共查询到19条相似文献,搜索用时 250 毫秒
1.
甾体激素对C6细胞摄取甘氨酸的快速作用 总被引:3,自引:0,他引:3
目的 :探讨甾体激素对C6细胞摄取甘氨酸快速作用的非基因组织机制。方法 :应用液体闪烁技术 ,通过检测C6细胞在加入了甾体激素和 /或其它试剂后摄入标记甘氨酸量的改变 ,确定甾体激素的作用。结果 :C6细胞高亲合力的甘氨酸依赖于钠离子和氯离子。皮质酮 ,孕酮 ,地塞米松可快速抑制这种摄取 ,雌二醇 ,脱氧皮质酮无显著的抑制作用 ,表明甾体激素作用有特异性。皮质酮的作用在 10 4 -8~ 10 -6 mmol/L范围内效应与浓度成正相关。皮质酮偶联牛血清蛋白后作用依然存在。RU38486 能部分阻断皮质酮的效应。细胞外液钙离子缺乏时皮质酮的作用基本消失。结论 :虽然皮质酮 ,孕酮 ,地塞米松神经胶质细胞和神经元摄取甘氨酸的快速作用不一样 ,但均是非基因组机制。 相似文献
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在离体家兔AVN区标本上,用微电极技术研究了Ⅲ类抗心律失常新药UK-68798对AN,N,NH,H4种细胞的电生理效应。浓度5×10-9至5×10-6mol/L的UK-68798对4种细胞的动作电位幅值(APA)、静息膜电位(RP)皆无影响。对AVN的自搏率有剂量依赖性减慢作用,但不改变A-H传导时间。在5×10-8-5×10-6mol/L剂量范围,此药使动作电位时程(APD50)和(APD90)发生剂量依赖性延长。4种细胞中以N细胞的APD50和APD90延长百分率最高。各种细胞APD90延长百分率的排列次序为N<AN<H<NH,当浓度为5×10-6mol/L时的延长百分率分别为95±26%(N),75±22%(AN),63±26%(H),46±26%(NH)。在UK-68798的作用下,4种细胞的有效不应期(ERP)也发生剂量依赖性延长,但不存在像APD延长百分率那样的差别。此外,4种细胞ERP所相当的复极化膜电位未受药物影响,从而避免了由于兴奋性恢复的不均一性,使AVN区成为折返性心律失常的发源地. 相似文献
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白介素-2促进RC-4B/C垂体瘤细胞系增殖 总被引:6,自引:0,他引:6
采用细胞培养方法,以^3H-TdR掺入率反映细胞增殖水平,研究了白介素-2(IL-2)对大鼠源的RC-4B/C垂体瘤细胞系增殖的影响,并初步分析了IL-2的作用机制。结果表明:⑴IL-2(10-1000U/ml)剂量依赖性地显著提高RC-48/C细胞^3H-TdR掺入率。⑵特异性酪氨酸蛋白激酶(PTK)抑制剂tyrphostin(1μmol/L)可明显降低RC-4B/C细胞^3H-TdR掺入率,并 相似文献
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应用PCR-SSCP技术并结合Southern印迹杂交从基因水平对正常和 ̄3H-TdR恶性转化小鼠胚胎成纤维细胞NC3H10和TC3H10中neu基因进行研究。Southern印迹杂交结果表明恶性转化的TC3H10细胞neu基因出现重排和扩增,SSCP分析未发现TC3H10细胞neu基因跨膜区突变。上述结果说明TC3H10细胞neu基因结构异常可能在跨膜区外,neu基因异常在 ̄3H-TdR诱导的细胞恶性转化过程中可能有重要的作用,EGF可促进neu基因表达增高,研究发现在EGF持续作用下,NC3H10细胞neu基因甲基化水平无显著变化,说明EGF可能是通过其它途径调控neu基因表达增高的,排除了EGF通过改变neu基因甲基化水平而调控neu基因表达的可能性。 相似文献
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应用PCR-SSCP技术并结合Southern印迹杂交从基因水平对正常和^3H-TdR恶生转化小鼠胚胎成纤维细胞NC3H10和TC3H10中neu基因进行研究,Southern印迹杂交结果表明恶性转化的TC3H10细胞的neu基因出现重排和扩增,SSCP分析未发现TC3H10细胞neu基因跨膜区突变,上述结果说明TC3H10细胞neu基因结构异常可能在跨膜区外,neu基因异常在^3H-TdR诱导的 相似文献
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转TK基因的人结肠癌细胞对多种原药敏感性的研究 总被引:2,自引:0,他引:2
构建了含有单纯疱疹病毒胸苷激酶基因(HSV-TK)的重组逆转录病毒载体LTKSN,经PA317细胞包装后,感染人结肠癌细胞株LoVo.用G418筛选到稳定表达HSV-TK基因的细胞克隆LoVo/LTKSN,LoVo/LTKSN与野生型LoVo细胞相比,生长曲线无明显差异,细胞形态亦无改变,细胞毒试验证明LoVo/LTKSN对GCV的敏感性很高,半杀伤浓度IC50为0.5μmol/L,比野生型细胞提 相似文献
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一氧化氮抑制内皮素促血管平滑肌细胞增殖作用的信号转导途径 总被引:6,自引:0,他引:6
培养的家兔胸主动脉血管平滑肌细胞(VSMC)分别以内皮素(ET-1)、一氧化氮(NO)前体L-Arg和NO供体SIN-1刺激,或用ET-1+L-Arg、ET-1+SIN-1联合刺激,测VSMC^3H-TdR掺入、丝裂素活化蛋白激酶(MAPK)活性及蛋白激酶C(PKC)活性的改变,以研究NO抑制ET-1促VSMC增殖作用的信号转导途径。结果表明:(1)ET-1 10^-8mol/L单独刺激,^3H- 相似文献
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Corticosterone acutely prolonged N-methyl-d-aspartate receptor-mediated Ca2+ elevation in cultured rat hippocampal neurons 总被引:2,自引:0,他引:2
Takahashi T Kimoto T Tanabe N Hattori TA Yasumatsu N Kawato S 《Journal of neurochemistry》2002,83(6):1441-1451
This work reports the first demonstration that corticosterone (CORT) has a rapid and transient effect on NMDA receptor-mediated Ca2+ signaling in cultured rat hippocampal neurons. Using single cell Ca2+ imaging, CORT and agonists of glucocorticoid receptors were observed to modulate the NMDA receptor-mediated Ca2+ signals in a completely different fashion from pregnenolone sulfate. In the absence of steroids, 100 micro m NMDA induced a transient Ca2+ signal that lasted for 30-70 s in 86.1% of the neurons prepared from postnatal rats (3-5 days old). After pre-treatment with 0.1-100 micro m CORT for 10-20 min, NMDA induced extremely prolonged Ca2+ elevation. This prolonged Ca2+ elevation was terminated by the application of MK-801 and followed by washing out of CORT. The proportion of CORT-modulated neurons within the NMDA-responsive cells increased from 25.1 to 95.5% when the concentration of CORT was raised from 0.1 to 50 micro m. Substitution of BSA-conjugated CORT produced essentially the same results. When hippocampal neurons were preincubated with 10 micro m cortisol and 1 micro m dexamethasone for 20 min, a very prolonged Ca2+ elevation was also observed upon NMDA stimulation. The CORT-prolonged Ca2+ elevation caused a long-lasting depolarization of the mitochondrial membrane, as observed with rhodamine 123. In contrast, incubation with 100 micro m pregnenolone sulfate did not considerably alter the time duration of NMDA-induced transient Ca2+ elevation, but caused a significant increase in the peak amplitude of Ca2+ elevation in hippocampal neurons. These results imply that high levels of CORT induce a rapid and non-genomic prolongation of NMDA receptor-mediated Ca2+ elevation, probably via putative membrane surface receptors for CORT in the hippocampal neurons. 相似文献
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Sato S Osanai H Monma T Harada T Hirano A Saito M Kawato S 《Biochemical and biophysical research communications》2004,321(2):510-513
We examined the rapid effects of corticosterone (CORT) on N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ signals in adult mouse hippocampal slices by using Ca2+ imaging technique. Application of NMDA caused a transient elevation of intracellular Ca2+ concentration followed by a decay to a plateau within 150s. The 30min preincubation of CORT induced a significant decrease of the peak amplitude of NMDA-induced Ca2+ elevation in the CA1 region. The rapid effect of CORT was induced at a stress-induced level (0.4-10microM). Because the membrane non-permeable bovine serum albumin-conjugated CORT also induced a similar rapid effect, the rapid effect of CORT might be induced via putative surface CORT receptors. In contrast, CORT induced no significant effects on NMDA-induced Ca2+ elevation in the dentate gyrus. In the CA3 region, CORT effects were not evaluated, because the marked elevation of NMDA-induced Ca2+ signals was not observed there. 相似文献
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Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus 总被引:4,自引:2,他引:2
This study attempts to determine if the cochlear nucleus (CN) contains glycinergic synaptic endings. The uptake and release of exogenous radiolabeled glycine were measured in vitro in the three major subdivisions of the guinea pig CN: anteroventral, posteroventral, and dorsal. A kinetic analysis of [3H]glycine uptake revealed the presence in each CN subdivision of a high- and a low-affinity uptake mechanism. The high-affinity mechanism had a Km of 25.2-30.5 microM and a Vmax of 3.8-4.8 nmol/10 mg of cell water/5 min, whereas the low-affinity mechanism had a Km of 633-718 microM and a Vmax of 26.6-37.1 nmol/10 mg of cell water/5 min. At steady state, the high-affinity mechanism accumulated 10 microM [3H]glycine from the medium, achieving tissue concentrations that were 13-24 times that in the medium. The high-affinity uptake was dependent on the temperature and on the concentrations of NaCl and glucose in the incubation medium. It exhibited a high degree of substrate specificity, as determined by the effects of structural analogues of glycine on the uptake of [3H]glycine. Each CN subdivision also contained two mechanisms mediating [14C]glycine release. One was activated by depolarizing electrical stimuli, produced a rapid transient release of [14C]glycine, and was dependent on the presence of extracellular Ca2+. The other was continuous, producing a slow spontaneous efflux of [14C]glycine. Released glycine could be removed primarily by uptake, because during release measurements, the amount of [14C]glycine detected in the medium decreased when glycine uptake activity was optimized. The electrically evoked, Ca2+-dependent release and the high-affinity uptake of glycine may mediate the synaptic release and inactivation of glycine, respectively. These findings, therefore, support the presence of glycinergic synaptic endings in each CN subdivision. 相似文献
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Calcium influx mediated by nicotinic receptors and voltage sensitive calcium channels in SK-N-SH human neuroblastoma cells 总被引:2,自引:0,他引:2
L Noronha-Blob R Gover J Baumgold 《Biochemical and biophysical research communications》1989,162(3):1230-1235
When SK-N-SH human neuroblastoma cells were exposed to nicotine (NIC) or KCl they showed a dose-dependent transient increase (2- to 4-fold) in intracellular Ca2+ concentration ([Ca2+])i as detected by quin-2 fluorescence, with half maximal effects (EC50) observed at 13 microM and 26 mM, respectively. Tubocurarine and 1-isodihydrohistrionicotoxin potently blocked the NIC-evoked (IC50 congruent to 1 microM and 0.3 microM, respectively), but not the high [K+]o-evoked [Ca2+]i accumulation. The KCl-induced response was inhibited by verapamil and diltiazem (IC50 = 1.4 and 10.9 microM, respectively). Tetrodotoxin (3 microM) and tetraethylammonium (10 microM) had no effect on [Ca2+]i accumulation induced by either agent. Increases in [Ca2+]i could be evoked sequentially by NIC and KCl in the same cells suggesting independent mechanisms of Ca2+ entry. In a Ca2+-free medium, no response to either KCl or NIC was observed. However, when Ca2+ ions were restored, [Ca2+]i accumulation was enhanced to the same extent as cells suspended in a Ca2+-containing buffer. Long-term (18 hr) pretreatment of SK-N-SH cells with pertussis (100 ng/ml) or cholera toxins (10 nM) had no effect on NIC or KCl-induced [Ca2+]i accumulation. Together, these data demonstrate the presence of NIC receptors and voltage-sensitive Ca2+ channels on SK-N-SH neuroblastoma cells, through which [Ca2+]i may be modulated. 相似文献
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H A Pershadsingh E B Stubbs W D Noteboom M L Vorbeck A P Martin 《Biochimica et biophysica acta》1985,821(3):445-452
The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids. 相似文献
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Rapid Elevation of Calcium Concentration in Cultured Dorsal Spinal Cord Astrocytes by Corticosterone
Junwei Zeng Min Li Zhi Xiao Yuanshou Chen Quanzhong Chang Hong Tian Huan Jin Xiaohong Liu 《Neurochemical research》2013,38(2):382-388
In addition to the classic genomic effects, increasing evidence suggests that GC can generate multiple rapid effects on many tissues and cells through nongenomic pathway. In the present study, the effects of corticosterone (CORT) on the intracellular calcium concentration ([Ca2+]i) in cultured dorsal spinal cord astrocytes were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of [Ca2+]i. CORT (0.01–10 μM) caused a rapid increase in [Ca2+]i with a dose-dependent manner in cultured dorsal spinal cord astrocytes. The action of CORT on astrocytic [Ca2+]i was blocked by pertussis toxin (a blocker of G protein activation, 100 ng/ml), but was unaffected by RU38486 (glucocorticoid receptor antagonist, 10 μM). In addition, cycloheximide (protein-synthesis inhibitor, 10 μg/ml) pretreatment could not impair the CORT-evoked [Ca2+]i elevation. Furthermore, Ca2+ mobilization induced by CORT was abolished by chelerythrine chloride (protein kinase C inhibitor, 10 μM), but was not impaired by H89 (protein kinase A inhibitor, 10 μM). These observations suggest that a nongenomic pathways might be involved in the effect of CORT on [Ca2+]i in cultured dorsal spinal cord astrocytes. In addition, our results also raise a possibility that a putative pertussis toxin-sensitive mGCR (G-protein-coupled membrane-bound glucocorticoid receptor) and the downstream activation of protein kinase C may be responsible for CORT-induced Ca2+ mobilization in cultured dorsal spinal cord astrocytes. 相似文献
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Williams JB Mallorga PJ Lemaire W Williams DL Na S Patel S Conn PJ Conn JP Pettibone DJ Austin C Sur C 《Analytical biochemistry》2003,321(1):31-37
Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [14C]glycine was time dependent and saturable with a Michaelis-Menten constant (Km) of 27+/-3 microM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, respectively. The [14C]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2+/-1.7 microM and fully inhibited by ALX-5407 (IC50=522 +/-83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors. 相似文献
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目的研究皮质酮对大鼠海马神经元的毒性作用及NMDA受体亚基表达的影响.方法以体外原代培养的大鼠海马神经元为研究对象,根据影响因素,即给予的不同浓度皮质酮和其它因素分为8个组:对照组、10-7mol/L皮质酮组(简称10-7组)、10-6mol/L皮质酮组(简称10-6组)、10-5mol/L皮质酮组(简称10-5组)、10-6 高糖组、10-5 高糖组、10-6mol/L MK801组和10-5mol/L MK801组,镜下观察不同浓度皮质酮作用下海马神经元形态学的变化,并采用MTT方法测量各组细胞存活率,利用免疫细胞化学结合图象分析对原代培养海马神经元NMDA受体亚基的表达进行观察.结果 10-6、10-5浓度的皮质酮对海马神经元影响较大,细胞存活率较对照组明显降低,但10-6 高糖组、 10-5mol/L 高糖组、10-6mol/L MK801及10-5mol/L MK801 4个组,分别与相同皮质酮浓度处理组比较,细胞存活率显著提高.10-6和10-5组海马神经元上NMDA受体亚基表达较对照组明显降低.10-7mol/L浓度的皮质酮对上述指标影响不大.结论过量的皮质酮对大鼠海马神经元具有损伤作用,NMDA受体参与了此过程,NMDA受体拮抗剂和高浓度葡萄糖可保护海马神经元. 相似文献