersed brain cells mimic the intact cerebral cortex with respect to the metabolism of thyroid hormone and the regulatory mechanisms which defend cerebrocortical T3 levels. The vigorous metabolism of both T4 and T3 by these cultures may explain some of the difficulties in demonstrating thyroid hormone-dependent biochemical changes at physiologically relevant levels of thyroid hormone.     

Production of thrombin and antithrombin III by brain and astroglial cell cultures.

There is increasing evidence that some proteases and protease inhibitors are produced within the central nervous system. It has been proposed that the balance between these two classes of proteins may be an important modulator of brain cell growth and differentiation. Here we report that antithrombin III (ATIII) is produced in brain and primary astroglial cultures. In addition, we show that human astroglial cultures contain prothrombin mRNA, and secrete a thrombin-like protein that makes complexes with antithrombin III.     

Expression and translocation of protein kinase C isoforms in rat microglial and astroglial cultures.

Cellular distribution and activation by phorbol myristate acetate (PMA) of classical (alpha, betaI, betaII,gamma), novel (delta, epsilon, theta, eta), and atypical (zeta, iota) protein kinase C (PKC) isoforms were studied in cultured rat neonatal microglial and astroglial cells by Western blot analysis. Among the classical isoforms, only betaII was expressed in microglia and astrocytes in the same abundance. The expression of betaI in microglia was less abundant, while PKCalpha was not detectable in this cell type. PKCgamma was absent in both cell populations. A different pattern of expression was also found for novel and atypical isoenzymes: Both cell types expressed delta, theta, eta, zeta, and iota isoforms, but PKCepsilon was absent in microglia and the expression of PKCzeta and PKCiota in these cells was low compared to astrocytes. The pattern of PKC distribution in cytosolic and particulate fractions as well as activation by short (10 min) and prolonged (4 hr) PMA treatment in both cell types were similar. On the whole, in comparison with astrocytes, PKC in microglial cells was less expressed, both in terms of number of isoforms and level of expression. The microglial profile of PKC isoforms differed from that of rat peritoneal macrophages, which did express PKCalpha. Preliminary evidence suggests that the ability of PMA to enhance cyclic AMP responses in astrocytes, but not in microglia, is related to the different pattern of expression of PKCalpha and PKCepsilon in the two cell types.     

Arachidonic acid lipoxygenation may mediate interleukin-1 stimulation of nerve growth factor secretion in astroglial cultures

Interleukin-1β (IL-1) stimulates by about fivefold NGF secretion from rat neonatal cortical astrocytes in primary culture. We investigated the possible intracellular second messenger mechanisms involved in the IL-1 induced NGF secretion. Basal NGF secretion did not require extracellular Ca²⁺, whereas Ca²⁺ was necessary for the maximal NGF secretion stimulated by IL-1 (10 units/ml). The protein kinase C activator TPA stimulated by sixfold NGF secretion, but in this case, TPA acted synergistically with IL-1 to increase NGF secretion. Treatment of cells with the phospholipase A₂ inhibitor mepacrine (30 μM) inhibited basal (by 50%) and IL-1 stimulated (by 80%) NGF secretion. Indomethacin, a cyclooxygenase inhibitor, produced a slight increase in basal NGF secretion at low concentrations, while PGE₂ (10 μM) inhibited basal and IL-1 stimulated NGF secretion. In contrast, treatment of cells with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, blocked in a concentration-dependent manner (IC₅₀ = 10 μM) IL-1 stimulation of NGF secretion. The leukotriene LTB₄ increased basal NGF secretion and this effect was not additive with IL-1 when both agents were added at saturating concentrations, indicating a common mechanism of action for these two agents. Thus, one possible mechanism by which IL-1 stimulates NGF secretion from astrocytes is by activation of the phospholipase A₂-lipoxygenase pathway. © 1993 Wiley-Liss, Inc.     

Primary cultures from defined brain areas; effects of seeding time on cell growth, astroglial content and protein synthesis

The influence of seeding time on cell growth, astroglial content and on protein synthesis during cultivation was determined in primary cultures from 3 phylogenetically different brain areas from rat cerebral cortex, striatum and brainstem. Brainstem cultivated from 17-day-old embryos and all the cultures studied from the 3 brain areas of newborn and 7-day-old rat showed a similar increase in total and water-soluble protein during cultivation. Glial fibrillary acidic protein (GFAp, alpha-albumin) levels increased with age in all cultures studied. There was a rapid increase in GFAp (alpha-albumin) between 1 and 2 weeks in cultures from newborn and between 2 and 3 weeks in brainstem cultures from 17-day-old embryos, these increases being slower thereafter. Incorporation of [3H]valine into soluble protein was lower in 3-week-old cultures than in 1- and 2-week-old cultures derived from newborn and 7-day-old rat brain. The incorporation rates were similar in comparisons of the various cultures. Similar results were obtained from embryonic cultures, although the decrease in incorporation rate was between 3 and 4 weeks. The efficiency of incorporation (% TCA-precipitated material/total [3H]activity) was higher in 2- and 3-week-old than in 1-week-old cultures from newborn and 7-day-old rats and in 3- and 4-week-old cultures of brainstem from 17-day-old rat embryos. These findings suggest a cell differentiation during cultivation. The results show that seeding time has a variable influence on cultures from the different brain areas studied concerning cell growth, astroglial content and probably differentiation during cultivation. Embryonic cell cultures seem, in general, to develop one week later than neonatal and postnatal ones. Cultures of newborn rat cells from cerebral cortex, striatum and brainstem show many similarities in the above parameters during cultivation. This is also the case for brainstem cultures from embryonic rat.     

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.     

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.     

Corticotropin-releasing hormone and dexamethasone do not alter secretion of immunoreactive beta-endorphin from dissociated fetal hypothalamic cell cultures

Corticotropin-releasing hormone (CRH) and glucocorticoids are major regulators of the hypothalamic-pituitary-adrenal (HPA) axis controlling secretion of beta-endorphin and other pro-opiomelanocortin (POMC)-derived peptides from pituitary. Although previous work has shown that CRH stimulates secretion of beta-endorphin from adult hypothalamic explants, and that glucocorticoids can inhibit basal and stimulated secretion of POMC-derived peptides from pituitary, the role of glucocorticoids on hypothalamic beta-endorphin secretion is not known. Studies were performed to assess the effects of CRH and dexamethasone, a potent glucocorticoid, on secretion of immunoreactive (IR) beta-endorphin from dissociated fetal hypothalamic cell cultures. CRH (10(-9)-10(-6) M) did not stimulate secretion of IR-beta-endorphin from hypothalamic cells which did release IR-beta-endorphin upon potassium-induced depolarization. However, CRH did stimulate IR-beta-endorphin secretion from fetal hypothalamic explants which were similar to hypothalamic tissue from which dissociated hypothalamic cell cultures were derived. Exposure of cells to dexamethasone (10(-6) M) did not inhibit basal or potassium-stimulated release of IR-beta-endorphin. These results indicate that: (1) dissociated fetal hypothalamic cells in culture do not exhibit a functional CRH receptor coupled to stimulation of IR-beta-endorphin secretion; (2) exposure of hypothalamic cells to dexamethasone does not inhibit basal nor depolarization-induced release of IR-beta-endorphin; and (3) dissociated fetal hypothalamic cells may have limited utility in elucidating specific regulatory relationships because of in vitro conditions and/or cytoarchitectural relationships.     

Dynamic morphological responses of mouse astrocytes in primary cultures following medium changes

Within 5 minutes of changing the medium of 3-4 week old mouse astrocyte cultures, dramatic morphological alterations were seen in the cultures at the phase microscope level. These alterations included the appearance of membrane ruffles, followed by the formation of phase light vacuolar regions. Ultrastructurally, these ruffles could be seen as phagocytic arms, and the vacuoles appeared as blisters of the superficial cell layer. These morphological responses were due to the introduction of new serum proteins. Colloidal gold-labelled serum proteins were used to visualize the dynamics of the macromolecular uptake following this medium change. Some proteins were taken up by phagocytosis, whereas others were associated with coated vesicles and endosomal vesicles. The majority of these gold-labelled proteins were concentrated in vacuoles (presumably lysosomes) and were localized in the superficial cellular sheets. Inner cellular sheets contained little colloidal gold-labelled serum proteins, but displayed prominent pinocytotic profiles. Glucose consumption in these cultures remained constant at 1.6 mumoles/mg protein/hr. Glycogen content varied among individual cells, but it remained constant in the cultures at 60 nmoles bound glucose/mg protein throughout the 48 hour examination period. These results suggest that dense cultures of primary astrocytes may act similarly to the glia limitans.