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1.
Masayuki Hara Yoshihiro Matsuda Nobuaki Okumura Keisuke Hirai Hachiro Nakagawa 《Journal of neurochemistry》1989,52(3):909-912
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. 相似文献
2.
Glucose Transport in Astrocytes: Regulation by Thyroid Hormone 总被引:7,自引:4,他引:3
Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate-saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid-hormone-free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these "hypothyroid" cells markedly increased the net rate of 2-deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes. 相似文献
3.
Insulin Is Released from Rat Brain Neuronal Cells in Culture 总被引:4,自引:4,他引:0
Derrel W. Clarke Laura Mudd Frederick T. Boyd Jr. Michael Fields† Mohan K. Raizada 《Journal of neurochemistry》1986,47(3):831-836
Depolarization of neuronal cells in primary culture from the rat brain by potassium ions in the presence of calcium or by veratridine caused a greater than three-fold stimulation of release of immunoreactive insulin. HPLC of the released insulin immunoreactivity from the neuronal cultures comigrated with the two rat insulins. The depolarization-induced release of insulin was inhibited by cycloheximide and was specific for neuronal cultures since potassium ions failed to cause the release in comparably prepared astrocytic glial cells from the rat brain. Prelabelling of neuronal cultures with [3H]leucine followed by depolarization resulted in the release of radioactivity that immunoprecipitated with insulin antibody. The release of [3H]insulin was biphasic. These observations suggest that neuronal cells from the brain have the capacity to synthesize insulin that could be released under depolarization conditions. 相似文献
4.
The effect of chronic hyperglycemia on the glucose transporter moiety of the blood-brain barrier and cerebral cortex was studied in rats 3 weeks after the administration of a single intravenous dose of streptozotocin (60 mg/kg), using specific [3H]cytochalasin B binding methods. Streptozotocin-treated rats developed hyperglycemia, as well as polydipsia and polyuria, and failed to gain weight. The density of D-glucose-displaceable cytochalasin B binding sites in the brain microvessels of streptozotocin-treated hyperglycemic rats was increased by about 30% compared with those of control rats, without change in the affinity of binding. Chronic hyperglycemia had no effect on the density or affinity of specific binding of cytochalasin B to cerebral cortical membranes. These findings do not support the hypothesis that glucose transporters in brain microvessels comprising the blood-brain barrier are "down-regulated" in chronic hyperglycemia. 相似文献
5.
Characteristics of the β-Adrenoreceptor from Neuronal and Glial Cells in Primary Cultures of Rat Brain 总被引:1,自引:1,他引:0
Stephen P. Baker Colin Sumners Joseph Pitha† Mohan K. Raizada 《Journal of neurochemistry》1986,47(4):1318-1326
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. 相似文献
6.
Developmental Changes in the Activity of Enzymes of Purine Metabolism in Rat Neuronal Cells in Culture and in Whole Brain 总被引:1,自引:3,他引:1
Sara Brosh Oded Sperling Yael Bromberg Yechezkel Sidi 《Journal of neurochemistry》1990,54(5):1776-1781
The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain. 相似文献
7.
Effects of Organotins on Rat Brain Astrocytes in Culture 总被引:5,自引:1,他引:4
Abstract: The interaction of triethyltin (TET) and trimethyltin (TMT) with rat brain astrocytes in vitro was investigated. Both compounds are highly neurotoxic after in vivo application, cause neurobehavioral changes, and elicit neuronal and glial responses in the CNS. In this study, 5-week-old cultures were exposed to TMT or TET (0.1–2.5 µM) for 24 h. A concentration-dependent cytotoxicity was observed for both agents by vital dye uptake assay using neutral red (NR). The order of potency for half-maximal cytotoxicity (NR-50) was TET (0.7 µM)> TMT (2.5 µM), in agreement with results found after in vivo administration. TET and TMT caused similar morphological changes: large holes extending through the plasma membrane appeared initially in the flattened cell bodies, cytoplasmic extensions were retracted, and long cellular processes formed. Later, the cell bodies rounded up and had only a few extremely long and thin processes. Indirect immunofluorescence staining using anti-vimentin and anti-glial fibrillary acidic protein (GFAP) antibodies revealed that the orderly array of the intermediate filament system was severely disturbed. At lower concentrations, an increased bundling was observed, and at higher concentrations the disassembly of the intracellular framework was seen, and cellular staining appeared rather diffuse. Western blot analysis of cellular extracts was carried out to determine the protein levels of GFAP and vimentin. In this culture system, TET and TMT caused an almost two-fold increase in the levels of GFAP at concentrations around and below NR-50, indicating that astrocytes react to organotics independently of neuronal signals. Concomitantly, TET (0.7 µM) and TMT (2.5 µM) led to a 63% decrease in the activity of ecto-5′-nucleotidase, which in addition to its enzymatic function, represents a transmembrane cell surface protein and has been implicated in cellular adhesion and cell communication processes. Thus, triorganotins directly affect astrocytes in culture and alter their functional stage even in the absence of neurons. 相似文献
8.
用细胞松弛素B抑制红细胞膜上葡萄糖运输蛋白(GluT-1)对葡萄糖的运输,观察到对阴离子运输有促进作用。当GluT-1结合其底物分子-葡萄糖后同样加快了阴离子运输速率.另一方面,实验办给出了葡萄糖跨膜运输特性和Cl-离子浓度的关系,表明随着Cl-离子浓度的加大能使葡萄糖运输过程中Km减小、V(max)增大。这些结果表明了在完整红细胞膜上阴离子交换蛋白Band3和GluT-1之间存在着双向联系,即一种膜蛋白的构象改变能影响另一种膜蛋白的功能。 相似文献
9.
William M. Pardridge Paul D. Crane Lawrence J. Mietus William H. Oldendorf 《Journal of neurochemistry》1982,38(2):560-568
Abstract: Recent studies indicate the lumped constant (LC), which defines the relative rates of brain utilization of glucose and 2-deoxyglucose (2-DG), doubles to values > 1.0 under conditions of hypoglycemia. Since changes in the LC should be predictable given the kinetic parameters of blood-brain barrier (BBB) transport and brain phosphorylation of glucose and 2-DG, the present studies were designed to measure the necessary kinetic parameters. The carotid injection technique was used to determine cerebral blood flow and the Km , Vmax , and K D of glucose and 2-DG transport through the BBB in seven brain regions in rats anesthetized with 50 mg/kg i.p. pentobarbital. Regional glucose transport through the BBB was characterized by an average Km = 6.3 m m , average Vmax = 0.53 μmol min−1 g−1 , and average K D = 0.022 ml min−1 g−1 . The nonsaturable route of transport of glucose represented on the average 40% of the total glucose influx into brain regions at an arterial glucose concentration of 10 m m . In addition, the rate constants of phosphorylation of glucose and 2-DG were measured for each region. Substitutions of the measured kinetic parameters for sugar transport and phosphorylation into equations defining the LC confirm the observation that the LC would be expected to vary under extreme conditions such as hypoglycemia and to exceed values of 1.0 under these conditions. 相似文献
10.
Glucose Transporter in Plasma Membranes of Cultured Neural Cells, as Characterized by Cytochalasin B Binding 总被引:2,自引:2,他引:0
Identification of hexose transporter sites by cytochalasin B binding was conducted with a centrifugation assay. The determination of KD and Bmax values by LIGAND computer analysis provided binding data that are similar in primary astrocytes (238 nM and 14 pmol/mg protein) and neuroblastoma cells (179 nM and 13.6 pmol/mg protein). In contrast, only an insignificant number of transporter sites was detectable in C6 glioma cells, irrespective of whether membrane fractions were obtained by a two-phase polymer system or by a latex phagocytosis technique yielding inside-out plasma membranes. The latter membrane preparation was utilized to identify and quantitate the transporter molecules at the inner membrane surface of primary astrocytes, i.e., 160 nM (KD) and 5.8 pmol/mg protein (Bmax), respectively. 相似文献
11.
Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex 总被引:2,自引:2,他引:0
Matthijs Lopes-Cardozo Orla M. Larsson Arne Schousboe† 《Journal of neurochemistry》1986,46(3):773-778
Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain. 相似文献
12.
The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of less than 60 nM and linear at greater than 60 nM. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated diffusion. Moreover, biotin uptake in astrocytes grown in biotin-restricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10-fold) in the Vmax of the biotin uptake without any change in the apparent Km. 相似文献
13.
Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors. 相似文献
14.
目的:探讨高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响及作用机制。方法:SD大鼠胰岛细胞原代培养,不同浓度的葡萄糖处理后,采用MTT比色法检测细胞存活率,Hoechst-PI染色观察细胞凋亡,电镜观察细胞超微结构改变,RT-PCR方法检测Bax及Bcl-2相关基因mRNA表达水平。结果:5.5 mmol·L~(-1),11.1 mmol·L~(-1)和22.2 mmol·L~(-1)葡萄糖处理后,胰岛细胞活性分别下降到78.08%±2.29%、58.39%±3.13%和36.05%±2.63%,与阴性对照组比较差异具有统计学意义(P0.05);Hoechst-PI染色结果显示随着葡萄糖作用浓度的增加,凋亡细胞数量也增加;RT-PCR显示胰岛细胞经不同浓度葡萄糖处理后,Bax mRNA的表达明显上调,Bcl-2 mRNA表达下调(P0.05);电镜观察结果显示随着葡萄糖作用浓度的增加,胰岛细胞超微结构损害程度依次加重。结论:高浓度葡萄糖能明显引起胰岛细胞活性的下降,诱导凋亡反应的发生,凋亡机制与Bcl-2家族蛋白相关。 相似文献
15.
Carrier-Mediated Transport of Thyroid Hormones into Rat Glial Cells in Primary Culture 总被引:2,自引:1,他引:1
Jacques Francon Françoise Chantoux Jean-Paul Blondeau 《Journal of neurochemistry》1989,53(5):1456-1463
The uptake of 3,3',5-[3'-125I]triiodo-L-thyronine ([125I]L-T3) and of L-[3',5'-125I]thyroxine ([125I]L-T4) by cultured rat glial cells was studied under initial velocity (Vi) conditions. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The following respective values of Km (microM) and Vmax (fmol/min/microgram of DNA) were obtained at 25 degrees C: 0.52 +/- 0.09 and 727 +/- 55 for L-T3 and 1.02 +/- 0.21 and 690 +/- 85 for L-T4. Ki values (microM) for the inhibition of [125I]L-T3 uptake by unlabeled analogues were as follows: L-T4, 0.88; 3,3',5'-triiodo-L-thyronine, 1.4; 3,3'-diiodo-L-thyronine, 2.9; 3,3',5-triiodo-D-thyronine, 4.8; and triiodothyroacetic acid, 5.3. These values indicate that the uptake system is stereospecific. Unlabeled L-T3 was a better competitor than unlabeled L-T4 for the uptake of [125I]L-T4, an observation suggesting that both hormones were taken up by a common carrier system. L-T3, and L-T4 uptake was pH dependent, a finding suggesting that the phenolic unionized form of the hormones was preferentially taken up. L-T3 uptake was studied in the presence of various inhibitors; the results suggest that uptake was independent of the transmembrane Na+ gradient and of the cellular energy. Compounds that inhibited cellular uptake but were without effect on L-T3 binding to isolated nuclei also inhibited L-T3 nuclear binding in intact cells, an observation suggesting that uptake could be rate limiting for the access of L-T3 to nuclear receptors when transport is severely inhibited. 相似文献
16.
Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells 总被引:1,自引:1,他引:0
Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the alpha-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an "adipocyte" form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport. 相似文献
17.
Chaya Brodie 《Journal of neurochemistry》1990,55(1):186-191
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. 相似文献
18.
Glucose Deprivation Results in a Lactate Preventable Increase in Adenosine and Depression of Synaptic Transmission in Rat Hippocampal Slices 总被引:4,自引:1,他引:3
John C. Fowler 《Journal of neurochemistry》1993,60(2):572-576
Abstract: The effect of glucose deprivation on adenosine levels and on synaptic transmission was investigated in rat hippocampal slices. Incubation of hippocampal slices either in glucose-free medium or in the presence of the glucose transport inhibitor cytochalasin B (50 μ M ) increased bath adenosine levels and depressed the extracellularly recorded synaptic potential or population spike. The addition of lactate (10 m M ), a precursor for mitochondrial ATP generation, prevented the elevation in adenosine and the depression of the population spike. These results indicate that the neuroinhibitory modulator adenosine is elevated during glucose deprivation and contributes to the hypoglycemic depression of synaptic transmission. The increase in adenosine during glucose deprivation can be prevented by providing substrate for mitochondrial ATP generation. The present results indicate an interaction between lactate and adenosine such that an increase in lactate may contribute to a decline in adenosine production. 相似文献
19.
Abstract: Understanding the mechanism of brain glucose transport across the blood-brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis-Menten kinetics. These models predict that the steady-state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K t . In experiments where steady-state plasma glucose content was varied from 4 to 30 m M , the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 m M , the brain glucose level approached 9 m M , which was significantly higher than predicted from the previously reported K t of ∼4 m M ( p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible Michaelis-Menten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower K t of 0.6 ± 2.0 m M , which was consistent with the previously reported millimolar K m of GLUT-1 in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis-Menten kinetics. 相似文献
20.
Susan J. Vannucci 《Journal of neurochemistry》1994,62(1):240-246
Abstract: Two glucose transport proteins, GLUT1 and GLUT3, have been detected in brain. GLUT1 is concentrated in the endothelial cells of the blood-brain barrier and may be present in neurons and glia; GLUT3 is probably the major neuronal glucose transporter. Of the few studies of glucose transport in the immature brain, none has quantified GLUTS. This study used membrane isolation and immunoblotting techniques to examine the developmental expression of GLUT1 and GLUT3 in four forebrain regions, cerebral microvessels, and choroid plexus, from rats 1–30 days postnatally as compared with adults. The GLUT1 level in whole brain samples was low for 14 days, doubled by 21 days, and doubled again to attain adult levels by 30 days; there was no regional variation. The GLUT3 level in these samples was low during the first postnatal week, increased steadily to adult levels by 21–30 days, and demonstrated regional specificity. The concentration of GLUT1 in microvessels increased steadily after the first postnatal week; the GLUT1 level in choroid plexus was high at birth, decreased at 1 week, and then returned to near fetal levels. GLUT3 was not found in microvessels or choroid plexus. This study indicates that both GLUT1 and GLUT3 are developmentally regulated in rat brain: GLUT1 appears to relate to the nutrient supply and overall growth of the brain, whereas GLUT3 more closely relates to functional activity and neuronal maturation. 相似文献