甾体激素对神经母细胞株SK-N-SH细胞摄取甘氨酸的快速作用

本研究观察了ＳＫ－Ｎ－ＳＨ细胞摄取甘氨酸的一般情况,并进一步研究了甾体激素对ＳＨ－Ｎ－ＳＨ细胞摄取甘氨酸的快速作用。结果显示：ＳＫ－Ｎ－ＳＨ细胞高亲和力的甘到摄取是Ｎａ＾＋和Ｃｌ＾－依赖性的。皮质酮、孕酮和地塞米松对这种摄取有快速促进作用,雌二醇和脱氧皮质酮无明显作用,表明甾体激素快速作用有特异性。皮质酮作用在１０＾－９￣１０＾－６ｍｏｌ／Ｌ范围内呈浓度依赖性。此质酮快速作用不受蛋白质合成抑制影响     

糖皮质激素快速抑制高钾离子诱导嗜铬细胞瘤细胞内游离钙浓度升高的作用及其特征

本研究应用钙离子特异荧光指示剂Fura-2/AM,使用Miracal影像系统(Miracal Image System)检测了糖皮质激素对高钾离子升高嗜铬细胞瘤细胞(PC12细胞)内游离钙浓度([Ca2+]i)作用的影响.结果表明:(1)皮质酮抑制高钾离子诱导PC12细胞[Ca2+]i升高与其预处理细胞时间的长短有关,预处理3 min时,皮质酮开始产生抑制作用;预处理5 min时,其呈现的抑制作用最强;预处理25 min时,抑制作用基本消失.(2)皮质酮在10-8～10-5 mol/L范围内时可呈剂量-效应关系,抑制高钾离子诱导的PC12细胞[Ca2+]i升高,皮质酮浓度为10-5 mol/L时,其产生的抑制作用最大;在10-9mol/L时抑制作用不明显.(3)其它甾体激素如皮质醇、地塞米松、孕酮、雌二醇、睾酮、醛固酮在浓度为10-5 mol/L时,也能抑制高钾离子引起的PC12细胞[Ca2+]i升高,但在同一浓度时作用强度有所不同.胆固醇浓度为10-5 mol/L时,对高钾离子诱导PC12细胞钙浓度升高没有抑制作用.在同一浓度时它们的作用强度顺序大致为:孕酮=皮质醇＞地塞米松＞睾酮＞醛固酮=雌二醇＞＞胆固醇.(4)在皮质酮浓度为10-5mol/L时不能抑制由细胞外无钙到有钙过程引起的PC12细胞[Ca2+]i升高.     

0.7 MPa(7ATA)高气压下大鼠脊髓突触体甘氨酸摄取的变化

目的:研究氮麻醉时甘氨酸神经递质功能变化。方法:制备脊髓突触体,用同位素方法观察0.7MPa(7ATA)高压空气环境中大鼠脊髓突触体摄取甘氨酸的情况。结果:0.7MPa(7ATA)时甘氨酸摄取速度减慢,达到饱和摄取量时间延长,最大饱和摄取量下降。甘氨酸摄取的Vm减小,Km增加。在加入10-7mol.L-1皮质酮后,可增加0.7MPa(7ATA)高压空气时甘氨酸摄取Vm。结论:在高气压下发生氮麻醉时,甘氨酸摄取转运体的功能降低,与甘氨酸亲和力下降;皮质酮有助于高亲和力甘氨酸转运体功能恢复。     

皮质酮对大鼠下丘脑薄片室旁核神经元自发电活动的影响

本实验应用离体大鼠下丘脑薄片技术,用玻璃微电极细胞外记录、观察了下丘脑室旁核神经元的自发电活动,以及皮质酮对自发电活动的影响。在36个下丘脑薄片上观察了104个室旁核神经元的自发电活动,其放电形式主要有三种:慢而不规则(50个,占48.1%)、快速连续(44个,占42.5%)和周期性放电(10个,9.4%)。在进行皮质酮灌流的实验中,这104个单位有25个在皮质酮(10~(-7),10~(-6)mol/L)作用后,自发放电明显减少,有8个单位出现兴奋效应;其余的没有观察到明显反应。上述33个产生反应的神经元其反应特点是:潜伏期短、反应的程度与皮质酮浓度有关、糖皮质激素胞液受体阻断剂 RU38486可以阻断这种反应。结果表明糖皮质激素可以快速影响下丘脑薄片内某些室旁核神经元的电活动,为甾体激素的快速非基因机制作用提供了新的证据,提示室旁核神经元膜上有糖皮质激素受体存在。     

孕酮通过细胞膜机制刺激下丘脑GnRH释放

孕酮刺激下丘脑释放促性腺激素释放激素(GnRH)的分子机制不同于经典的甾体激素两步作用原理,可能通过其它的细胞机制发挥作用。为了探讨孕酮作用于下丘脑细胞可能存在的细胞膜机制,作者将孕酮连接在牛血清白蛋白(BSA)分子上,观察它们在用雌激素维持的去卵巢未成年大鼠的离体视前区-下丘脑前区-下丘脑内侧基底区(POA-AHA-MBA)低冷灌流系统中对 GnRH 释放的影响,结果表明,孕酮-3-羧甲基肟 BSA(P-3-BSA)可使 POA-AHA-MBA 释放 GnRH 逐渐增加,4h 后达到高峰;而孕酮-11α-半琥珀酰-BSA(P-11-BSA)和11-去氧可     

神经活性甾体激素的快速中枢效应

过去认为甾体激素只能由神经系统外的内分泌组织产生。甾体激素的作用原理为经典的基因作用机制。近年来的研究发现脑组织也能产生神经源性的神经甾体激素,而且神经细胞膜表面存在甾体激素结合位点。神经活性甾体激素和脑源性神经甾体激素可通过调制配基门控离子通道受体、Ｇ蛋白耦联受体产生快速中枢效应。     

甾体激素对神经元作用的新机制

甾体激素作用的核机制长期以来人们普遍认为甾体激素通过如下机制发挥作用:甾体激素与其在细胞浆内的受体结合形成激素受体复合物,该复合物活化后,转位到细胞核内,与核蛋白和 DNA 上的特定接受位点结合,抑制特异的基因组,加快某些特异 DNA 的转录速率。最近有资料表明,雌激素及孕激素等甾体的受体位于细胞核内,而糖皮质激素受体在胞浆及胞核内均存在。甾体激素作用的这种基因机制被认为同样适用于神经组织。     

酪氨酸对人离体滋养层细胞孕酮与hCG分泌的影响

本文观察三种剂量(2×10~(-5)mol/L,2×10~(-4)mol/L和2×10~(-3)mol/L)的酪氮酸对离体培养的滋养层细胞孕酮及hCG分泌的影响,并对其抑制效应的机理作了初步探讨。实验结果表明,三种剂量的酪氨酸均可抑制滋养层细胞孕酮分泌(P<0.01),但是,在孕酮分泌受酪氨酸抑制的同时,未见对hCG分泌发生影响(P>0.05),进一步观察了酪氨酸对滋养层细胞3β-羟甾脱氢酶活性的影响,结果表明,酪氨酸能显著抑制3β-羟甾脱氢酶活性,提示酪氨酸对滋养层细胞孕酮生成的抑制作用与抑制3β-羟甾脱氢酶活性有关。     

神经活性甾体对神经元的作用

神经活性甾体是指神经组织中具有活性的甾体激素,根据甾体激素的作用机制可分为三类：（1）通过细胞表面离子通道型受体介导产生效应,这些受体包括GABAA受体,NMDA受体等。（2）通过G蛋白偶联的膜受体指导第二信使反应,再通过DNA结合蛋白,调节基因表达产生效应,（3）通过细胞内受体介导调控基因的表达产生效应,甾体激素的这些效应尤其是对离子通道型受体和G蛋白偶联型受体的调节作用,已引起重视。     

不同靶器官的糖皮质激素细胞受体的功能相同吗?

近年来发现,大鼠的胸腺、肾、白脂肪及肝等不同靶组织的皮质激素受体具有相同的生物学功能:1)不同靶组织结合的活性相同2)与其它甾体激素如孕酮、睾丸酮、雌二醇、地塞米松的竞争抑制物的亲和性以及与这些甾体激素的结合情况也彼此相同。然而,不同的靶细胞对同一种皮质激素却表现出不同效应,例如地塞米松能刺激肾细胞合成磷酸烯醇丙酮酸羰激酶,但激素对脂肪细胞中的该酶的合成有抑制作用,这可能反映了激素对不同靶细胞的作用除通过与受体的结合外,还有其它的作用途径     

Rapid Elevation of Calcium Concentration in Cultured Dorsal Spinal Cord Astrocytes by Corticosterone

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.     

Steroid Inhibition of Neural Micro vessel Morphogenesis In Vitro: Receptor Mediation and Astroglial Dependence

Steroid hormones alter several aspects of microvascular function within the CNS. Both microvessel formation and blood-brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte-endothelial interactions, we studied their effects on astroglial-induced microvessel morphogenesis in vitro. C6 astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary-like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6-induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80-100% at 1 microM. Tetrahydrocortisone and 17 alpha-hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone-mediated inhibition. Astroglial cells were further implicated as the effectors of steroid-mediated inhibition because none of the steroids inhibited astroglial-independent capillary-like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor-mediated mechanism.     

Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus

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.     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

Nongenomic effects of progesterone on the contraction of muscle cells from the guinea pig colon

Progesterone (PG) affects muscle cells by genomic mechanisms through nuclear receptors and by nongenomic mechanisms through unidentified pathways. This study aimed to determine the pathways mediating its nongenomic actions. Experiments were performed in dissociated muscle cells from guinea pig colons. Nongenomic actions were defined as those occurring within 10 min of PG exposure. PG blocked the contraction to CCK-8 and NKA (10(-7) M) but did not impair ACh (10(-7) M) and KCl (2.5 x 10(-2) M)-induced contraction. Both CCK-8 and NKA contract muscle cells by releasing calcium from intracellular stores, whereas ACh and KCl can utilize extracellular calcium. PG also blocked the contraction induced by inositol 1,4,5-trisphosphate, thapsigargin, and caffeine, agents that contract muscle cells by releasing calcium from storage sites. The nongenomic actions of PG were transient because they were absent 1 h after the first PG dose, remaining unresponsive after a second PG dose was administered. Furthermore, PG had no effect on the contraction induced by CCK-8 and thapsigargin in muscle cells from animals pretreated with daily intramuscular PG for 4 days. Cytosolic incorporation experiments of [(3)H]PG showed that pretreatment with unlabeled PG significantly reduced the radiolabeled PG incorporation in the cytosol. We conclude that the nongenomic actions of PG on colonic muscle cells transiently blocked calcium release from storage sites, and this response became rapidly desensitized. This effect does not appear to be specific to PG because other steroid hormones such as aldosterone and testosterone can also induce it.     

Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C

Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [³H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.     

ACh受体在皮质酮引起的大鼠延髓腹外侧区前交感神经元反应中的作用

目的：研究乙酰胆碱（ACh)受体在皮质酮（CORT）对大鼠头端延髓腹外侧区（RVLM）前交感神经元快速效应中的作用,探讨糖皮质激素在交感心血管活动调节中的非基因组机制。方法：本研究采用细胞外记录和微电泳等方法观察CORT对氨基甲酸乙酯麻醉大鼠RVLM前交感神经元的作用,观察分别给予ACh受体拮抗剂阿托品（ATR）、筒箭毒（d-TC)或六烃季铵（C6）后CORT对RVLM前交感神经元的影响。结果：在RVLM共记录到33个前交感神经元,CORT能导致25（76％）个前交感神经元快速兴奋,且具有剂量依赖性,余8个前交感神经元没有反应;其中被CORT兴奋的10个单位微电泳ART后神经元的放电明显下降,但对CORT导致的兴奋作用没有明显的影响。分别向7和6个被CORT兴奋的前交感神经元微电泳d-TC和C6后,单位放电没有变化,同时对CORT导致的兴奋作用无影响。结论：CORT对RVLM前交感神经元具有快速的兴奋作用,这种作用可能并不通过ACh受体介导。