Characterization of the Thyroid Hormone Transport System of Cerebrocortical Rat Neurons in Primary Culture

Abstract: The uptake of 3',3,5-triiodo- l -thyronine (T₃) and l -thyroxine (T₄) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25°C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The K _m of T₃ uptake was very similar to that of T₄, and did not vary significantly from day 1 to 4 in culture (310–400 n M ). The maximal velocity ( V _max) of T₃ uptake nearly doubled between day 1 and 4 of culture (41 ± 3 vs. 70 ± 5 pmol/min/mg of DNA, respectively). The V _max of T₄ uptake did not change (28 ± 8 and 31 ± 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T₃ or T₄ uptakes were the same (T₃ > T₄ > 3',5',3-triiodo- l -thyronine > 3',3,5-triiodo- d -thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T₄ was a stronger competitor of labeled T₄ uptake than of labeled T₃ uptake, whereas unlabeled T₃ had the same potency for both processes. These results suggest that T₃ and T₄ are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T₃ uptake increases during neuronal maturation.     

Regulation by Thyroid Hormones of Glucose Transport in Cultured Rat Myotubes

Primary cultures of skeletal muscle obtained from neonatal rats possess a saturable process for active glucose uptake, the myotubes having a relatively high affinity for the substrate with a Km of 1 mM. The expression of the glucose transport system was most apparent after fusion of single myoblasts to multinucleated myotubes [3-4 days in vitro (DIV)], at which time glucose uptake increased sharply to reach plateau values at about 6-8 DIV. Treatment of the cells at age 6 DIV with triiodothyronine or thyroxine caused a marked increase in glucose uptake beginning 4 h after treatment and reaching a maximum at 24 h. Thyroid hormone-induced increase in glucose uptake was not reduced by either tetrodotoxin or verapamil, thus indicating that the effect was not secondary to the ability of the hormone to increase contractile activity. The effect of thyroid hormones was eliminated completely by inhibition of protein synthesis. The results indicate that thyroid hormones play an important role in regulation of glucose transport in skeletal muscle.     

Thyroid Hormones Reorganize the Cytoskeleton of Glial Cells Through Gfap Phosphorylation and Rhoa-Dependent Mechanisms

Thyroid hormones (3,5,3′-triiodo-l-thyronine, T₃; 3,5,3′,5′-l-tetraiodothyronine, T₄; TH) play crucial roles in the growth and differentiation of the central nervous system. In this study, we investigated the actions of TH on proliferation, viability, cell morphology, in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and actin reorganization in C6 glioma cells. We first observe that long-term exposure to TH stimulates cell proliferation without induce cell death. We also demonstrate that after 3, 6, 12, 18, and 24 h treatment with TH, C6 cells and cortical astrocytes show a process-bearing shape. Furthermore, immunocytochemistry with anti-actin and anti-GFAP antibodies reveals that TH induces reorganization of actin and GFAP cytoskeleton. We also observe an increased in vitro ³²P incorporation into GFAP recovered into the high-salt Triton insoluble cytoskeletal fraction after 3 and 24 h exposure to 5×10⁻⁸ and 10⁻⁶ M T₃, and only after 24 h exposure to 10⁻⁹ M T₄. These results show a T₃ action on the phosphorylating system associated to GFAP and suggest a T₃-independent effect of T₄ on this cytoskeletal protein. In addition, C6 cells and astrocytes treated with lysophosphatidic acid, an upstream activator of the RhoA GTPase pathway, totally prevented the morphological alterations induced by TH, indicating that this effect could be mediated by the RhoA signaling pathway. Considering that IF network can be regulated by phosphorylation leading to reorganization of IF filamentous structure and that alterations of the microfilament organization may have important implications in glial functions, the effects of TH on glial cell cytoskeleton could be implicated in essential neural events such as brain development.     

Effect of Thyroid Hormones on Benzodiazepine Receptors in Neuron-Enriched Primary Cultures

The effect of administration of thyroid hormones on central benzodiazepine receptors was investigated using neuron-enriched primary cultures obtained from the neopallium of 16-day-old embryonic rats. Addition of L-triiodothyronine for 3 days decreased the maximal number of benzodiazepine receptor binding sites without any change in affinity at 10(-5) and 10(-6) M. L-Thyroxine administered for 3 days had the same effect at 10(-5) M. No significant change was observed over periods of less than 3 days, a finding indicating that this inhibition was not a direct in vitro effect. This down-regulation seems to be a direct modulatory effect of thyroid hormones on cerebral cortical neurons. Addition for 3 days of D-thyroxine and D-triiodothyronine, which are physiologically inactive isomers of the thyroid hormones, did not induce any significant alterations in benzodiazepine receptors. The decrease in number of cerebral cortical neuronal benzodiazepine receptors due to L-isomers of thyroid hormones may be related to the convulsions and anxiety observed in thyrotoxicosis in humans.     

甲状腺激素对适应性免疫细胞的作用

摘要：甲状腺激素（Thyroid hormones,THs）参与免疫功能的调节,在固有免疫和适应性免疫中发挥着重要作用。THs异常分泌所致的免疫功能失调被认为参与了格雷夫斯病和桥本甲状腺炎等自身免疫性疾病的发生发展。目前,THs在固有免疫细胞（中性粒细胞、巨噬细胞、树突状细胞、自然杀伤细胞、肥大细胞）中的作用已得到了较好的阐明,但THs对适应性免疫细胞（T淋巴细胞与B淋巴细胞）的影响等方面的研究仍未引起足够的重视。因此,本研究从适应性免疫细胞的角度出发,重点讨论了THs对这些细胞的发育、分化及功能等方面的影响,为进一步理解THs调节免疫功能的作用提供新视角。     

Estrogen Receptor Is Expressed in Different Types of Glial Cells in Culture

Abstract: Estrogens derived from the aromatization of androgens are believed to be responsible for the induction of the sexual differentiation of the CNS interacting with specific estrogen receptors (ER) present in developing neurons. However, the brain cellular distribution of ER is not so well documented. The aim of this study was to investigate the qualitative and quantitative expression of ER mRNA in well characterized cultures of rat type 1 and type 2 astrocytes and of oligodendrocytes by polymerase chain reaction. A series of amplifications with a set of primers spanning along the entire ER mRNA was utilized in the different types of glial cells, in a positive control (uterus), and in a negative control (SK-N-BE cell line) previously shown to be devoid of ER. The data obtained show that ER mRNA is expressed in all three types of glial cell analyzed in almost equal amounts, which are 25–50 times lower than those in the uterus. The mRNA expressed in the glia is homologous with that expressed in the uterine tissue.     

激素对植物细胞悬浮培养代谢产物的影响研究进展

激素是调节植物细胞生长发育和代谢产物形成的主要物质。综述了在植物细胞悬浮培养中,激素对细胞生物量和代谢产物含量的影响的研究进展。内容包括外源激素的种类、浓度、配比对悬浮培养细胞生物量和代谢产物含量的影响,内源激素检测技术的发展历程、内源激素的含量变化及其对悬浮培养细胞生物量和代谢产物含量的影响,内源激素和外源激素对植物细胞悬浮培养影响的相互作用关系以及新品种激素影响作用的相关研究。     

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 ¹ and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 ². Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 μm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.     

Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Characteristics of the β-Adrenoreceptor from Neuronal and Glial Cells in Primary Cultures of Rat Brain

The cellular characteristics of the beta-adrenoreceptor in glial and neuronal cells from the newborn rat brain were determined by (-)-[125I]iodocyanopindolol binding. In membranes from both cell types, the binding was saturable and from competition assays the potency series of (-)-isoproterenol greater than (-)-epinephrine = (-)-norepinephrine greater than (+)-isoproterenol was observed. 5'-Guanylyl-imidodiphosphate reduced the affinity of (-)-isoproterenol for the beta-adrenoreceptor from glial cells but had no effect on agonist affinity in neuronal cells. Chronic treatment of both cell types with (-)-isoproterenol reduced the receptor content and the capacity of the agonist to increase the cellular cyclic AMP content. However, the receptor recovery after chronic agonist treatment was faster in glial cells (72 h) than neuronal cells (120 h) and was blocked by cycloheximide. Treatment of both types with the irreversible beta-blocker bromoacetylalprenololmentane (2 microM) reduced the receptor content by 78% but no receptor recovery was observed for 120 h after the initial receptor loss. The data indicated that the majority of beta-adrenoreceptors in both cell types are the beta-1 subtype, but show some differences in receptor-agonist interactions. Furthermore, these CNS cells may be useful models for regulatory studies on the beta-adrenoreceptor.     

Regulation of Glial Cell Line-Derived Neurotrophic Factor Release from Rat C6 Glioblastoma Cells

Abstract: We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24-h treatment with fibroblast growth factor-1, −2, or −9 elevated secreted GDNF protein levels five- to 10-fold. The proinflammatory cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α, and lipopolysaccharide elevated GDNF release 1.5- to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13-didecanoate (10 n M ) activation of protein kinase C, the Ca²⁺ ionophore A23187 (1 µ M ), okadaic acid (10 n M ) inhibition of type-2A protein phosphatases, nitric oxide donors (1 m M ), and H₂O₂ (1 m M )-induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 µ M ) or dibutyryl cyclic AMP (1 m M ) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 µ M ). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.     

Purification and Initial Characterization of Rat B49 Glial Cell Line-Derived Neurotrophic Factor

Abstract: The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of ∼33–45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is ∼16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC₅₀ in midbrain cultures at ∼40 pg/ml, or 1 p M , and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.     

A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

新生大鼠雪旺细胞原代培养方法改良

对常用的阿糖胞苷处理及差速贴壁法进行大鼠雪旺细胞原代培养及纯化的方法进行改进。先用阿糖胞苷处理杀死大部分的成纤维细胞,再用抗-Thy-1.1抗体和兔补体处理去除残余成纤维细胞,获得纯化的雪旺细胞。此外,我们对抗-Thy-1.1抗体和兔补体的浓度、处理时间等都进行了改进,避免了由于雪旺细胞状态不好而引起的大量雪旺细胞死亡。此方法能够将雪旺细胞的纯度由90%提高到99%。     

大鼠脑微血管内皮细胞的分离与原代培养

为了建立大鼠脑微血管内皮细胞体外培养模型,探索纯度较高的大鼠脑微血管内皮细胞分离和原代培养的方法并进行形态学观察。采用2～3周龄的SD大鼠,解剖得到大脑皮质,两次酶消化及牛血清白蛋白或葡聚糖和Percoll梯度离心获得较纯的脑微血管段后,接种于涂布基质的培养皿进行原代培养;培养的细胞采用相差显微镜形态学观察、透射电镜观察及Ⅷ因子相关抗原免疫组化检测鉴定。结果发现,培养12h即可见细胞从贴壁的脑微血管段周围长出,细胞呈短梭形,区域性单层生长,5～7天内皮细胞融合,内皮细胞纯度达90％以上;内皮细胞的贴壁和生长有赖于所涂布的基质,纤连蛋白／Ⅳ型胶原优于鼠尾胶和明胶;Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性,透射电镜观察可见相邻内皮细胞间存在紧密连接结构。提示该方法能成功进行纯度较高的大鼠脑微血管内皮细胞原代培养,可用于脑微血管内皮的生理、生化及药理学研究,亦可用于构建大鼠血脑屏障模型。     

糖基化终产物对原代培养乳鼠肾脏细胞损伤的影响

糖基化终产物(AGEs)在糖尿病肾病的发生发展过程中起着重要的作用.但目前其作用机制还不太清楚.通过体外乳鼠肾脏细胞的原代培养,探讨AGEs对肾细胞的损伤作用及可能的作用机制.取出生3天的SD大鼠的乳鼠肾脏进行体外原代细胞培养,并取传代到4-6代的细胞进行实验研究.分别用不同浓度的AGEs(0、1.2、2.5、5、10、20 mg/ml),不同的作用时间(6、12、18、24 h)作用于体外培养的肾细胞,用MTT法检测AGEs对肾细胞的增殖情况,用酶试剂盒法检测AGEs对肾细胞培养液中乳酸脱氢酶(LDH)、β-N-乙酰氨基葡萄糖苷酶(NAG)的含量,以及肾细胞内还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的含量.实验结果表明随着AGEs作用肾细胞时间的延长和浓度的增加,细胞存活率、细胞内GSH含量和SOD活性均逐渐下降,而细胞培养液中LDH和NAG的含量则逐渐升高,与正常培养的对照组细胞相比差异非常显著(P＜0.001),并且AGEs对细胞的作用与其浓度和作用时间呈显著的量效关系.实验结果说明AGEs对原代培养的肾细胞有明显的损伤作用,并随着AGEs作用浓度的增加和作用时间的延长对肾细胞的损伤越来越严重,实验结果也表明.肾细胞对AGEs的作用很敏感,其损伤细胞的途径和作用机制可能是由于改变了肾细胞膜的通透性和降低肾细胞抗氧化能力,该实验研究也进一步提示了AGEs是导致糖尿病肾脏并发症发生的重要原因之一.     

Effect of Serum Lipoproteins on Growth and Sterol Synthesis in Cultured Rat Brain Glial Cells

Cells dissociated from brains of 1-day-old rats were cultured in medium containing either lipoprotein-deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin-precipitable sterols, and from the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low-density lipoproteins (LDL), high-density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).     

Glial Cell Line-Derived Neurotrophic Growth Factor Inhibits Apoptotic Death of Postnatal Substantia Nigra Dopamine Neurons in Primary Culture

Abstract: Glial cell line-derived neurotrophic factor (GDNF) was identified on the basis of its ability to enhance the development of embryonic mesencephalic dopamine neurons. It remains unknown whether GDNF is a physiologically relevant trophic factor for these neurons. We have shown that natural cell death among dopamine neurons of the substantia nigra occurs largely postnatally. To investigate whether GDNF may have the ability to support these neurons during their period of natural cell death, we have used a postnatal primary culture model. We find that GDNF is able to support the viability of postnatal nigral dopamine neurons by inhibiting apoptotic death. This ability of GDNF shows both regional specificity for the nigra and cellular specificity for the dopamine phenotype. Among eight other neurotrophic factors previously reported to support embryonic dopamine neurons, GDNF was unique in this ability. Thus, GDNF meets this criterion for a physiologically relevant trophic factor for dopamine neurons of the substantia nigra.